Replicating Vaccines

Birkhäuser Advances in Infectious Diseases
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Philip R. Dormitzer l Christian W. Mandl l
Rino Rappuoli
Editors
Replicating Vaccines
A New Generation
Editors
Dr. Philip R. Dormitzer Dr. Christian W. Mandl
Novartis Vaccines & Diagnostics Novartis Vaccines & Diagnostics, Inc.
Sydney St. 45 Massachusetts Ave. 350
02139 Cambridge Massachusetts 02139 Cambridge Massachusetts
USA USA
[email protected] [email protected]
Dr. Rino Rappuoli

Novartis Vaccines & Diagnostics
S.r.l.
Via Fiorentina 1
53100 Siena
Italy
[email protected]
ISBN 978 3 0346 0276 1 e ISBN 978 3 0346 0277 8

DOI 10.1007/978 3 0346 0277 8
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Preface
Live vaccines were the first vaccines to be used in prevention of infectious diseases,
and they are among the most successful vaccines in the more than 200-year history
of modern vaccination. To just mention a few of the most prominent examples: live
vaccines against smallpox (vaccinia virus, from which the term vaccine is derived),
yellow fever (17D strain), polio (Sabin), and tuberculosis (Bacillus Calmette-
Guérin BCG) have literally changed the course of history. These vaccines cause
mild or subclinical infections, which closely mimic natural infection by the wild-
type pathogens. In many cases, this kind of vaccination elicits long-lasting and
comprehensive immune responses without a need for booster immunizations or the
inclusion of immune-stimulatory substances (adjuvants). However, live vaccines
can often also be burdened by the threat of causing vaccine-induced disease, which
may particularly affect immunocompromised individuals or result from spontane-
ous genetic reversions to a more virulent phenotype. These risks, even if very small,
are considered increasingly unacceptable nowadays within a society that expects
preventive medicine to be essentially risk-free.
Safety considerations together with an exploding increase of scientific capabil-
ities for recombinant expression and characterization of proteins have shifted the
field of vaccine science towards the development of subunit vaccines during the
past decades. This development is accompanied by a rapid growth of our under-
standing of the innate and adaptive immune response and has led to new types of
immune-stimulatory substances. These substances could overcome the limitations
of subunit vaccines by shaping and enhancing the immune response. The capability
to sequence entire genomes has produced the field of reverse vaccinology, which
allows identification of new protein subunit vaccine components. The tremendous
advances in understanding protein structure have recently created the new area of
structural vaccinology, which introduces the concept of rationally designed vaccine
antigens.
Do these advances in protein vaccines and adjuvant design mean that the area
of replicating vaccines is coming to an end? We do not think so, and this book
provides ample evidence to support this conclusion.
v
vi Preface
Essentially, the same technological advances that are propelling the develop-
ment of new recombinant and subviral vaccines are also guiding the rational design
of a new generation of replicating vaccines, which will combine the intrinsic
immunological strengths of this type of vaccine with a flawless safety profile.
Molecular biology and immunology provide a deep understanding of pathogenic
determinants and pathogen host interactions as well as the ability to specifically
modify these factors. Historically, live vaccines were either derived from apatho-
genic natural strains or attenuated by methods of serial laboratory passages in
various host cells, leading to an undirected process of genetic adaptations. The
molecular mechanisms of attenuation were mostly unknown at the time these
vaccines were first widely used. In fact, in many cases, the basis for attenuation
of currently used live attenuated vaccines still is not fully understood. However, we
now witness a quantum leap of technological capacities to specifically modify the
genetic make-up of viruses and bacteria. This ability enables the generation of
rationally designed live vaccines and, beyond that, the development of completely
new types of replicating vaccines, such as vectored vaccines, single-round infec-
tious vaccines, or replicon vaccines. These approaches are linked by the fact that
microbial genome amplification and protein expression take place in the vaccine,
but the production and spread of infectious progeny as well as the vaccines’
interaction with the host defense system are specifically modified to achieve a
maximum of vaccine safety and immunogenicity.
This book’s intention is to span and illustrate with specific examples a large
spectrum of replicating vaccines. We do not attempt to cover the entire field of new
approaches. A complete enumeration would be an almost impossible goal, given
the enormous wealth of creativity that shapes the development of new replicating
vaccines. However, we do intend to provide the reader with a range of typical
examples to paint a comprehensive picture of the existing and arising technologies.
The topics included range from established or recently introduced live vaccines to
novel exploratory approaches, including vectored and replicon based vaccines. In
this context, we chose to apply the term “replicating” more broadly than has been
done by most authors. Traditionally, “replicating” is considered synonymous with
“infectious”, describing a microorganism capable of infecting, multiplying, and
spreading in a host. Thus, replicating, infectious, and live vaccines were clearly
separated from nonreplicating vaccines such as inactivated whole virus, subunit, or
subviral particle vaccines. However, an entire class of new approaches, including
self-replicating nucleic acids (replicons), single-round infectious particles, or con-
ditionally replicating agents, does not fully fit either of these two traditional
definitions. These novel, rationally designed agents can undergo limited or partial
processes of the microbial replication cycle, but they either do not spread to new
cells or spread restricted by certain growth conditions or for only a very few
replication cycles. However, all of these new approaches share the property of
genome replication and protein expression in the host. Growing evidence suggests
that the immune response elicited by such vaccines closely mimics that of more
typical, classic live vaccines. For these reasons, we extend the meaning of “repli-
cating vaccine” to also include vaccines that undergo partial, limited, or defective
Preface vii
pathogen replication cycles, and we have included such vaccines within the scope
of this book, “A new generation of replicating vaccines”. These new types of
replicating vaccines promise to carry the successful concept of live vaccines into
a new era by combining the immunological strengths of live vaccines with the
safety of noninfectious protein vaccines.
The book is structured into four sections, each devoted to another group or aspect
of replicating vaccines. Part I provides an overview of existing and recently intro-
duced live vaccines, highlighting their strengths as well as some limitations and
concerns. These articles illustrate both the tremendous potential for live vaccine
approaches as well as the existing need for improvement with some of these vaccines.
Part II is devoted to the rational design and genetic modifications of microorgan-
isms to generate attenuated vaccine strains. The capability to genetically mani-
pulate bacterial and viral genomes has recently increased by technological leaps in
DNA sequencing and synthesis capacities and the establishment of reverse genetics.
Part III summarizes implications of our increased understanding of host
pathogen interactions on the development of live vaccines. This includes the
molecular analysis of host tropism and innate immune mechanisms. Insights into
how microorganisms interact with cellular components and counteract the host cell
defense mechanisms have resulted in a multitude of new approaches for attenuated
strain development. These approaches include the directed alteration of host tro-
pism, the generation of increased vulnerability to the host defense system, and the
generation of microorganisms that are readily propagated in the laboratory but
cause only abortive infections upon inoculation into the vaccine. Part IV highlights
some of the above mentioned new types of replicating vaccines that carry the
concept of live vaccines a step further. These vaccines include single round
infectious particles (pseudo-infectious), vectored vaccines, replicons, and chimeric
live vaccine strains.
The next decade will see some members of the new generation of replicating
vaccines progress through clinical trials, achieve licensure, and benefit human
health. As with all new technologies, there will be many challenges to be addressed,
including issues of production, stability, safety, and efficacy. It will be exciting to
watch and participate in these new developments, which ultimately will fulfill the
promise of creating a safe and friendly life insurance for the twenty-first century.
Acknowledgment
The Editors are thankful to Diana Boraschi, who has professionally coordinated the
preparation of this volume. Her relentless attention has made possible the realization
of this book, which will provide important insights to the scientific community for
future global vaccination strategies.
Cambridge, MA, USA Philip R. Dormitzer

Cambridge, MA, USA Christian W. Mandl
Siena, Italy Rino Rappuoli
April 2010
Contents
Part I Today’s Live Attenuated Vaccines
Live Vaccines and Their Role in Modern Vaccinology . . . . . . . . . . . . . . . . . . . . . 3

Gordon Dougan, David Goulding, and Lindsay J. Hall
Live Attenuated Vaccines: Influenza, Rotavirus and Varicella

Zoster Virus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15
Harry B. Greenberg and Ann M. Arvin
Classical Live Viral Vaccines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 47

Thomas P. Monath
Part II Genetically Attenuated Micro-Organisms as Vaccines
Recombinant Live Vaccines to Protect Against the Severe Acute

Respiratory Syndrome Coronavirus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 73
Luis Enjuanes, Jose L. Nieto-Torres, Jose M. Jimenez-Guardeño,
and Marta L. DeDiego
Live-Attenuated Shigella Vaccines. Is Encouraging Good Enough? . . . . . . 99

Yves Germani and Philippe J. Sansonetti
New Generation BCG Vaccines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 119

Michael V. Tullius and Marcus A. Horwitz
Part III Manipulating Host-Pathogen Interactions to Make Vaccines
Basic Science Paves the Way to Novel Safe and Effective Pestivirus
Vaccines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 173
Norbert Tautz and Gregor Meyers
ix
x Contents
Live Attenuated Influenza Virus Vaccines: NS1 Truncation

as an Approach to Virus Attenuation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 195
Natalie Pica, Peter Palese, and John Steel
An Attenuated HSV-1 Live Virus Vaccine Candidate That

is Replication Competent but Defective in Epithelial Cell-to-Cell
and Neuronal Spread . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 223
Elizabeth E. Zumbrun and Harvey M. Friedman
Live Attenuated Vaccines for Respiratory Syncytial Virus . . . . . . . . . . . . . . . 237

Michael N. Teng
Live Attenuated Cholera Vaccines: Flagella and Reactogenicity . . . . . . . . 261

D. Ewen Cameron and John J. Mekalanos
Part IV New Types of Replicating Vaccines
Replication-Defective Herpes Simplex Virus Mutant Strains

as Genital Herpes Vaccines and Vaccine Vectors . . . . . . . . . . . . . . . . . . . . . . . . . 285
David M. Knipe
Nucleic Acid-Based Infectious and Pseudo-Infectious Flavivirus

Vaccines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 299
Justin A. Roby, Roy A. Hall, and Alexander A. Khromykh
Application of Cleavage Activation Mutants of Influenza Virus

as Live Vaccines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 321
Juergen Stech and Hans-Dieter Klenk
Alphavirus Particle-Based Vaccine Vectors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 331

Scott J. Balsitis, Clayton W. Beard, and Peter W. Mason
Recombinant, Chimeric, Live, Attenuated Vaccines

Against Flaviviruses and Alphaviruses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 349
Thomas P. Monath
Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 439
Contributors
Ann M. Arvin Departments of Medicine, Pediatrics and Microbiology and Immu-

nology, Stanford University School of Medicine, Stanford, CA 94305, USA
Scott J. Balsitis Novartis Vaccines and Diagnostics, 350 Mass Ave, Cambridge,
MA 02139, USA
Clayton W. Beard Novartis Vaccines and Diagnostics, 350 Mass Ave,

Cambridge, MA 02139, USA
D. Ewen Cameron Department of Microbiology and Molecular Genetics, Harvard

Medical School, 200 Longwood Avenue, Boston, MA 02115, USA
Marta L. DeDiego Centro Nacional de Biotecnologia (CNB), CSIC, Campus

Universidad Autonoma, Darwin 3, Cantoblanco, 28049 Madrid, Spain
Gordon Dougan The Wellcome Trust Sanger Institute, The Wellcome Trust
Genome Campus, Hinxton, Cambridge CB10 1SA, UK, [email protected]
Luis Enjuanes Centro Nacional de Biotecnologia (CNB), CSIC, Campus Univer-

sidad Autonoma, Darwin 3, Cantoblanco, 28049 Madrid, Spain, L.Enjuanes@cnb.
csic.es
Harvey M. Friedman Infectious Disease Division, Department of Medicine,

University of Pennsylvania School of Medicine, 502, Johnson Pavilion,
Philadelphia, PA 19104-6073, USA, [email protected]
Yves Germani Unitié Pathogénie Microbienne Moléculaire INSERM U786/

Réseau International des Instituts Pasteur, Institut Pasteur, 28 rue du Dr Roux,
Cedex 15, 75724 Paris, France, [email protected]
xi
xii Contributors
David Goulding The Wellcome Trust Sanger Institute, The Wellcome Trust
Genome Campus, Hinxton, Cambridge CB10 1SA, UK
Harry B. Greenberg Joseph D. Grant Medicine and Microbiology & Immunology,

Stanford University School of Medicine, 291 Campus Drive LKSC Bldg Room
LD3C02, Stanford, CA 94305-5101, USA, [email protected]
Lindsay J. Hall Alimentary Pharmabiotic Centre, Biosciences Institute, Universi-

ty College Cork, Cork, Ireland
Roy A. Hall Centre for Infectious Disease Research, School of Chemistry and
Molecular Biosciences, University of Queensland, Brisbane, QLD 4072, Australia
Marcus A. Horwitz Division of Infectious Diseases, Department of Medicine,

School of Medicine, University of California Los Angeles, 10833, Le Conte
Avenue, Los Angeles 90095, CA, USA, [email protected]
Jose M. Jimenez-Guardeño Centro Nacional de Biotecnologia (CNB), CSIC,

Campus Universidad Autonoma, Darwin 3, Cantoblanco, 28049 Madrid, Spain
Alexander A. Khromykh Centre for Infectious Disease Research, School of

Chemistry and Molecular Biosciences, University of Queensland, Brisbane, QLD
4072, Australia, [email protected]
Hans-Dieter Klenk Institute of Virology, Philipps-University, Marburg,

Germany, [email protected]
David M. Knipe Department of Microbiology and Molecular Genetics, Harvard

Medical School, 200 Longwood Avenue, Boston, MA 02115, USA, david knipe@
hms.harvard.edu
Peter W. Mason Novartis Vaccines and Diagnostics, 350 Mass Ave, Cambridge,
MA 02139, USA, [email protected]
John J. Mekalanos Department of Microbiology and Molecular Genetics,

Harvard Medical School, 200 Longwood Avenue, Boston, MA 02115, USA,
[email protected]
Gregor Meyers Institut für Immunologie, Institute for Animal Health, Friedrich-
Loeffler-Institut, Paul-Ehrlich-Str. 28, 72076 Tübingen, Germany, gregor.
[email protected]
Thomas P. Monath Kleiner Perkins Caufield & Byers LLC and Harvard School
of Public Health, 295 Townsend Hill Road, Townsend, MA 01469, USA,
[email protected]
Contributors xiii
Jose L. Nieto-Torres Centro Nacional de Biotecnologia (CNB), CSIC, Campus

Universidad Autonoma, Darwin 3, Cantoblanco, 28049 Madrid, Spain
Peter Palese Department of Microbiology, Mount Sinai School of Medicine, New

York, NY 10029, USA, Department of Medicine, Mount Sinai School of Medicine,
New York, NY 10029, USA
Natalie Pica Department of Microbiology, Mount Sinai School of Medicine, New

York, NY 10029, USA, [email protected]
Justin A. Roby Centre for Infectious Disease Research, School of Chemistry and
Molecular Biosciences, University of Queensland, Brisbane, QLD 4072, Australia
Philippe J. Sansonetti Unitié Pathogénie Microbienne Moléculaire INSERM

U786, Institut Pasteur, 28 rue du Dr Roux, Cedex 15, 75724 Paris, France
Juergen Stech Friedrich-Loeffler-Institut, Greifswald Insel Riems, Germany
John Steel Department of Microbiology, Mount Sinai School of Medicine, New

York, NY 10029, USA
Norbert Tautz Institute for Virology and Cell Biology, University of Lübeck,
Ratzeburger Allee 160, 23562 Lübeck, Germany, [email protected]
Michael N. Teng Divisions of Translational Medicine and Allergy & Immunology,

Department of Internal Medicine, Nanomedicine Research Center, University of
South Florida College of Medicine, 12901, Bruce B. Downs Blvd, Tampa, FL
33612, USA, [email protected]
Michael V. Tullius Division of Infectious Diseases, Department of Medicine,

School of Medicine, University of California Los Angeles, 10833, Le Conte
Avenue, Los Angeles 90095, CA, USA
Elizabeth E. Zumbrun Center for Aerobiological Sciences, US Army Medical

Research Institute of Infectious Diseases, Fort Detrick, Frederick, MD 21702, USA
Part I
Today’s Live Attenuated Vaccines
Live Vaccines and Their Role in Modern
Vaccinology
Gordon Dougan, David Goulding, and Lindsay J. Hall
Abstract Since the invention of vaccination by Jenner live vaccines have been key
components of immunization programs. However, in the modern era the justifica-
tion for the role of live vaccines is worth re-evaluating. Here we discuss, using
specific examples, about how the use and development of live vaccines will be
managed in the genomics era.
1 Introduction
The use of living microorganisms as a basis for immunization has been central to
the development of vaccines. Indeed, the very first recognized vaccine against
smallpox, developed by Edward Jenner, was based on a live inoculum containing
poxviruses. Live vaccines offer a potential advantage in that they can theoretically
stimulate the immune system in a manner that more closely mimics that encoun-
tered during infection where immunity is acquired more “naturally” (Fig. 1). This is
in contrast to the use of the inactivated or purified components of the infecting
agent. Of course, this simplistic view does not completely hold up to the rigors of
modern scientific appraisal but there are kernels of truth in this hypothesis. Indeed a
significant proportion of vaccines registered for use in humans over the years are
“live”. However, it would be fair to say that in the modern world legislators and many
scientists would prefer to replace all live vaccines with nonliving antigens. Thus, the
approach is gradually falling out of favor as we learn more about how to stimulate
appropriate immunity using better and more defined vaccine formulations. The trends
working against the live vaccine approach include the requirement for a better
G. Dougan (*) and D. Goulding,

The Wellcome Trust Sanger Institute, The Wellcome Trust Genome Campus, Hinxton, Cambridge
CB10 1SA, UK
e mail: [email protected]
L. J. Hall
Alimentary Pharmabiotic Centre, Biosciences Institute, University College Cork, Cork, Ireland
P.R. Dormitzer et al. (eds.), Replicating Vaccines, 3

Birkh€auser Advances in Infectious Diseases,
DOI 10.1007/978 3 0346 0277 8 1, # Springer Basel AG 2011
4 G. Dougan et al.
Fig. 1 A transmission electron micrograph showing attenuated salmonella living inside a human
cell
definition of vaccine composition and higher safety standards. Both of these factors
work against a live approach as antigen definition is significantly more challenging in
a live entity and it is difficult to convince legislators and scientists alike that anything
living does not have the capacity to evolve a more virulent phenotype, especially as so
many modern vaccines harbor some element in an immunocompromised state.
Nevertheless, many licensed vaccines are based on live antigens, including rubella
and Sabin polio, and appraisal of the field is still warranted.
2 Live Vaccines, a Brief History
Originally, the microorganisms on which live vaccines were developed originated

from two sources. One approach was to identify a related microbe in a host other
than humans and “adapt” this to humans for vaccination purposes, sometimes using
a passage through an intermediary host. The theory behind this approach is that
many pathogens exhibit a phenomenon known as host restriction or adaptation, in
that they are more virulent in one host species than another. In the case of Jenner’s
smallpox vaccine he reputedly exploited material harboring a Poxvirus adapted to
cattle to immunize humans against smallpox. Indeed, he was able to demonstrate
immunity to smallpox using a direct challenge [1]. The smallpox vaccine in use
today is based on Vaccinia virus, which we know is related to Variola, the cause of
smallpox, but is genetically very distinct. Modern molecular studies have analyzed
Live Vaccines and Their Role in Modern Vaccinology 5
the differences in the genetic makeup of Vaccinia versus Variola viruses and they
are indeed significant and there are, thus, many reasons why Vaccinia is attenuated
in humans [2, 3]. Indeed, the Vaccinia virus used for vaccination may have acquired
further attenuating mutations as it has been passaged over the years and here lies
another potential problem with live vaccines, genetic drift.
The second approach is to passage a microbe that has a virulent form in humans
over a long time period in an alternative host or even through some sort of in vitro
system. In fact, a combination of both approaches is often employed. Indeed BCG,
the current tuberculosis vaccine based on virulent Mycobacterium bovis, was
obtained using extensive in vitro passage through laboratory media. BCG is
known to differ genetically from parental M. bovis, although the direct ancestral
M. bovis was lost. Genome analysis on BCG has identified a series of potentially
attenuating lesions in the M. bovis genome. Further, it has been shown that different
vaccine lots of BCG stored in different companies or geographical locations have
accumulated different sets of genetic lesions, illustrating the perils of genetic drift.
Indeed, this drift may in part explain differences in efficacy observed with BCG-
based vaccination programs over the years [4 6].
Although the above approaches were extremely productive over the past century
or so for providing a source of vaccines the approach is now obsolete and more
rigorous genetic and quality control approaches will be required to generate any
future live vaccines suitable for licensing. The modern science of genomics will
demand a full genetic validation and a rigorous seed lot system for any vaccine for
use in humans and it is from this perspective that we will continue this review.
3 A Brief Summary of the Modern Perspective
Before continuing it is worth providing a brief summary of the state of play in the
live vaccine field. At the present time we have several live vaccines that are still
extensively used as well as others that have a significant “track record” in humans,
including several based on different poxviruses. Thus, the existing live vaccines are
an accepted class that, because of a long safety record, do not have to fit into the
potential requirements for any completely new live vaccine. BCG will serve as an
example of such a vaccine. Even though these vaccines are likely to be used long
into the future they could be subjected to a more rigorous quality regimen. For
example, it is possible that genome sequencing or functional genomic studies could
be exploited to improve the reproducibility of the manufacture of these vaccines at
different sites and by different companies. Such an approach could be undertaken in
an attempt to tackle issues such as genetic drift. Vaccine production lots could be
sequenced to identify mutant lots, although such an approach would be resisted by
some manufactures as a “can of worms” that could potentially be opened!
It is difficult to imagine that any new live vaccine would escape such rigorous
analysis in terms of quality. Using modern approaches it will be possible to validate
the genetic and biochemical make up of live vaccine lots at a level that was
6 G. Dougan et al.
previously unimaginable. Routine whole genome sequencing could be relatively

easily applied to vaccine lots and even functional genetic studies such as RNA
sequencing and proteomics could be applied. Further, longer term studies at a whole
genome level on genetic stability (down to the accumulation of SNPs, indels and
rearrangements) could be requested. What we are alluding to here, is a better
validation of the genetic composition of any live vaccines. Thus, live vaccine
design in the future will move towards a more rational approach.
4 Rational Attenuation
As illustrated above, scientists previously relied on natural selection to drive the

generation of attenuated microbes suitable for use as live vaccines. This natural
selection could have occurred truly in the wild as pathogens adapted to a lifestyle in
different host species. Alternatively natural selection could have been artificially
driven by scientists forcing a pathogen to adapt to a novel environment such as a
laboratory growth medium. We know from modern genome studies that such
adaptations can be rapid and almost continuous as different genetic and selective
forces are brought to bear on genomes. For example, a range of different genetic
changes accumulate as even laboratory adapted Escherichia coli K12 are passaged
on laboratory media [7, 8]. We know from studies on the vaccine strains of polio
that simple SNPs were responsible for both attenuation and the pattern of reversion
to virulence as the virus was reintroduced into the human host during live vaccina-
tion [9, 10]. We also know that some viruses, particularly those with RNA-based
genomes, exhibit a significant level of natural genetic flux. This flux presents
challenges to defining a consensus genome and here quasi-populations have to be
taken into account. Thus, taking all these factors into consideration, we can
retrospectively monitor the stability of mutations as well as explore the potential
value of any mutation as attenuators.
As we continue to build up our understanding of the molecular basis of virulence
we increase our ability to select mutations as candidates for creating attenuated
forms of any pathogen. We can explore the role that individual genes or even
nucleotides play during the infection process and hence have an opportunity to
interfere with infection at particular stages of the process. Obviously, this will have
a knock on effect for immunity as we can explore the ongoing immune response
during infection and try to identify key points at which immunity is stimulated prior
to the onset of clinical disease. Pathogens will try to modulate immunity to gain an
advantage in the host and we can explore methods whereby we can hijack these
characteristics and modify them to self defeat the pathogen.
All of these approaches can be summarized by the term “rational attenuation”.
By this, we can consider introducing defined mutations into the genome of a
pathogen that generate a rationally attenuated microbe that is highly immunogenic,
induces protective immunity but does so without endangering or incapacitating the
recipient vaccinee. This term is useful as a definition but is not accepted by all
workers in the field as the term rational is open to philosophical scrutiny. Never-
theless, we will use it to describe modern approaches to attenuation.
5 Creating a Rationally Attenuated Vaccine
Any new live vaccine will most likely have to be fully characterized in terms of
genomic architecture (at the genome sequence level) and the genetic basis for
attenuation will have to be fully defined. Hence, in these cases rational attenuation
will involve the generation of defined attenuating mutations on a fully sequenced
genetic background. It may be conceptually difficult to achieve this goal starting
with an avirulent form of microbe, such as a commensal as they are already
attenuated. However, methods to demonstrate safety and a consistent level of
attenuation/immunity will be required. Commensals, by their own definition, are
bacteria that colonize an individual without normally causing disease. Working
with these vaccine vehicles would be expected to circumvent some of the safety and
environmental issues associated with wide scale immunization regimens based on
genetically engineered attenuated pathogens. Some commensals, however, do have
the capacity to cause disease, particularly if their host is compromised in some way
[11]. Indeed, commensals which are not pathogenic in humans can harbor genes
encoding potentially toxic proteins, as disease manifestation often involves the
coordinated interaction of many genes, and toxins alone are not sufficient for the
expression of full virulence. The use of molecular approaches such as whole genome
sequencing and comparative genomics is therefore of value to identify any potential
“pathogenic” loci such as those encoding toxins, which can then be removed by
reverse genetics prior to their use as vaccines. In contrast, by starting with a fully
virulent host microbe it should be possible to demonstrate a clear degree of attenua-
tion using model systems but safety studies in humans will always be required.
There are now a significant number of different candidate vaccines that have
been created and tested based on some form of rational attenuation. Some of these
were created before whole genome sequencing became routine, but for viruses in
particular, this approach has been in place for some time. There are many specific
examples of such live vaccines presented in more detail throughout this volume so
it is not appropriate to go through multiple examples here. The authors, instead,
focus on their area of particular expertise.
6 Creating Rationally Attenuated Live Enteric Bacterial

Vaccines
There has been an historical interest in the generation of attenuated enteric bacterial
strains that can form the basis of live oral vaccines. This is in part driven by the
observation that full protection against many mucosal pathogens in the intestine
(and potentially at other body surfaces) requires some form of mucosal immunity,
8 G. Dougan et al.
often coupled with a systemic immune response [12]. Again, this is not universally
accepted but it is certainly a factor that has driven the field. Enteric pathogens can
target different sites in the intestine and can exhibit differences in their abilities to
invade tissues. For example Vibrio cholerae primarily targets the small intestine
whereas Shigella species target the colon. V. cholerae is hardly invasive at all
whereas pathogens such as Salmonella typhi, the cause of typhoid, are highly
invasive and cause primarily systemic diseases. Nevertheless, many attempts
have been made to create attenuated vaccine strains with differing degrees of
genetic definition.
7 Typhoid as an Example
The only licensed live oral typhoid vaccine is based on a S. typhi strain known as
Ty21a, which was created empirically using chemical mutagenesis. Although the
genetic basis of attenuation was thought by some to be a mutation in the galE gene
it was subsequently shown that galE mutants of S. typhi can retain some significant
degree of virulence and can cause typhoid [13 15]. These observations stimulated a
search for a rational approach to genetic attenuation in S. typhi and a myriad of
candidate attenuating genes have been identified leading to candidate vaccines
[16 19]. One of the first rationally attenuated S. typhi was a strain harboring
mutations in both the chorismate (aro) and purine (pur) pathways. The chorismate
pathway is the only biosynthetic route by which bacteria can synthesize the
aromatic ring whereas the purine pathway is required for nucleic acid biosynthesis.
As the availability of aromatic compounds and purines is limiting in the human host
these mutants starve in vivo and are consequently attenuated. However, S. typhi aro
pur double mutants were found to be over attenuated and consequently poorly
immunogenic in humans, properties that were reproduced in mice [20]. In contrast,
S. typhi harboring two defined deletion mutations in the aro pathway were subse-
quently shown to be less attenuated and more immunogenic in humans [21].
Unfortunately, they were also somewhat reactogenic and viable bacteria were
found in the blood of volunteers. Hence, further mutations were required to create
an immunogenic but less aggressive strain.
A S. typhi strain known as M-01ZH09 has recently completed phase II studies as
a single-dose live oral typhoid vaccine and is being prepared for an efficacy study in
a typhoid endemic region. M-01ZH09 harbors mutations in the chorismate pathway
(two independent aro mutations) and in a gene ssaV involved in adaptation to
survival in macrophages (a gene in the Salmonella Pathogenicity Island SPI2). The
SPI2 mutation (ssaV) was added because this mutation destroyed the ability of
bacteria to survive in macrophages/blood and this addition rationally removed the
vaccinaemial phenotype [17, 22]. Hence, attenuation is driven by starvation for
essential nutrients and by a missing step in the pathogenic process (survival in
macrophages) [18]. M-01ZH09 was created after a series of experiments in mice, in
other animals and in human volunteers [17, 18, 23]. Hence, in this case a rational
approach benefited live vaccine design. A critically important feature of any live
vaccine is that excessive replication and potential virulence is not present when the
immunizing strain enters an immunocompromised, or indeed any form of compro-
mised host. Hence, it would be desirable to build in mutations, which are attenuat-
ing even in the absence of a vigorous immune response. Significantly, the SPI2
mutations described above for S. typhi are attenuated even in severely compromised
hosts such as those defective in an interferon response [18].
Similar rational attenuation approaches have been exploited with other enteric
pathogens. V. cholerae strains lacking active cholera toxin have been created and
these have shown some promise as live oral cholera vaccines [24, 25]. However,
issues such as poor immunogenicity in the field have compromised the wide scale
use of these vaccines, at least to date. Many laboratories have tried to create
Shigella (dysentery) vaccines based on attenuated Shigella species. Although this
work has been in progress in one form or another for over 50 years no vaccine of
this type has been licensed. This is, in part, because it has proved impossible to
design a Shigella vaccine which is both immunogenic and nonreactogenic [26]. At
the moment all immunogenic vaccine candidates have proved to be too reactogenic
in early human studies [27]. Thus, we need to dissect immunogenicity away from
reactogenicity to move this stalled field forwards.
8 Mice Are Not Men
Moving vaccination regimens between species presents problems at the best of

times but there are particular problems in the case of live vaccines. Many pathogens
and even microbes in general exhibit some degree of host adaptation or even
restriction. Hence live vaccines worked up in a particular model species may
struggle when moved into the human target. Of course these problems are not
restricted to live vaccines, a good illustration is the relative failure of DNA
vaccination in humans, but it is real. Most candidate vaccines are developed to a
significant degree in mice before being transferred to humans either directly or
through an intermediate clinical test species such as nonhuman primates. A way
around this problem is to use a surrogate vaccine species adapted to the nonhuman
to develop the vaccine and then transfer the knowledge gained to the target human
adapted species. A good example of this approach is with Salmonella vaccines
where much of the vaccine development is performed using the surrogate species
S. typhimurium before transferring the developed approach (including selection
of attenuating mutations) into the human restricted S. typhi. With viruses compara-
tors would be cattle versus human RSV, cattle versus human rotaviruses and
SIV/HIV based vaccines. Surrogate approaches are fraught with potential problems
but in general they are the only obvious route available where a good immuno-
logical correlate of protective immunity is not available. Host restriction barriers
are generally multi-factorial and can involve immunomodulation, potentially
compromising immunological readouts [28, 29]. Another property of any live
10 G. Dougan et al.
vaccine that is difficult to predict between species is reactogenicity. The level of

attenuation can differ significantly between compromised hosts and unlike humans,
animals cannot articulate their feelings. Careful monitoring of animals, including
gait, temperature and physiology can help predict where potential reactogenicity
problems might exist.
9 The Delivery of Antigens by Live Vectors
Live vaccines were originally designed to elicit protection against one species. This
could be the same pathogen in terms of species, as with live polio vaccine or it could
be an immunologically related pathogen as with BCG and smallpox vaccine.
However, through genetic engineering it is possible to consider any live vaccine
as a potential delivery vehicle for any antigen from any pathogen. This is the live
vector concept. Antigens from pathogenic viruses, bacteria and even helminths
have been expressed in heterologous live vaccine vehicles. The heterologous
antigen can be delivered as an expressed antigen or even in the form of DNA/
RNA designed to be expressed in the host [30 33]. Immunogenicity can primarily
be targeted at the heterologous antigen and not the vehicle. Alternatively, when
utilizing a live vector the aim can be to induce immunity against both the delivery
vehicle and the targeted heterologous antigen. This is particularly attractive as you
can obtain a “2-for-1” vaccine against disease.
Live vaccine delivery vehicles have been built on derivatives of viruses, bacteria
or even parasites [34 36]. Many are designed to undergo a limited period of
replication within the host but others are essentially nonreplicative and serve as a
means to target antigen to the correct tissue or intracellular target, optimal, for
example, for inducing cytotoxic T cell responses [37 39]. A further consideration
for heterologous antigen delivery is the mode of expression of the heterologous
antigen. Expressing a foreign antigen can impact on the competitiveness or fitness
state of any replicating entity and it is important that this does not unduly impact on
immunogenicity. This issue has been extensively investigated in live bacterial
vaccine delivery systems. Here factors such as gene stability (chromosomal versus
plasmid location), timing of expression and the eventual location of the expressed
antigen (inside or outside the cell) have been considered (Fig. 2). One approach has
been to exploit the use of promoters that only become significantly activated once
the vaccine vehicle has entered the host (so-called in vivo inducible). This can
potentially enhance stable expression and immunogenicity of foreign antigens,
while reducing the metabolic load during vaccine preparation. Different delivery
vehicles may require distinct forms of “fine tuning” in order to yield optimal
immunogenicity [31, 40]. A good example is the use of the anaerobically induced
nirB promoter in S. typhi [41].
One of the potential advantages of exploiting a live vehicle for antigen delivery
is their ability to potentially induce both local and cellular immune responses in the
host. Many vehicles can stimulate IgA production when delivered mucosally and
Fig. 2 A Salmonella
typhimurium expressing the
Vi capsular antigen of S. typhi
as a clearly visible example of
a bacterial vector engineered
to express a heterologous
antigen. The Vi antigen is
detected by immunogold
labeling of anti Vi antibodies
some can also promote potent cytotoxic responses to carried antigens. These are
attractive features and ones which have encouraged the more recent work on this
approach to vaccination. Live vehicles have also been exploited in conjunction with
other vaccination regimens, including DNA vaccination, in so called prime-boost
approaches. Prime boosting has proved to be particularly useful for generating
effective immune responses against challenging pathogens such as HIV, M. tuber-
culosis and malaria [33, 42 45]. One of the aims of using a prime boost approach is
to obtain a mixed immunological response in the vaccine, including potentially
humoral and cellular immunity or simply to bias immunity to a Th1 rather than a
Th2 response [46]. An alternative approach has been to incorporate the expression
of host immunological effectors or regulators such as cytokines from the vaccine
vehicle, although there are specific safety issues associated with the delivery of
immunologically active self antigens [47 49]. Nevertheless, this approach con-
tinues to receive attention especially within the field of cancer immunotherapy.
10 Conclusions
Live vaccine development continues to be an area of significant investigation both in

the academic and industrial vaccine communities. Immunization with live vaccines
can potentially stimulate key components of the cellular immune response. Safety
remains an issue for serious consideration and a holistic genomic approach may be
needed for the future quality control of live vaccines.
Acknowledgments This work was supported by The Wellcome Trust.

12 G. Dougan et al.
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Live Attenuated Vaccines: Influenza, Rotavirus
and Varicella Zoster Virus
Harry B. Greenberg and Ann M. Arvin
Abstract Since vaccinia virus was first used to protect against smallpox in the
eighteenth century, live attenuated vaccines have proved to be highly effective in
reducing the morbidity and mortality caused by many human viral pathogens.
Contemporary live viral vaccines are designed using several different strategies
to achieve attenuation. These basic principles and approaches are illustrated by
vaccines to prevent rotavirus, influenza and varicella-zoster virus infections that are
described in this chapter. As shown from the experience with these three vaccines,
contemporary live attenuated viral vaccines have had a major impact on disease
caused by these ubiquitous human pathogens.
1 Introduction
The value of live viral vaccines was established historically by the recognition that
inoculation with vaccinia virus protected against smallpox. In this case, a closely
related but much less virulent pathogen of cattle elicited protective immunity in
people. Contemporary live viral vaccines are designed using several different strate-
gies to achieve attenuation. These basic principles and approaches are illustrated
by vaccines to prevent rotavirus, influenza and varicella zoster virus (VZV) infec-
tions that are described in this chapter. In the case of VZV, influenza and one of
the current rotavirus vaccines, attenuation is accomplished through laboratory
H.B. Greenberg (*)

Joseph D. Grant Medicine and Microbiology & Immunology, Stanford University School of
Medicine, 291 Campus Drive LKSC Bldg Room LD3C02, Stanford, CA 94305 5101, USA
A.M. Arvin
Departments of Medicine, Pediatrics and Microbiology and Immunology, Stanford University
School of Medicine, Stanford, CA 94305, USA

16 H.B. Greenberg and A.M. Arvin
manipulations of a naturally occurring “wild type” parental strain recovered from

an infected individual. For one of the rotavirus vaccines, a “Jennerian” approach,
similar to that used for smallpox, has been used. In all cases the goal of attenuation
is to preserve sufficient replicative capacity so that the vaccine virus will induce a
robust and broad adaptive immune response similar to the natural infection, but
not the illness expected, after inoculation of the wild type virus. For this purpose,
the attenuated virus must retain infectivity at the site of inoculation, whether the
vaccine is given by oral or intranasal mucosal inoculation, as is the case for the live
attenuated rotavirus and influenza virus vaccines, or by subcutaneous injection of
VZV vaccines. Some live attenuated viral vaccines are associated with mild
symptoms, such as fever or rash in some recipients but the tropisms of the parent
virus that typically damage host cells are disrupted. VZV vaccines are derived from
a clinical isolate that was attenuated by the traditional approach of sequential
passage in human and nonhuman cells and by adapting the virus to grow at low
temperature. The rotavirus vaccine make by GSK was also attenuated by multiple
passage of wild type human rotavirus in cell culture. The other rotavirus vaccine
and the influenza vaccine’s attenuation relies on the inherent capacity of these
viruses to undergo reassortment. This strategy can be adapted to achieve recombi-
nants that have genes from a related, nonhuman virus as in the case of rotavirus or in
which genes from virulent strains are inserted into a “backbone” consisting of genes
that have acquired attenuating mutations by cold adaptation or other methods, as
was done to create live attenuated influenza vaccines. The ability of the vaccine
virus to replicate in the human host becomes restricted as a consequence of these
laboratory manipulations. However, it is critical that live attenuated vaccine viruses
retain their genetic stability, to assure both that adaptive immunity comparable to
that elicited by the wild type virus is maintained and that replication in the host does
not produce a reversion to the virulence of the wild type virus. Since live attenuated
viral vaccine strains may be transmissible, genetic stability must also be retained
despite replication in secondary contacts. Once attenuation has been achieved, the
development of live attenuated viral vaccines requires defining the optimal infec-
tious dose and dosage regimen to elicit adaptive immunity against the pathogen.
Finally, their potential to provide protection against infection must be confirmed in
large-scale efficacy trials. As shown from the experience with rotavirus, influenza
and VZV vaccines, live attenuated viral vaccines have major benefits for reducing
the morbidity and mortality caused by these ubiquitous human pathogens.
2 Influenza
2.1 Introduction
Influenza is the major cause of epidemic and pandemic severe respiratory disease in
people of all ages in all areas of the world. Influenza is also an important natural
pathogen of other animal species, including birds, horses and pigs. Natural infection
Live Attenuated Vaccines: Influenza, Rotavirus and Varicella Zoster Virus 17
with wild-type influenza virus elicits a long-lasting immune response that protects
the individual from influenza illness following re-exposure to the same, or very
similar, strain of influenza but not from influenza strains that are antigenically
distinct from the infecting strain. As influenza virus evolves it undergoes genetic
changes in all its genes, including those encoding the major antigens on the virion
surface [the hemagglutinin (HA) and neuraminidase (NA) glycoproteins], which
are targets of protective immunity. Because the virus can undergo antigenic drift
and shift, it may cause multiple symptomatic infections throughout a lifetime.
Inactivated influenza vaccines were first put into use over 50 years ago for military
personnel and have been in general use for more than 30 years. Although inacti-
vated vaccines are generally safe and effective, there is room for improvement,
especially in very young children and elderly adults and in situations where the
vaccine strain is “antigenically mismatched” with the circulating strain. In order
to address some of the deficiencies of the inactivated influenza vaccine, live
attenuated influenza vaccines (LAIV) have been developed.
2.2 Virology, Epidemiology and Pathogenesis
The influenza viruses are members of the Orthomyxoviridiae family, characterized

by a negative sense, single-stranded, eight segment RNA genome surrounded by a
lipid membrane. The two major surface glycoproteins, the HA and NA, are inserted
into the outer lipid membrane and determine the serologic classification of
the specific viral strain. Three genera of influenza virus (A, B, and C) circulate in
humans but types A and B cause most of the morbidity and mortality and these two
types are those that are currently incorporated into the various licensed vaccines.
Influenza naturally infects humans as well as several other animal species including
avian species including poultry, pigs and horses. These animal hosts often serve
as a reservoir for the evolution of new pandemic strains via gene reassortment
interactions.
Worldwide, influenza viruses cause considerable morbidity and mortality every
year, with an estimated 35,000 deaths in adults >50 years old and an average of
114,000 excess hospitalizations each year in the United States [1]. The seasonal
winter outbreaks of influenza result from antigenic drifts that occur every few years
in each of the three (or four) major influenza viruses, the two A strains (H3N2,
H1N1), and one or more B strains, that are now circulating and the introduction of
new susceptibles into the population. Influenza viruses are spread by inhalation of
viral particles aerosolized by coughing and sneezing [2]. The pathogenesis
of influenza virus infection begins with infection of the respiratory mucosal epithe-
lium. Influenza is an acute febrile illness associated with myalgias, headache,
cough, rhinitis, and otitis media, which is usually self-limited but can progress
to pneumonia. Generally, the risk of influenza morbidity and mortality is highest in
persons >65 years old, young children under 5 years, and persons with chronic
cardiac or pulmonary disease or immunocompromising conditions. As was seen in
the recent pandemic of variant H1N1 virus, pandemic strains can occasionally
cause increased morbidity or mortality in healthy young adults as opposed to the
elderly. Because first encounters with influenza often cause lower respiratory tract
infection, the hospitalization rate for influenza is 100 per 100,000 children aged 0 4
years [1] High infection rates in children of school age also facilitate influenza
spread. Influenza pandemics result from antigenic shifts associated with reassort-
ment events or emergence of new strains from avian reservoirs, as occurred in 1918,
1957, 1968 and 2009. Under these conditions, an influenza virus with an HA and/or
NA that had not previously infected humans and that can infect, cause disease and
be transmitted efficiently, is introduced into a large naı̈ve population. At any given
time, the potential risks of a new pandemic are virtually impossible to estimate
accurately but such a pandemic constitutes a major public health emergency as
occurred with the recent emergence of the novel variant H1N1 strain in 2009.
2.3 Immunology
Immune responses to a wild-type influenza infection are robust and leave the
individual with a strong immunological memory that prevents the same or an
antigenically similar variant from causing disease for decades. The response can
be measured in many different compartments, including IgG and IgA antibodies in
the serum, secretory IgA in the nasal secretions, and T, B and NK cells in the
peripheral blood as well as various lymphoid tissues, especially those in the
respiratory tract. Functional antibodies that neutralize the virus or prevent it from
binding its cognate receptor are designated hemagglutination inhibiting (HAI)
antibodies and can be found in the serum and occasionally in nasal secretions.
Cellular immune responses and additional antibody responses target a variety of
regions on the viral HA and NA surface glycoproteins and other proteins encoded
by the virus, particularly M, NP, and NS. The quantity of HAI and the amount of
neutralizing antibody in serum have been correlated with the extent of protection
from disease; some evidence indicates that the serum titer of antibodies to NA is
also correlated with protection. Despite the presence of these multiple components
of immunological memory and effector function and substantial information corre-
lating some of these responses with protection, the fundamental role each has in
preventing illness following re-exposure to influenza remains to be elucidated.
The immune response to inactivated influenza vaccine has been extensively
studied [2]. The immune response to vaccination with LAIV has been studied in
several different settings and the immune response is qualitatively similar but
quantitatively less than that elicited by natural infection. After LAIV immunization,
mucosal IgA, serum HAI, and neutralizing antibodies and cellular T and B cell
responses are observed. Lower responses are not surprising given that the vaccine
stimulates immunity by replication in the upper respiratory tract, the site of
replication of the wild-type virus. The level of replication of the LAIV strain is
significantly reduced compared to that of wild-type virus. Despite evidence for
vaccine-induced immunity in both local and systemic compartments, the specific

functional role of any particular immune response and validated correlates of
LAIV-induced protection from influenza disease in vaccinated individuals have
not been defined.
LAIV elicits the most robust immune response in young children, particularly
those that are seronegative for influenza at the time of vaccination [3 5]. Serocon-
version rates, measured by the presence of HAI antibody, are as high as 80 90% in
young children after two doses of vaccine. Seroconversion rates are lower for
children or adults that have preexisting antibody at the time of vaccination. The
presence of antibody at the time of immunization may limit the extent of replication
of the vaccine in the upper airways, evidenced by lower rates of shedding, and may
mask the boosting of the immune response using relatively crude measures of
immunogenicity such as HAI antibody in the serum. In children, LAIV induces
nasal secretion of IgA and production of circulating IgG and IgA antibody secreting
cells (ASC) 7 10 days after immunization [6, 7]. Children 6 36 months of age have
measurable IFNg-secreting cells in their PBMCs by 13 days after LAIV; these
responses were not evident in children vaccinated intranasally with heat-inactivated
LAIV or intramuscularly with inactivated vaccine [8]. In a study of children aged
5 9 years, blood was collected at 10 and 28 days post vaccination and stimulated
with the A/H3N2 strain ex vivo. Both CD4 and CD8 influenza-specific T cell
frequencies were increased in these children compared to their prevaccination
values. These increases were greater than those observed for children vaccinated
with the seasonal trivalent inactivated influenza vaccine (TIV) in the same study
and the CD8+ T cells induced by LAIV underwent a number of specific phenotypic
changes [9 11].
Vaccine studies often rely on correlate markers to demonstrate that the vaccine
will perform as expected under the conditions being studied. A robust correlate of
protection is an immunological marker that when present coincides with protection
from disease upon subsequent exposure to the wild-type virus and the lack of which
correlates with susceptibility to illness. Due to high rates of efficacy demonstrated
for LAIV combined with the difficulty in using traditional serum-based influenza
assays to measure an immune response in adults, these markers have been difficult
to identify for LAIV. Virtually all adults have had multiple encounters with wild-
type influenza and/or have been vaccinated for influenza. Thus, most adults have
readily measurable levels of influenza antibody in their serum prior to vaccination.
In contrast to studies in young seronegative children, vaccination of adults with
LAIV does not usually produce a measurable increase in serum HAI antibody titers.
Recent studies on T cell immunity following vaccination showed similar results
[9, 11]. The levels of prevaccination influenza-specific CD4 and CD8 cells increase
with age of the subjects and adults have significantly higher baseline quantities than
children [11]. The level of prevaccination influenza specific CD4 T cells seems to
be a critical determinant of whether or not vaccinees experience a subsequent rise in
either CD4 or CD8 T cells. In contrast to the T cell and HAI responses, adults
generally have increased influenza-specific antibody-secreting B cells in the blood
7 10 days post LAIV vaccination. Although only 16% of adults have a serological
response measured by a fourfold or greater increase of HAI antibody following

immunization, approximately 80% of the subjects have a measurable increase in
the number of influenza-specific IgG-secreting antibody-secreting cells in the
peripheral blood. This was true for individuals who had been vaccinated in the
prior year as well as those who were not [6, 7]. These data demonstrated that LAIV
elicits a readily detectable B cell response in most adults, which is consistent with
the clinical experience that LAIV is highly efficacious in an adult population aged
18 49 [12, 13].
2.4 Vaccine Development, Composition, and Mechanism

of Attenuation
Development of live, attenuated vaccines based on the cold-adapted (ca), attenu-

ated ca A/Ann Arbor/6/60 and ca B/Ann Arbor/1/66 backbones has spanned several
decades. The vaccine contains three vaccine strains, two attenuated influenza A
strains and one attenuated influenza B strain. These vaccine strains are genetic
reassortants each harboring two gene segments from the currently circulating wild-
type virus conferring the appropriate antigens (e.g., A/H3N2, A/H1N1, or B) and
six gene segments of live, attenuated influenza A or influenza B donor virus or
master donor virus (MDV). The resulting 6:2 genetic reassortant combines the
attenuation inherent to the MDVs with the antigens needed to elicit a neutralizing
immune response that should prevent disease caused by currently circulating
strains of influenza. LAIV is used for active immunization of subjects from ages
2 through 49 and is currently manufactured in specific pathogen-free embryonated
chicken eggs. The three constituent attenuated influenza A and B strains are
blended and filled into sprayer devices used to deliver the vaccine liquid into the
nasal passages.
In the 1960s, investigators set out to attenuate influenza virus for vaccine use
through a process designated as cold-adaptation. Forcing the virus to replicate
efficiently at lower than normal temperatures resulted in changes to its genetic
makeup making it less fit to replicate at normal and elevated body temperatures,
thereby attenuating the strain. Biological characterization of ca A/Ann Arbor/6/60
and ca B/Ann Arbor/1/66 demonstrated that the resulting viruses are cold adapted,
as defined by ability to replicate to titers at 25 C that were similar to titers obtained
at 33 C. The strains are also temperature sensitive (ts), as defined by replication of
the virus at 39 C that was debilitated compared to its replication at 33 C [14]. The
spectrum of temperatures at which the ca virus replicated well was lower than the
wild-type viruses that caused disease. Further characterization of ca A and B/Ann
Arbor strains in the highly susceptible ferret model demonstrated that these strains
were attenuated (att) compared to wild-type influenza viruses and were unable to
replicate in the lung tissues of ferrets or elicit signs of influenza-like illness (ILI)
[15]. These two strains provide the genetic background of all LAIV strains,
imparting their ca, ts, and att properties to the vaccine.
Sequence analysis and comparison of the genomes of ca A/Ann Arbor/6/60 and

ca B/Ann Arbor/1/66 to their respective parental strains confirmed that a number of
changes had accumulated during cold passage. The introduction of reverse genetics
enabled biological traits to be associated with specific nucleotides without having to
account for potential problems caused by constellation effects. Five nucleotide
positions distributed between the PB1, PB2, and NP gene segments of A/Ann
Arbor/6/60 controlled both the ts and att properties [16]. Studies with B/Ann
Arbor/1/66 revealed that three positions (two in PA and one in NP) control the ts
phenotype, an additional two nucleotides in M control the att phenotype, and
another subset of three changes in PA and PB2 are responsible for the ca phenotype
[17, 18]. When the minimal set of mutations are made in divergent influenza strains
the biological traits transferred; thus, the fundamental mechanisms restricting the
replication of these vaccine strains at elevated temperatures are a result of the
complex genetic signatures that affect multiple points of the replication cycle to
provide a robust and stable set of attenuating changes to the viruses.
Following intranasal administration, the vaccine virus infects and replicates in
epithelial cells of the upper respiratory tract resulting in an immune response.
Characterizing the genetic stability of the vaccine in humans was important for
understanding the properties of the vaccine. In a study of genetic stability, 98
children, 9 36 months of age, were vaccinated with LAIV and nasal swab samples
were taken at frequent and regular intervals. Of the children in the study, 86% shed
at least one of the three strains in the vaccine. The ca and ts phenotypes were
preserved in all the shed viruses tested [19]. Of the isolates, 54 were chosen
at random and their genomes sequenced in their entirety and compared to the
sequences of the strains used to vaccinate the children. These analyses revealed
that some genetic changes occurred in a majority of shed isolates and in some
cases the mutations were shared by multiple isolates [20]. Interestingly, in most
cases, the change(s) evident in the virus that was shed were representative of
changes that existed in the bulk vaccine material. Despite the presence of these
mutations, all isolates invariably retained their characteristic biological attenuated
properties.
A corollary concern associated with genetic stability and vaccine shedding is the
potential for person-to-person transmission of the virus. The study of the genetic
stability of the vaccine in children was also designed to assess the probability of
transmission of the vaccine virus. In addition to the 98 children vaccinated
with LAIV, 97 children attending the same day care center received placebo.
Nasal swabs were obtained at frequent intervals from each child and the presence
of vaccine virus was assessed. Vaccine virus was recovered from 80% of the
vaccinated children and from only one placebo recipient [19]. The influenza
B vaccine virus recovered from the placebo recipient was shed on only 1 day.
The transmitted vaccine isolate was shown to retain its characteristic ca and ts
properties and exhibited the attenuation phenotype in ferrets and the placebo child
from whom LAIV was recovered had no symptoms of influenza. These results
indicated a 0.58% probability of vaccine transmission occurring from a single
contact of a vaccinated young child with an unvaccinated young child [19].
The likelihood of transmission from a vaccinated adult is expected to be substan-

tially lower because LAIV shedding is much less than in children.
2.5 Safety and Efficacy
Vaccines derived from ca A/Ann Arbor/6/60 and ca B/Ann Arbor/1/66 have been
extensively characterized in clinical studies. Prior to the mid 1990s, monovalent
and bivalent forms of these vaccines were evaluated in over 15,000 subjects in
a number of different clinical studies, many sponsored by the NIH [21]. Studies
of commercially produced, frozen and refrigerator stable, trivalent formulations
of LAIV (Flumist®, MedImmune) have been conducted in a wide range of settings
in individuals from 6 months to over 80 years of age. These studies have been done
both before and after licensure.
LAIV has reproducibly prevented ILI caused by all three currently circulating
influenza types. A meta-analysis of placebo-controlled studies showed that the
mean efficacy of two doses in previously unvaccinated young children was 77%,
with efficacies of 85%, 76%, and 73% against A/H1N1, A/H3N2, and B, respec-
tively. The mean efficacy of one dose in previously vaccinated children was 87%
[8, 22 30]. A single dose of vaccine, while not optimal, has been shown to provide
a high degree of clinical efficacy among previously unvaccinated young children
[26, 31].
Three studies were conducted in which LAIV and TIV were compared. The
largest of these included over 8,000 children. LAIV was shown to reduce the burden
of illness by nearly 55% compared to TIV. Of note, the A/H3N2 strains circulating
in this study were antigenically mismatched to the two vaccines and the children
vaccinated with LAIV had 79% fewer cases of modified ILI compared to the TIV
group [24]. In two other studies, one conducted in children with recurrent respira-
tory illness and the other in older children with asthma, LAIV was also shown to be
more efficacious than TIV [22, 27]. Immunity elicited by LAIV may provide for a
larger margin of error for antigenic mismatching than occurs after inactivated
vaccine administration. LAIV has been shown to provide protection against signifi-
cantly antigenically drifted variants in several clinical settings. In 1997 1998,
children were immunized with a trivalent blend of LAIV containing the A/Wuhan/
359/95 (H3N2) strain. The H3N2 virus that subsequently circulated was A/Sydney/
05/97, which has an H3 that is quite distinct from the antigen contained in the
vaccine. Despite this mismatch, the vaccine conferred efficacy greater than 85%
against the A/Sydney/05/97 virus [24]. During the same season, LAIV was also
shown to protect adults against the drifted H3 strain [12]. In a direct comparison study
of LAIV and TIV in children, LAIV reduced modified ILI caused by an antigenically
drifted A/H3N2 strain by 79% compared to TIV [23].
Two placebo controlled field studies in adults have been reported using either
effectiveness endpoints [12] or culture confirmed prevention of ILI in adults
60 years or older [32]. In a series of field studies in young adults, TIV was more
efficacious than LAIV; however, both groups suffered less illness than observed in
the placebo group. In a study conducted in the 2007 2008 influenza season with
1,952 subjects, the inactivated vaccine was shown to have an efficacy of 72%
compared to a placebo and LAIV had an efficacy of 29% compared to a placebo
[33 35]. These two vaccines have also been studied in military personnel. In a
retrospective cohort analysis, LAIV was more effective than TIV at preventing
influenza illness in recruits and TIV was slightly more effective in nonrecruits [36].
In an analysis of more than a million nonrecruits, TIV was more effective at
lowering health care encounters for pneumonia and influenza than LAIV and the
latter was shown effective in only one of the three seasons analyzed. However,
LAIV was effective in the subset of vaccine-naı̈ve service members at levels similar
to TIV [37]. The less robust results of these studies in adults compared to studies in
children, where LAIV has appeared to be more efficacious than TIV, may reflect the
interaction of LAIV with the already flu-experienced immune system of the adult
host. Presumably the higher level of preexisting immunity in adults is more
restrictive for immune responses to the replication dependent LAIV than for the
parenterally administered, non replicating TIV.
Safety is obviously a critical issue when evaluating a live viral vaccine. In
controlled studies, the most common adverse events in children 2 years of age or
older given LAIV were runny nose or nasal congestion, low-grade fever, decreased
activity and decreased appetite. In the youngest children, who received two doses of
vaccine, no significant differences were observed following the second dose. In
adults, the most common adverse events were runny nose/nasal congestion, cough,
and sore throat, which were all short lived. In a large safety database study using the
Northern California Kaiser Hospital system, a 3.5-fold increase in asthma events
was noted within 42 days of vaccination in the prespecified age stratum of 18 35
months [38]. The observation was further investigated in the large efficacy study of
LAIV and TIV in young children. In the age stratum less than 24 months of age
(6 23 months), 3.2% of children in the LAIV group had medically attended
wheezing events within 42 days of vaccination compared to 2.0% in the TIV
group. This difference was significant. There was no significant difference in
rates after 42 days in this age group or in the children 24 months of age or older
[1, 23]. These finding led to the licensure of LAIV for children over the age of
2 years.
2.6 Issues for the Future
The utilization of the live virus vaccine technology continues to be refined and
improved. Recent studies in children should encourage greater use of this vaccine in
this highly susceptible and vulnerable population. The current manufacturing
methods used to make LAIV, like those used to produce the inactivated vaccine,
are based on production technologies that are over 50 years old. More modern
production methods, including manufacturing in cell culture substrates, are needed
and are being developed. In addition, the generation of the 6:2 reassortant viruses
used to initiate vaccine seed strain production is being refined and integrated with
the use of reverse genetics technology. Finally, the attributes that make this vaccine
effective in young children are being further explored and LAIV strategies are
being developed for pandemic solutions where the advantages over inactivated TIV
for producing large amounts of vaccine are substantial.
3 Rotavirus
3.1 Introduction
Rotaviruses are the most frequent cause of severe diarrheal disease in young
children worldwide and are also ubiquitous enteric pathogens of many other
mammalian and avian species. By 5 years of age, 1 in 50 children worldwide will
have been hospitalized and 1 in 205 will have died from rotavirus-associated
causes. Virtually all of these deaths occur in children living in developing countries.
The worldwide morbidity and mortality associated with rotavirus makes it one of
major vaccine-preventable causes of infant mortality. Natural infection with wild-
type rotavirus elicits immunity that efficiently protects from subsequent severe
illness, irrespective of the rotavirus serotype. In the past decade, two live attenu-
ated, orally administered rotavirus vaccines have been developed and introduced in
many countries; both have proven safe and effective. Several third generation
candidates are in late-stage development. In the USA, the introduction of one of
the currently licensed rotavirus vaccines has been associated with a remarkable
decline in rotavirus illness. However, the overall impact of the licensed rotavirus
vaccines on disease in extremely poor countries, where they are needed the most,
remains to be determined.
3.2 Virology, Epidemiology and Pathogenesis
Although human rotaviruses (RVs) were discovered in the intestines of children

with diarrhea only 36 years ago [39], substantial progress has been made in our
understanding of their role in human disease. Rotaviruses are the most important
cause of severe watery diarrhea in all regions of the developed and less developed
world. During diarrheal illness, rotaviruses are shed in the stool in great quantity
(in amounts as high as 1010–11 particles per gram of stool), which allowed the
rapid development of sensitive and specific antigen detection diagnostics. Based on
results from these simple diagnostics tests, it soon became clear that RVs were the
cause of approximately 20 30% of severe diarrheal disease requiring hospitaliza-
tion in children under the age of 5, worldwide [40]. Recent estimates indicate that
there are over 114 million rotavirus diarrheal episodes annually; these lead to
approximately 24 million clinic visits, 2.4 million hospitalizations (40% of all
diarrheal hospitalizations), and over 500,000 deaths in children under 5 years
of age [41].
The burden of disease from rotavirus infection is not restricted to the less
developed world. Studies from Europe indicate that approximately half of severe
gastroenteritis in children less than 5 years of age is caused by rotavirus. In studies
from the US, 50% of children hospitalized or treated in the emergency department
for gastroenteritis were infected with rotavirus [42]. These data lead to the estimate
that one of every 150 children under 3 years of age will be hospitalized and 1 of 11
will be seen as an outpatient in an emergency department for treatment of rotavirus
disease. In the US, rotavirus is estimated to cause 20 40 deaths, 55,000 70,000
hospitalizations, and 410,000 physician visits annually [43]. The overall costs of
rotavirus disease in the US are thought to exceed a billion dollars annually.
Rotavirus disease occurs with high frequency around the world, in both temper-
ate and tropical climates and in both developed and less developed countries. The
large quantity of virus that is shed probably explains why improvements in hygiene
in the developed world have not reduced the incidence of infection. In temperate
climates, prior to the introduction of vaccines (see below), rotavirus disease
occurred seasonally in the cooler dryer months of the year [44]. In the US, waves
of rotavirus infection tend to start in the southwest in the fall and end in the
northeast in the spring, whereas in Europe infections tend to spread from south to
north over generally the same time frame [45]. Of note, a recent study predicts that
wide-spread vaccination will alter this seasonal trend [46]. The seasonality of
rotavirus infections fluctuates far less in tropical climates but the highest numbers
of infections occur in the coolest and driest months of the year [47].
Rotaviruses, like other members of the Reoviridae family, have a double
stranded (11 segment) RNA genome and icosahedral symmetry. The viral serotype
is determined by its two surface proteins, VP4 (P type) and VP7 (G type) [48]. Both
of these proteins are the targets of neutralizing and protective antibodies. Due to its
segmented RNA genome, the genes encoding VP4 and VP7 segregate relatively
independently. At least eleven distinct human VP4 P types and ten VP7 G types
have been isolated [49]. However, only a small number of P and G type combina-
tions are encountered with any significant frequency in people and just four
combinations, P(8)G1, P(8)G2, P(8)G3, and P(4)G2, account for over 90% of all
isolates. Serotypic diversity does change over time and based on geography,
especially in the less developed world. In the last decade, isolation of P(8)G9 and
viruses has been more frequent. The relationship between serotypic diversity and
protective immunity is still not well understood but it seems clear that a significant
level of heterotypic immunity is produced following an initial rotavirus infection.
Because of this immunity, severe episodes of illness after the primary infection are
relatively uncommon [50].
From studies of animals and experimental infection of adult volunteers, the
incubation period for rotavirus is usually less than 48 h. It was thought that in immu-
nocompetent children, rotavirus infection was restricted to the mature enterocytes
on the tips of the small intestinal villi. However, recent studies in humans and
animals indicate that this paradigm is not correct; most rotavirus infections are
associated with some level of viremia and systemic replication [51]. The clinical
relevance of the findings of extraintestinal spread and replication of rotavirus is
still unclear and the great bulk of rotavirus replication clearly occurs in the mature
villus tip cells of the small bowel. The pathologic changes in the intestines
of children infected with rotavirus include shortening and atrophy of the villi,
mononuclear infiltration in the lamina propria and distended cisternae of the
endoplasmic reticulum. A direct relationship between the extent of enteric histo-
pathologic changes and disease severity has not been demonstrated. In a mouse
model, rotavirus disease is associated with very modest histopathology.
3.3 Immunology
Studies of natural rotavirus infection in man and animals demonstrated the existence
of acquired immunity both to recurrent disease and, to a lesser extent, reinfection
following primary infection [50]. Passive transfer studies of monoclonal antibodies in
mice demonstrated that neutralizing antibody to either VP4 or VP7 could transfer
either homotypic or heterotypic protection, depending on the antibody specificity
in vitro [40]. Interestingly, other studies in mice have shown that non-neutralizing
IgA antibodies to the antigenically conserved VP6 protein can also mediate protec-
tion, apparently via an antiviral effect occurring during transcytosis [52]. This novel
intracellular neutralization event could help explain the well-documented clinical
observation of heterotypic immunity following primary infection with a single
rotavirus serotype. Several studies in the mouse model indicated that that B cells
were the critical determinant of protection from reinfection after infection whereas
CD8+ T cells were responsible for restricting the course of viral shedding during
primary infection [53]. CD4+ T cells aid CD8+ T cells and B cells and apparently can
mediate active protection via an IFNg-dependent pathway after immunization with
recombinant VP6.
Rotavirus-specific fecal IgA responses occur in the majority of children after a
symptomatic infection. They peak from 1 to 4 weeks after infection and then
decline rapidly. It is reasonable to hypothesize that the transient presence of
mucosal immunity after primary infection contributes to the absence of sterilizing
immunity to reinfection. Secondary and subsequent rotavirus infections tend to
boost the fecal IgA response and in many children eventually induce sustained,
protective fecal anti-rotavirus IgA levels [54]. Studies of human neutralizing
antibody responses against rotavirus have shown that upon first exposure to rotavirus,
children develop higher homotypic than heterotypic antibody levels [53], although
both types of response are usually present. Studies in animal models and humans
indicate that the presence of intestinal antibodies is probably the primary protective
effector mechanism against rotavirus. Protective humoral immunity in several, but
not all, animal models is associated with the presence of neutralizing antibodies
directed at VP4 and/or VP7. In studies performed in day care centers and orphanages
where antibodies to rotavirus were measured very shortly before a rotavirus outbreak,
intestinal and/or serum antibody levels correlated with protection against rotavirus
reinfection [55]. Levels of rotavirus-specific antibodies (stool IgA in particular) were
correlated with protection in some but not all studies involving naturally infected as
well as vaccinated children. In vaccine studies, some investigators [56] found a
correlation between the presence of neutralizing antibodies and protection; however,
the percentage of children with detectable serotype specific neutralizing antibody
titers is always significantly less than the percentage of children protected by
vaccination [57]. Thus, although serotype specific neutralizing antibodies seem to
play a role in protection, it seems likely that heterotypic antibodies or against other
proteins or other mechanisms also play a role in immunity.
Recent studies have also drawn attention to the possible importance of the innate
immune response and interferon in regulating rotavirus immunity. In the gnotobi-
otic porcine model, probiotic treatment with Lactobacillus acidophilus significantly
enhanced both B and T cell responses to attenuated live virus infection [58]. It has
been shown that levels of type I and II IFN are elevated in rotavirus-infected
children and animals [59, 60]. Both type I and II interferon are able to limit
rotavirus infection in vitro and, in early studies, IFNa administration successfully
alleviated RV diarrhea in cattle and pigs. The IFN-regulatory factor 3 (IRF3)
interacts with the RV protein NSP1, clearly linking RV infection to innate immu-
nity [61]. NSP1 also inhibits activation of NFkB by a novel mechanism involving
targeted degradation of an F-box protein of the E3 ligase complex [62, 63]. Studies
in vivo demonstrated that the systemic virulence of selected strains of rotavirus was
enhanced and a lethal biliary and pancreatic disease induced when interferon
signaling was abrogated during rotavirus infection [64]. Hence, innate immunity
plays a critical role in modulating rotavirus infection in vitro and in animal model
systems but the role in humans remains largely unexplored.
3.4 Vaccines Development, Composition and Mechanism

of Attenuation
Two live attenuated, orally administered rotaviral vaccines are currently licensed
and in use in many countries around the world [48, 65 67]. Both vaccines have
been shown to be safe and effective as well as cost-effective in developed and
developing countries. The aim of anti-rotavirus vaccination strategies is to repro-
duce the level of immunity induced following natural infection. Natural infection,
either symptomatic or asymptomatic, efficiently prevents subsequent severe rotavirus
disease but does not necessarily prevent reinfection or mild illness. Based on the
observation that animal rotaviruses appear to be substantially restricted for growth,
pathogenicity, and transmission in heterologous hosts such as humans (host range
restriction), the initial strategy for rotavirus vaccine development was a modified
Jennerian approach using either live simian/human or bovine/human rotavirus
reassortants as vaccines. In this approach, an animal origin rotavirus that is

restricted for growth in humans is reassorted with a human rotavirus and reassor-
tants are isolated with genomes that are primarily animal in origin but which
contain genes encoding VP4 or VP7 from the human parent. Such reassortants
are expected to induce protective immunity to human rotaviruses because of the
presence of human surface proteins (VP4 or VP7) but not to cause severe disease
because most of their genome was derived from the animal rotavirus parent which
is restricted for growth in the human host. Of note, these vaccines are fully
replication competent in cell culture and they are derived from potentially virulent
animal strains but they have undergone considerable cell culture passage which, in
itself, could also be a cause of attenuation. The first modified Jennerian vaccine
of this type was a quadrivalent rhesus vaccine (RotashieldTM, Wyeth/Lederle)
consisting of four monoreassortants between human G types 1 through 4 and the
simian RRV strain [68]. The vaccine contained components for expression of these
G types based on the assumption that immunity to the four most common human
rotavirus types would be needed to induce a high level of efficacy. This vaccine was
shown to be highly immunogenic and efficacious in multiple phase III studies in the
USA, Finland and Venezuela and was licensed for use in the United States. Of note,
the level of efficacy substantially exceeded the level expected based on the type-
specific neutralizing antibody response induced by the immunization and this
finding later was repeated with the bovine rotavirus based modified Jennerian
vaccine. Shedding studies of the RRV-based vaccine indicated that it was shed in
moderate quantity and was able to transmit with reasonable efficiency to unvacci-
nated children in the environment. The consequences of this shedding and trans-
mission were never evaluated but it did not appear that the virus gained virulence
during passage in humans. Unfortunately, after licensure and administration to
almost one million American children, the RRV-based vaccine was withdrawn
from the market because of its strong temporal association of the first dose of
vaccine (which was licensed to be given orally at 2, 4, and 6 months of age) with
intussusception in older (over 3 months of age) children receiving their first dose.
Interestingly, the impact of this vaccine on the total attributable risk of intussuscep-
tion, especially if initially administered to children only at 2 months of age or
below, remains unknown but was likely to be very small. At the time the vaccine
was withdrawn from manufacture, it was undergoing efficacy evaluation in
less developed countries which were never completed. However, immunogenicity
studies carried out in Bangladesh indicated that the RRV-based vaccine appears to
have been more immunogenic in this setting than either of the two currently
licensed second generation vaccines.
Subsequently, a second generation pentavalent modified Jennerian vaccine
called RotaTeqTM (Merck), based on mono or di-reassortants derived from the
mixed infection of a bovine rotavirus (WC3) and human rotavirus strains which
provided human G1, G2, G3, G4 (VP7), and P[8] (VP4) was introduced [65]. This
vaccine contained five (rather than four) separate reassortants based on the theoretical
consideration that immunity to all five serotypic species would be most efficient
at inducing protective immunity. The basis of attenuation for this vaccine, like the
preceding RotashieldTM product, is presumed to be the inherent host range restriction

linked to its primarily bovine-origin genome. The specific genetic and mechanistic
basis for host range restriction of the two reassortant-based vaccines is not well
understood, although several studies indicate that this restriction is linked, at least in
part, to rotaviral protein NSP1 and host specific inhibition of the innate immune
system [69]. Studies of viral shedding have demonstrated that, in general, the bovine-
based reassortant vaccine is highly restricted for replication in humans and is shed in
a very limited fashion compared to wild-type human rotavirus or to the RRV-based
vaccine although no head-to-head comparison was ever done. Nevertheless, recent
studies have demonstrated that severely immunodeficient children can become
chronically and symptomatically infected with this vaccine [70]. As with the RRV
vaccine, this vaccine is administered orally in a three dose regimen.
The other currently licensed vaccine (RotarixTM, GSK) is a more traditionally
constructed, live attenuated vaccine. A virulent G1P human rotavirus strain (89-12)
was multiple passaged in monkey kidney cell culture in order to acquire a suitable
level of attenuation to reduce virulence [66]. The rationale underlying the develop-
ment of this monovalent vaccine was that a single natural rotavirus infection,
either symptomatic or asymptomatic, very effectively provides protective immu-
nity to subsequent severe disease, irrespective of serotype. Therefore, it seemed
logical that a single attenuated human rotavirus strain might do the same. As with
the Merck vaccine, the molecular basis for attenuation of this vaccine candidate is
unknown although presumably, in this case, point mutations introduced during
multiple passage in cell culture are responsible for the attenuation. Since the actual
molecular basis for attenuation of the two currently licensed vaccines remains
unknown, it is difficult to comment definitively on their level of genetic stability
except to say that, to date, evidence for reversion to virulence has not been found.
This vaccine is also administered orally, but in this case only two doses of vaccine
are given.
3.5 Safety and Efficacy
The two second generation vaccines have been evaluated for safety and efficacy in
studies representing a wide variety of socioeconomic conditions in several
countries. In very large field studies both were shown to be safe and effective.
Protection rates in developed or moderately developed countries provided by both
vaccines are very similar; rates vary from 70 80% against any rotavirus disease
to 90 100% against severe gastroenteritis. Interestingly, to date, no appreciable
advantage of the multivalent over the monovalent vaccine has been observed. Large
pre- and post-licensure studies have shown that these vaccines are not associated
with intussusception if the first dose is administered to children under the age of
3 months [71]. The effectiveness of the pentavalent Merck vaccine in preventing
rotavirus-associated gastroenteritis and hospitalizations was demonstrated in the
United States and the monovalent vaccine effectively prevented rotavirus-associated
deaths in Mexico [72 74]. Since successful efficacy and/or immunogenicity trials
in a variety of countries around the world have been completed, a general WHO
global recommendation for the use of these vaccines was issued in June 2009 [75].
Of note is the fact that recent studies of the two new vaccines in very poor areas of
Africa and Asia indicate that these vaccines are less effective (49.5 76.9% protec-
tion rates against severe disease) in these countries than has been observed in
developed countries [76]. However, they are still very cost-effective in terms of
number of severe rotavirus-induced diarrheas prevented: Overall in African trials,
Rotarix prevented 3 out of 5 episodes of severe rotavirus-induced disease per 100
vaccinated children [75].
3.6 Issues for the Future
Although two safe and effective live attenuated rotavirus vaccines are now avail-
able, several important practical issues are not yet resolved. Exactly how important
the moderate decrease in efficacy of these vaccines in very poor countries is and
whether it can be circumvented in some straightforward way is not known. Second,
the two current vaccines cost too much to be affordable without substantial sub-
sidies and cheaper vaccine products will be required to serve the global needs in the
long term. The two vaccines are currently highly effective but rotaviruses certainly
have the ability to evolve serotypically and it remains to be seen if new strains of
human rotavirus will emerge that are less effectively countered by immunity
elicited by the current vaccines. Finally, as with all vaccines, rare and unexpected
adverse events are always possible and continued vigilance regarding safety,
especially in very immunosuppressed children is necessary.
Several third generation rotavirus vaccines are in various stages of development.
A pentavalent live attenuated reassortant vaccine based on another bovine strain
(UK) is currently undergoing evaluation in several less developed countries includ-
ing China, India and Brazil. This vaccine has been shown to be highly efficacious in
phase two trials in Finland. Monovalent human rotavirus vaccines derived from
naturally attenuated strains recovered from human infants in India and Australia are
in various stages of evaluation. The Indian strain (116E) appears to be highly
immunogenic in preliminary studies [77]. Several groups have proposed that
some form of parenterally administered inactivated vaccine might be safer vis-a-
vis the risk of intussusception. Such a vaccine might be more immunogenic and
hence more effective in some less developed regions where efficacy of the live virus
vaccines seems to be restricted [76]. No data from human studies is currently
available to evaluate the utility of this strategy. Finally, the quadravalent rhesus
rotavirus-based vaccine is currently undergoing a re-evaluation in a phase three
efficacy trial in Africa based on the early data indicating that this vaccine appeared
to be more immunogenic than the current two commercial vaccines and hence,
might be substantially more efficacious in a less developed setting. The results of
this interesting trial should be available in the next year or two.
4 Varicella Zoster Virus
4.1 Introduction
VZV is a human alphaherpesvirus, most closely related to herpes simplex viruses

(HSV) 1 and 2. VZV has the typical morphology of herpes virus particles and while
its double stranded DNA genome is the smallest of the human herpesviruses,
at least 70 gene products are encoded [78]. VZV causes varicella (chickenpox) as
the primary infection and establishes life-long persistence in sensory ganglia;
reactivation from latency produces the clinical syndrome referred to as zoster
(shingles) [79, 80].
4.2 Epidemiology
The molecular epidemiology of VZV has been investigated extensively based on

the detection of single nucleotide polymorphisms in selected VZV open reading
frames and by whole VZV genome sequencing of more than 20 isolates [81, 82].
VZV circulates as five distinct clades that exhibit varying predominance in different
geographical areas but overall, the genetic diversity of the virus is limited. While
clades 1 and 2 are the most divergent, genome sequences were 99.83% identical,
with only188 site differences [83]. Sequences of nine clade 1 viruses showed 99.9%
identity compared with Dumas strain, which is the first VZV genome that was
sequenced [84]. Clade 1 viruses are most common in Europe and North America,
clade 2 viruses are predominant in Asia and clade 5 is most prevalent in Africa. The
Oka virus used to derive VZV vaccines is a clade 2 strain. As expected, immigration
has redistributed European, African and Asian clades.
VZV skin lesions contain high concentrations of infectious virus during both
primary and recurrent infections, which results in a highly successful strategy for
persistence in the human population. Whereas the prevalence of other human
herpesviruses has declined in developed countries, varicella epidemics continue
to produce high infection rates. Episodes of zoster in older individuals provide a
constant mechanism for reintroducing the virus, causing varicella in naı̈ve indivi-
duals who are in close contact and who then spread the virus to other susceptibles.
In temperate climates, VZV is acquired almost universally during childhood; attack
rates are substantially lower in tropical areas. Before varicella vaccine was intro-
duced, the incidence of varicella in the United States was ~four million cases per
year, reflecting the number of children in the annual birth cohort. Secondary
bacterial infections and VZV encephalitis were the most common morbidities;
hospitalization rates were estimated to be 2 5 per 1,000 cases and approximately
100 fatal cases were reported annually [85]. The estimated incidence of herpes
zoster is >1 million cases per year in the United States and complications, espe-
cially post-herpetic neuralgia, are frequent in older individuals [80]. While the
morbidity caused by VZV in healthy children and adults is significant, illness

associated with this ubiquitous pathogen can be much more severe in immunocom-
promised patients. Children who are immunodeficient because of underlying
disease or immunosuppressive therapies may develop progressive varicella; the
risk of VZV reactivation is much higher in immunocompromised children and
adults, and whether or not it is manifest as cutaneous zoster, reactivation may
cause life-threatening disseminated VZV infection.
4.3 Pathogenesis of Primary and Recurrent VZV Infection
As defined clinically, the events in primary VZV infection include respiratory

inoculation, viremia and the appearance of vesicular skin lesions. Studies of VZV
pathogenesis using the severe combined immunodeficiency (SCID) mouse model
show that VZV exhibits a marked tropism for T cells in human thymus/liver
xenografts in vivo; VZV is also highly infectious for human tonsil T cells, particu-
larly those in the subpopulation of activated, memory CD4 T cells, in vitro [86, 87].
VZV is readily transferred into skin xenografts when infected tonsil T cells are
injected into the circulation of SCID mice. Infected T cells exit capillaries and
initiate replication in epidermal cells, which progresses over a 10 21 day period
until the lesion reaches the skin surface; cell cell spread of VZV in skin is
modulated by a potent innate response of the epidermal cells surrounding the
newly forming lesion [88]. Induction of the IFN pathway and upregulation of
NFkB signaling are prominent in adjacent cells. These observations suggest a
model of primary VZV pathogenesis in which the virus infects respiratory epithelial
cells, enters T cells in tonsils and other lymphoid tissues of the Waldeyer’s ring
and initiates a T cell-associated viremia which transports the virus to skin sites of
replication; after a period of subclinical lesion formation during the incubation
period, the characteristic varicella exanthem appears.
In the course of primary infection, VZV gains access to cranial nerve and dorsal
root ganglia and as suggested by recent evidence, to autonomic enteric ganglia as
well [89]. Access is presumed to occur via retrograde transfer along neuronal axons
from skin lesions, T cell viremia or both. VZV-infected T cells transport the virus
into DRG xenografts in the SCID mouse model [90]. Persistent infection is estab-
lished in neurons for the life of the host. Abortive replication limited to ganglia or to
subclinical skin replication may occur; however, when VZV reactivation causes
zoster, VZ virions are presumed to move by anterograde axonal transport to the skin
dermatome where vesicular lesions appear. In contrast to HSV, VZV reactivation
can destroy neurons and satellite cells in the affected ganglia [80]; cell cell spread
with fusion of neurons and surrounding satellite cells is observed in VZV-infected
DRG xenografts in the SCID model [91]. Recurrent VZV may also lead to viremia.
Migrating T cells may become infected during trafficking through skin or ganglion
sites of VZV replication, allowing viral transport to lungs, liver, brain and other
organs in immunocompromised patients.
4.4 Host Response
In addition to innate cellular responses, NK cells appear to be critical since NK cell

deficiencies are associated with severe, often fatal primary VZV infection. In the
healthy host, VZV-specific immunity emerges in parallel with the appearance of
skin lesions during primary VZV infection; both VZV antibodies and VZV-specific
CD4 and CD8 T cells are induced [92]. However, cell-mediated immunity is
necessary to resolve varicella, as shown by the risk of progressive infection in
immunocompromised children who developed VZV IgG and IgM antibodies but
failed to mount a VZV-specific T cell response [93]. Adaptive CD4 and CD8 T cells
and IgG antibodies that recognize various VZV proteins persist but their peptide
specificity is not well-characterized. VZV antibodies that bind envelope glycopro-
teins exhibit neutralizing activity. The extent of both antibody and T cell mediated
memory immunity to VZV may be determined by the initial clonal expansion or by
secondary stimulation from varicella exposures or subclinical reactivations, or
by all of these mechanisms. Circulating VZV memory T cell frequencies are
~0.1 0.2% in immune adults [94].
While reported, symptomatic second episodes of varicella are rare, even among
severely immunodeficient patients. A clinical history of varicella is often unreli-
able; individuals with apparent second cases had no evidence of prior infection
when specimens obtained before the episode were available [95]. Protection from
varicella illness appears to be induced regardless of the clade that caused the initial
infection, which can be explained by the highly conserved VZV genome. An
important caveat is that the incidence of subclinical VZV reinfection is not
known although it has been proved, using molecular methods, that VZV reactiva-
tions in the same individual were caused by viruses of different VZV clades [96].
Protection from clinically apparent reinfection may be mediated by neutralizing
antibodies present at respiratory sites of inoculation or by a rapid humoral and T
cell response if replication is initiated. Adults in close contact with children who
have varicella exhibit boosts in both VZV antibody and cell-mediated responses.
Administering passive antibodies to VZV within 4 days after exposure of a naı̈ve
host can prevent varicella or modify its severity, as demonstrated by varicella zoster
immune globulin prophylaxis in immunocompromised children and newborns.
Experiments in the SCID mouse model show that infection may be blocked when
antibody to the glycoprotein, gH, which has potent neutralizing activity, is given
shortly after inoculation of skin xenografts [97].
In one of the most well-established immunologic correlates known, zoster
in older adults and immunocompromised patients is associated with reduced T
cell proliferation and production of IFN-g and other cytokines by peripheral blood
mononuclear cells stimulated with VZV antigen and with fewer circulating VZV-
specific CD4 and CD8 T cells [92, 98]. In contrast, VZV IgG antibody titers are not
related to the risk of reactivation; passive antibody adminstration did not alter
zoster severity in clinical studies done before antiviral drugs were available.
However, antibodies may contribute to modulating cell cell spread of VZV in
skin and possibly in the affected ganglion. Symptomatic zoster is associated with
a dramatic increase in the VZV-specific T response; IgG, IgM and IgA antibody
titers are also boosted but the resolution of zoster, like varicella, requires cellular
immunity. Of interest, hematopoietic cell transplant recipients may have subclinical
reactivation, detected by the presence of VZV DNA in peripheral blood mononu-
clear cells and recover VZV-specific T cell responses without clinical zoster.
4.5 VZV Vaccines
VZV is the only human herpesvirus for which vaccines are licensed. The live
attenuated varicella and zoster vaccines are made from the attenuated Oka virus
[99, 100]. Inactivated Oka-derived vaccines have also been evaluated in immuno-
compromised and healthy patients [101].
Composition. The VZV Oka vaccine seed stock was derived from a clinical
isolate, the parent Oka (pOka) virus, which was recovered from a varicella skin
lesion; pOka was passaged in guinea pig and human fibroblasts at low temperature.
VZV vaccines contain infectious VZ virions made in cells approved for manu-
facturing live viral vaccines; Oka-derived vaccines also contain viral and host cell
proteins and DNA because VZV replication is very highly cell-associated. Oka
vaccines are currently manufactured by Biken, Merck and GlaxoSmithKline. Not
surprisingly, because of the extreme cell association of VZV, Oka vaccines made
by all three manufacturers represent mixtures of VZV genomes. Multiple single
nucleotide polymorphisms are identified; some are shared in the various vaccine
preparations but others are not and wild type markers are also present [102 104].
The pediatric vaccines, Varivax (Merck) Varilrix (Glaxo) and Okavax (Biken)
contain approximately 1,300 pfu of Oka vaccine virus.
Mechanism of attenuation. Attenuation of pOka was achieved empirically by
tissue culture passage and verified clinically by the administration of Oka vaccine
preparations to susceptible children in Japan [100]. The experience showing atten-
uation of the Biken Oka vaccine was confirmed in trials of varicella vaccines made
from vaccine Oka seed stocks by Merck in the U.S. and by GlaxoSmithKline in
Europe.
Investigations in the SCID mouse model demonstrate that vaccine Oka has
reduced virulence in skin compared to pOka. In contrast, pOka and vaccine Oka
do not differ in their infectivity for T cells and DRG xenografts in vivo [79, 86, 90,
105]. These experiments suggest that attenuation of vaccine Oka in skin is intrinsic,
resulting from genetic changes accumulated during tissue culture passage in fibro-
blasts rather than simply because the vaccine is given by a subcutaneous route of
inoculation. The evidence that this attenuation is tissue/cell type specific for skin
but not T cells or DRG is consistent with the capacity of Oka vaccine to cause a
varicella-like rash in immunocompromised patients and its potential to establish
latency in the sensory ganglia of healthy vaccinees [106, 107]. Experiments with
pOka/vOka chimeric viruses showed that attenuation in skin was conferred by
different segments of vaccine Oka in the chimera, suggesting that multiple VZV
genes have relevant mutations [108]. Identifying mutations that might contribute to
attenuation by full genome sequencing is challenging because as noted, varicella
vaccines contain mixtures of variants that have various genetic differences [109].
Vaccine Oka mutations do not alter its susceptibility to inihibition by acyclovir and
related antiviral drugs.
Measles mumps rubella varicella (MMR-V) multivalent vaccines. Vaccine
Oka is also used as a component of a multivalent vaccine containing live attenuated
measles, mumps and rubella (MMR-V) [110, 111]. This formulation requires a
higher titer of vaccine Oka than the single component vaccine. The Merck vaccine
(ProQuad) contains not less than 3.99 log 10 pfu of vaccine Oka; the GlaxoSmith-
Kline vaccine (PriorixTetra) contains not less than 3.3 log 10 pfu.
Higher potency vaccines for zoster. Higher potency live attenuated Oka vaccines
have been developed and evaluated for their potential to increase VZV cellular
immunity in healthy older adults in the U.S. [112]. Dose-finding studies were done
using VZV-specific T cell proliferation and responder CD4 T cell frequencies as the
endpoint before a large scale efficacy study was undertaken [98]. High potency
vaccines boosted VZV T cell responses among 55 87-year-old subjects to ranges
observed in younger adults, ages 35 40 years, who had naturally acquired VZV
immunity. The infectious virus content of the high potency zoster vaccine manu-
factured by Merck Inc. is ~20,000 pfu, which is more than 14-fold more than
Oka/Merck pediatric vaccines. This higher infectious virus content is presumed to
be necessary because zoster vaccine recipients have pre-existing VZV immunity
and because immunosenescence diminishes the antiviral T cell responses of older
individuals.
Inactivated VZV vaccines. Heat inactivation can reduce the infectious virus
content of varicella vaccine to undetectable levels. Heat inactivated vaccine was
used to assess effects on T cell responses in healthy elderly individuals [113] and
for immune reconstitution and zoster prevention in hematopoietic cell transplant
patients [101].
Clinical experience with the efficacy and safety of varicella vaccines. The
development of live attenuated VZV vaccines in the U.S. and Europe was first
undertaken to protect children with leukemia from varicella [99]. The capacity of
vaccine Oka to cause varicella-like illnesses has limited use in immunocompro-
mised children. However, trials of live attenuated varicella vaccines in healthy
children led to their introduction as a routine childhood vaccine in North America,
Australia and some Europe and Asian countries [99, 114]. Pre-licensure evaluations
demonstrated that these vaccines induced both humoral and cell-mediated immu-
nity against VZV, with antibody titers and VZV T cell proliferation responses in the
range of those observed after natural VZV infection in childhood. Immunogenicity,
measured by serologic and cell-mediated responses, correlated with infectious virus
and antigen content. Age was also a factor; a two dose regimen was required to
achieve >90% seroconversion rates in those over 12 years old.
The efficacy of varicella vaccine was demonstrated in a small placebo control-
led trial in the U.S., leading to licensure in 1995 [99]. Extensive post-licensure
surveillance has supported that the vaccine is effective and safe in healthy children
and adults. The recommendation to vaccinate all children at 12 18 months of age
and all susceptible older children and adults has had a major impact on varicella
incidence, hospitalizations and deaths among the pediatric population and also
among persons in older age groups, most of whom benefit indirectly [85, 115].
The annual incidence of varicella decreased by more than 80% when a coverage
rate of ~90% was achieved in 2005; hospitalization rates for varicella complications
decreased by 88% and age-adjusted mortality was reduced by 66%.
Although the initial recommendation was to give a single dose to children under
12, reports of breakthrough infection in vaccinated children remained relatively
common. Efficacy analyses during outbreaks and in surveillance sites showed a
single dose vaccine effectiveness rate of ~85%. Although breakthrough varicella
cases were typically mild, these cases were a source of VZV transmission to other
susceptibles and interfered with the public health objective of varicella control.
Therefore, a two dose regimen for all age groups was implemented in the U.S. in
2007 [116]. Whether this pattern reflects waning immunity is debated [117, 118].
However, the single dose regimen is associated with lower seroconversion rates by
the most sensitive assay for VZV IgG antibodies, suggesting that primary vaccine
failure accounts for many cases of apparent breakthrough varicella in vaccine
recipients [119].
Reports about varicella vaccine adverse events to the U.S. vaccine adverse event
reporting system (VAERS) showed a rate of 2.6/100,000 doses during the first 10
years after licensure [120, 121]. Varicella vaccine can cause a mild, self-limiting
rash in healthy recipients within the first 6 weeks [122, 123]. Some children with
severe undiagnosed immunodeficiencies have developed progressive infection
caused by Oka vaccine virus; however, treatment with acyclovir has been effective
in most cases.
Zoster after vaccination. Zoster can occur in vaccinated individuals. Using
sequence differences between Oka and most North American VZV isolates, it has
been possible to demonstrate that these cases can be due to either vaccine or wild
type VZV [109]. Zoster caused by wild type VZV has been reported in vaccine
recipients with no history of breakthrough varicella, indicating that infection can be
acquired subclinically, reach neurons and establish latency in sensory ganglia
[106]. When evaluated in vaccinated immunocompromised children, zoster was
significantly less common than zoster following natural infection. More recently,
prospective studies in healthy children demonstrated that the incidence of zoster
was 4 12 fold less in vaccinated children under 10 years of age compared to those
with natural infection [124] and zoster was rare (27.4 cases/100,000 person years)
in 170,000 vaccinated children [125]. Vaccine-related cases of zoster have been
mild although cases of meningitis and meningoencephalitis have been reported
[107].
Information about how commonly Oka vaccine virus establishes latency in
sensory ganglia is limited and consists of VZV DNA sequence analysis of skin
lesion specimens from vaccinated people with clinical zoster. However, recent
evidence from a postmortem study suggests that vaccine Oka persists in multiple
ganglia for years after vaccination, as observed with wild type VZV and latency
was established without vaccine-associated skin lesions [89]. Whether viral load
is reduced compared to wild type VZV is not known. These observations are
consistent with the capacity of vaccine Oka virus to cause viremia in immuncom-
promised children and with the evidence that its T cell tropism is intact in the SCID
mouse model. The potential for super-infection with wild type VZV in vaccinated
individuals along with the persistence of the vaccine virus in ganglia may also
permit genetic recombination of wild type and vaccine viruses. Recombination of
Oka and wild type VZV has been demonstrated by direct sequencing of VZV DNA
from zoster lesions in a previously vaccinated individual.
MMR-V. MMR-V vaccine was licensed in the U.S. based on comparability of the
VZV antibody titers against viral glycoproteins, as measured by ELISA. Although
the mechanism is not known, MMR-V has been associated with an increased
incidence of febrile seizures from 7 to 14 days after the first vaccine dose; rates
were 4/10,000 for MMR and 9/10,000 for MMR-V [126]. These observations led to
the recommendation to offer an option of giving MMR and varicella vaccines at
different sites or to give MMR-V, along with counseling parents about the rare
possibility of febrile seizures within 2 weeks after vaccination.
Live attenuated varicella vaccine in high risk patients. Varicella vaccine
has been used in clinical practice to immunize children with HIV infection against
severe varicella and zoster when their CD4 T cell counts were >15 25%; a recent
report found an 82% effectiveness against varicella and 100% effectiveness
against zoster in a review of carefully monitored children with HIV [107, 127].
Varicella vaccine has also been given to children with leukemia in remission
and solid organ transplant recipients as a safer option than risking natural infection
[98]. The rationale is that antiviral therapy can be given if varicella-like illness
occurs.
Varicella vaccine issues for the future. Whether the two dose regimen intro-
duced in 2007 will reduce the incidence of breakthrough varicella in childhood
requires continued surveillance. Like many viral infections, varicella is more severe
in adults. Therefore, as has been true for other childhood viral vaccines, it is important
to maintain active surveillance programs to be sure that protection is sustained.
Whether those with vaccine-induced immunity will need booster doses is difficult
to predict; robust immune responses elicited by a two dose regimen in early child-
hood may prove to be as long-lasting as natural immunity. Whether intermittent
re-exposure to varicella is necessary to maintain natural immunity is not known
but interrupting varicella epidemics will obviously reduce such contacts. Based on
the assumption that some boosting of memory immunity is required, whether by
exogenous re-exposure or endogenous restimulation through subclinical reactivation,
some models predict a higher incidence of zoster among those with natural infection,
as a consequence of varicella vaccine programs. However, surveillance studies show
no increase in zoster [100]. New information indicating that Oka vaccine latency
occurs frequently may mean that vaccine-related zoster will be a concern as vaccine
recipients become older. If so, as described below, zoster can also be prevented by
vaccination. Oka vaccine latency may also result in recombination with wild type
VZV in vaccine recipients who are super-infected. However, it seems unlikely that
reversion to wild type patterns of VZV virulence will occur.
Clinical experience with the efficacy and safety of zoster vaccine. The associa-
tion of zoster-related morbidity in older adults and immunocompromised patients
with declining memory T cell immunity along with experience confirming the
efficacy and safety of live attenuated varicella vaccines set the stage for developing
zoster vaccines [112]. In an early proof of concept study, immunization with a heat-
inactivated Oka/Merck vaccine preparation was associated with a reduction in the
incidence of zoster from 33 to 13% during the first year after autologous hemato-
poietic cell transplantation when the vaccine was given as one dose before and three
doses after transplantation [101]. Comparing vaccine recipients with matched
controls who were unvaccinated showed that VZV specific CD4 T cell responses
were reconstituted much earlier among vaccinees, despite their severely immuno-
compromised state. No vaccine-related adverse effects were observed in recipients
of this heat inactivated VZV vaccine. By showing a correlation between restoring
VZV T cell immunity and reduced zoster incidence, this study provided direct
evidence of the role of cell-mediated immunity in preventing the progression of
VZV reactivation to symptomatic zoster.
After dose finding studies, a large placebo-controlled trial was done to evaluate
high potency live attenuated Oka vaccine preparations, ranging from 18,700 to
60,000 pfu (median 24,600 pfu) for effects on zoster incidence and severity [128].
Enrolment targeted healthy adults who were >60 years old. Among the 38,546
participants, the median age was 69 in both the vaccine and placebo cohorts; 6.6%
of vaccine and 6.9% of placebo recipients were 80 years old. Intensive surveil-
lance for zoster was carried out for an average of 3 years; cases were determined
by laboratory confirmation and each episode was assessed using pre-established
criteria for zoster severity, post-herpetic neuralgia, and health quality of life. The
primary endpoint of the study was a zoster burden-of-illness score, representing
a composite index reflecting the incidence and severity of zoster. This score
was significantly lower in the vaccine cohort compared to the placebo cohort
(P < 0.001); the effect was independent of sex or age <70 vs. >70 years. Post-
herpetic neuralgia (PHN) is the most common debilitating complication of zoster in
older individuals. PHN rates were 0.46 cases per 1,000 person-years in the vaccine
cohort and 1.38 cases in the placebo cohort (P < 0.001); the effect of vaccination
on PHN rates was also independent of sex and age stratification. The study was
designed with the incidence of zoster per 1,000 person-years as a secondary
endpoint. The zoster incidence was 5.42 in the vaccine group and 11.12 per 1,000
person-years in the placebo group (P < 0.001). This difference represents a 51.3%
efficacy of the high potency vaccine for zoster prevention among individuals >60
years. When the data was stratified by age cohorts, vaccine efficacy for zoster
prevention was 63.9% among those <70 years old vs. 37.6% in those who were
>70 years old (P < 0.001). Thus vaccine recipients in the older cohort were more
likely to develop zoster despite immunization. Nevertheless, participants in the
vaccine group who were >70 years old experienced less severe zoster than those
>70 years in the placebo group. The impact of vaccination on zoster severity was
greater among those >70 years, who were at a higher risk for a more severe episode.
Although the vaccine was less effective for zoster prevention in the older age
cohort, the benefit of reduced zoster severity maintained vaccine efficacy, assessed
from the burden of illness, at 55.4% in healthy adults >70 years old. Overall, the
burden of illness score was reduced by 61.1% and PHN incidence was 66.5% lower
in men and women >60 years old who were vaccinated.
Serious adverse events were uncommon and rates were equivalent in the vaccine
and placebo cohorts. Even though the vaccine contained high concentrations of
infectious vaccine Oka, all episodes of zoster were confirmed to be wild type VZV
when lesion specimens were tested by PCR and sequencing.
Immunogenicity of zoster vaccine. Zoster vaccine given to healthy older indivi-
duals in dose finding studies boosted VZV T cell responses above baseline, with
a half life of at least five years [112]. High potency Oka vaccine corrected
deficiencies in CD4 T cells that produced IFN-g or IL-2 and frequencies of
CD4 and CD8 effector memory T cells that responded to VZV antigen [94].
Some participants in the efficacy study of zoster vaccine were also evaluated for
effects on VZV cellular immunity as measured by responder cell frequencies and
ELISPOT assay. Responses were higher within the first 6 weeks when vaccine
and placebo recipients were compared and were higher in those who were less than
70 years old when vaccinated compared to those over 70 in the vaccine cohort.
Despite a decline by 12 months, VZV T cell responses continued to be above
baseline in the vaccine group for the 3 year follow-up period [94].
Zoster vaccine issues for the future. The experience with high potency Oka
vaccine demonstrates that symptomatic VZV reactivation can be prevented or its
consequences minimized in healthy older people by enhancing their VZV-specific
T cell responses. Importantly, safety was maintained despite the high inoculum of
vaccine virus. That this intervention diminishes the risk and consequences of VZV
reactivation is relevant to the potential for vaccine control of other herpesviruses
that also persist for the life of the host and although the effect is on reactivation
rather than active replication of a chronic infection, the zoster vaccine can be
viewed as a proof of principle that therapeutic vaccination is feasible. Among the
unresolved questions are the optimal age for giving zoster vaccine and how long
protection against zoster and post herpetic neuralgia will be maintained. More
information about protection in the very old is also needed. Whether the vaccine
virus reaches sensory ganglia when given to individuals with pre-existing VZV
immunity is not known. Given the evidence that super-infection can occur with
wild type VZV strains and in varicella vaccine recipients, it is possible that zoster
vaccination could lead to recombination events as has been observed in a few
instances after varicella vaccination. Whether inactivated Oka vaccine will reduce
zoster morbidity in immunocompromised patients in a larger placebo-controlled
trial is being evaluated.
Opportunities to improve live attenuated VZV vaccines. The VZV genome can
be mutated readily using cosmid and bacterial artificial chromosome methods and
the consequences of targeted mutations on VZV virulence in skin, T cells and DRG
can be evaluated in the SCID mouse model of VZV pathogenesis. Insights gained
about the molecular mechanisms of VZV pathogenesis have relevance to designing

“second generation” live attenuated VZV vaccines that have reduced potential
to infect T cells and neurons and are safer for immunocompromised patients.
Disrupting T cell tropism would be predicted to prevent vaccine virus delivery to
neuronal cells, which would block the establishment of vaccine virus latency in
sensory ganglia and eliminate the potential for vaccine virus recombination with
wild type VZV as well as vaccine-related zoster.
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children vaccinated with varicella vaccine in a prepaid health care plan in the United States,
2002 2008. Pediatr Infect Dis J 28:1069 1072
126. Prevention CfDCa (2008) Prevention of herpes Zoster: recommendations of the advisory
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127. Gershon AA, Levin MJ, Weinberg A, Song LY, LaRussa PS, Steinberg SP, Bartlett P (2009)
A phase I II study of live attenuated varicella zoster virus vaccine to boost immunity in
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128. Oxman MN, Levin MJ, Johnson GR, Schmader KE, Straus SE, Gelb LD, Arbeit RD,
Simberkoff MS, Gershon AA, Davis LE et al (2005) A vaccine to prevent herpes zoster
and postherpetic neuralgia in older adults. N Engl J Med 352:2271 2284
Classical Live Viral Vaccines
Thomas P. Monath
Abstract Classical, live viral vaccines have been developed by adapting viruses by
serial passages in animals, tissue or cell cultures during which multiple mutations
in the viral genome have accumulated. The majority of vaccines in use today were
developed in this way and a number of similar investigational vaccines are cur-
rently in development. The principal advantage of live vaccines is that they mimic
natural infection and induce durable immunity, including cytotoxic T cell responses
that are not generated by soluble proteins and inactivated vaccines. Recent studies
of gene activation following a live vaccine (yellow fever 17D) have shed light on
the role of innate immune responses in provoking strong, polyfunctional adaptive
immunity following the administration of live vaccines. The principal disadvantage
of live vaccines is that, occasionally, infection caused by the live vaccine causes
adverse events resembling the parental (virulent) virus. Such events can be due to
reversions in critical attenuating mutations or to host-specific susceptibility factors.
The attenuating mutations in live vaccines increase the inapparent: apparent infec-
tion ratio compared to the parental virus, but overt infection (adverse events) while
far less frequent than in natural infection can still occur. This problem is inconse-
quential for infections that are typically mild or self-limited, such as measles, mumps,
rubella, and varicella, but can be devastating for infections that are frequently lethal
such as yellow fever or in individuals who are immunocompromised. Despite these
issues, live vaccines have had dramatic benefits in reducing the incidence of the most
important infections of humankind, and in one case (smallpox) a live vaccine helped
eradicate a viral disease.
T.P. Monath
Kleiner Perkins Caufield & Byers LLC and Harvard School of Public Health, 295 Townsend Hill
Road, Townsend, MA 01469, USA

48 T.P. Monath
1 Introduction
Classical live viral vaccines are those developed by empirical methods, mostly by
adapting the virus to replicate in a host different from that in which the virus grows
naturally. Almost all viral vaccines in use today (Table 1) were developed in this
way, and they have contributed to the successful control of many major diseases.
Each of these vaccines has its own unique history, indications for use, and
biological characteristics, the details of which are beyond the scope of this brief
review. Therefore, this chapter will focus on the general principles underlying the
development, safety, and immunogenicity of classical live vaccines and illustrate
the same with specific examples.
2 Timelines for Vaccine Development
Figure 1 displays the evolution of development of approved live viral vaccines,

beginning with a description, by Edward Jenner in 1796, of the use of cowpox for
protection against smallpox to a modern smallpox vaccine produced in cell culture
Table 1 Viral vaccines approved for use

A. Available as live vaccines
Adenovirus 4, 7 (military use)a
Junı́n (Argentine hemorrhagic fever, available in Argentina)
Measles
Measles mumps rubella
Measles mumps rubella varicella
Mumps
Rotavirus
Rubella
Smallpox (vaccinia)
Varicella
Yellow fever
Zoster
B. Available as live and inactivated vaccines
Hepatitis A (live vaccine available in China)
Influenza [live vaccine available in US (Flumist®) and Russia]
Japanese encephalitis (live SA14 14 2 available in Asia)
Poliomyelitis
C. Available only as inactivated or subunit vaccines
Hepatitis B
Rabies
Human papillomavirus
Tick borne encephalitis
Kyasanur forest disease (India)
Hemorrhagic fever with renal syndrome (Korea, China)
a
In registration
Classical Live Viral Vaccines 49
Fig. 1 Year in which currently approved vaccines were first tested in humans, establishing proof
of concept and initial data on safety and immunogenicity
(ACAM2000) first tested in humans 206 years later. Jenner referred to the material
used in his studies as “vaccine”, a term derived from the Latin word for cow
(vacca). One hundred years later, Louis Pasteur applied the term “vaccination” to
the use of other agents for inducing prophylactic immunity. Jenner’s invention
(use of an animal virus that was poorly adapted for growth in humans but capable
of evoking cross-protective immune responses), often referred to as Jennerian
vaccination, is still en vogue; for example, the first human rotavirus vaccines
were developed using bovine (e.g., Nebraska Calf Diarrhea Virus) or simian
rotaviruses and veterinarians have long used measles vaccine to protect animals
against the antigenically related canine distemper virus. Vaccinia virus used for
smallpox vaccination in modern times is not cowpox, but a different orthopoxvirus
(closer to horsepox than cowpox) which arose during uncontrolled passages in
animals and humans prior to the establishment of standardized manufacturing in the
middle of the 20th Century.
The development of all other live viral vaccines awaited the availability of tools
for isolating and efficiently growing obligate intracellular viruses and for identify-
ing, quantitating, and characterizing them in the laboratory. These tools first
became available in the late 1920s and early 1930s, when yellow fever, influenza,
and polio viruses were shown to infect laboratory rodents (Theiler, Armstrong and
others), chorio-allantoic membranes of the developing chick were demonstrated to
be susceptible to fowlpox virus (Woodruff and Goodpasture), and various relatively
crude tissue culture systems were developed for growing viruses (Maitland, Carrell,
and others). Use of the embryonated egg for virus propagation enabled the isolation
of causative agents of viral diseases, for example mumps virus (in 1934) and the
production of live vaccines, including vaccinia (1933) and yellow fever (1936).
Cell culture methods developed by Enders, Weller, and Robbins in 1948 enabled
50 T.P. Monath
the propagation of mumps, measles, polio, and other viruses for vaccine production.
The advent of molecular biology in the 1960s has allowed the rational design of new
live viral vaccines, including reassortant rotavirus vaccines, chimeric, live vaccines,
and many other new generation vaccines described elsewhere in this book.
140 years had elapsed since Jenner’s smallpox vaccine was described, before a
new live, attenuated vaccine for humans was developed (Fig. 1). Following the lead
of Pasteur, who had “fixed” rabies virus neurovirulence by serial passage in rabbit
brain and had thereby reduced its virulence for dogs, Max Theiler (Rockefeller
Institute, New York) passed the French strain of yellow fever virus (a virus
evolutionarily adapted to primate hosts) by serial passage in mouse brain until it
lost its ability to cause hepatitis in monkeys, while retaining a relatively high level
of neurotropism. The virus fixed by intracerebral passage up to 176 times was used
to prepare a vaccine from a clarified suspension of mouse brain tissue. In 1931,
Sawyer, Kitchen, and Lloyd used Theiler’s French neurotropic virus for human
immunization [1]. Human immune serum was added to the vaccine, since the virus
was thought to be insufficiently attenuated for direct application. By 1936, Max
Theiler and Hugh Smith had developed a safer, live attenuated yellow fever vaccine
(the 17D strain) by serially passaging another wild-type virus (the Asibi strain) in
cultures of minced mouse embryos and embryonated chick embryos; this vaccine
was shown in human trials to be safe and highly immunogenic [2], and the 17D
vaccine remains in wide use, with over 600 million doses distributed over ensuing
decades. Other early attempts were abortive and did not result in approved products.
About the same time as yellow fever vaccine was being tested, Smorodintsev et al.
in the Soviet Union immunized volunteers with a mouse-adapted strain of influenza
virus given by the respiratory route [3], and in 1935, Kolmer prepared a live vaccine
candidate against poliomyelitis that had been attenuated by serial passage in
monkey spinal cord tissue (the vaccine proved unsafe, causing cases of polio at
an incidence of 1 in 1,000) [4].
Live, attenuated viruses represent the most widely used and effective approach
to human vaccination. Of the 16 licensed vaccines against human viral diseases
available today, 12 (75%) are live, attenuated vaccines and four (25%, polio,
influenza, Japanese encephalitis, and hepatitis A) are used as either inactivated or
live, attenuated products (Table 1).Only four widely available, approved viral vac-
cines for humans (hepatitis B, human papillomavirus, rabies, tick-borne encephalitis)
and two regional vaccines (Kyasanur Forest disease, hemorrhagic fever with renal
syndrome) are available solely as inactivated or recombinant subunit vaccines.
Classical, live, attenuated investigational vaccines against respiratory syncytial
virus, parainfluenza, dengue, West Nile, Venezuelan equine encephalitis, Rift Valley
fever, and chikungunya are in various stages of development. Although considered
too risky for human immunization, a live attenuated HIV vaccine (or the SIV
equivalent) has demonstrated the feasibility (in the monkey model) of prophylactic
immunization against this most challenging disease [5]. The success of live vaccines
is based on a suite of recognized advantages, described in the next section and in
Table 2. The obstacles to development of live vaccines include (1) the difficulty in
finding the correct balance of attenuation (safety, tolerability) and immunogenicity,
Table 2 Advantages of live viral vaccines

Replicate in host, expand antigenic mass
Combine roles of delivery vehicle, adjuvant, and antigen
Often target antigen presenting cells
Wide dissemination of antigen
Contains all antigens of pathogen of interest in native configuration
Strong innate immune response, cytokine environment similar to pathogen of interest
Intracellular replication, MHC I presentation, cytotoxic CD8þ T cell response
Durable response, large pool of memory B and CD8þ T cells
which is still largely an empirical exercise though facilitated today by knowledge of

molecular determinants of virulence and immunity, and (2) the inability to propagate
certain viruses in a suitable substrate. The regulatory pathway for live vaccines is
increasingly difficult, and it is certain that the time for the development of some
older vaccines, such as smallpox, rubella and polio would have been much longer
if undertaken in today’s regulatory environment due to concerns over safety. The
principal problems with live viral vaccines are the potential for serious adverse events
related to active infection in individuals with acquired or hereditary susceptibility
factors; genetic instability of the virus; lack of thermostability and (historically),
contamination with adventitious viruses during manufacture.
3 Development of Classical Vaccines
The principle that attenuation of virulence would occur by passage of a virus in an

unnatural host was established first by Pasteur, who found that the virulence of
rabies virus became attenuated for dogs after serial passages in rabbit brain. The
same principle was applied by Theiler in developing yellow fever vaccine by
adaptation in mouse brain or in chick embryo tissue culture [for which he received
the only Nobel Prize (1951) for a virus vaccine [6]]; by Koprowski et al. in adapting
poliovirus type 2 to mice and cotton rats [7]; by Enders et al. in adapting measles
virus by passage in human kidney, human amnion, and chick embryo cells [8]; and
by Maassab et al. by adapting influenza virus in primary chick kidney cells [9], to
name but a few examples. In some cases, tissue culture passages were done at
suboptimal temperature for virus growth as a means of selecting a temperature
sensitive virus population that would be less fit for replication in humans at higher
body temperature. This approach was employed in the development of measles,
influenza, varicella, and the (investigational) respiratory syncytial virus vaccines.
The attenuating mutations that occurred in oral polio vaccine strains during passage
also rendered them temperature sensitive. The passage history of some vaccines
was quite elaborate; in particular, the development of oral polio vaccines by Sabin
involved meticulous plaque purification and neurovirulence testing to select candi-
date strains. Theiler monitored the virulence of yellow fever virus at many points
52 T.P. Monath
during its passage series in minced chick embryo tissue culture by inoculating
monkeys intracerebrally and found that neurovirulence and viscerotropism were
lost between the 89th and 114th passages (Fig. 2) [10]. The passage history of
the SA14-14-2 Japanese encephalitis vaccine involved some peculiar empirical
passages in rodents by different routes (Fig. 2). Those viruses for which no animal
model existed for human virulence, such as measles, mumps, rubella, and varicella
posed the problem that attenuation could not be readily assessed as the passage
series in tissue cultures progressed. The decision to stop a passage series and
perform clinical tests to determine whether the correct balance of attenuation and
efficacy had been reached was essentially a “judgment call” and resulted from a
series of trials and errors [11].
4 Genetic Basis of Attenuation
For many vaccines, including measles and mumps, the genetic basis of attenuation
is unknown. Comparison of the genomic sequences of classical live vaccine strains
and their virulent parents, and the use of infectious cDNA clones, in which muta-
tions or reversions that occurred in vaccine strains are investigated for their
phenotypic effects, have provided descriptive insights into the molecular basis for
attenuation of some vaccines. However, due to the large number of mutations that
have arisen during the long series of passages in the derivation of live vaccines
and the multigenic nature of virulence, it has often not been possible to pin-point
the role of specific determinants. For example, the 17D and parental Asibi strains
of yellow fever differ at 20 amino acids and four nucleotide changes in the 30
noncoding region (NCR). It has not yet been determined precisely which mutations
are responsible for the reduced virulence of the 17D vaccine strain [12], although
gene switching experiments have shown that the mutations in both the structural
genes [principally the envelope (E) glycoprotein] and the nonstructural region are
important [13]. Rubella vaccines and their progenitors have been sequenced; the
TO-336 vaccine differed from its parent at 21 nucleotides but the phenotypic
changes associated with mutations are unknown and no consistent pattern of muta-
tions occurred in other vaccine strains [14]. Similarly, varicella vaccine (Oka) and its
wild-type parent have been compared; a number of sequence differences have been
found but the determinants responsible for the attenuation remain undefined [13].
In contrast, precise mapping of the molecular determinants of the live poliovirus
vaccines has been accomplished. For example, in the case of poliovirus types 2,
attenuation is dependent on only two nucleotide changes, one in the 50 NCR and a
threonine!isoleucine mutation in a capsid gene VP1, at position 143 [15]. Poliovirus
types 1 and 3 also have a critical attenuating mutation in the 50 NCR (as well as
attenuating mutations in the capsid genes.) The polio type 1 vaccine has many more
attenuating mutations in the capsid (and one in a nonstructural gene) consistent with
the better safety record of that serotype [16].
a Asibi virus (wild- b SA14 (wild-type)

type) mosquito pool
18 passages in 100 passages in

minced mouse primary hamster
embryo kidney cells
P18
P100
5 plaque
purifications chick
embryo cells
58 passages
Minced chick
embryo
P76
One mouse
passage IP
inoculation
P106
145 passages in
minced chick
Attenuation occurs
embryo with brain Spleen virus
between P89 and 114
and spinal cord plaque purified in
removed chick embryo cells
P221
One mouse
passage SC
12 passages in inoculation
embryonated eggs
P232
Skin virus passed
17D vaccine in chick embryo
cells
6 passages in
hamsters by oral
route; spleens
passaged in chick
embryo, plaque
P115 purified
5 passages in
infant mice, using
virus from skin and
peripheral lymph
nodes
2 plaque
purification
passages in
primary hamster
kidney cells
P122
SA14-14-2 vaccine
Fig. 2 Passage histories of A. yellow fever 17D and B. the live, attenuated Japanese encephalitis
SA14 14 2 vaccine, leading to development of attenuated vaccines
54 T.P. Monath
5 Advantages of Live Viral Vaccines
Most naturally acquired viral infections induce a durable state of immunity against
reinfection with the homologous virus, which is often life-long. If this immunity is
not complete, at least disease expression is modified and less severe. Live viral
vaccines mimic this attribute of natural infection, whereas inactivated or subunit
vaccines are generally associated with short-lived immunity. The reasons for this
difference are not completely understood. Some key factors are the activation of
innate immunity by the active infection and the production of type I interferons
promoting a strong adaptive immune response, a brisk CD4þ helper T cell res-
ponse, and a large pool of memory B and CD8þ T cells. Unlike inactivated or
subunit vaccines, replicating live vaccines cause an expansion of antigenic mass and
present all of the epitopes of the native virus to antigen presenting cells. Antigen
processing proceeds in a Class-I-dependent manner, identical to that following natural
viral infection. Both humoral and cellular responses are evoked efficiently. Finally, live
viral vaccines can be delivered by the natural (e.g., oral, intranasal) route of infection
and elicit mucosal immunity protecting the portals of entry of virus infection.
The extraordinary efficacy of yellow fever 17D vaccine has spurred studies
aimed at understanding the ability of this vaccine to rapidly elicit strong, life-long
immunity in virtually 100% of individuals [17] after a single dose of 100 plaque
forming units or less [12]. The adaptive immune response includes early production
of IgM antibodies that last for 18 months or more [18], robust neutralizing antibody
production that persist life-long [19], a balanced Th1 and Th2 helper cell response,
and a CD8þ T cell response [20]. The gene activation signatures following yellow
fever 17D vaccination and interaction of the virus with immune cells have been
studied in detail [21 23]. Yellow fever 17D virus targets dendritic cells (DCs) as an
early site of replication, though productive replication in these cells appears to be
limited [24, 25]. The virus activates myeloid and plasmacytoid DCs to produce
proinflammatory cytokines (IL-12p40, IL-6 and interferon-a) via toll-like receptors
(TLR) 2, 7, 8, and 9 [26]. Other proinflammatory mediators induced in peripheral
blood mononuclear cells of vaccinees include IL-1a and CXC-chemokine ligand 10
(CXCL10) [20]. The activation of multiple TLRs is responsible for the mixed
Th1 and Th2 helper T cell response seen after 17D immunization. Production of
interferon-a by plasmacytoid DCs requires TLR-mediated activation of the mam-
malian target of rapamycin (mTOR), a regulator of cytokine expression, and
activation of downstream mediators of mTOR, p70 ribosomal S6 protein kinases
[24].The innate immune receptors RIG-I and melanoma differentiation-associated
gene 5 (MDA-5) are stimulated also, as well as the genes that regulate these
signaling pathways (e.g., RIG-I-like RNA helicase). Transcription factors that
regulate expression of interferon-a are activated (e.g., IRF7, ETS, and STAT1).
In addition, genes in the AIM2 (inflammasome) pathway and the complement
pathway are switched on. Importantly, gene signatures correlate with the magnitude
of the B and T cell responses in individual subjects. Complement protein C1qB,
eukaryotic translation factor 2 alpha kinase 4 (EIF2AKA) and solute carrier family 2,
member 6 (SLC2A6) correlated with CD8þ responses [20, 26]. Activation of TNF
receptor superfamily, receptor 17 (TNFRSF17), a receptor for B cell activating
factor (BAFF), was a correlate of the magnitude of the antibody response. Overall,
the gene signatures indicate a marked up-regulation of the innate immune system
that persisted for about 2 weeks after vaccination. This response determined, in
turn, the strength and persistence of the adaptive polyfunctional immune responses
to 17D vaccine. It is likely that analogous patterns of gene activation occur
following natural infection, and that they will also be uncovered when other live
viral vaccines are subjected to study.
An obvious control for studies of gene activation and the role of innate immune
activation in adaptive immune responses would be a comparison of the latter
following the administration of an inactivated or subunit vaccine against the same
virus. Inactivated (subunit) vaccines tend to elicit Th2 oriented helper T cell
responses, weak or absent CD8þ cytotoxic T cell responses, and B cell responses
that are relatively short-lived. How these differences are reflected in the gene
activation signatures following immunization with inactivated versus live virus
vaccines remains to be determined.
Live vaccines have the potential advantage of being administered via the natural
route of infection, and of stimulating mucosal as well as systemic immunity. The
live attenuated intranasal vaccine against influenza is immunogenic and effective,
given as a single dose to adults or two doses to children. Unlike the parenteral
(inactivated) influenza vaccine, the intranasal vaccine does not efficiently elicit
rises in hemagglutination-inhibiting antibodies, indicating that other mechanisms,
which more closely mimic the responses to natural influenza infection, underlie
protective immunity [27]. The live vaccine is able to evoke CD8þ T cells [28] and
secretory IgA antibodies [29], whereas inactivated vaccine does not. It is likely
that mucosal immunity and T cells in the respiratory tract play an important role
in vaccine induced protection against infection in the upper and lower airway
epithelium.
Similarly, oral polio vaccine is associated with the production of local immunity
that not only protects the host but also restricts virus replication in the pharynx and
gut and reduces transmission of wild-type viruses, a benefit particularly important
in the developing world. Mucosal immunity is evoked in a high proportion of
neonates even after a single dose of oral vaccine. Oral polio vaccine is more
efficient than inactivated vaccine in inducing mucosal immunity [30].
The application of measles vaccine by aerosol induces higher systemic and
mucosal antibody responses than subcutaneous inoculation [31].
6 Benefits of Live Virus Vaccine Utilization
The public health impact of live virus vaccines can hardly be exaggerated. Smallpox
vaccine was critical first to the prevention and control of smallpox worldwide, and
then, as part of a strategy of surveillance and containment, vaccination contributed
56 T.P. Monath
to the eradication of the disease (the first and only example of the eradication of a
human infectious disease). The vaccine protects against illness not only if given
before exposure but also postexposure when administered during the first week.
The success of poliovirus vaccination is almost as spectacular. Oral polio
vaccine was introduced into routine childhood immunization worldwide [32], and
this approach combined with mass campaigns has led to significant declines in polio
and elimination of the disease in many parts of the world, particularly the Western
Hemisphere. Overall vaccine coverage rates approach 80% [33]. Seroconversion
rates in industrialized countries are >95% after three doses, but immunogenicity
is substantially less in developing countries (~73%) [34] due to interference by
immunity to heterologous enteroviruses or ingestion of maternal (milk) antibody.
The vaccine not only induces serum neutralizing antibodies that protect the central
nervous system but also affords mucosal protection and a reduction in the potential
for transmission. Eradication of polio may be feasible, but the use of oral (live)
vaccine will need to be replaced with inactivated vaccine because of the problem of
persistence and transmission of the live vaccine virus and recombinant viruses.
Measles vaccine is highly effective, resulting in >95% seroconversion rates and
efficacy under field conditions of >90% [35]. The vaccine is generally given as a
stand-alone vaccine in the Expanded Program of Immunization, or is combined
with mumps and rubella in developed and some developing countries. Overall
vaccine coverage worldwide approaches 80% [31]. Some countries, e.g., Scanda-
navia, have been able to eliminate measles altogether through vaccination. Because
of the high infectiousness of the virus, where immunization coverage is incomplete
or lapses, the disease has reappeared.
Rubella vaccination has led to dramatic declines in the incidence of congenital
rubella syndrome, and indeed this disease can be eradicated where immunization
practices are vigorously applied.
Two arthropod-borne diseases, yellow fever and Japanese encephalitis, have been
successfully controlled through routine vaccination and mass campaigns in many
endemic regions. Like the yellow fever vaccine, the live attenuated SA14-14-2
Japanese encephalitis vaccine induces protective immunity after a single dose
in >90% of vaccines, although immunity may be less persistent.
7 Problems with Live Vaccines
7.1 Shedding, Viremia, and Recombination
With the exception of oral polio vaccine and vaccinia virus, shedding of live viral
vaccines is considered to be brief in duration and low in magnitude, with very low
risk of transmission to contacts.
Smallpox vaccine (vaccinia virus) is delivered by epidermal scarification and
causes a cutaneous lesion that sheds virus for prolonged periods (~3 weeks) [36],
until scabbing is complete. This can be the source of infection of direct contacts or
persons exposed to fomites from contaminated clothing or dressings, who in turn
may develop severe adverse events if they have underlying risk factors such as
eczema or immune suppression. The application of dressings to limit shedding and
precautions to limit fomite spread is recommended [37].
Between 70 and 90% of infants undergoing primary immunization with oral
polio vaccine excrete virus in feces [38]. This is a frequent source of infection for
family members and nonfamilial contacts. As discussed below, shedding and
transmission of virus assumes greatest importance when neurovirulent revertant
virus [causing vaccine-associated paralytic poliomyelitis (VAPP)] is spread to
immunologically competent or immunodeficient contacts. Individuals with B cell
deficiency can become life-long carriers of vaccine derived polioviruses, posing a
significant challenge for the eradication of the disease [39].
The cold-adapted live intranasal influenza vaccine is shed in nasal secretions,
but at a very low level. Shedding of virus following vaccination occurs transiently
(for ~1 day) versus 5 days or more in the case of natural infection, and viral loads
are ~1,000-fold lower than in natural infections [40, 41]. The risk of transmission is
thus exceedingly small. When monovalent influenza A vaccine was administered to
subjects without immunity to the vaccine strain, 40 80% shed virus with relatively
low peak titers between 1.5 and 3.0 log TCID50/mL [42]. A theoretical concern for
use of the live, avian (H5N1) influenza vaccine in the context of active transmission
is that the vaccine virus could reassort with wild-type virulent H5N1 virus and
produce a virulent virus adapted for interhuman transmission.
The likelihood of horizontal transmission of measles vaccine virus is considered
very low, although shedding in respiratory secretions following subcutaneous
vaccination has been documented occasionally [43]. Measles virus is shed in urine
for 10 days or more after natural infection, and shedding may be prolonged in immune
deficient children [44]. Viral RNA or antigen has also been detected in urine of a high
proportion of vaccinees [45].
After natural infection, mumps virus is shed in respiratory secretions, principally
before the onset of parotitis [46]. Shedding of live mumps vaccine has been
documented, and transmission to susceptible contacts recorded, principally in the
case of less attenuated vaccine strains such as Urabe and Leningrad-3 [47, 48].
Skin lesions are the source of shedding and potential transmission of the varicella
vaccine virus. The vaccine causes skin lesions in ~3% of healthy vaccinees, and these
are exceedingly mild. Transmission to unimmunized contacts is extremely rare, and
only a few documented cases have been reported. However, in leukemic children
vaccination may be associated with increased number of skin lesions and potential for
shedding. In one study, 23% of contacts were infected and 17% developed a rash
[49]. Tertiary spread is exceedingly rare.
Yellow fever 17D produces a minimal viremia in humans, and the levels (< 2.0
log10 PFU/mL) are ~ 10,000 times lower than observed in wild-type yellow fever
infections, and are insufficient to infect mosquitoes [11]. Moreover, yellow fever
17D are incapable of infecting mosquitoes after oral infection [50]. The live,
attenuated Japanese encephalitis SA14-14-2 vaccine is infectious for mosquitoes,
58 T.P. Monath
but the low viremia levels in vaccinated subjects precludes infection and transmis-
sion. Viremia following yellow fever 17D vaccination is the source of neuroinva-
sion in rare cases of yellow fever vaccine associated neurotropic adverse events
(YEL-AND).
7.2 Genetic Instability
The replication of RNA viruses is error-prone resulting in rapid adaptation

and evolution, and these viruses characteristically have a quasi-species nature
(heterogenous mixtures of sequence variants). High mutation rates are believed to
be due to the absence of proofreading enzymes in viral RNA-dependent RNA
polymerases or reverse transcriptases. The generally accepted average incidence
of mutations is 10 4 10 5 per nucleotide per round of RNA replication [51, 52].
However, this rate may differ among RNA viruses; for example one estimate of the
mutation rate in the yellow fever 17D polymerase gene was as low as 5.7 10 8 per
copied nucleotide, suggesting this virus was more genetically stable than most [53].
An additional feature of RNA viruses is the occurrence of “hot spots” for mutation
in different functional areas of the genome; these may be related to adaptation for
growth in a particular cell type (see the chapter “Recombinant, chimeric, live,
attenuated vaccines against flaviviruses and alphaviruses” for examples).
The genome sequence and biological characteristics of the subpopulations in
quasi-species vaccines can differ greatly, and selection of a more virulent subpopu-
lation in the vaccinated host can lead to adverse consequences. For example, the
Urabe strain of mumps vaccine (discontinued due to neurovirulence) contains
subpopulations that differ in the sequence of the hemagglutinin-neuraminidase
gene and the propensity to cause meningitis post-vaccination [54]. The yellow
fever 17D vaccine is also a mixed population, and it contains subpopulations with
different plaque morphology and reactivity with monoclonal antibodies [11]. This
“magic sauce” of virion populations appears to have the right balance of attenuation
and efficacy, which could be perturbed with a few passages, since over-attenuation
and a higher frequency of serious adverse events occurred during early develop-
ment when passage level was not controlled by a seed lot system. Specifically,
a high incidence of postvaccinal encephalitis was associated with uncontrolled
passage of yellow fever 17D sub-strains during the early years of vaccine manufac-
ture [55]. The problem was resolved when stabilization of passage level during
vaccine manufacture was instituted in 1941. Similarly, when the Japanese enceph-
alitis SA-14-14-2 vaccine underwent some additional passages and was manufac-
tured in a new substrate (primary dog kidney) [56] instead of the standard primary
hamster kidney cell substrate, it was overattenuated when tested clinically.
The quasi-species nature of oral poliovirus vaccine is especially problematic
due to revertants in the 50 NCR mutations that are critical to attenuation [57, 58].
Revertants associated with an increase in neurovirulence and failure to pass the
monkey safety test occur at a low frequency (0.1% of the total virus population) but
can increase during large scale production. The most important adverse event
associated with the use of oral poliovirus vaccine is VAPP, which is most commonly
caused by the polio type 3 vaccine component. The incidence of VAPP following
primary vaccination is approximately 1 in 900,000 [59]. Immune deficiency, particu-
larly B cell deficiency is a risk factor for VAPP, due in part to uncontrolled replication
and an opportunity for the emergence of revertant strains. Analysis of viruses isolated
from VAPP patients shows that the majority retain genome sequences that are less
than 1% divergent from the original vaccines, but as would be expected sequence
divergence depends on the duration of time between vaccination and isolation.
Persistent infections occur particularly in immune deficient subjects. Most cases of
VAPP result from infection with revertants at determinants associated with increased
neurovirulence, particularly for polio 2 and 3 where attenuation is controlled by only
2 or 3 mutations, including the single 50 NCR mutations [60 62]. Recombination
events (with oral polio vaccine strains and heterologous enteroviruses) are also
implicated in VAPP.
Perhaps because of its lower mutation rate [63], yellow fever 17D vaccine has
been less frequently associated with adverse events arising as a result of mutation.
Deaths from postvaccinal encephalitis (yellow fever vaccine associated neurotropic
disease) are exceedingly rare but from one case (a 3 year-old girl in the US) virus
was recovered from the brain which exhibited increased neurovirulence for experi-
mental animals (mice and monkeys). The isolate differed from 17D vaccine at two
determinants in the E gene (E155 and E303) and in a nonstructural gene (NS4B76)
[63]. The mutation at E303 is very close to a determinant (E305) that distinguishes
17D vaccine from wild-type yellow fever virus and is located in Domain III of the E
protein, which contains ligands for cell-receptor interactions. It is possible that
mutations in this region could have altered tropism of the vaccine virus for neural
tissue.
Genetic instability at determinants controlling replication can result from selec-
tive pressure in vitro or in vivo. For example, mutations in loci controlling temper-
ature sensitivity (ts) can be favored under conditions of nonpermissive growth
temperatures [64]. Polioviruses isolated from patients with VAPP have lost the ts
phenotype and have increased fitness for growth in human intestine [39, 65].
The reader is referred to the chapter on recombinant, chimeric, live, attenuated
vaccines against flaviviruses and alphaviruses for further details on problems in
vaccine development associated with genetic stability of live RNA virus vaccines.
In contrast to RNA viruses, vaccines using live DNA viruses are inherently
stable [63].
7.3 Thermostability and Microbial Contamination
Most live vaccines are relatively unstable unless kept cold or lyophilized, and
vaccine failures have been caused by improper storage and handling [66]. The
instability of live vaccines has required the establishment of an elaborate cold-chain
60 T.P. Monath
infrastructure to support vaccine distribution in the Expanded Program of Immuni-

zation and has added significantly to the costs and complexity of childhood
immunization in developing countries. In the late 1970s efforts to improve the
stability of lyophilized measles and yellow fever viruses, two of the least thermo-
stable vaccines, resulted in significant improvements. However, the improvements
are incremental and have not approached the ideal of a liquid, heat-stable formula-
tion that would not require a cold-chain (this has been the subject of significant
research efforts stimulated by the Gates Grand Challenge grants). Moreover, once
reconstituted to the liquid state, infectivity (potency) of live vaccines is lost rapidly,
and the reconstituted vaccines must be administered within 1 h (yellow fever) or 8 h
(measles). The live, varicella vaccine is especially thermolabile, must be stored
lyophilized in a mechanical freezer, and used within 30 min after reconstitution.
Thermostability requirements have been established by the World Health Organi-
zation and differ across vaccines. In general, these requirements specify that the
vaccine must retain minimum potency and not lose more than a specified amount
(e.g., 0.5 or 1 log10) under accelerated conditions (exposure at 37 C temperature)
for a specified length of time which may be as short as 2 days (polio) or as long
as 30 days (smallpox). Excipients used by some manufacturers to stabilize live
vaccines, in particular hydrolyzed porcine gelatin [67], can be responsible for
allergic adverse events.
With one exception (smallpox vaccine), it is not possible to include antimicro-
bial agents in multidose vaccine vials due to the fact that these agents inactivate
viruses; this is another reason why the vaccine must be used quickly after reconsti-
tution. There have been a number of reported episodes of bacterial contamination
of improperly handled multidose vaccine vials, resulting in serious infections
and even deaths [12]. An exception is smallpox (vaccinia), which is not adversely
affected by glycerol phenol preservatives in the diluent used to reconstitute the
lyophilized vaccine, which is in a 100-dose container used for repeated percutaneous
vaccinations over 30 days after reconstitution.
7.4 Adventitious Viruses
It is obvious that live vaccines would contain any passenger virus that happened to
be introduced during manufacture or as a result of contamination of the original
isolate used to develop the vaccine. The control of such contamination has steadily
improved over the years, and currently stringent steps are taken to reduce the
possibility of such contamination and to detect it through the use of both compen-
dial and special testing procedures applied to cell banks, raw materials, virus seeds,
and vaccine lots. There has been an effort in the vaccines industry to eliminate
sources of such contamination by using continuous cell cultures which can be
readily controlled rather than primary cell cultures or tissues (from embryonated
eggs and animals) for virus propagation, and to remove bovine serum and other
animal-derived proteins from the manufacturing process. This movement was
accelerated after the bovine spongiform encephalopathy epizootic in the 1980s.

When a new strategic supply of smallpox vaccine was required (to protect against
biological attack), serum-free cell culture methods of manufacture were selected in
place of the original calf lymph production [68, 69]. The concern over animal
viruses contaminating vaccines has increased with the recognition that many
viruses would not be detected by standard testing methods, such as bovine and
porcine circoviruses, polyomaviruses, and caliciviruses. The early history of vaccine
development is replete with significant problems related to adventitious agents, and a
few illustrative examples will be given.
Embryonated hens’ eggs were used for the propagation of measles and yellow
fever viruses, beginning in the 1930s and primary chick embryo cells have been
used to prepare measles and mumps vaccines. Flocks from which eggs were
sourced during the early years were not controlled for adventitious virus infections.
In 1966, measles and yellow fever 17D vaccines were discovered to be contami-
nated with avian leukosis virus [70]. Although the presence of leukosis virus is
certainly undesirable because of the possibility of insertion of leukosis virus
oncogenes, there is no evidence to implicate the virus in human disease. The
question was addressed by a retrospective survey for cancer deaths in veterans
who had received yellow fever vaccine as early as World War II [71]. The incidence
of all cancers, lymphoma, and leukemia was not significantly different (and in fact
was lower) in persons vaccinated 5 22 years previously with 17D vaccine than in
those not vaccinated. All major manufacturers of 17D currently utilize eggs from
ALV and other specific pathogen-free (SPF) flocks. Vaccines manufactured in eggs
or chick embryo cells, including yellow fever 17D, test positive by the product-
enhanced reverse transcriptase (PERT) assay, reflecting the presence of defective
particles containing endogenous avian leucosis or retrovirus sequences [72]. No
evidence has been found for infectious or inducible replication competent retro-
viruses or for infection with avian leucosis or endogenous avian retrovirus in
humans [73].
The lack of thermostability of live viral vaccines has required the addition of
stabilizers, typically proteins, to improve shelf life and stability under conditions of
shipment, storage, and use in the field. In the early days of vaccine development,
pooled normal human serum was used as a stabilizing excipient added to vaccines.
Yellow fever 17D was originally prepared in this way. In 1937, Findlay and
MacCallum reported cases of acute hepatitis in persons who received 17D vaccine
[74]. Cases were also reported in Brazil during the vaccination campaigns between
1938 and 1940 and, after careful study, were attributed to an adventitious agent
in the vaccine rather than to hepatitis caused by 17D virus [75]. In 1942 a large
epidemic of jaundice occurred in US military personnel who had received 17D
vaccine, with 28,000 cases and 62 deaths [76]. The use of pooled serum for
stabilizing 17D vaccines was discontinued in Brazil in 1940 and in the USA in
1943. Subsequent retrospective serological studies confirmed that the responsible
agent was hepatitis B virus [77]. In addition to the acute deaths from fulminant
hepatitis B, the vaccinees had a 3.3-fold increased risk of later developing liver
cancer [78].
62 T.P. Monath
Fetal bovine serum (FBS) is commonly used during expansion of cell cultures
for virus propagation. A common contaminant of FBS is bovine viral diarrhea virus
(BVDV), a ruminant pestivirus. Before the routine control of raw materials and cell
banks for the presence of BVDV, many vaccine lots contained this adventitious
agent and some modern vaccines may still contain infectious virus or at least RNA
sequences [79, 80]. Some mammalian cells used for manufacturing, such as Vero
cells, are permissive to growth of BVDV [81]. BVDV is not a known human
pathogen, but antibodies to BVDV have been found in patients with HIV/AIDS
[82] and in children with congenital neurological conditions, and antigen has been
found in stools of subjects with infantile gastroenteritis [83]. BVDV has also been
recovered from human leukocytes [84].
Cache Valley virus, a mosquito-borne bunyavirus, has been recovered from FBS
and, on at least one occasion, it contaminated a biological manufacturing process
[85]. Cache Valley virus is teratogenic in sheep and may also be in humans. A
human case of severe multiorgan failure and encephalitis caused by natural infec-
tion with Cache Valley virus has been reported [86].
Poliovirus vaccines (and a parenteral adenovirus vaccine used by the military)
were originally manufactured in primary monkey kidney cell culture. Some lots
of vaccine were found to be contaminated with simian virus 40 (SV40), a polyoma-
virus of Asian macaques that causes a progressive multifocal leukoencephalopathy
(PML) syndrome in immunosuppressed monkeys and cancer in rodents. Since
many children were exposed to SV40 as a result of polio vaccination in the 1950s
and 1960s, long term surveys were organized to assess the risk of cancer. It was
initially concluded that no increased risk was associated with exposure [87] and this
conclusion was supported by a number of additional epidemiological studies.
However, many of these studies were flawed based on the fact that exposed and
unexposed populations could not be reliably differentiated. The concern about a
role of SV40 in human cancers remains, because many different laboratories have
reported finding SV40 in a variety of human cancer tissues, and in particular human
mesotheliomas [88, 89]. SV40 does not contaminate current poliovirus vaccines.
7.5 Adverse Events due to Unchecked Replication in the Host
Yellow fever 17D vaccine causes a syndrome of hepatitis, multiorgan failure,

cardiovascular collapse, and high lethality (>60%), similar to that caused by the
wild-type virus. Fortunately the incidence of yellow fever vaccine associated
viscerotropic adverse events (YEL-AVD) is low (between 1:50,000 and 250,000
depending on the occurrence of risk factors for this condition) [12, 90, 91]. Unlike
VAPP, the occurrence of this syndrome is not dependent on genetic instability,
mutation, or selection of a virus variant with enhanced pathogenicity [92, 93].
Instead, YEL-AVD occurs in individual patients who have one of a variety of
underlying, familial or acquired risk factors that permit unchecked replication
of 17D virus in vital organs. The occurrence of this syndrome and its severity
is analogous to progressive vaccinia in individuals with defects in their T cell
responses, who are unable to clear their live vaccine infections. Indeed, some
patients who developed YEL-AVD had previously undergone thymectomy and
likely had a defect in adaptive immunity [94]. However the underlying defects
leading to YEL-AVD appear to be more complex. One case report described
possible defects in chemokine receptor 5 (CCR5) and the CCR5 ligand RANTES
[95] and another case report described a possible defect in an allele of the 20 50 -
oligoadenylate synthetase 1 (OAS1) gene involved in interferon-specified antiviral
responses [96]. Mice deficient in type I interferon receptor genes develop a lethal
viscerotropic disease [97]. Thus, defects in innate immunity, so important to the
control of adaptive immune responses to yellow fever 17D vaccine, appear to be
implicated in some cases of severe adverse events.
7.6 Precautions and Contraindications
Live vaccines have a number of precautions and contraindications for use, aimed at
protecting individuals who have underlying risk factors for adverse events. Each
vaccine has its unique set of such precautions, but there are some common and
logical themes. Age is an established risk factor for increased incidence or severity
of many natural viral infections, and the same principle applies to vaccine viruses
derived from wild-type viruses. Very young infants (<6 months of age) are at
increased risk of wild-type yellow fever and of YEL-AND following 17D vaccine,
and are thus excluded from vaccination. This adverse event is likely due to
immaturity of the blood brain barrier. (Age may also be a factor limiting effective-
ness of live vaccines. Where the prevalence of immunity is high, efficacy of
vaccination in young infants (< 6 months) may be impaired due to maternally
acquired immunity. This is the case for measles vaccine). Advanced age may also
be a risk factor for adverse events; the reporting rate of both YEL-AND and YEL-
AVD is >twofold higher in persons > 70 years of age than in younger persons [90].
Pregnancy is a contraindication, mainly on theoretical grounds that the live vaccine
virus could infect the placenta or fetus, causing stillbirth or congenital malforma-
tion. Inherited or acquired immune deficiency or treatment with immunosuppres-
sive drugs or radiation is also a contraindication for use of most live vaccines.
Fortunately significant events have been rare; progressive vaccinia in T cell defi-
cient subjects, the occurrence of fatal YEL-AND in a patient with HIV/AIDS [98],
and rare cases of pneumonia, severe rashes and hepatitis following varicella
vaccination of immunocompromised patients [99] are illustrative examples.
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Part II
Genetically Attenuated Micro-Organisms
as Vaccines
Recombinant Live Vaccines to Protect Against
the Severe Acute Respiratory Syndrome
Coronavirus
Luis Enjuanes, Jose L. Nieto-Torres, Jose M. Jimenez-Guardeño,

and Marta L. DeDiego
Abstract The severe acute respiratory syndrome (SARS) coronavirus (CoV) was
identified as the etiological agent of an acute respiratory disease causing atypical
pneumonia and diarrhea with high mortality. Different types of SARS-CoV
vaccines, including nonreplicative and vectored vaccines, have been developed.
Administration of these vaccines to animal model systems has shown promise for
the generation of efficacious and safe vaccines. Nevertheless, the identification of
side effects, preferentially in the elderly animal models, indicates the need to
develop novel vaccines that should be tested in improved animal model systems.
Live attenuated viruses have generally proven to be the most effective vaccines
against viral infections. A limited number of SARS-CoV attenuating modifications
have been described, including mutations, and partial or complete gene deletions
affecting the replicase, like the nonstructural proteins (nsp1 or nsp2), or the
structural genes, and drastic changes in the sequences that regulate the expression
of viral subgenomic mRNAs. A promising vaccine candidate developed in our
laboratory was based on deletion of the envelope E gene alone, or in combination
with the removal of six additional genes nonessential for virus replication. Viruses
lacking E protein were attenuated, grew in the lung, and provided homologous
and heterologous protection. Improvements of this vaccine candidate have been
directed toward increasing virus titers using the power of viruses with mutator
phenotypes, while maintaining the attenuated phenotype. The safety of the live
SARS-CoV vaccines is being increased by the insertion of complementary modi-
fications in genes nsp1, nsp2, and 3a, by gene scrambling to prevent the rescue of a
virulent phenotype by recombination or remodeling of vaccine genomes based
on codon deoptimization using synthetic biology. The newly generated vaccine
L. Enjuanes (*), J.L. Nieto Torres, J.M. Jimenez Guardeno, and M.L. DeDiego
Centro Nacional de Biotecnologia (CNB), CSIC, Campus Universidad Autonoma, Darwin 3,
Cantoblanco 28049, Madrid, Spain

74 L. Enjuanes et al.
candidates are very promising, but need to be evaluated in animal model systems
that include young and aged animals.
1 The Disease
The SARS-CoV was identified as the etiological agent of an acute respiratory

disease causing atypical pneumonia and diarrhea with an average mortality of
10% [1]. SARS-CoV is a zoonotic virus that crossed the species barrier, most likely
originating from bats, and has been grown in other species, notably civets [2 4].
The virus emerged in Guangdong Province, China, in late 2002, and rapidly spread
to 32 countries [5 10]. After July 2003, only a few community-acquired and
laboratory-acquired SARS cases have been reported (http://www.who.int/csr/sars/
en/). Of the human CoVs (HCoVs), such as HCoV-229E, HCoV-OC43, SARS-
CoV, HCoV-NL63, and Hong Kong University 1 (HKU1)-CoV, SARS-CoV causes
the most severe disease [11, 12].
SARS-CoV like viruses are circulating in bats from different continents
[4, 13 15]. In addition, SARS-CoV could be used as a biological weapon, and
it has been declared as a category C priority pathogen by the National Institute of
Allergy and Infectious Diseases Biodefense (http://www.niaid.nih.gov/Biodefense/
bandc priority.html). A defined efficacious therapy is not yet available for SARS,
and the possibility of a reemergence of the virus due to the presence of a high number
of bats in different continents infected with SARS-CoV ancestors is a realistic one.
Therefore, the design of an effective and safe vaccine to protect against SARS is still
high priority.
2 Types of SARS-CoV Vaccines and Prospects of Protection

Against SARS by Vaccination
Several types of SARS vaccines have been developed, including inactivated

viruses, subunit vaccines, virus-like particles (VLPs), DNA vaccines, heterologous
expression systems, and live attenuated vaccines derived from the SARS-CoV
genome by reverse genetics (for recent reviews see [16 19]). This chapter will
focus on live attenuated vaccines, i.e., on the development of safe, live, recombinant
vaccines based on attenuated SARS-CoV, including biosafety safeguards that can be
engineered to assure attenuation.
A preliminary issue is whether previous and current attempts for developing a
vaccine to protect against SARS have provided promising results. We believe that
this is the case based on the results obtained with different types of vaccines
[17 19]. Nevertheless, the vaccines developed so far have not been tested in
humans for obvious reasons and, in addition, the animal models used to evaluate
Recombinant Live Vaccines to Protect Against the Severe Acute 75
the experimental vaccines do not fully reproduce the clinical signs observed in the
natural host. In addition, with few exceptions, the evaluation of these vaccines has
been made in young animals, and it has been shown that the outcome of challenge
experiments, although positive in young animals, frequently showed side effects
when performed in old mice [20, 21]. Recently, animal models have been consid-
erably improved, reproducing most of the pathology observed in humans [22, 23].
In particular, a mouse-adapted SARS-CoV model, selected after fifteen passages
in mice (SARS-CoV-MA15), reproduces most clinical signs observed in human
infections during the SARS epidemic in 2003, including death of infected mice.
This animal model is considered the best available. Therefore, vaccine candidates
developed so far may have to be reevaluated in this model using young and aged
mice.
2.1 Inactivated and Vectored Vaccines Developed

to Prevent SARS
Vaccines based on whole purified inactivated virus have the benefit of presenting a
complete repertoire of viral antigens, although inactivated vaccines do not in
general provide longlasting immunity. These vaccines provide good protection in
mice [24], hamster [25], and partial protection in ferrets [25, 26].
In rhesus monkeys, a formaldehyde-inactivated SARS-CoV vaccine showed
partial protection [27, 28]. An inactivated SARS-CoV vaccine was also adminis-
tered to humans. This vaccine was safe and induced neutralizing antibodies, but no
efficacy data have been reported [29]. Overall, inactivated vaccines, based on whole
purified virus, induced neutralizing antibodies, were apparently safe at least in
young animal models and provided good protection.
Subunit vaccines have the advantage of their simplicity, chemical definition, and
lack of potential variability [30]. In the case of SARS-CoV, a great advantage
is that well-defined S protein domains binding the cell receptor human angiotensin
converting enzyme (hACE2) have provided full immune protection [31 34]. This
concept is reinforced by the observation that monoclonal antibodies specific for the
receptor binding domain elicited protection in several animal models, including
African green monkeys [35 41].
In addition to S protein derived domains or peptides, protein 3a, a large protein
of SARS-CoV exposed on its envelope, also elicits virus neutralizing antibodies
[42] and could be useful in improving subunit vaccines. Furthermore, immunity
to SARS-CoV has also been demonstrated with virus-like particles (VLP) [43].
Overall, the results obtained with subunit vaccines strongly suggest that protection
against SARS by vaccination is feasible.
DNA vaccines are safe and nonexpensive but, often, are not very efficient in
large mammals. DNA vaccines induce SARS-CoV neutralizing antibodies and
protection in mice [44 47].
The use of viral vectors to protect against SARS has been extensively explored.
Adenovirus induced good protection in mice [25]. Modified vaccinia Ankara
(MVA) provides protection in mice [35] and ferrets [48] although induction of
antibody-dependent enhancement of disease (ADED) was reported. Adeno-asso-
ciated virus induces long-term protection against SARS-CoV [34]. Parainfluenza
virus elicits protection in hamsters and monkeys [49, 50]. Recombinant measles
viruses expressing the S protein of SARS-CoV induces neutralizing antibodies and
immune responses against SARS-CoV [51]. Newcastle disease recombinant virus
expressing the S protein of SARS-CoV induces neutralizing antibodies in African
green monkeys immunized via the respiratory tract [52]. A recombinant attenuated
vesicular stomatitis virus (VSV) protects mice against SARS-CoV challenge 4
months after vaccination [53, 54]. Venezuelan equine encephalitis (VEE) virus
expressing the S protein of SARS-CoV induces protection against challenge with
virulent virus in the mouse model [20]. Overall, these results indicate that there is a
very good prospect for the development of an efficacious and safe vaccine to
prevent SARS. Nevertheless, there are relevant aspects that need to be improved
in order to achieve a vaccine that can be fully protective and free of side effects both
in young and in elderly people.
3 The Virus
SARS-CoV is an enveloped, single-stranded positive-sense RNA virus with a

genome of 29.7 kb that belongs to Genus b of the Coronaviridae [55 57]
(Fig. 1a). The replicase gene is encoded within the 50 two-thirds of the SARS-CoV
genome, including two overlapping open reading frames (ORF) named ORFs 1a
and 1b. The latter is translated by a ribosomal frameshift upstream of the ORF 1a
stop codon [58, 59] (Fig. 1b). Translation of both ORFs in the cytoplasm of infected
cells results in the synthesis of two polyproteins, pp1a and pp1ab, that are processed
by two viral proteinases to yield 16 functional nonstructural proteins (nsps)
[60, 61]. These nsps are the components of the membrane-anchored replication
transcription complex [62]. All CoVs encode species-specific accessory genes in
their downstream ORFs, with a remarkably conserved order: replicase/transcriptase,
spike (S), envelope (E), membrane (M), and nucleocapsid (N). The lipid bilayer
envelope contains at least three proteins: E and M that coordinate virion assembly and
release, and the large peplomer S (Fig 1a). This glycoprotein is located on the virion
surface, conferring the virus characteristic corona shape. S is the main mediator of
host cell attachment and entry. SARS-CoV ORFs 3a, 6, 7a, and 7b encode additional
virus membrane proteins [63 67]. Other accessory proteins are 8a, 8b and 9b. The
functions for most of the accessory proteins are still unclear; however, it is known that
some of these proteins influence virus host interaction and viral pathogenesis
[68 70].
For SARS-CoV, hACE2 molecule serves as a receptor [71]; CD209L has also
been implicated as an alternative receptor in entry [72].
b 7b
3b M 7a 8b 9b
L REP 1a REP 1b S 3a E 6 8a N
An
nsp: 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16
p9 p87 X PL2pro TM TM 3CL TM RdRp HEL ExoN NendoU MT
Fig. 1 SARS CoV structure and genome organization. (a) Schematic diagram of SARS CoV
structure. S, spike protein; M, membrane protein; E, envelope protein; N, nucleoprotein; 3a, 6, 7a,
and 7b, accessory structural proteins. (b) Schematic representation of SARS CoV genome. Rep 1a
and 1b, replicase genes; 3b, 6, 8a, 8b, and 9b, nonessential genes. Other genes as in (a). In the
bottom boxes, the putative functional open reading frames of the SARS CoV replicase
are indicated. Nsp, replicase nonstructural proteins; p9 and p87, tentative amino terminus replicase
polypeptides; X, ADP O ribose 10 phosphatase; PL2pro, papain like cysteine protease 2; TM,
putative transmembrane domains; 3CL, 3C like main protease; RdRp, RNA dependent RNA
polymerase; HEL, helicase; ExoN, 30 to 50 exoribonuclease; NendoU, Nidoviral uridylate specific
endoribonuclease; MT, putative ribose 20 O methyltransferase
4 Generation of Recombinant SARS-CoV Vaccines Based

on the Deletion or Modification of Genes
Live attenuated viruses have generally proven to be the most effective vaccines
against viral infections. The production of effective and safe live attenuated vac-
cines for animal CoVs has not been satisfactory, largely because vaccine strains
are insufficiently immunogenic and, in addition, may recombine, resulting in novel
viruses with increased virulence [73 75]. Several groups, including ours, have
described modifications to the SARS-CoV that are attenuating. These “domesti-
cated” viruses may be useful platforms to develop inactivated or live vaccines.
In general, for RNA viruses, it is essential to develop a reverse genetic system
to develop a virus with an attenuated phenotype. This is certainly the case for
coronaviruses that have the largest genome known (around 30 kb) for an RNA
virus, increasing the technical difficulty of generating an infectious cDNA. We have
developed efficient transmissible gastroenteritis CoV (TGEV) and SARS-CoV
reverse genetics systems, by inserting infectious cDNA clones of these viruses
into bacterial artificial chromosomes (BACs) [76 80] (Fig. 2a). In this system, the
a T by C T by A
10338 11163
7b
L 3b M 7a 8b 9b
CMV REP 1a REP 1b S 3a E 6 8a N
An
pBAC-SARS-CoV-URB*
b GENES DELETED IN THE RECOMBINANT VIRUSES
ENVELOPE: E
ACCESSORY : 6, 7a, 7b, 8a, 8b, 9b
COMBINATION OF BOTH: E, 6, 7a, 7b, 8a, 8b, 9b
Fig. 2 Structure of the infectious SARS CoV cDNA cloned in a bacterial artificial chromosome
and the derived deletion mutants. (a) Schematic structure of the SARS CoV infectious cDNA of
the Urbani strain, cloned in a bacterial artificial chromosome (BAC). The infectious cDNA is
expressed under the control of the cytomegalovirus promoter (CMV); L leader; the cDNA includes
genetic markers (T10338C and T11163A), introduced to differentiate the engineered clone from
the wild type Urbani strain genome; acronyms on the top of the bar indicate gene names, as in
Fig. 1. (b) Table with the genes deleted in three SARS CoV recombinant viruses. Deletion mutants
without E gene led to viruses with attenuated phenotypes
genomic RNA is expressed in the cell nucleus under the control of a cytomegalovirus
promoter (first amplification by the cellular polymerase II), with subsequent amplifi-
cation in the cytoplasm by the viral RNA-dependent RNA polymerase. This reverse
genetics system is highly efficient because it implies two amplification steps. In
addition, cDNA stability in the BAC is very high. Soon after the BAC technology
was applied to assemble an infectious coronavirus cDNA clone, alternative strategies
were developed, including (1) a system to assemble a full-length cDNA construct of
the TGEV genome by using adjoining cDNA subclones that have unique, flanking,
interconnecting junctions [81]. Transcripts derived from the TGEV cDNA assembled
using this approach can be used to derive infectious recombinant virus; (2) a system
in which the cloning vector is a poxvirus. Using the genome of this poxvirus
including the genome cDNA copy as a template, the viral genome is transcribed
in vitro, and infectious virus is recovered from transfected cells [82]; (3) a modified
procedure was described in which the coronavirus genomic RNA is transcribed inside
cells using a poxvirus genome as a template. To this end, the viral genome is cloned
under the control of T7 promoter, and the poxvirus DNA including the infectious
cDNA is transfected into cells that are infected with a poxvirus expressing T7
polymerase [83]. The generated transcript reconstitutes an infectious CoV.
In the case of SARS-CoV, several genes have been deleted in order to generate
viruses with attenuated phenotypes. Nevertheless, deletion of one or more accessory
genes did not significantly attenuate SARS-CoV [88]. Fortunately, we showed that
deletion of the E gene, encoding the envelope protein, led to a viable SARS-CoV,
indicating that E protein is not essential for virus replication. Interestingly, viruses
lacking E protein are attenuated, grow in the lung, and are immunogenic in different
animal models [79, 85 87].
Modification or deletion of other SARS-CoV genes has also been considered in
the design of vaccines to prevent SARS. Some of these genes (nsp1, nsp14, S, and
N) are essential for virus replication, while others (3a, 6, 7a, 7b, 8a, 8b, 9b) are
nonessential for virus growth in cell culture or in vivo. The design of SARS
vaccines based on deletion of SARS-CoV genes is described below. Nevertheless,
most attention is given to the deletion or modification of E, nsp1, nsp2, and 3a
genes.
SARS-CoV deletion mutants lacking each of ORFs 3a, 3b, 6, 7a, or 7b did not
significantly influence in vitro and in vivo replication efficiency in the mouse model
[88, 89]. All recombinant viruses replicated to similar wild-type levels, suggesting
that either the group-specific ORFs play a limited role in in vivo replication
efficiency or that the mouse model used in the evaluation does not meet the
requirements to discriminate the activity of group-specific ORFs in disease [88].
In fact, it was unexpected that the deletion of ORFs such as 3a, 7a, and 7b which
encode structural proteins [64, 67, 88, 90, 91] would show little influence on virus
replication in the mouse model. Only deletion of ORF 3a showed a minor decrease
(below tenfold) in virus growth. Furthermore, deletion of combinations of genes,
such as deletion of ORFs 3a and 3b, and ORF6, showed a 10 30-fold titer reduction
in Vero cells, but showed a limited effect on virus growth in the murine model at
day 2 postinfection. Moreover, the simultaneous deletion of larger combinations of
group-specific genes such as 6, 7a, 7b, 8a, 8b, and 9b has led to the production of an
infectious SARS-CoV deletion mutant that propagated in cell culture with a titer
similar to that of the parental wild-type virus and was not attenuated in transgenic
mice that expressed the SARS-CoV receptor (hACE2) [85]. Therefore, the effect of
SARS-CoV gene deletions needs to be tested in more relevant animal models.
Interestingly, the deletion of the E gene alone, or in combination with the
removal of genes 6 9b, led to mutant viruses that seem to be promising vaccine
candidates [79, 85 87], and is described next.
4.1 Vaccines Based on the Deletion of E Protein
The E gene was nonessential for the genus b MHV CoV [92], although elimination
of this gene from the MHV genome reduced virus growth in cell culture more than
1,000-fold. In contrast, for the group 1 TGEV coronavirus, expression of the E gene
product was essential for virus release and spread. Propagation of E gene deleted
TGEV (TGEV-DE) was restored by providing E protein in trans [93, 94].
A recombinant SARS-CoV (rSARS-CoV) that lacks the E gene, generated from a
BAC (Fig. 2b), was recovered in Vero E6 cells with a relatively high titer (around
106 pfu/ml) and also from Huh-7 and CaCo-2 cells with low titers, indicating that
SARS-CoV E protein is not essential for virus replication in cell culture [79].
Electron microscopy observation of Vero E6 cells infected with the SARS-CoV
wt ΔE Δ[6-9b] Δ[E,6-9b]
wt wt ΔE ΔE
Fig. 3 Electron microscopy of SARS CoV and envelope protein deletion mutants. (a) Extracel
lular viruses released from cells infected with the SARS CoVs indicated at the top. (b) Micro
graphs of wt and SARS CoV DE mutants in the budding process. In cells infected with the wt
virus, 5% of the virions in the final budding step were found bound to the cell, whereas in the E
protein deleted viruses, this number was increased to 16%, suggesting that absence of E protein
led to a delay in the “pinch off step”
wt or the DE deletion mutant showed a higher efficiency of assembly and release for
the wt virus (Fig. 3). In this respect, SARS-CoV-DE behaves like MHV, although
SARS-CoV-DE grows to a considerably higher titer. Vaccine viability and efficacy
require the production of viruses with high titers. Interestingly, adaptation of the
rSARS-CoV-DE virus to grow in Vero cells after 16 passages led to an increase of
virus titers reaching values almost identical to those displayed by the full-length
virus (around 107 pfu/ml) [87]. This titer is close to those required for a competitive
live attenuated vaccine.
4.2 Evaluation of SARS-CoV-DE Vaccine Candidate

in Different Animal Model Systems
While SARS-CoV infects and replicates in several species, including mice, ferrets,
hamsters, and nonhuman primates, most of these animals only develop inapparent
or mild disease [95]. An ideal animal model that completely reproduces human
clinical disease and pathological findings has not been identified. To evaluate the
rSARS-CoV-DE vaccine candidate, we have used three animal model systems:
hamster, transgenic mice expressing the hACE2 receptor for human SARS-CoV,
and conventional mice challenged with the mouse-adapted virus [22, 23, 79, 85 87,
96 98]. These animal model systems are complementary.
The hamster model has been used to study SARS-CoV-DE virus pathogenicity,
because it demonstrates elements present in human cases of SARS-CoV infections
including interstitial pneumonitis and consolidation [79, 96, 97]. The hamster
model reproducibly supports SARS-CoV replication in the respiratory tract to a
higher titer and for a longer duration than in mice or nonhuman primates. Virus
replication in this model is accompanied by histological evidence of pneumonitis,
and the animals develop viremia and extrapulmonary spread of virus [96]. Although
overt clinical disease is absent, the hamster model is a useful model for the
evaluation of SARS-CoV infection. Titers of recombinant SARS-CoV (rSARS-
CoV) achieved in the respiratory tract of hamsters were similar to those previously
reported for the wild-type virus [96] and were at least 100-fold higher than titers of
the rSARS-CoV-DE virus, suggesting that this mutant virus is attenuated. Histo-
pathological examination of lungs from infected hamsters showed reduced amounts
of viral antigen and pulmonary inflammation in rSARS-CoV-DE infected than in
rSARS-CoV infected animals, indicating that rSARS-CoV-DE is attenuated in vivo
[79]. In fact, reduction of SARS-CoV titers in patients has been associated with a
considerable reduction in pathogenicity and increase in survival rates [99, 100].
rSARS-CoV-DE immunized hamsters remained active following wild-type virus
challenge while mock immunized displayed decreased activity [86].
The transgenic mice model is based on the production of mice expressing the
hACE2, the receptor for human SARS-CoV. Transgenic mice models have been
obtained in different laboratories by expressing the hACE2 under the control of
different promoters [98, 101]. These mice develop moderate respiratory disease, but
overwhelming neurological disease with 100% mortality after intranasal infection
with SARS-CoV. As such, they are very useful to assess attenuation and vaccine
safety and efficacy. We previously showed that infection of these highly susceptible
mice with rSARS-CoV-DE, or rSARS-CoV with E and several group-specific
protein genes 6, 7a, 7b, 8a, 8b, and 9b deleted (rSARS-CoV-[DE,6 9b]) resulted
in neither weight loss nor death, even after inoculation with very high virus
doses [85].
The mouse-adapted SARS-CoV model used in the evaluation of the rSARS-
CoV-DE and rSARS-CoV-D[E,6 9b] was based on the recent isolation of a
SARS-CoV adapted to growth in mice or rats [22, 102, 103]. This model provided
a useful system for vaccine evaluation because some strains of mice and rats
infected with these viruses develop severe respiratory disease and even death.
A mouse-adapted strain was isolated after 15 passages through the lungs of
BALB/c mice (MA15 strain) and, unlike the parental Urbani strain of virus,
intranasal inoculation with this virus results in signs of respiratory disease with
substantial mortality [22]. We showed that immunization with rSARS-CoV-DE or
SARS-CoV-D[E,6 9b] almost completely protected BALB/c mice from fatal
100
80
SURVIVAL, %
60
ΔE
40
ΔE, 6-9b
20 PBS
0
0 2 4 6 8 10 12 14
TIME POST-INFECTION, days
Fig. 4 Protection induced by DE mutants against an adapted SARS CoV in mice. Six week old
Balb/c mice were immunized with 12,000 pfu of rSARS CoV DE (red circles), rSARS CoV D
[E,6 9b] (green squares), or PBS (black triangles) and challenged at day 21 post immunization
with 1 105 pfu of the mouse adapted Urbani strain of SARS CoV (MA15). Mice were moni
tored daily for survival
respiratory disease caused by mouse-adapted SARS-CoV (Fig. 4), and partly

protected hACE2 transgenic mice from lethal disease [87].
In summary, the immunogenicity and protective efficacy of rSARS-CoV-DE has
been shown in the three animal model systems described above, hamsters, highly
susceptible transgenic mice expressing the hACE2 receptor for human SARS-CoV
and conventional mice challenged with the MA15 virus. Interestingly, both homol-
ogous and heterologous protection was observed. In fact, hamsters and hACE2
transgenic mice immunized with rSARS-CoV-DE developed high serum neutraliz-
ing antibody titers and were protected from replication of homologous (SARS-CoV
Urbani) and heterologous SARS-CoV (GD03) in the upper and lower respiratory
tract [86, 87]. The relevance of this observation is that the GD03 strain of SARS-
CoV is one of the serologically most divergent human SARS-CoV identified, in
relation to the Urbani strain. In addition, it has been shown that the GD03 strain is
closely related to the isolates obtained from animals and if SARS-CoV were to
reemerge, it would probably have an animal origin. Despite being attenuated in
replication in the respiratory tract, rSARS-CoV-DE virus is an immunogenic and
efficacious vaccine in hamsters and two mouse models.
4.3 SARS-CoV E Gene Is a Virulence Gene
E gene deletion mutants SARS-CoV-DE and SARS-CoV-D[E,6 9b] were attenuated

in two animal model systems, hamster and transgenic mice, expressing the ACE-2
receptor, as indicated above. In fact, infection with both deletion mutants led to no
weight loss, death, or lung immune histopathology, in contrast to infection with
virulent SARS-CoV [79, 85 87] (Fig. 5). In addition, a more refined test for virus
CLINICAL DISEASE LETHALITY
110 100
STARTTING WEIGHT, %
∆E
ΔE,6-9b
100 75
SURVIVAL, %
∆E
90 ΔE,6-9b 50
80 wt 25
Δ6-9b
0 Δ6-9b
70
2 4 6 8 2 4 6 8
TIME POSTINOCULATION, days
TIME POSTINOCULATION, days
Fig. 5 Effect of SARS CoV envelope E protein deletion on virus virulence. (a). Clinical disease.
Six week old hACE2 transgenic mice were inoculated with 12,000 pfu of rSARS CoV DE (red
squares), rSARS CoV D[E,6 9b] (green squares), wild type rSARS CoV (black circles), or
rSARS CoV D[6 9b] (blue circles) and monitored daily for weight loss (left) and survival (right)
virulence was performed with hamsters using the activity wheel, and no decrease of
hamster activity was detected 7 days after hamster infection with the SARS-CoVs
lacking the E gene, in contrast to those infected with a virus with full-length genome.
Furthermore, rSARS-CoV-DE did not infect the brain of infected transgenic mice, in
contrast to the wt virus. Overall, these data indicate that E is a virulence gene [79, 85].
The potential mechanism of E gene product in virulence has been investigated in
our laboratory. We have shown that the expression of E gene drastically reduced the
expression of genes involved in stress and unfolded protein responses [104]. A
reduction in stress responses has been associated with a decrease in the innate and
specific immune responses [105 108]. As a consequence, we have postulated that
deletion of the E gene leads to an increased immune response to the virus, reducing
its apparent pathogenicity.
4.4 Future Improvement of rSARS-CoV-DE Vaccine
Three complementary strategies are being applied to improve the rSARS-CoV-DE

vaccine:
4.4.1 To Increase Virus Titers While Maintaining the Attenuated Phenotype
To generate an efficient inactivated or live modified vaccine, virus titers need to be

high in order to obtain an economically competitive vaccine. To increase virus
titers, we propose a novel approach based on previous findings showing that
coronavirus genomes encoding a mutated nsp14 30 -50 -exonuclease (ExoN) display
a mutator phenotype [109]. The engineered SARS-CoV with a mutated or deleted E

protein will be modified to include an ExoN that causes the accumulation of
mutations throughout the viral genome. The mutated viruses will be passed in
cell culture by infecting cells with the highest virus dilution possible. These
dilutions should contain only those mutant viruses with the highest titer. Therefore,
we expect that serial passages of these dilutions will select virus clones with high
titers. Once the desired virus titers have been achieved, it will be confirmed that
the high titer viruses are still attenuated in vivo. Virus evolution will be reverted
to standard levels by replacing the mutator nsp14 by the native one using the
infectious cDNA clone [110]. Selected viruses will be tested for protection as
previously described.
4.4.2 Deletion of a Second Gene That Interferes with Host-Immune Response
We have previously shown that rSARS-CoV-DE elicited protective immune

responses [86, 87]. At the same time, we and others have also shown that it
was possible to delete additional nonessential genes to generate viable SARS-
CoV [85, 88]. Some of the additionally deleted genes are involved in the inhibition
of IFN activation [68, 111]. We propose to delete some of these genes and
determine whether removal of any of them increases the immune response to the
vaccine candidate.
4.4.3 Construction of rSARS-CoV Mutants with Modified E Protein (E*)

Eliciting Higher Immune Responses to the Virus Than rSARS-CoV
Without E Protein
SARS-CoV E protein reduced stress, unfolded protein, and immune responses to the
virus. We have postulated that efforts to enhance assembly (and levels of viral
protein) without diminishing the stress response, which is increased in the absence
of E, might increase immunogenicity without compromising safety. As a conse-
quence, we propose the construction of rSARS-CoV mutants with modified E
protein (E*) eliciting higher immune responses to the virus than rSARS-CoV-DE.
In these mutants, an E* coding gene fully functional in virus morphogenesis is
inserted within the viral genome. The approach is based on the previous identifica-
tion of host proteins binding SARS-CoV E protein, influencing virus-induced stress
response and the immune response to the virus. E protein ligands were identified
by co-immune precipitation and mass spectrometry studies, as we have previously
reported [112], and by yeast two-hybrid technologies [113]. The effect of these
proteins on the stress and immune response has been identified. We propose to
modify specific E protein domains, in order to prevent virus host cell interactions
that counteract the induction of a strong immune response by rSARS-CoV vaccines.
4.5 Live SARS-CoV Vaccines Based on Viruses Attenuated by

Modification of Structural or Nonstructural Proteins
We will focus on the modification of three SARS-CoV proteins, as previous

findings on these proteins indicate that they are not fully essential for virus viability,
and that their modification may lead to attenuated viruses.
4.5.1 Modification of the Replicase nsp1 Gene
Most of the experimental information on the influence of coronavirus replicase

protein modification in attenuation has been obtained changing nsp1 and nsp2
[114 119]. In the case of SARS-CoV, it has been shown that nsp1 significantly
inhibited IFN-dependent signaling by decreasing the phosphorylation levels of
STAT1 while having little effect on those of STAT2, JAK1, and TYK2 [115].
A modification of SARS-CoV nsp1 (mutations R124S and K125E) resulted in a
virus that replicated as efficiently as wild-type virus in cells with a defective IFN
response, while its replication was strongly attenuated in cells with an intact IFN
response [115]. Thus, it is likely that nsp1 mutants will lose virulence and have a
reduced pathogenicity.
Alternatively, mutations or deletions in the nsp1 gene could be introduced,
similar to those described in the MHV replicase [114, 116] that led to an attenuated
CoV phenotype. These types of mutants could be investigated for their relevance in
the generation of attenuated SARS-CoV phenotypes that could be tested for vaccine
candidates.
4.5.2 Modification of Replicase nsp2 Gene
Deletion of nsp2 in MHV and SARS-CoV viruses caused 0.5 1 log10 reductions in
peak titers in single-cycle growth assays, as well as a reduction in viral RNA
synthesis and growth [117, 119]. These findings indicate that nsp2 is not essential
for virus replication and that its deletion may lead to viruses with an attenuated
phenotype. In addition, recent studies with MHV and HCoV-229E suggest that this
protein may have functions in pathogenesis [117, 120]. Therefore, nsp2 seems a
promising candidate to complement the safety of a rSARS-CoV-DE vaccine.
4.5.3 Modification of Protein 3a
This O-glycosylated accessory protein of 274 amino acids forms a K+-permeable

channel-like structure [91]. It is not essential for growth in tissue culture cells, but
deletion of the 3a gene leads to a small (5 10-fold reduction) virus titer reduction
both in vitro and in vivo [88]. Protein 3a may also be involved in triggering high
levels of proinflammatory cytokine and chemokine production [121 123], and its
deletion may reduce SARS-CoV virulence. Gene 3a maps at a distal position from
genes nsp1 or nsp2. Therefore, a recombination event that restores the wild
phenotype for gene 3a and genes nsp1 or nsp2 in one event seems very unlikely.
5 Development of a SARS-CoV Vaccine by Modification

of the Transcription-Regulating Sequences
Coronavirus transcription is regulated by highly conserved sequences preceding

each gene. These transcriptional regulatory sequences (TRSs) are almost identical
to sequences located at the 50 end of the genome, just downstream of the leader
sequence. The TRS preceding each gene encodes a complementary sequence in the
newly synthesized RNA of negative polarity. These RNAs have to hybridize with
the TRS located next to the leader in the process of discontinuous RNA synthesis,
typical of CoVs. An alternative approach for developing safer, recombination-
resistant live coronavirus vaccines has been developed by Baric’s group [84]. The
novel procedure involves the modification of the TRSs in a SARS-CoV vaccine
strain, to a sequence incompatible with the TRS of any known circulating CoVs.
It was postulated that recombinant events between wt coronaviruses and TRS
remodeled SARS-CoV would result in genomes containing incompatible mixed
regulatory sequences that block expression of subgenomic mRNAs. Using a molec-
ular clone, the SARS-CoV TRS network was remodeled from ACGAAC to
CCGGAT [84]. This rewiring of the genomic transcription network allows efficient
replication of the mutant virus, icSARS-CRG. The icSARS-CRG recombinant
virus replicated to titers equivalent to wt virus and expressed the typical ratios of
subgenomic mRNAs and proteins. It has been shown that this vaccine candidate
provides protection against challenge with virulent SARS-CoV.
6 Potential Side Effects of SARS-CoV Vaccines
Previous studies using animal CoVs have provided experimental evidence for
humoral [124 133] and T cell mediated responses to animal coronaviruses that
exacerbate disease [134], as previously summarized [17]. This safety concern was
increased in the case of SARS-CoV by two studies. In one report [135], antibodies
that neutralized most human SARS-CoVs also enhanced virus entry mediated by
two civet cat SARS-CoVs. These viruses had S glycoproteins related to the SARS-
CoV GD03 isolate. In a second report, it has been shown that the administration of
MVA-based SARS-CoV S vaccine into ferrets, but not MVA alone, followed by
live SARS-CoV challenge, resulted in enhanced hepatitis [136]. Nevertheless, these
side effects have not been described in other studies with SARS-CoV in mice,
hamster, ferrets, and African green monkeys [24, 35, 36, 44, 96, 137 140].
In general, immunization with vaccine candidates has resulted in the absence of
side effects. Nevertheless, there are still three concerns that remain unaddressed.
One is that specific viral proteins, such as SARS-CoV N expressed by a Venezuelan

Equine Encephalitis (VEE) virus vector has resulted in enhanced immunopathology
following viral challenge [20], similar to the immune pathology observed following
vaccination with formalin-inactivated respiratory syncytial virus (RSV) [141 143].
A second main concern is the observation that SARS-CoV vaccines that provide
protection in the absence of side effects in young mice show immunopathological
complications in aged mice [20]. A third consideration is that most vaccine candi-
dates have been tested in animal models that do not fully reproduce the clinical
symptoms observed in humans, and, with one exception, no phase I clinical trials in
humans have been performed. Therefore, SARS vaccine candidates would require
additional rigorous clinical and immunological evaluation, using the SARS-CoV
mouse-adapted virus model, and potential side effect assessment both in young and
in aged animals.
7 Future Trends to Increase Biosafety of Live Modified

SARS-CoV Vaccines
Live virus vaccine formulations should include rational approaches to minimize the
potential reversion to the wt phenotype and simultaneously resist recombination
repair. In principle, a combination of SARS-CoV genome modifications could lead
to viruses with an attenuated phenotype that could be considered safe and effective
vaccine candidates.
While rSARS-CoV-DE or the selected rSARS-CoV-E* will be attenuated, in
principle, reversion to the virulent phenotype could take place by the reintroduction
of the E gene into the virus, by recombination with a closely related coronavirus
present in the environment. Furthermore, it cannot be excluded that compensatory
mutations increasing virus fitness could cause reversion to the virulent phenotype.
To minimize these possibilities, additional modifications have to be introduced into
the final vaccine candidate, including the modifications of ORFs encoding proteins
nsp1, nsp2, or 3a, described above. The advantage of combining deletions or muta-
tions in the E protein with those in nsp1 or nsp2 ORFs reside in that these genes
map into distal positions of the genome (more than 20 kb 50 separation), making it
very unlikely that a single recombination event could restore the wt virus phenotype.
In addition, other creative reorganizations of the virus genome have been described
that could increase SARS-CoV safety (described below).
7.1 Gene Scrambling to Prevent the Rescue of a Virulent

Phenotype by Recombination
CoVs have a characteristic, strictly conserved genome organization with genes

occurring in the order 50 -Pol-S-E-M-N-30 . MHV virus mutants with the genes
encoding the structural proteins located in a different order were constructed, and
it was shown that the canonical coronavirus genome organization is not essential for
in vivo replication [144]. Some of the mutants showed an attenuated phenotype.
Interestingly, rearrangement of the viral genes may be useful in the generation of
CoV with reduced risk of generating viable viruses by recombination with circulat-
ing field viruses. In fact, potential recombination between viruses with different
gene orders most likely will lead to nonviable viruses lacking essential genes.
7.2 Vaccines Based on Codon Deoptimization of Viral Genome
As a result of the degeneracy of the genetic code, all but two amino acids in the
protein coding sequence can be encoded by more than one synonymous codon. The
frequencies of synonymous codon used for each amino acid are unequal and have
coevolved with the cell’s translation machinery to avoid excessive use of subopti-
mal codons, which often correspond to rare or otherwise disadvantaged tRNAs
[145, 146]. This results in a phenomenon termed “synonymous codon bias” which
varies greatly between evolutionarily distant species [147]. While codon optimiza-
tion by recombinant methods has been widely used to improve cross-species
expression, the opposite direction of reducing expression by intentional introduc-
tion of suboptimal synonymous codons has seldom been chosen [146].
De novo gene synthesis with the aim of designing stably attenuated polioviruses
and SARS-CoV is a novel strategy to construct virus variants containing synthetic
replacements of virus coding sequences by deoptimizing synonymous codon usage.
Infection with equal amounts of poliovirus particles revealed a neuroattenuated
phenotype and a striking reduction of the specific infectivity of poliovirus particles
[145]. Similar attempts have been made by Baric’s group to design SARS-CoV
vaccines. These vaccine candidates provide protection in the mouse model system
after challenge with virulent virus (Ralph Baric, personal communication). Due to
the distribution effect of many silent mutations over large genome segments,
codon-deoptimized viruses should have genetically stable phenotypes, and they
may prove suitable as attenuated substrates for the production of vaccines.
8 Concluding Remarks
The production of effective and safe vaccines for animal coronaviruses, previously
reported, has not been satisfactory [17, 18, 73, 74]. In contrast, the production of
inactivated, subunit, vaccines based on DNA and recombinant vectors or vaccines
generated by reverse genetics using SARS-CoV genomes seem more promising.
Vaccine candidates need to be tested in the SARS-CoV mouse-adapted model, and
in macaques, in all cases using both young and aged animals. Later, the absence of
side effects and safety has to be assessed in human phase I clinical trials.
Vaccine manufacturers have the tendency to use well-defined inactivated vac-

cines. Unfortunately, this approach has limited efficacy and elicits immune responses
with relatively short immunological memory. A possible balance between efficacy
and safety is the development of RNA replication-competent propagation-defective
vaccine candidates, based on viral replicons that can generate one-cycle viruses using
packaging cell lines [148].
Acknowledgements This work was supported by grants from the Comisión Interministerial de
Ciencia y Tecnologı́a (CICYT) Bio2007 60978, the Consejerı́a de Educación y Cultura de la
Comunidad de Madrid S SAL 0185/06, Ministerio de Ciencia e Innovación (MICINN) Project
PROFIT, CIT 010000 2007 8, Fort Dodge Veterinaria, and the European Communities (Frame
VII, EMPERIE project HEALTH F3 2009 223498, and PLAPROVA project KBBE 2008
227056). MLD, JLN, and JMJ received fellowships from the Department of Education and Science
of Spain. The work is also supported by a grant from the National Institutes of Health (US) RO1
AI079424 01A1, W000151845 (LE).
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Live-Attenuated Shigella Vaccines.
Is Encouraging Good Enough?
Yves Germani and Philippe J. Sansonetti
Abstract Several strategies have been used to develop vaccines against Shigella
infection. Among these, the most tested has been the construction of live attenuated,
orally administered vaccine candidates in which defined mutations were introduced
in specific genes. Two major options exist: (1) altering key metabolic pathways
affecting bacterial growth in tissues or (2) knocking out virulence genes selected
upon their expected capacity to affect one or several key steps of the infectious
process. In certain cases, the two options have been combined.
Live-attenuated Shigella vaccine candidates have shown great promise. They
elicited, in general, significant immune responses when administered orally to
volunteers. They have the capacity to confer protection by eliciting both mucosal
and systemic immune responses, particularly the intestinal production of secretory
IgAs directed against the O-antigens, a series of complex surface sugars accounting
for the bacterial serotypes, which are known to mediate protection following natural
infection. These responses have been measured by evaluating antibody-secreting
cells, serum antibody levels, and fecal IgA to O-antigens and individual virulence-
related protein antigens for a dozen of vaccine candidates. Live-attenuated vaccines
also offer the potential to elicit strong IFN-g responses, a cytokine that is
known to provide protection against Shigella infection. With regard to possible
T-cell mediated responses, much basic research is still warranted to optimize
vaccine approaches. Owing to the wide range of Shigella serotypes and subtypes,
there is a priori a need for a multivalent vaccine representing the prevalent species
and serotypes. The barrier to the use of live vaccine candidates against shigellosis is
Y. Germani (*)
Unitié Pathogénie Microbienne Moléculaire INSERM U786/Réseau International des Instituts
Pasteur, Institut Pasteur, 28 rue du Dr Roux, Cedex 15, 75724 Paris, France
P.J. Sansonetti
Unitié Pathogénie Microbienne Moléculaire INSERM U786, Institut Pasteur, 28 rue du Dr Roux,
Cedex 15, 75724 Paris, France

100 Y. Germani and P.J. Sansonetti
the issue of multivalency and indications for an average to poor immune responses
observed in infants and children in endemic areas. In addition, identification of the
correlates of protection is needed to accelerate the development of these vaccines.
1 Introduction
More than 100 years after Shiga’s discovery, shigellosis is still a global human
health problem and there is neither a licensed vaccine nor a consensus as to the
mechanism[s] of host immunity to Shigella and optimal vaccine strategy. Still,
advances in our understanding of the molecular mechanisms of virulence of
Shigella have enabled the development of a new generation of live-attenuated
candidate vaccines. But progress in attaining a balance achieving safe and effective
Shigella vaccines remains a challenge.
Bacillary dysentery is endemic throughout the planet, although essentially a major
health concern in its most impoverished areas with substandard hygiene and unsafe
water supplies. Various surveys carried out in treatment centers show that Shigella is
associated with 5 15% of cases of diarrhea and 30 50% of cases of dysentery [1].
The incidence of disease declines with the duration of stay in high-risk settings [2].
Shigellosis can be caused by any serotype belonging to four groups: group
A (Shigella dysenteriae with 17 serotypes), group B (Shigella flexneri with 14
serotypes and subserotypes), group C (Shigella boydii with 20 serotypes), and
group D (Shigella sonnei with a single serotype).
The ability of Shigella to cause diarrheal illness is restricted to human and higher
nonhuman primates (NHP). The disease is characterized in its classical forms, by
a short period of watery diarrhea with intestinal cramps and general malaise,
followed by the appearance of a dysenteric syndrome that comprises intestinal
cramps and tenesmus, leading to permanent emission of bloody, often mucopuru-
lent stools. Shigella species cause bacillary dysentery by invading the large intesti-
nal epithelium in which they promote strong inflammation in human and NHP [3].
Acute complications may occur in absence of quick antibiotic treatment, such as
toxic megacolon, peritonitis, and septicaemia that is mostly observed in severely
malnourished children. Conversely, repeated shigellosis episodes may lead to
severe malnutrition, thus a vicious circle.
The serotype 1 of S. dysenteriae (i.e., the Shiga bacillus) emerges as one of
particular concern, due to expression of the Shiga toxin, a potent cytotoxin that not
only aggravates intestinal lesions but also causes major systemic complications
such as the Hemolytic Uremic Syndrome (HUS). When poor conditions are con-
centrated in a single epidemiological crisis, like in refugee camps, the attack and
mortality rates may be quite high, as observed in Goma, Zaire, in 1994 in the course
of a S. dysenteriae 1 epidemic [4].
Projections based on methodologically convincing epidemiological studies from
the three previous decades allowed, back in 1999, to evaluate the number of cases of
shigellosis to 165 million per year, with a death rate ranging between 500,000 and
1.1 million, 69% being children below 5 years in the developing world [5]. These
Live Attenuated Shigella Vaccines. Is Encouraging Good Enough? 101
impressive figures have undoubtedly led the community to realize that shigellosis is
a high-impact disease, particularly in the poorest populations.
Current figures may not be that high, however, although the epidemiological
situation is evolving and figures are lacking in key areas, particularly in Africa.
Recent surveys indicate that, in general, the incidence of diarrheal diseases remains
stable worldwide, although mortality shows a sustained decrease, being currently
evaluated at an incidence of 4.9/1,000 per year [6]. A recent epidemiological survey
conducted in six Asian countries [1] has established that shigellosis was likely to
be following a similar trend with a stable incidence of cases [4.6% of cases of
diarrhea], and decreased severity and mortality. The rationale for this change in
disease profile is still unknown. Several pieces of explanation may be proposed,
such as better nutrition and hygiene paralleling economic development of the Asian
continent, absence of current epidemics of S. dysenteriae 1, better education of
mothers, improvement of primary health care, and extended use of oral rehydration
solution (ORS) and antibiotics.
The issue of antibiotic (multi)resistance is likely to be an important one, however.
Beyond the possibly positive impact of free, uncontrolled use of antibiotics on the
disease profile at this stage, one may soon face a new crisis associated with massive
multidrug resistance. In some areas, the prevalence of strains resistant to all first-line
antibiotics, including fluoroquinolones, reaches 5% and is clearly on the rise [7].
Shigella infection appears to be more ubiquitous in impoverished Asian popula-
tions than previously thought, and new antibiotic-resistant strains of different
species and serotypes are emerging in this part of the world [1]. It is also unlikely
that the epidemiological situation in Asia can be generalized, thus a need for an
exhaustive evaluation of the incidence of shigellosis, particularly in the sub-
Saharan part of the African continent. Current economic stagnation and frequent
social instability are creating conditions for shigellosis to remain a leading cause of
morbidity and mortality. In order to facilitate such studies, there is a need for
efficient and durable surveillance networks benefiting from good microbiological
expertise and novel quick, reliable, and robust diagnostic tools such as immuno-
chromatographic dipsticks that could be used directly on fecal samples [8].
All elements considered, including the permanent risk of massive re-emerging
epidemics, the need for a Shigella vaccine clearly remains. Its major target would
be the pediatric population of the developing world, essentially infants around the
age of 1 year, and possibly also the elderly population that represents the other peak
of disease susceptibility. Such a vaccine could also benefit travelers to high-risk
areas, particularly those working or intervening in these areas, e.g., members of
nongovernmental organizations (NGOs), army personnel.
2 The Need for an Epidemiologically Valid Shigella Vaccine
Shigella flexneri is endemic in developing countries and accounts for most Shigella
infections worldwide [9]. Pandemics of S. dysenteriae 1 dysentery, as they occurred
in Central America from 1968 to 1972 [10], South Asia in the 1970s [11], Central
Africa in the 1980s [12], and East Africa in the 1990s [13, 14], profoundly influence
the global mortality burden that can be attributed to Shigella [5, 14]. During
S. dysenteriae type 1 epidemics, all age groups are affected, but in endemic areas,
the incidence of shigellosis peaks during the first 5 years of life and declines
thereafter, suggesting that immunity develops after repeated exposures during
childhood [15]. The lack of S. dysenteriae 1 endemicity results in lowbackground
immunity in populations, so epidemics of S. dysenteriae 1 dysentery affect adults
and children alike, and the target ages for the use of a Shiga vaccine would be
similarly broad [10 12].
S. sonnei incidence tends to increase in countries where living standards
improve, thus dominating as an endemic strain in Western countries. This serotype
persists in these transitional countries causing sporadic diarrhea and occasional
outbreaks in epidemiological niches [such as day-care centers] [16, 17]. Shigellosis
due to S. boydii or S. dysenteriae serotypes other than type 1 is uncommon
[1, 5, 18]. Shigella is also a primary cause of traveler’s diarrhea in individuals
from industrialized countries visiting developing areas [19]. They mainly acquire
S. sonnei and S. flexneri infections [20].
Owing to the wide range of Shigella serotypes and subtypes, there is a need for a
multivalent vaccine representing prevalent species and serotypes. Furthermore, the
protective performance of a Shigella vaccine in any particular setting will depend
on the representation of serotypes in the vaccine and the relative epidemiological
occurrence of different serotypes in this setting. Thus, knowledge of the distribution
of serotypes among clinical isolates is a key in designing new vaccines for public
health programs.
Ideally, an epidemiologically valid Shigella vaccine would provide protection
at least against S. dysenteriae 1, the dominant S. flexneri serotypes and S. sonnei
[5, 21, 22]. The WHO has set it at the top of its priority list, along with ETEC, for
the development of a vaccine, and this has recently emerged as a “Shigella-ETEC
vaccine initiative” by the Bill & Melinda Gates Foundation.
3 Pathogenesis
Shigella is a pathovar whose pathogenic characteristics have been acquired follow-

ing acquisition of a large virulence plasmid, a series of adaptive mutations in the
chromosome, as well as acquisition of bacteriophages and pathogenicity islands.
The virulence plasmid supports a large pathogenicity island that encodes a type III
secretion system and some protein effectors (i.e., Ipa [invasion plasmid antigen]
proteins) that account for invasion of epithelial cells [23, 24]. Once bacteria have
penetrated into cells, they lyse the phagocytic vacuole and escape into the cyto-
plasm where they move thanks to their capacity to induce polar nucleation and
assembly of actin filaments via the plasmid-encoded outer membrane autotransporter
protein IcsA/VirG [25]. Motility allows cell-to-cell spread, thus plays a major role in
epithelial invasion [26]. Chromosomal sequences account for development of the
infectious process in invaded mucosal tissues, a striking example being the aerobactin
operon encoding an iron-chelating complex in S. flexneri and S. sonnei that is
essential for bacterial growth in tissues [27]. Shigella enterotoxins have also been
identified. ShET1 is encoded by the chromosome of S. flexneri 2a [28, 29] and Sen is
encoded by the virulence plasmid of the various subgroups [30, 31].
4 Immune Response to Natural Infection

with Wild Shigella spp.
Wild-type Shigella infection confers protective immunity and prevents disease

during subsequent exposures. An individual convalescent from S. flexneri 2a
infection is protected against reinfection only with the homologous serotype.
Compelling evidence of serotype-specific natural immunity comes from the longi-
tudinal study of a cohort of children in whom primary Shigella infection conferred
76% protective efficacy against reinfection with the same serotype [18]. This has
been the basis for resumed interest in LPS determinants for immunization, even by
the parenteral route [32].
Lipopolysaccharide (LPS) O-side chains, the major bacterial surface antigens,
are the main target of hostadaptive immunity. Of great relevance to vaccine
development is the observation that this immunity is serotype specific. Adult
volunteers experimentally infected with either S. sonnei or S. flexneri were signifi-
cantly protected against illness only following rechallenge with the homologous
strain (64 74% protective efficacy) [33, 34].
Antibody response to the somatic antigens of Shigella appear early in infection
and follow the typical course for anti-LPS antibodies, that is, an IgM response that
peaks within weeks and decreases slowly. Anti-LPS antibodies are elicited upon
infection, both locally as secretory IgA and systemically as serum IgG. Antibody-
mediated protection has been shown to be mostly serotype specific [35], pointing to
the O-specific polysaccharide moiety of LPS, also termed O-antigen (O-Ag), as the
target of the protective immune response. Indeed, Shigella serotypes are defined by
the structure of their O-Antigen repeating unit [36].
This has been a strong incentive to consider that protection was an achievable goal
with an oral vaccine reproducing key steps of the natural infectious process. Still,
natural protection is not absolute and rather short lasting, and again, essentially
serotype specific [37] not necessarily “good news” for Shigella vaccine development.
5 Rational Selection of Genes to Develop Live-Attenuated

Deleted Mutants and Clinical Trials
Live Shigella vaccine candidates can be administered by the oral route, thus avoiding
the need for needle injection. They are easier to manufacture than other potential
formulations. Clinical trials in adult human volunteers have been invaluable for
evaluating candidate Shigella vaccines. A challenge dose of 10 1,000 virulent

organisms, preceded by a bicarbonate buffer to reduce gastric acidity, is sufficient
to consistently induce the symptoms of shigellosis [34].
5.1 Pioneer Vaccine Candidates
In the attempts to develop a live-attenuated vaccine as reported in [38], David Mel

from the Military Medical Institute (Belgrade) conducted controlled phase III field
trials on 36,000 adults and children in Yugoslavia in the 1960s [39]. Using
streptomycin-dependent (SmD) mutants of S. flexneri and S. sonnei, he showed
that oral vaccination was an achievable goal. He also showed that multiple strains
could be mixed in a combination vaccine and that protection was serotype specific
[39 41]. To produce this pioneer vaccine candidate, Shigella strains were serially
cultivated on streptomycin-containing media until they acquired streptomycin
resistance and dependence. SmD, i.e., inability to grow in the absence of exogenous
streptomycin [42], lost their ability to cause purulent keratoconjunctivitis in Guinea
pigs (i.e., Sereny test). This vaccine candidate was administered in four doses
(2 1010, 3 1010, 4 1010, and 5 1010 CFU) over 11 days. It was clinically
rather well tolerated in adults, healthy children, and debilitated institutionalized
children [43 47]. Only a small percentage of recipients showed vomiting following
administration of the first dose [39, 41, 45 48]. Protection endured for 1 year.
A single booster dose extended protection for an additional year [41].
The pioneer SmD vaccine strains, however, had drawbacks because the bases of
attenuation were unknown and occasional lots reverted to streptomycin dependency
even though the revertants remained negative in the Sereny test. Furthermore,
difficulties occurred in the large-scale manufacture and process control [47, 49].
Nevertheless, this first vaccine candidate provided proof of concept for future
multivalent vaccines aiming to confer broad-spectrum protection, and this has
remained the gold standard over the years, in spite of the possible reversion of the
mutation, stressing the need for an association of attenuated mutations consisting in
gene deletions, whose selection needs to be rationally based on the increasing
knowledge in the pathogenic mechanisms of Shigella [50].
Another pioneering work was the construction of a mutant-hybrid S. flexneri 2a
vaccine strain by Formal et al. [51]. The ability of this hybrid mutant to propagate in
the lamina propria was diminished even if epithelial invasion still occurred [52].
In response to this, a Shigella flexneri 2a mutant that had lost the ability to invade
intestinal epithelial cells has been selected. The xylose rhamnose region of Escher-
ichia coli, which diminishes the ability to propagate in the lamina propria (even if
the epithelial invasion occurs) was transferred into it. This mutant-hybrid vaccine
candidate was well tolerated and immunogenic but conferred only partial protection
[45, 48].
Another vaccine strain was developed by Istrati following successive passages
[53, 54]. The genetic basis of the attenuation of the S. flexneri 2a strain T32-Istrati
was subsequently shown to correspond to a large deletion of 32-MDa in the

virulence plasmid encompassing part of the pathogenicity island, icsA/virG and
sen. When evaluated in Romania, S. flexneri 2a strain T32-Istrati was shown to be
well tolerated and protective [55, 56]. That was confirmed in a randomized placebo-
controlled field trial performed in China [54]. Interestingly, this vaccine candidate
was also reported to provide protection against shigellosis due to S. flexneri 1b and
S. boydii [rev. in 37].
5.2 Rational Selection of Genes
Following these encouraging initial results, attempts were indeed made at rationally
attenuating virulence of candidate strains representing the most frequently isolated
serotypes, such as S. flexneri 2a and S. sonnei, as well as S. dysenteriae serotype 1,
due to severity of cases.
Because there is only a small margin between the risk caused by moderate
attenuation and poor immunogenicity by a strong attenuation, two major strategies
have been considered:
1. Altering key metabolic pathways affecting bacterial growth in tissues or
2. Knocking out virulence genes selected upon their expected capacity to affect
one or several key steps of the infectious process.
The consequences of introducing different mutations into wild-type Shigella
strains have been evaluated in clinical trials.
To develop a live bivalent S. flexneri 2a and S. sonnei vaccine, a hybrid strain
expressing both S. flexneri 2a and S. sonnei O-antigens was developed in China
(Lanzhou Institute) [57, 58] by introducing a S. sonnei form I plasmid with
deletions of ipa and virF into S. flexneri 2a T32 [59]. In S. sonnei, the O-antigen
is encoded by the rfb locus located on the form I invasion plasmid. This bivalent
vaccine was evaluated in large numbers of volunteers [54]. Its protective efficacy
was 61 65% against S. sonnei 57 72%, and 48 52% against heterologous Shigella
serotypes. Although the use of three high doses of vaccine strain (> 2 1010 CFU)
remains a practical problem, this vaccine was licensed in China in 1997, but no
other clinical trial was performed outside China [58].
More recent vaccine candidates have combined both approaches. aro D and A
mutations were initially considered because they abrogate synthesis of aromatic
amino acid, thus impairing growth of bacteria in vivo. An S. flexneri aro mutant
(SFL124) expressing the S. flexneri group antigen Y was constructed [60 62] in an
attempt to obtain cross protection across the S. flexneri serotypes. The advantage
of SFL124 is the possibility to convert it to other S. flexneri serotypes, using
glucosylating and/or acetylating phages [63 68]. This mutant appeared too attenu-
ated when tested in medical students in Vietnam, thus very well tolerated by
volunteers in clinical trials, but weakly immunogenic [60, 62]. This vaccine candidate
raised an important issue regarding the bases of its attenuation. It is likely that the
wild-type strain that had been selected was already weakly pathogenic; therefore,
its further attenuation by aro mutation likely caused insufficient colonization
potential and poor immunogenicity.
A recent review has stressed the need to confirm full pathogenicity of the strains
that serve as a basis for vaccine construction [37]. This is ethically complicated, but
the mere isolation from a patient may not guarantee that the isolate shows “optimal”
pathogenicity.
Other metabolic mutations have been considered, particularly guaAB that intro-
duces a severe auxotrophy impairing synthesis of nucleic acids [69], as well as
mutations impairing the strain’s capacity to scavenge ferric iron (Fe3+), a property
required to compete for vital Fe3+, via the production of siderophores (i.e.,
aerobactin or enterochelin), against iron-chelating molecules of mucosal surfaces
(i.e., lactoferrin) or tissues (i.e., transferrin) [27]. The most recent Shigella vaccine
candidates have undergone a combination of metabolic and virulence mutations.
This combination can lead to various degrees of attenuation. Current vaccine
candidates, on these bases, can fall into the category of weakly attenuated or
strongly attenuated strains.
In the category of weakly attenuated candidates belong icsA/virG-based mutants.
IcsA/VirG is an outer membrane protein of Shigella that nucleates cellular actin,
thereby allowing intracellular motility and cell-to-cell spread of the microorganism.
Mutation in this gene impairs the capacity of Shigella to spread extensively in the
epithelium, away from its initial site of entry [25]. It has been shown that such
mutants were directly targeted to colonic solitary nodules, the actual inductive sites of
the mucosal immune response [70].
Combined with a deletion of the aerobactin operon (iuc iut), in S. flexneri 2a,
icsA/virG has provided a vaccine candidate, SC602 (a derivative of wild-type
strain Pasteur Institute S. flexneri 2a 494), that has undergone phase I and II
clinical trials (Walter Reed Army Institute of Research and US Army Institute
for Research in Infectious Diseases) whose results were considered encouraging in
Western volunteers [71, 72]. In brief, when fed to North American volunteers,
dosages > 106 CFU were unacceptably reactogenic, with fever or significant
constitutional symptoms and diarrhea in about 15% of the recipient volunteers.
But the strain was strongly immunogenic, eliciting a high percentage of circulating
plasmocytes producing anti-LPS IgA by the ELISPOT assay. By contrast, at a
dosage level of 104 CFU, adverse clinical reactions were uncommon and mild, yet
the induced immune response remained moderately robust [71]. When vaccinees
who had received a dose of 104 CFU as vaccine inoculum were challenged with
a wild-type pathogenic S. flexneri strain of similar serotype, they appeared
fully protected against dysentery, and subsequent studies carried out in the USA
and Israel demonstrated the absence of accidental transmission [71]. These
data showed that in the experimental challenge model, even a single dose of
an engineered vaccine strain could confer significant protection against severe
shigellosis. They also demonstrated the difficulty of finding a proper balance
between clinical acceptability and immunogenicity in adult volunteers in devel-
oped countries.
In a trial in Bangladesh, SC602 was well tolerated (in all age categories, including
1-year-old infants with inocula up to 107 CFU), colonization appeared limited, and
immunogenicity very weak [73]. One possible explanation for the poor performance
in Bangladesh could be that the volunteers had higher background immunity due to
previous exposure. On the other hand, several possibly combined hypotheses may
account for this issue: the protective role of breast feeding against the vaccine strain
in infants, the high level of innate stimulation of the intestinal mucosa by recurrent
enteric infections in a highly endemic zone, thereby severely affecting the capacity of
the vaccine strain to colonize the mucosa, the high exposure of children, at an early
age, to multiple enteric pathogens, including the most prevalent serotypes of Shigella,
thus a quickly acquired status of adaptive immunity. In any event, these observations
are important to consider because they are very unlikely to apply only to this
particular category of vaccine candidate. Considering at least two oral doses as a
possible solution, it would require a second phase II study in similar epidemiological
conditions and thus SC602 awaits further evaluation.
The WRAIR developed a S. flexneri 2a vaccine strain with deletions in the icsA/
virG, sen, and set genes. This intranasal WRSF2G11 vaccine candidate showed
higher immunogenicity and protective efficacy than strain SC602 [74], probably
because a DicsA/virG-based vaccine, which lacks enterotoxin genes, has lower
levels of reactogenicity without hampering immune responses.
A DicsA/virG S. sonnei vaccine candidate was constructed by scientists at WRAIR
(WRSS1), although the virulence of this strain had not been demonstrated in
volunteers. The form I plasmid of most wild-type S. sonnei strains is highly unstable
[33, 75]. This invasiveness plasmid is required for expression of O-antigen by this
serotype. In contrast with most of wild S. sonnei strain, investigators selected this
strain because its form I invasiveness plasmid was stable [33, 75]. In a Phase I trial,
a strong O-antigen specific IgA antibody-secreting cells and moderate interferon
(IFN)-g responses in peripheral blood mononuclear cells were observed [73], but
low-grade fever or mild diarrhea was recorded in 22% of North American vaccinees
given a single dose of 106 CFU of WRSS1. Another phase I trial was performed in
adults with a single dose of 103, 104, or 105 CFU [76]. At the two lower dosage
levels, the vaccine was well tolerated (1 of 30 subjects developed moderate diarrhea
and five mild diarrhea). At 105 CFU, 2 of 15 subjects developed fever and four
experienced moderate diarrhea. At the 104 CFU dose, all subjects manifested
IgA anti O-antigen antibody secreting cell responses and 73% of the vaccinees
showed more than 50 IgA anti O-antigen antibody secreting cells per 106 PBMC.
This dosage level provided the better balance of immunogenicity and clinical
acceptability. Recently, Collins et al. [77] administered two Shigella sonnei
vaccines, WRSs2 and WRSs3, along with WRSS1 to compare their rates of
colonization and clinical safety in groups of five rhesus macaques. The primate
model provides the most physiologically relevant animal system to test the validity
and efficacy of vaccine candidates. In this pilot study using a gastrointestinal model
of infection, the vaccine candidates WRSs2 and WRSs3, which have undergone
additional deletions in the enterotoxin and LPS modification genes, provided better
safety and comparable immunogenicity to those of WRSS1.
Recently, a S. dysenteriae 1 vaccine candidate (SC599, Institut Pasteur) has been

tested in phase I and II trials (Saint George Vaccine Institute, London, UK, and
Centre de Vaccinologie Cochin-Pasteur, Paris, France). The vaccine was well
tolerated with only mild constitutional symptoms and was minimally shed in
stool; however, serum antibody titers were modest or absent [78, 79]. In this strain,
three deletions have been introduced: DicsA/virG, Dent fep fes (genes encoding the
enterochelin system), and DstxA, the gene encoding the catalytic subunit of Shiga
toxin. Unlike its S. flexneri and S. sonnei DicsA/virG counterparts, this strain has
shown good tolerance, limited systemic immunogenicity (as judged by seric IgM,
IgG, and IgA titers), and average to good mucosal immunogenicity as judged by
percentage of anti-LPS IgA measured by ELISPOT, in comparison to SC602 and
WRSS1 [80].
WRSd1 is another S. dysenteriae 1 vaccine strain (deletion of icsA/virG, fnr, and
stxAB genes) developed by WRAIR [81] from the wild-type strain 1617 that was
isolated during a dysentery outbreak in Guatemala in 1969. This oral vaccine
was evaluated (40 volunteers). Immunogenicity is poor; the reactogenic dose was
minimal [9], suggesting that the fnr gene affects gastrointestinal tract colonization.
WRSS1, SC602 and WRSd1, are attenuated principally by the loss of the IcsA/
VirG protein. One drawback has been the reactogenic symptoms of fever and
diarrhea experienced by the volunteers, that increased in a dose-dependent manner.
WRSs2 and WRSs3, second-generation IcsA/virG-based S. sonnei vaccine candi-
dates, are expected to be less reactogenic while retaining the ability to generate
protective levels of immunogenicity. Besides the loss of IcsA/VirG, WRSs2 and
WRSs3 also lack plasmid-encoded enterotoxin ShET2-1 and ShET2-2. WRSs3
further lacks MsbB2 that reduces the endotoxicity of the lipid A portion of the
bacterial LPS. Studies in cell cultures and in gnotobiotic piglets demonstrate that
WRSs2 and WRSs3 have the potential to cause less diarrhea due to loss of ShET2-1
and ShET2-2 as well as alleviate febrile symptoms by loss of MsbB2 [102].
In the absence of clear correlates of protection, it is currently difficult to
anticipate the potential of this family of vaccines for the future. This is a particu-
larly important issue, as the serotype-dependent nature of protection would neces-
sitate further construction of strains, particularly S. flexneri 3a, 1b, and 6, in order to
cover a broader spectrum of serotypes [82], and testing of a combination of these
strains to address issues such as interference. Only a phase III efficacy trial
conducted in an endemic area may provide the final piece of information required
to validate the approach.
Several attenuated derivatives from the S. flexneri 2a wild strain 2457T were
constructed at the Center for Vaccine Development (University of Maryland).
Lessons have also been learnt from clinical trials with the series of attenuated
derivatives of this S. flexneri 2a strain 2457T. CVD candidate 1203 harbors dele-
tions of aroA and icsA/virG [83]. In a Phase I clinical trial in adults, CVD 1203 was
well tolerated at a dose of 106 CFU, but highly reactogenic and giving a strong
immune response at 108 CFU or higher doses [61]. The reactogenicity was probably
correlated to the high tumor-necrosis factor-a concentrations assayed in stools and
serum. To achieve a satisfactory degree of attenuation, alternative mutations were

evaluated.
CVD 1204 carries deletions in guaAB. Tested in a phase I trial, CVD 1204
showed strong immune responses in North American adults but induced a reacto-
genicity [62].
In our effort to obtain more attenuated candidate, CVD 1207 [31] was devel-
oped. This candidate belongs to the category of strongly attenuated strains under-
going a guaBA [69] mutation combined with icsA/virG [83], set [28], and sen [84]
mutations, thereby knocking out the genes encoding two putative enterotoxins of
this serotype. CVD 1207was very well tolerated in phase I trials with escalating
single doses of up to 108 CFU [85]. At 109 and 1010 CFU, only a few volunteers
experienced mild diarrhea [85]. But its immunogenicity was insufficient.
CVD 1208, another isogenic derivative of strain 2457T that carry deletions
in guaAB, sen, and set [85], has been tested in a phase I trial [85, 86]. These
strains differ from isogenic CVD 1207 by having an intact icsA/virG gene. This
design permitted researchers to assess the impact of inactivating ShET1 and ShET2.
The tolerance appeared excellent, including the lack of residual diarrhea, validating
the elimination of Sen and Set expression, thereby allowing administration of vaccine
doses up to 109 CFU without side effects. At such doses, systemic and mucosal
responses reached good levels, similar to those observed with SC602 with a 104 CFU
inoculum, if one tries a comparison [71]. The enterotoxin-negative strain CVD 1208
was considered a highly attractive vaccine candidate that reflects the desired balance
of clinical acceptability and robust immunogenicity. This is clearly an alternative
option that also needs to be validated in further trials, including in the field.
Recently, Simon et al. [87] evaluated B memory responses in healthy adult
volunteers who received one oral dose of live-attenuated Shigella flexneri 2a
vaccine. Oral vaccination with live-attenuated S. flexneri 2a elicits B[M] cells to
LPS and IpaB, suggesting that B[M] responses to Shigella antigens should be
further studied as a suitable surrogate of protection in shigellosis.
6 Alternative Strategy: Hybrid Live Vector Shigella Vaccines
Attempts in the late 1970s to construct hybrid live-attenuated vaccine strains were
based on the new specific knowledge of pathogenesis describing the importance of
host cell invasion, immune responses to the Ipa proteins and the role of mucosal,
cell-mediated immune as well as systemic immune responses in protection. In this
strategy, the objective was to express Shigella O and antigens (Ipa for instance) to
maintain the invasive phenotype of Shigella in well-tolerated E. coli or attenuated
Salmonella enterica typhi backgrounds.
The vaccine strain PGAI 42-1-15 was constructed by introducing into E. coli O8
the genes encoding synthesis of group- and type-specific O-antigens of S. flexneri 2a.
Phase I showed PGAI 42-1-15 was well tolerated in US adult volunteers; the vaccine
strain was excreted for several days [88], but the vaccine failed to protect volunteers
challenged with 104 CFU or 102 CFU virulent S. flexneri 2a strain 2457T [88].
Reason of this failure was unclear, but one hypothesis is that to be protective, an
E. coli live vector strain must also have the capacity to invade epithelial cells.
An invasive E. coli live vector EcSf2a-1 strain was constructed at WRAIR. The
invasion plasmid of S. flexneri 5 and the genes encoding synthesis of group- and
type-specific O-antigens of S. flexneri 2a were introduced into E. coli K12 [89].
At a dose of 109 CFU, 31% of vaccinees developed fever, diarrhea, or dysentery. It
was acceptably reactogenic at lower dosage levels but failed to protect vaccinees
against experimental challenge with S. flexneri 2a [89]. The aroD-deleted deriva-
tive strain EcSf2a-2 was constructed to diminish reactogenicity [90]. EcSf2a-2 which
retained the ability to invade epithelial cells caused adverse reactions. Instead of a
strong immunogenicity, only 36% of vaccines were protected against illness during
experimental challenge [90].
Investigators at WRAIR developed an oral Salmonella Typhi live vaccine strain
Ty21a expressing the S. sonnei O polysaccharide [91]. This 5076-1C live vector
strain was abandoned because it showed lot-to-lot variability in its immunogenicity
and ability to protect volunteers in challenge studies against wild-type S. sonnei
strain 53G [33, 92, 93].
7 Vaccine Candidates in Less Developed Countries
Live bacteria have generally been used in attempts to induce mucosal immunization
against enteric pathogens, as they are thought to be more immunogenic than killed
cells and, in some cases, could offer the possibility of immunization with a single
dose. But the intestinal microbiology and physiology of the healthy population
living in developing countries differ significantly from that of populations living in
industrialized countries. This has significant influence on the design of a live-
attenuated vaccine. Commonly, persons (mainly the pediatric population) living
in impoverished areas usually have heavy colonization in their proximal small
intestines. This state is accompanied by a local inflammatory state [94, 95] and
involves heightened activity by the innate immune system. These changes in the
intestine may contribute to the observed blunting of immune responses to orally
administered attenuated vaccines [94 96]. For example, some enteric vaccines
(cholera, polio, rotavirus) that are reactogenic at low dose in adult volunteers living
in developed countries have exhibited lower immunogenicity in volunteer subjects
living in developing countries [97 99]. Their immunogenicity is generally success-
fully enhanced by increasing the number of vaccine organisms per dose or by
administering additional doses of vaccine [97].
However, regarding bacillary dysentery, although vaccine candidate SC602 admi-
nistered at a low dose (104 CFU) had been reactogenic, had been heavily excreted and
conferred protection in North American adult volunteers, during phase I and II trials
in Bangladesh, none of adults and children who ingested up to 106 CFU excreted the
vaccine strain or mount a significant immune response [100]. One hypothesis is that
Bangladeshi volunteers have decreased levels of accessible iron in tissues compared
with subjects living in developed countries, so that SC602 is more crippled in
individuals living in developing countries. Furthermore, the immune response in

these volunteers may also be depressed by micronutrient deficiencies, especially
zinc or vitamin A [101]. Vaccination strategies in the under developed countries
setting need also to include consideration of the possible impact of maternal immu-
nity on conferring passive immunity to infants [58].
8 Conclusion
Vaccination offers the greatest hope as an effective and sustainable strategy against
shigellosis. An oral Shigella live-attenuated vaccine would have distinct advantages
over subunit vaccines for use in developing countries. It provides an ideal solution
if it is easy and cheap to manufacture, safely stored, and distributed without losing
viability. Results from the phase I and II clinical trials of live-attenuated Shigella
vaccine candidate (CVD 1208, SC602, SC599, WRSS1, WRSd1) are promising.
They are safe and immunogenic, and SC602 protects against dysentery. Adverse
effects (mild fever, diarrhea) sometimes observed have been reduced by elimina-
tion of the sen and set genes from the vaccine strain CVD1208. Among the issues,
one is to obtain the strongest possible immunogenicity combined with the highest
level of cross protection, in the simplest possible vaccine preparation. Furthermore,
optimal storage conditions to maintain the stability of the vaccine at different
temperatures and packaging of the vaccines in single-dose format remain to be
developed. Another major question is to define the optimal serotype numbers to
be introduced in an oral vaccine, considering the increasing serotype diversity
observed depending on the region considered.
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28:1642 54
New Generation BCG Vaccines
Michael V. Tullius and Marcus A. Horwitz
Abstract Bacille de Calmette et Guérin (BCG) was attenuated from a virulent

strain of Mycobacterium bovis a century ago and has since been administered as an
anti-tuberculosis (TB) vaccine to more than four billion people, making it the most
widely used vaccine of all time. Although BCG provides significant protection
against disease and death due to childhood and disseminated forms of TB, the
efficacy of BCG against adult, pulmonary disease is inconsistent. Thus, despite near
universal vaccination with BCG in TB endemic areas, TB remains a heavy burden
worldwide, especially in developing nations. In recent years, BCG has been utilized
in two major vaccine development strategies. First, BCG has been used as a vector
to express foreign antigens in studies aimed at developing new vaccines against a
variety of viral, parasitic, and bacterial pathogens, and against cancer and allergic
diseases. More recently, in a new vaccine paradigm, BCG has been used as a
homologous vector to overexpress native mycobacterial antigens in studies aimed
at developing improved vaccines against TB. As a vaccine vector, BCG has several
major advantages including a very well-established safety profile, high immunoge-
nicity (excellent CD4þ and CD8þ T-cell responses and strong TH1-Type immune
responses), and low manufacturing cost. As a vector for recombinant TB vaccines,
BCG has the additional advantages of providing a broad array of relevant myco-
bacterial antigens in addition to the recombinant antigens, moderate efficacy to
begin with, high acceptability as a replacement vaccine for BCG in TB endemic
countries, and the capacity to express M. tuberculosis proteins in native form and
release them in a way that results in their being processed similarly to M. tuberculosis
proteins. In addition to the overexpression of native proteins to improve their
immunogenicity and protective efficacy against TB, recombinant BCG vaccines
have been developed that express immunomodulatory cytokines or have been
M.V. Tullius and M.A. Horwitz (*)

Division of Infectious Diseases, Department of Medicine, School of Medicine, University of
California Los Angeles, 10833, Le Conte Avenue, Los Angeles, CA 90095, USA

120 M.V. Tullius and M.A. Horwitz
engineered for enhanced antigen presentation. Several new recombinant BCG

vaccines against TB have demonstrated improved protective efficacy against
M. tuberculosis, M. bovis, and M. leprae in small animal models. Against non-TB
targets, results have been variable, but several recombinant BCG vaccines have
demonstrated excellent immunogenicity and protective efficacy; stable and high-
level expression of foreign antigens in recombinant BCG, in a way that will make
them available for proper processing and presentation, have been recurrent challenges.
1 Introduction
Bacille de Calmette et Guérin (BCG), an attenuated strain of Mycobacterium bovis,

was developed as a vaccine against human tuberculosis (TB), which is primarily
caused by the closely related species Mycobacterium tuberculosis. Although its
efficacy against TB is suboptimal, BCG is the most widely used vaccine of all time,
having been administered to more than four billion people since 1921. In addition to
its use as a prophylactic agent against TB, BCG has also been used as a therapeutic
agent in the treatment of bladder cancer.
BCG is an intracellular bacterial parasite that survives and multiplies in mono-
nuclear phagocytes. Akin to M. tuberculosis, inside the human macrophage, BCG
resides in an endosomal compartment that does not fuse with lysosomes [1, 2] and is
not highly acidified [3].
In the past two decades, BCG has been used as a vaccine vector for a number of
purposes. First, it was used as a heterologous vector to express foreign antigens of a
variety of pathogens, such as HIV and Borrelia burgdorferi, the agent of lyme
disease [4 9]. Subsequently, in studies initiated in this laboratory, it was used in a
new vaccine paradigm as a homologous vector overexpressing native antigens, so
as to improve its protective efficacy against TB [10]. Most recently, a variety of
additional modifications of BCG have been explored in an effort to improve its
protective efficacy against TB. These include endowing it with immunomodulatory
cytokines capable of directing the immune response; altering its life cycle in
antigen-presenting cells by engineering it to escape its phagosome and enter the
cytoplasm, thereby enhancing class I MHC antigen presentation, and arming it
with a protease to enhance its ability to present antigens on MHC molecules. This
chapter will focus on these new approaches to improving the immunogenicity and
protective efficacy of BCG against TB and on the use of BCG as a vector for new
recombinant vaccines against non-tuberculous pathogens.
2 A Brief History of BCG
Albert Calmette and Camille Guérin developed BCG as a TB vaccine by passaging

M. bovis 230 times between 1906 and 1919 on medium consisting of pieces of
potato cooked and soaked in ox bile containing 5% glycerine, after which the
New Generation BCG Vaccines 121
organism lost its pathogenicity for animals. Their strategy was essentially an
amalgam of Jenner’s smallpox vaccine strategy centered on using a less-virulent
species (at least for humans) closely related to the target pathogen and Pasteur’s
anthrax and rabies vaccine strategy centered on the attenuation of pathogens by
culture under nonnatural conditions. The vaccine was first administered to children
in 1921. Although controlled studies were not conducted at that time, the vaccine
was believed effective in reducing the mortality of TB in children below estimated
levels prior to its use, and the vaccine gained wide acceptance in Europe and
subsequently elsewhere [11].
Modern molecular analyses have elucidated genetic differences between BCG
and M. tuberculosis. Many of these differences, involving about 3% of the ~4,000
genes of these organisms and clustered in gene segments or Regions of Difference,
reflect genomic differences between M. bovis and M. tuberculosis [12, 13]. In
addition, during its original attenuation from M. bovis, the original BCG strain
lost a gene segment containing nine open reading frames known as Region of
Difference 1 or RD1 [13]. After BCG was distributed to different countries and
strains propagated and maintained separately, genetic differences among BCG
strains evolved including gene deletions and duplications [12, 13]. Genealogically,
strains with relatively few subsequent gene deletions are characterized as “early”
strains, and include BCG Russia, Moreau, and Japan, and strains that have
acquired additional deletions are characterized as late strains, and include BCG
Tice, Connaught, Pasteur, Glaxo, and Danish.
Controlled studies conducted in 1937 and afterwards demonstrated that BCG
was efficacious in reducing the incidence and mortality of TB in children and in
reducing the incidence of disseminated forms of TB such as meningitis and miliary
TB [14, 15]. However, the efficacy of BCG against adult pulmonary TB was incon-
sistent, ranging from 35% to þ80% [16]. A large meta-analysis calculated the
overall efficacy of BCG against adult TB at 50% [14], but this figure disguises the
fact that trials tended to divide into those demonstrating either high or low efficacy,
rather than conform to a normal distribution. Trials in nontropical regions of the
world have tended to have high efficacy and trials in tropical regions of the world
have tended to have low efficacy [14].
In 2007, the most recent year for which data are available, there were an
estimated 9.27 million cases of tuberculosis worldwide [17]. Most of these cases
occurred in populations where BCG vaccination is near universal and hence these
cases can be viewed as vaccine failures. Thus, whatever the efficacy of BCG, there
is considerable room for improvement of this century-old vaccine.
3 Why Is the Efficacy of BCG so Inconsistent?
A number of hypotheses have been advanced as to why BCG protects well against
TB in some trials and not in others. They are as follows.
3.1 Prior Exposure to Atypical Environmental Mycobacteria That

Mask or Interfere with the Immune Response to BCG
In support of a direct antagonistic effect of environmental mycobacteria, Brandt

et al. [18] reported that mice pre-sensitized with a mixture of three environmental
bacteria (Mycobacterium avium, Mycobacterium scrofulaceum, and Mycobacterium
vaccae) clear BCG more rapidly and are less well protected against M. tuberculosis
challenge. On the other hand, in a different experimental murine model, pre-
sensitization with M. vaccae and M. scrofulaceum before BCG vaccination
enhanced protection against M. tuberculosis challenge, and pre-sensitization with
M. avium had no influence on the protective efficacy of BCG [19].
3.2 Malnutrition That Interferes with the Development

of a Protective Immune Response
In support of this idea, McMurray et al. have demonstrated that, in contrast to well-
nourished guinea pigs, protein-deficient guinea pigs fail to develop mature well-
organized granulomas; moreover, when vaccinated with BCG, the protein-deficient
guinea pigs are less well protected against M. tuberculosis aerosol challenge than
well-nourished guinea pigs [20].
3.3 Use of Different BCG Strains
Some have postulated that some strains of BCG are more attenuated than others and
that they consequently induce an inferior protective immune response [12, 21];
Brosch et al. postulated that evolutionarily early strains, with fewer gene deletions,
may be more potent than evolutionarily late strains. Against this idea, a study in this
laboratory showed that an evolutionarily early strain (BCG Japan) and several
evolutionarily late strains of disparate genealogy (BCG Tice, Connaught, Pasteur,
and Danish) were comparably efficacious in protecting against M. tuberculosis
aerosol challenge in the demanding guinea pig model of pulmonary tuberculosis
[22]. Moreover, a large meta-analysis failed to find differences in efficacy among
strains of BCG or for that matter between BCG and M. microti (vole bacillus) [14, 23].
3.4 Exposure to Helminths
Helminth infection is highest in tropical regions of the world. Helminths induce TH2-
type immune responses, which may interfere with the capacity of BCG to induce
a protective TH1 type of immune response against M. tuberculosis. In support of
this, Elias et al. found that worm-infected volunteers have significantly diminished
TH1-type responses compared with dewormed controls [24].
3.5 High Levels of IL-4
Rook et al. have proposed that high levels of IL-4 in tropical regions, in part as a result
of helminth infection, interfere with the protective immune response to BCG [25].
4 Recombinant BCG Overexpressing Native Proteins

as Vaccines Against Tuberculosis
4.1 Rationale for the Choice of BCG as Vector
In addition to recombinant BCG vaccines, many different types of vaccines have

been evaluated for efficacy against tuberculosis in animal models including subunit
vaccines, DNA vaccines, and attenuated M. tuberculosis. With the exception of
attenuated M. tuberculosis, none of these vaccines matches, let alone surpasses,
the efficacy of BCG in the most challenging animal models. Because of the risk of
reversion to virulence, attenuated M. tuberculosis vaccines are burdened with a
significant safety concern; multiple gene deletions will almost certainly be required
to obtain regulatory approval of these vaccines, and with these additional deletions,
the vaccines are likely to exhibit reduced efficacy. Thus, at present, improving BCG
offers the greatest potential for a vaccine that is more efficacious and at least as safe
as BCG.
Aside from its moderate efficacy to begin with, BCG has several major advan-
tages as a vector for new recombinant BCG vaccines against tuberculosis (Table 1).
First, it has a very well-established safety profile, having been administered to over
Table 1 Advantages of BCG as a vector for recombinant vaccines

General advantages
Well established safety profile administered to >4 billion people
Live vaccine high immunogenicity
Unaffected by maternal antibodies can be given in a single dose at birth
Elicits excellent CD4+ and CD8+ T cell responses
Elicits strong TH1 Type immune responses
Relatively inexpensive to manufacture
Additional advantages for recombinant TB vaccines
Provides a broad array of shared mycobacterial antigens in addition to recombinant antigens
Moderate efficacy against M. tuberculosis to start with
High acceptability as a replacement vaccine for BCG in high incidence countries
Similar intracellular compartment to M. tuberculosis and hence similar antigen processing
Expresses M. tuberculosis proteins in native form
four billion persons. Serious adverse effects are exceedingly rare except in immu-
nocompromised individuals, for whom the vaccine is not recommended. Second,
as a live vaccine, it has high immunogenicity. Third, and of great importance,
modified versions of BCG that are superior to BCG have very high acceptability as
a replacement vaccine for BCG in regions of the world where the burden of
tuberculosis is greatest; for all of its shortcomings, BCG is life preserving in such
parts of the world, especially in infants, and health care workers are reluctant to
accept an alternative vaccine in a vaccine trial that is not clearly at least as
efficacious as BCG. In such parts of the world, recombinant BCG is considered
“BCG+” and is therefore readily acceptable as an alternative to BCG in vaccine
trials, provided of course that it has demonstrated sufficient safety and efficacy in
preclinical studies. Fourth, BCG occupies the same intraphagosomal compartment
as M. tuberculosis in host cells, and consequently processes and presents antigens
similarly. In human mononuclear phagocytes, both BCG and M. tuberculosis
multiply extensively if not exclusively within a phagosome; a small minority of
M. tuberculosis may exit the phagosome at very late stages of infection (Clemens
and Horwitz, unpublished data) as has been reported in myeloid cells [26], possibly
as a prelude to lysis of the host cell, but the majority of mycobacterial multiplica-
tion takes place in an endosome-like compartment that favors antigen presentation
via class II MHC molecules. Fifth, BCG expresses M. tuberculosis proteins in
native form. Nonmycobacterial vectors may express highly conserved M. tuberculosis
proteins in native form, but a mycobacterial host is frequently required to express
proteins unique to mycobacteria, such as the mycolyl transferases, in native form [27].
Finally, BCG can be manufactured cheaply; cost is of course a significant consider-
ation in the developing regions of the world where tuberculosis has the highest
prevalence.
4.2 rBCG30
rBCG30, the first vaccine demonstrated more potent than BCG against tuberculosis,
is also the first vaccine of any kind incorporating the strategy of utilizing a
homologous vector to overexpress native antigens [10, 28]. This is a powerful
vaccine strategy that combines the approach of using a live attenuated homologous
vector with the approach of immunizing with key immunoprotective antigens of
the target pathogen. In the case of rBCG30, the live attenuated homologous vector
is BCG and the key native antigen is the M. tuberculosis 30 kDa major secretory
protein, a mycolyl transferase also known as the alpha antigen or Antigen 85B.
4.2.1 Rationale for Selecting the M. tuberculosis 30 kDa Protein

for a Recombinant BCG Vaccine
The rationale for using BCG as a homologous vector is discussed above. The
rationale for overexpressing the major secretory protein of M. tuberculosis is
derived from the Extracellular Protein Hypothesis for vaccines against intracellular
pathogens [29 34]. This hypothesis holds that proteins secreted or otherwise
released by intracellular pathogens are key immunoprotective antigens because
they are available for proteolytic processing by the host cell and presentation on
the host cell surface as MHC–peptide complexes, thus allowing the immune system
to generate a population of T cells capable of recognizing the MHC–peptide
complexes and exerting an antimicrobial effect against the host cell. The hypothesis
further holds that appropriate immunization of a naı̈ve host with such proteins
incorporated into a vaccine allows the immune system to generate a functionally
equivalent population of T cells later capable of recognizing and exerting an
antimicrobial effect against host cells infected with the target intracellular patho-
gen. Finally, the hypothesis holds that among the extracellular proteins released by
intracellular parasites, the most abundant ones will figure most prominently
because they would provide the richest display of MHC–peptide complexes on
the host cell surface.
The M. tuberculosis 30 kDa mycolyl transferase is the most abundant protein
released by M. tuberculosis, making up almost one-quarter of the total extracellular
protein released [33]. The 30 kDa protein (Antigen 85B) is highly homologous with
two other mycolyl transferases of ~32 kDa mass Antigen 85A and Antigen 85C
[35]. The 30 kDa protein is not only the major protein secreted into broth culture but
also among the major proteins of all types expressed by M. tuberculosis in infected
human macrophages [36]. The 30 kDa protein is highly immunogenic and, when
administered as a purified protein with adjuvant, it induces strong cell-mediated and
protective immunity in the guinea pig model of pulmonary tuberculosis [33].
4.2.2 Construction of rBCG30
rBCG30 is a recombinant BCG Tice strain overexpressing the 30 kDa protein from
plasmid pMTB30 [10], derived from the Mycobacterium Escherichia coli shuttle
vector pSMT3 [37]. The plasmid pMTB30 contains the full-length M. tuberculosis
30 kDa protein gene and flanking 50 and 30 regions including the promoter region.
rBCG30 expresses approximately 5.5-fold the amount of 30 kDa protein that
the parental BCG strain expresses. The M. tuberculosis and BCG 30 kDa proteins
are nearly identical, differing from each other by one amino acid. Other commonly
used BCG strains including Connaught, Glaxo, Japanese, Copenhagen, and
Pasteur produce amounts of 30 kDa protein comparable to that produced by BCG
Tice [28].
4.2.3 Preclinical Studies
rBCG30 was tested in the guinea pig model of pulmonary tuberculosis, a model
noteworthy for its resemblance to human disease clinically, immunologically, and
pathologically, and the gold standard among small animal models of tuberculosis.
BCG protects well in this model, in which animals are immunized and then
challenged with M. tuberculosis by aerosol. Compared with sham-immunized
animals, BCG-immunized animals are protected against weight loss, a hallmark
of TB, and death, and they have significantly less lung pathology and a lower
burden of M. tuberculosis in the lung and spleen (~1.5 2 logs fewer 10 weeks after
challenge).
Despite the fact that the 30 kDa protein is a relatively abundant secreted protein
of BCG, parental BCG induces negligible immune responses to the protein in
guinea pigs. In contrast, rBCG30 induces strong cell-mediated immunity, manifest
by cutaneous delayed-type hypersensitivity to the 30 kDa protein, and humoral
immunity, manifest by high serum antibody titer to the 30 kDa protein.
Paralleling these immune responses, in guinea pigs immunized with BCG or
rBCG30 and challenged 10 weeks later by aerosol with virulent M. tuberculosis
Erdman strain, rBCG30 induces greater protective immunity than BCG. Ten weeks
after challenge, rBCG30-immunized guinea pigs had fewer CFU in the lung
(0.8 0.1 log fewer) and spleen (1.1 0.1 log fewer) than BCG-immunized
animals [34]. In a survival study, rBCG30-immunized guinea pigs survived
significantly longer than BCG-immunized animals (Fig. 1) [28]. Remarkably few
rBCG30 organisms are required to induce strong cell-mediated and protective
immunity [38].
Fig. 1 rBCG30 immunized animals survive longer than BCG immunized animals after
M. tuberculosis aerosol challenge. Animals in groups of 20 or 21 were sham immunized or
immunized with BCG or rBCG30 Tice, and 10 weeks later challenged by aerosol with virulent
M. tuberculosis. A group of uninfected animals served as controls. Sham immunized animals died
most rapidly; BCG immunized animals survived significantly longer than sham immunized
animals; and rBCG30 immunized animals survived significantly longer than BCG immunized
animals. Thirty five percent of rBCG30 immunized animals survived to the point where
uninfected control animals began to die off. Reproduced with permission of the American Society
for Microbiology from Horwitz et al. [28].
In addition to enhanced protective efficacy against M. tuberculosis, rBCG30

induces greater protective immunity than BCG against M. bovis, the primary
agent of tuberculosis in domesticated animals, in the guinea pig model and against
M. leprae, the agent of leprosy, in a murine model (see below) [39, 40].
In preclinical safety studies, rBCG30 was well tolerated. rBCG30 is cleared at
the same rate as BCG in guinea pigs, i.e., rBCG30 and BCG are comparably
avirulent [28]. No adverse effects were observed in guinea pigs or mice in safety
studies conducted by the Aeras Global TB Vaccine Foundation.
4.2.4 Clinical Studies
rBCG30 was tested in a Phase 1 human study, the first live recombinant BCG
vaccine against tuberculosis to enter clinical trials [41]. The trial was double
blinded with volunteers randomized to rBCG30 or parental BCG Tice. There was
no significant difference between the two vaccines in clinical reactogenicity.
rBCG30, but not BCG, induced significantly increased Antigen 85B-specific
immune responses including significantly increased lymphocyte proliferation,
interferon-g (IFNg) secretion, IFNg enzyme-linked immunospot responses, direct
ex vivo CD4þ and CD8þ T-cell IFNg responses, and CD4þ and CD8þ memory
T cells capable of expansion. Moreover, in a novel assay of effector cell function,
rBCG30 but not BCG significantly increased the number of antigen-specific T cells
capable of inhibiting the growth of intracellular mycobacteria (Fig. 2). Thus,
rBCG30 was well tolerated and more immunogenic than BCG.
4.3 rBCG Expressing Other Native M. tuberculosis Proteins
Recombinant BCG expressing other native M. tuberculosis proteins and M. tuber-

culosis fusion proteins have been constructed and evaluated. Noteworthy studies in
which protective efficacy has been investigated are discussed below.
4.3.1 rBCG/Antigen 85A
Sugawara and colleagues studied the protective efficacy of a recombinant BCG

overexpressing Antigen 85A in guinea pigs, cynomolgus monkeys, and rhesus
monkeys [42 44]. Cynomolgus and rhesus monkeys exhibit different susceptibility
to M. tuberculosis [45]. The rhesus monkey is highly susceptible to progressive
infection culminating in death, whereas the cynomolgus monkey is relatively
resistant and able to contain low challenge doses [45].
Compared with guinea pigs vaccinated with parental BCG before aerosol chal-
lenge with M. tuberculosis, guinea pigs vaccinated with rBCG/Antigen 85A
showed a trend toward fewer CFU in the lung and spleen [42].
In cynomolgus monkeys vaccinated before intrathecal challenge with
M. tuberculosis H37Rv, both the parental and recombinant vaccines protected
Fig. 2 Phase I human trial of rBCG30: rBCG30 but not BCG immunized recipients show
increased Ag85B specific T cell inhibitory activity against intracellular mycobacteria. In a double
blind Phase 1 human trial in which recipients were vaccinated with BCG Tice or rBCG30 Tice,
peripheral blood mononuclear cells were harvested from ten recipients of each vaccine prevacci
nation and on days 56 and 112 postvaccination and stimulated with recombinant 30 kDa Antigen
85B (Ag85B) protein for 7 days. These Ag85B specific expanded T cells were then cocultured
with BCG infected autologous macrophages for 3 days. The macrophages were lysed; viable CFU
of BCG were enumerated on Middlebrook agar plates; and the percent inhibition mediated by
Ag85B specific T cells vs. medium rested T cells was calculated. Shown are the median values
(points), mid 50% values (boxes), and nonoutlier ranges (whiskers). *, p < 0.05 comparing pre
and postvaccination responses by Wilcoxon matched pairs test. **, p < 0.05 comparing rBCG30
and BCG vaccination groups by Mann Whitney U test. In other assays, rBCG30 but not BCG
immunized recipients showed significantly increased Antigen 85B specific lymphocyte prolifera
tion, interferon g (IFNg) secretion, IFNg enzyme linked immunospot responses, direct ex vivo
CD4þ and CD8þ T cell IFNg responses, and CD4þ and CD8þ memory T cells capable of
expansion. Reproduced with permission of the University of Chicago Press from Hoft et al. [41].
relative to sham-immunized controls, reducing CFU in the lung and spleen by ~2

logs. Compared with cynomolgus monkeys vaccinated with the parental BCG
vaccine, monkeys vaccinated with rBCG/Antigen 85A had fewer CFU in lung
sections, but not in spleen sections [43].
In rhesus monkeys vaccinated before intrathecal challenge with M. tuberculosis
H37Rv, monkeys vaccinated with rBCG/Antigen 85A had significantly fewer CFU
of M. tuberculosis in the lung and spleen than monkeys vaccinated with parental
BCG [44].
4.3.2 rBCG/Antigen 85C
Jain et al. [46] investigated a recombinant BCG vaccine overexpressing Antigen

85C, a member of the 30 32 kDa Antigen 85A, B, C family of mycolyl transferases.
Compared with BCG, the vaccine gave enhanced protection in the guinea pig model
against high-dose aerosol challenge with M. tuberculosis H37Rv, including signifi-

cantly reduced CFU in the lung and spleen, reduced pathology in the lung, liver, and
spleen, and reduced pulmonary fibrosis.
4.3.3 rBCG/ESAT-6 ( CFP10)
Pym et al. evaluated a recombinant BCG vaccine complemented with the RD1
region that is missing in BCG, having been deleted from all BCG strains during its
attenuation from M. bovis [47]. The vaccine secretes both ESAT-6 and CFP10, two
proteins encoded by the RD1 region. The vaccine was tested in both the mouse and
guinea pig models. Compared with mice immunized with BCG before intravenous
or aerosol challenge with M. tuberculosis, mice immunized with the recombinant
vaccine had comparable numbers of M. tuberculosis in the lung but fewer CFU in
the spleen; differences in the spleen were significant in two of four experiments. In
a single guinea pig study, animals immunized with the recombinant vaccine before
aerosol challenge with M. tuberculosis had comparable numbers of M. tuberculosis
in the lung but fewer CFU in the spleen than animals immunized with control BCG.
The recombinant vaccine, however, was more virulent than BCG in severely
immunocompromised SCID mice [48], and clinical development of the vaccine
has not proceeded.
Brodin et al. studied a potentially safer version of the vaccine constructed in
Mycobacterium microti [49]. In SCID mice, the recombinant M. microti vaccine
complemented with the RD1 region was less virulent than the recombinant BCG
vaccine complemented with the RD1 region but still much more virulent than a
BCG control. In a mouse model, in which immunized mice were aerosol challenged
with M. tuberculosis, mice vaccinated with the recombinant M. microti strain had
significantly fewer CFU in the spleen than mice immunized with the control BCG
vaccine at two of three time points. In the guinea pig model, the recombinant
M. microti vaccine and control BCG vaccine were comparably protective.
Bao et al. studied two recombinant BCG vaccines expressing ESAT-6 in a
murine model [50]. One recombinant BCG secreted ESAT-6 and one expressed
ESAT-6 as part of a nonsecreted fusion protein. There was no significant difference in
protective efficacy between either of the two recombinant BCG vaccines and BCG.
4.3.4 rBCG/38 kDa Protein
Castanon-Arreola et al. investigated a recombinant BCG vaccine overexpressing a

secreted M. tuberculosis 38 kDa glycoprotein and reported that mice immunized
with the recombinant vaccine before challenge with either M. tuberculosis H37Rv
or M. tuberculosis Beijing strain survived longer than mice immunized with the
parental BCG Tice vaccine [51].
4.3.5 rBCG/19 kDa Protein
Rao et al. investigated a recombinant BCG vaccine overexpressing a 19 kDa

lipoprotein of M. tuberculosis and found that it abrogated the protective effect of
BCG [52]. Compared with splenocytes of mice immunized with BCG, splenocytes
of mice immunized with the recombinant vaccine exhibited an enhanced TH2-type
immune response to BCG sonicate (increased IL-10 and decreased IFNg and
IgG2a:IgG1 ratio). In guinea pigs, BCG but not the recombinant vaccine induced
immunoprotection against subcutaneous M. tuberculosis challenge.
4.4 rBCG Expressing M. tuberculosis Fusion Proteins
4.4.1 rBCG/72f
Kita et al. investigated a recombinant BCG secreting a hybrid of two proteins

(Mtb39 þ Mtb32) named 72f in the cynomolgus monkey model [53]. The recombi-
nant vaccine induced immune and protective responses but they were not significantly
different from BCG controls.
4.4.2 rBCG/Antigen 85B-ESAT-6
Shi et al. tested recombinant BCG vaccines secreting fusion proteins of Antigen
85B and ESAT-6 in a mouse model [54]. The amount of the fusion protein secreted
was not quantitated but appeared to be small on the Western blots on which it
was detected. Splenocytes from mice immunized with the recombinant vaccines
produced significantly more IFNg in response to M. tuberculosis culture filtrate
proteins than splenocytes from mice immunized with control BCG. However, there
was no significant difference between the recombinant vaccine and BCG in protec-
tive efficacy.
Xu et al. evaluated recombinant vaccines expressing Antigen 85B, an Antigen
85B-ESAT-6 fusion protein, and an Antigen 85B-ESAT-6-mouse IFNg fusion
protein [55]. The biological activity of the mouse IFNg was not evaluated and
was not likely active. In the mouse model, the recombinant vaccines appeared to
give slightly better protection than BCG in the lung but not the spleen at late time
points.
4.4.3 rBCG/Antigen 85B-Mpt64190–198-Mtb8.4
Qie et al. investigated a recombinant BCG vaccine expressing a fusion protein of

Antigen 85B, an immunodominant peptide of Mpt64 and Mtb8.4 [56]. In a murine
model, the recombinant vaccine had comparable or slightly better efficacy than
BCG.

as Vaccines Against Leprosy
BCG has shown efficacy against leprosy in addition to TB, but as with TB,
protection is inconsistent. Two recombinant vaccines have been compared with
BCG for efficacy against leprosy in murine models of leprosy.
5.1 rBCG30
Gillis et al. immunized BALB/c mice with BCG, rBCG30, or a recombinant BCG
vaccine carrying plasmid pNBV1 encoding the M. leprae 30 kDa Antigen 85B
(rBCG30ML), and then challenged the animals 2.5 months later by administering
viable M. leprae into each hind foot pad [40]. Seven months later, the number of M.
leprae per foot pad was enumerated. In addition, splenocytes and lymph node
cells from immunized animals were evaluated for lymphocyte transformation
to M. tuberculosis Purified Protein Derivative (PPD). All vaccinated groups showed
sensitization to PPD; splenocytes from mice immunized with rBCG30 and
rBCG30ML showed the highest responses. In the one experiment in which an
efficacy comparison was feasible, rBCG30 and rBCG30ML gave protection supe-
rior to BCG and the difference between rBCG30 and BCG was statistically signifi-
cant, as was the difference between the two rBCG30 groups combined and BCG.
5.2 rBCG/Antigen 85A, Antigen 85B, and MPB51
Ohara et al. examined the protective efficacy of a recombinant BCG vaccine over-
expressing Antigen 85A, Antigen 85B, and MPB51 [57]. C57Bl/6 mice vaccinated
with recombinant vaccine but not with the control BCG vaccine had significantly
reduced M. leprae in footpads 30 weeks after challenge with M. leprae. Compared
with unimmunized controls, BALB/c mice vaccinated with either the control BCG or
recombinant BCG had reduced numbers of M. leprae in footpads. While there was no
significant difference in the number of footpad M. leprae between mice immunized
with the recombinant BCG or control BCG, there was a trend toward fewer M. leprae
in the footpads of recombinant BCG-immunized mice.
6 Recombinant BCG Overexpressing Native Proteins and

Attenuated M. bovis as Vaccines Against Bovine Tuberculosis
M. bovis is the principal etiologic agent of tuberculosis in domesticated animals.

M. bovis infection of domesticated animals exacts a significant economic toll; for
example, in cattle it results in reduced fertility, milk production, and meat value
[58]. Control measures, where they can be afforded, center on testing animals for a
cell-mediated immune response to M. bovis antigens (indicative of exposure) and
culling herds of animals testing positive. One approach to reducing the incidence of
M. bovis infection in domesticated animals is vaccinating the domesticated animals
and/or the wild animals that serve as reservoirs of infection. BCG has been tested as
a vaccine in cattle, but as in humans, its efficacy is suboptimal [59] (<50%),
prompting a search for vaccines that are better. Standard tests for TB in domes-
ticated animals frequently rely on assessment of a cell-mediated immune response
to PPD. Since BCG vaccination can interfere with tests involving PPD, the use of a
BCG vaccine in domesticated animals may need to be coupled with the use of a
diagnostic test employing antigens absent in BCG but present in M. bovis, such as
members of the ESAT-6 family.
Two new generation vaccines have been compared with BCG for efficacy
against M. bovis infection.
6.1 rBCG30
rBCG30, described above, was tested in a guinea pig model of M. bovis infection in
which vaccinated animals were challenged with M. bovis by aerosol. Compared
with BCG, rBCG30-immunized animals had a lower burden of M. bovis in the lung
and spleen [39].
6.2 WAg533
WAg533 is a newly attenuated strain of M. bovis. It was tested for efficacy in

brushtail possums, an important reservoir of M. bovis infection in New Zealand;
vaccinated animals were challenged with M. bovis by aerosol. Compared with
animals immunized with BCG, animals immunized with WAg533 by three differ-
ent routes (conjunctival/intranasal, oral, and subcutaneous) had reduced severity of
illness and lower CFU burdens in the lung and spleen [60].
7 Recombinant BCG Overexpressing Native Proteins and

Additionally Attenuated for Safety in HIV-Positive Persons
The tuberculosis and AIDS pandemics are closely intertwined. Approximately 12

million people throughout the world are infected with both M. tuberculosis and
HIV, or about a third of all persons infected with HIV. These coinfected people
have the greatest susceptibility to developing active tuberculosis, the major oppor-
tunistic infection in AIDS patients.
BCG is an extremely safe vaccine in immunocompetent people, but it can cause

serious and even fatal disseminated disease in immunocompromised individuals,
including AIDS patients. The World Health Organization has advised against
administering BCG to HIV-positive infants because of their increased risk of
disseminated BCG infection [61]. This has created a need for a vaccine that is
safe and effective in HIV-positive persons, especially infants. Since the HIV status
of infants in high-risk regions of the world is often unknown, such a vaccine may be
a prudent choice for all infants of unknown HIV status in regions of the world where
HIV prevalence is high.
Tullius et al. approached this problem by developing versions of rBCG30 that
are readily grown in the laboratory but are replication limited in the host [62].
Although their replication is limited, it is nevertheless sufficient to induce a strong
immunoprotective response.
7.1 rBCG(mbtB)30
rBCG(mbtB)30 is the first vaccine that is safer than BCG in the SCID mouse and
yet more potent than BCG in the guinea pig model of pulmonary tuberculosis [62].
rBCG(mbtB)30 was rendered siderophore dependent by deletion of the gene mbtB,
which encodes an enzyme necessary to produce the iron siderophores mycobactin
and exochelin. The vaccine grows normally in vitro in broth culture and in human
macrophages provided the iron-loaded siderophore mycobactin is provided, and
during in vitro growth, it is able to store iron mycobactin. This stored iron
mycobactin allows the vaccine to multiply for several divisions in vivo, sufficient
to induce cell-mediated and protective immunity. In the SCID mouse, rBCG
(mbtB)30 is much safer than BCG. In the guinea pig, rBCG(mbtB)30 is cleared
much faster than BCG; nevertheless, in contrast to BCG, it induces a strong cell-
mediated immune response to the 30 kDa protein. Most importantly, rBCG(mbtB)
30 induces protective immunity that is significantly greater than that induced by
BCG (Fig. 3).
7.2 rBCG(panCD)30
rBCG(panCD)30 was rendered pantothenate dependent by deletion of the panCD

genes [62]. This vaccine can multiply in vitro in broth culture or human macro-
phages in the presence of high concentrations of pantothenate, but multiplication is
highly limited in vivo. In the SCID mouse, rBCG(panCD)30 is much safer than
BCG. In the guinea pig, rBCG(panCD)30 is cleared very rapidly, allowing high
doses to be administered safely. In guinea pigs administered high doses of rBCG
(panCD)30, protection is comparable to BCG.
a b
c d
e f
Fig. 3 rBCG(mbtB)30, a replication limited but highly immunoprotective TB vaccine designed

specifically for HIV positive persons. (a) Siderophore dependence of rBCG(mbtB)30 grown in
broth culture. rBCG(mbtB)30 was cultured in medium containing 0.01 mg/ml mycobactin J and
washed before inoculation into broth containing 0 100 ng/ml mycobactin J (as indicated to the
right of the graph). Growth of the parental BCG strain (grown without mycobactin) is shown for
comparison. (b) Preloading of rBCG(mbtB)30 with mycobactin iron results in greater residual
growth in human THP 1 macrophages. rBCG(mbtB)30 was cultured in medium containing 0.01, 1,
or 10 mg/ml mycobactin J (as indicated to the right of the graph) and washed before addition to
THP 1 monolayers. CFU were enumerated 0, 3, and 7 days after infection. Data are the mean log
CFU SE for duplicate wells. The number of bacterial generations over the 7 day course of
infection is indicated to the far right of the growth curves (data are means for two independent
experiments). (c) Attenuation of rBCG(mbtB)30 in SCID mice. SCID mice in groups of 20 were
injected with 106 CFU of BCG or rBCG(mbtB)30, or sham treated with PBS via the tail vein, and
survival was monitored over a 40 week period. SCID mice vaccinated with rBCG(mbtB)30
survived significantly longer than mice vaccinated with BCG (p < 0.0001). (d) Limited replica
tion of rBCG(mbtB)30 in guinea pigs. Guinea pigs in groups of 24 were immunized by intradermal
administration of 106 CFU of BCG or iron mycobactin loaded rBCG(mbtB)30. At 1 15 weeks
after immunization, as indicated, three animals per group were euthanized, and CFU of BCG and
rBCG(mbtB)30 in the lungs, spleen, and inguinal lymph nodes were assayed. Data are mean log
CFU SE. The limit of detection was 1 log CFU. (e) Immunogenicity of rBCG(mbtB)30 in guinea
pigs. Guinea pigs in groups of six were immunized by intradermal administration of 103 CFU BCG
8 Recombinant BCG Expressing Immunomodulatory

Cytokines
Cytokines play a central role in the workings of the immune system, potentiating
some responses and dampening others. Recognizing this, investigators have
attempted to beneficially modulate immune responses to vaccines by incorporating
cytokines into them. Early studies by O’Donnell et al. and Murray et al. explored
the effect of recombinant BCG secreting cytokines on immune responses in mice
[63, 64]. O’Donnell et al. constructed a recombinant BCG secreting IL-2 and
showed that, compared with wild-type BCG, it induced enhanced splenocyte
secretion of interferon-g (IFNg) [64]. Murray et al. constructed BCG secreting a
number of different cytokines and studied a variety of immune responses in mice
immunized with the constructs. Compared with splenocytes from mice immunized
with parental BCG, splenocytes from mice immunized with BCG secreting inter-
leulin-2 (IL-2), granulocyte macrophage colony stimulating factor (GM-CSF),
or IFNg, but not BCG secreting interleukin-4 (IL-4) or interleukin-6 (IL-6),
had increased lymphocyte proliferation and increased production of cytokines,
especially IFNg, IL-2, and IL-10, upon stimulation with PPD [63].
Later studies have focused on the efficacy of cytokine-secreting BCG vaccines
in protection against tuberculosis, and in the therapy of bladder cancer and allergy.
8.1 Cytokine-Secreting Vaccines for Tuberculosis
8.1.1 rBCG/GM-CSF
Ryan et al. studied mice immunized with BCG secreting murine GM-CSF [65].
Compared with mice immunized with control BCG, mice immunized with BCG
secreting GM-CSF had greater numbers of antigen-presenting cells (CD11cþ
ä
Fig. 3 (continued) or rBCG30 or with 106 CFU of iron mycobactin loaded BCG mbtB or rBCG
(mbtB)30, or were sham immunized with PBS. Ten weeks after immunization, the animals were
skin tested by intradermal administration of highly purified M. tuberculosis 30 kDa major secre
tory protein (Antigen 85B), and the degree of induration was assessed 24 h later. Data are mean
diameters of induration SE. *, p 0.01; **, p 0.001 (ANOVA; compared with BCG
immunized guinea pigs). (f) Protective efficacy of rBCG(mbtB)30 in guinea pigs. Guinea pigs in
groups of 15 (except for the sham immunized group of nine animals) were immunized by
intradermal administration of BCG, rBCG30, BCG mbtB, or rBCG(mbtB)30 as in (e) above.
Ten weeks after immunization, the animals were challenged with a low dose aerosol of
M. tuberculosis Erdman strain. Ten weeks after challenge, the animals were euthanized and
CFU of M. tuberculosis in the lungs and spleen were assayed. Data are mean log CFU SE.
Open symbols indicate sham immunized animals and groups immunized with BCG strains
not overexpressing the 30 kDa protein. Closed symbols indicate groups immunized with BCG
strains overexpressing the 30 kDa protein. For (e) and (f), one representative experiment of three
is shown. Reproduced with permission of the American Society for Microbiology from Tullius
et al. [62].
MHCIIþ cells) in draining lymph nodes; increased numbers of IFNg-secreting spleen

cells stimulated ex vivo with BCG lysate 5 and 17 weeks after immunization; and ~1
log fewer CFU in the spleen after aerosol challenge with M. tuberculosis.
8.1.2 rBCG/IFNg and rBCG30/IFNg
Tullius et al. have constructed rBCG and rBCG30 expressing various forms of
human IFNg, including strains encoding monomeric and covalently linked dimeric
forms, and studied their effect on antigen presentation in human monocytes.
Infection of human monocytes with these constructs results in upregulation of
class I and II MHC molecules and enhanced presentation on an MHC class II
molecule of a peptide of the 30 kDa Antigen 85B major secretory protein (Tullius
and Horwitz, unpublished studies).
8.1.3 rBCG/IL-2
Young et al. studied the capacity of recombinant BCG secreting murine IL-2 to
counter a Type 2 immune response in mice and to alter the immune profile of
immunosuppressed mice [66]. As noted above, one theory as to why BCG is
sometimes poorly effective is that it is administered in a setting in which a TH2
type of immune response predominates, e.g., as a result of helminth infection. The
investigators showed that rBCG/mIL-2 but not control BCG can induce a TH1
profile in mice immunosuppressed with dexamethasone and in IL-4 transgenic
mice. Compared with BCG-immunized mice, the rBCG/mIL-2–immunized mice
exhibit greater splenocyte proliferation and IFNg production in response to PPD
and a higher IgG2a:IgG1 antibody ratio (consistent with a TH1-type immune
response) in both types of mice.
In a separate study, Young et al. investigated the protective efficacy of rBCG/
mIL-2 in mice [67]. Although the IL-2–secreting BCG vaccine induced a longer
lasting splenic lymphocyte proliferative response to PPD, a higher IFNg level in
response to PPD, and a higher IgG2a:IgG1 antibody ratio than control BCG, the
recombinant vaccine was not more protective against M. bovis aerosol challenge
than control BCG.
Slobbe et al. studied a BCG vaccine secreting cervine IL-2 in outbred red deer
[68]. rBCG/cIL-2 induced a smaller delayed-type hypersensitivity (DTH) response to
PPD than parental BCG. RT-PCR studies of lymphocytes from the deer revealed that
IL-2 and IFNg levels were similar in deer vaccinated with rBCG/cIL-2 or parental
BCG but that IL-4 levels were reduced in the deer vaccinated with rBCG/cIL-2.
8.1.4 rBCG/IL-18
Young et al. evaluated a recombinant BCG secreting murine IL-18 [67]. Disappoint-
ingly, this vaccine induced significantly less IFNg in splenocytes of immunized mice
that control BCG, and paralleling this finding, it was significantly less protective than
control BCG.
8.1.5 rBCG/IL-15
Tang et al. studied a recombinant BCG vaccine secreting a fusion protein of

Antigen 85B and murine IL-15 [69]. Whether the IL-15 portion of the fusion
protein was biologically active was not investigated. In any case, the investigators
report that the vaccine was cleared more rapidly than a control recombinant vaccine
secreting only Antigen 85B and that mice immunized with the recombinant vaccine
secreting the fusion protein, compared with mice immunized with the control
vaccine, had greater absolute numbers of CD4þ and CD8þ T cells in lung, spleen,
and peritoneal exudate cells, greater numbers of IFNg-secreting CD4þ cells to two
M. tuberculosis antigens, and a lower bacterial burden in the lung, but not the
spleen, after intratracheal challenge with M. tuberculosis.
8.2 Cytokine-Secreting Vaccines for Therapy of Bladder Cancer
In addition to its use as a vaccine against TB, BCG plays an important immuno-
therapeutic role in the treatment of superficial bladder cancer. Such treatment is
associated with induction of TH1 cytokines [70]. Approximately 30% of patients do
not respond to current BCG therapy and 50% of patients suffer a recurrence [71].
This has prompted investigators to explore new generation BCG vaccines secreting
TH1-inducing cytokines for the treatment of bladder cancer.
8.2.1 rBCG/IFNg
Arnold et al. tested the immunotherapeutic efficacy of rBCG expressing murine

IFNg in a mouse model [70]. They found that rBCG/mIFNg upregulated class I
MHC expression in murine bladder cancer cells to a greater extent than a BCG
control strain transfected with an empty vector. Intravesicular instillation of
rBCG/mIFNg resulted in greater recruitment of CD4þ T cells into the bladder
and increased expression of IL-2 and IL-4 compared with intravesicular instillation
of control BCG. Finally, rBCG/mIFNg but not BCG treatment of orthotopic bladder
cancer significantly prolonged survival compared with untreated animals; however,
while survival of rBCG/mIFNg-treated animals was greater than that of BCG-
treated animals, the difference did not reach statistical significance.
8.2.2 rBCG/IFNa
IFNa has been shown to improve the response of patients to BCG therapy [71],
prompting investigators to evaluate the immunogenicity of rBCG secreting IFNa
2B in vitro. Luo et al. found that rBCG/IFNa induced more IFNg and IL-2 from
human PBMC in vitro than BCG [72] and Liu et al. found that rBCG/IFNa induces
more potent PBMC cytotoxicity than BCG against human bladder cancer cell lines
and the effect was dose dependent [71]. The addition of neutralizing antibodies
against IFNa, IFNg, or IL-2 to PBMC cultures stimulated with rBCG/IFNa reduced
PBMC cytotoxicity against the cancer cells.
8.2.3 rBCG/IL-2
Yamada et al. studied the cytotoxic effect of recombinant BCG secreting murine
IL-2 on murine bladder cancer cells in vitro [73]. They constructed a recombinant
BCG secreting murine IL-2 fused to the signal sequence of the 30 kDa Antigen 85B
of BCG and reported that the fusion protein was functional. Peritoneal exudate cells
(PEC) incubated with rBCG/mIL-2 produced greater amounts of IFNg, TNFa, and
IL-12 and were more cytotoxic than PEC incubated with BCG. The enhanced
cytotoxicity was neutralized by the addition of anti–IL-2 antibody.
8.2.4 rBCG/IL-18
Luo et al. found that, compared with splenocytes from BCG-immunized mice,
splenocytes from mice immunized with rBCG/mIL-18 had increased IFNg,
TNFa, and GM-CSF levels and increased lymphocyte proliferation in response to
BCG antigens [74]. Mouse PECs (>90% macrophages) stimulated in vitro with
rBCG/mIL-18 also had greater cytolytic activity against a mouse bladder cancer
cell line than PEC stimulated with BCG.
8.3 Cytokine-Secreting Vaccines for Allergy
Antigen-specific TH2-type cells figure prominently in allergic reactions, and TH1

cells, via the secretion of IFNg, may counter allergic responses by dampening TH2
cell activity [75].
8.3.1 rBCG/IL-18
Biet et al. studied recombinant BCG secreting biologically active (increased NF-k
B) mouse IL-18 [75]. IL-18 acts synergistically with IL-12 to induce IFNg, and
since BCG itself induces IL-12, Biet et al. hypothesized that rBCG secreting Il-18
might exert a more potent dampening effect on TH2-type immune responses than
BCG [76]. rBCG/mIL-18 enhanced TH1 and diminished TH2-type immune responses
in mice. Compared with splenocytes from BCG-immunized mice, splenocytes from
rBCG/mIL-18 immunized mice had increased IFNg and GM-CSF production in

response to PPD. In contrast, rBCG/mIL-18 immunized mice had decreased serum
IgG to BCG antigens [75].
Biet et al. explored the therapeutic potential of rBCG/mIL-18 in a murine model
of pulmonary allergic inflammation, in which mice were sensitized to ovalbumin
[76]. The investigators found that lymph node cells from sensitized mice immu-
nized with rBCG/mIL-18 and challenged with ovalbumin produced more IFNg
when stimulated with ovalbumin than lymph node cells from sensitized mice
immunized with BCG and challenged with ovalbumin; in contrast, IL-5 production
was suppressed. Moreover, ovalbumin-sensitized mice immunized with rBCG/
mIL-18 had less bronchoalveolar eosonophilia after ovalbumin challenge than
BCG-immunized mice.
9 Recombinant BCG with Enhanced Antigen Presentation
In addition to engineering BCG that secretes various cytokines, two other approaches
have been used to improve antigen presentation. One approach is aimed primarily at
enhancing MHC class I antigen presentation and another approach is aimed at
enhancing MHC class II antigen presentation.
9.1 BCG with Altered Intracellular Pathway
Grode et al. engineered a recombinant BCG vaccine secreting listeriolysin, to

promote perforation of the phagosomal membrane, and with a deleted urease
gene, to reduce the pH in the phagosome to nearer the pH optimum of listeriolysin
[77]. The rationale for this vaccine was to increase class I MHC antigen presenta-
tion by allowing egress of BCG antigens to the cytosol. As this vaccine also was
proapoptotic in macrophages, it additionally promoted presentation of mycobacte-
rial antigens via cross-priming. In a murine model, this vaccine was more potent
than BCG against challenge with M. tuberculosis Beijing/W, reducing CFU in the
lung and spleen by 1 2 logs. The vaccine was tested for safety in SCID mice; mice
administered the recombinant vaccine intravenously survived significantly longer
than mice administered the parental BCG vaccine. In a study in the guinea pig
model, the vaccine was not more potent than BCG [78].
9.2 rBCG/Cathepsin S
Sendide et al. [79] reported that IFNg-induced surface expression of mature MHC
class II molecules is suppressed in THP-1 macrophages infected with wild-type
BCG compared with macrophages incubated with killed BCG, that the suppression
is correlated with reduced cathepsin S activity, and that the reduced cathepsin
S activity is mediated via BCG-induced IL-10 secretion. This prompted Soualhine
et al. to engineer and evaluate a recombinant BCG secreting the mature form
of human cathepsin S [80]. The rBCG/hCathepsin S strain had increased
IFNg-induced surface MHC class II expression and increased expression of an
MHC class II–Antigen 85B peptide complex. Whether a recombinant vaccine
secreting cathepsin S was capable of inducing improved protective immunity in
an animal model was not investigated.

and Escaping the Phagosome
Sun et al. [81] engineered a novel recombinant BCG vaccine that combined the
approach of overexpressing native antigens, first employed by Horwitz et al. [10],
with the approach of phagosome escape, first employed by Grode et al. [77]. To
expand the antigenic repertoire of the vaccine, the investigators engineered it to
overexpress, in addition to Antigen 85B (the antigen overexpressed in rBCG30,
described above), Antigen 85A and TB10.4, a low molecular mass protein in the
ESAT-6 family. Instead of using listeriolysin to promote phagosome membrane
lysis, these investigators used a mutant form of perfringolysin O. The vaccine,
designated AFRO-1, was safer than BCG in SCID mice. Vaccination of mice and
guinea pigs with AFRO-1 induced stronger immune responses to the overexpressed
antigens than vaccination with the parental BCG vaccine. In mice vaccinated
before aerosol challenge with the hypervirulent M. tuberculosis strain HN878
(Beijing-type clinical outbreak strain), the group vaccinated with AFRO-1 survived
significantly longer than mice vaccinated with the parental BCG vaccine.
11 Recombinant BCG Expressing Foreign Antigens
With the development of techniques to genetically manipulate mycobacteria [82 85],

researchers rapidly looked to exploit recombinant BCG as a multi-component vac-
cine vector by expressing foreign antigens from various pathogens [4 9, 86 89]. As
noted above, BCG possesses a number of potential advantages as a vaccine (Table 1):
it has been extensively used for many decades, it has a very good safety profile, and it
is inexpensive to produce [4, 90]. BCG is unaffected by maternal antibodies, so it can
be given in a single dose at birth, and it is capable of inducing a long-lasting cellular
immune response. Due to its intracellular location in the phagosome of macrophages
and dendritic cells, BCG primarily elicits a CD4þ cellular immune response through
MHC class II. However, BCG also elicits a humoral immune response, and potent
antibody responses to foreign antigens expressed by recombinant BCG have been

obtained in some cases (see Tables 2 6). Recombinant BCG expressing foreign
antigens can also elicit a CD8þ cytotoxic T-lymphocyte response through MHC
class I. This feature of recombinant BCG is particularly important for protection
against viral pathogens and those bacterial pathogens that invade the cytoplasm of the
host cell. Presentation of antigen through MHC class I may occur via cross-priming.
Recombinant BCG targeting viral (Table 2), parasitic (Table 3), and bacterial
(Table 4) diseases have been developed. Several studies have examined recombi-
nant BCG vaccines expressing toxins to enhance the adjuvant effect of BCG
(Table 5) and recombinant BCG vaccines against cancer and allergy as well
(Table 6). Despite the promise of recombinant BCG vaccines, results have been
decidedly mixed. Good to excellent immune responses and/or protection have been
achieved in a number of studies, but many studies have also demonstrated weak to
moderate immune responses and no protection or modest protective efficacy at best.
Many studies have used high doses (greater than the human dose of ~106 CFU
administered intradermally) and/or multiple doses of recombinant BCG in an
attempt to obtain better immune responses. Heterologous boosting of recombinant
BCG has been effective in generating a more potent immune response in several
studies [91, 96, 114]. While a boosting regimen that yields an effective vaccine has
benefit, it does negate one purported advantage of being able to give recombinant
BCG in a single dose at birth. The type of immune response that is obtained with
any particular rBCG vaccine cannot be predicted, and it is difficult to draw many
generalizations from the numerous immunological studies of rBCG vaccines due to
the large number of variables among studies (animal model, route of vaccination,
dose and timing of vaccine, method of growth and preparation of vaccine, vaccine
viability, expression level and cellular location of the foreign antigen, stability of
foreign antigen expression, etc.) [173 176]. However, the expression level of
the foreign antigen and its cellular location (intracellular, secreted, or membrane
anchored) have had profound effects on immune responses generated by recombi-
nant BCG vaccines in some studies. In general, higher expression levels and
targeting of the foreign antigen to the membrane or extracellular space have yielded
more potent vaccines [173, 175]. To achieve high expression of a foreign antigen,
episomal plasmids with strong promoters have typically been required. Unfortu-
nately, this has been associated with instability of expression of the foreign antigen
in more than a few studies. Integrated vectors are more stable but result in less
expression and often lower immune responses. High-level and stable expression
of foreign antigens in recombinant BCG have been rather difficult to achieve in
practice.
11.1 HIV
rBCG expressing HIV and SIV antigens were among the first rBCG multi-component
vaccines constructed. rBCG expressing Gag, Nef, Env, Pol, and RT, as com-
plete genes or as smaller fragments, have been developed and tested for their
Table 2 Viral diseases targeted by recombinant BCG vaccines
142
Disease (organism) Animal model Immunogenicity Protective efficacy References

Antigen
HIV and SIV
Gag, Pol, Env (HIV) BALB/c mice Weak Ab to Gag and Env [8]
IFNg secretion (SP) and CD8þ CTL (SP)
to Gag [only Gag tested]
Gag, Pol, Env, RT (HIV) BALB/c mice Weak Ab; CD8þ CTL (SP) [only Env [4, 6, 7]
tested]
HIVA (HIV) BALB/c mice No CD8þ T-cell response alone, but Protection against [91]
[synthetic construct containing ~73% a good response obtained after surrogate recombinant
of Gag fused to a multi-CTL heterologous boost Vaccinia virus
epitope from the Gag, Pol, Nef, challenge
and Env proteins]
Gag, Pol, Env, Nef (SIV) Rhesus macaques IgA and IgG secreted by PBMC; CTL [92]
(cocktail) (PBMC)
Gag, Env, Nef (SIV) BALB/c mice Mucosal IgA, serum IgG; CTL (SP) [93]
(cocktail)
Gag, Env, Nef (SIV) Cynomolgus macaques IFNg secretion and CTL (PBMC) No protection against rectal [94]
(cocktail) challenge
Gag, Nef (SIV) BALB/c mice Serum IgG; IFNg secretion (CD4þ [95]
(bivalent strain) T-lymphocytes, spleen)
Gag, Pol, Env (SIV) Rhesus macaques Weak IFNg ELISPOT and tetramer [96]
(cocktail) binding (PBMC) which could be
significantly enhanced with
heterologous boosting
Gag (HIV) BALB/c mice High titer Ab induced in 3 of 7 mice [97]
[p17gag B-cell epitope (aa 92–110)
fused to M kansasii a-antigen]
Gag (HIV) Chacma baboons No Ab; no or weak IFNg ELISPOT [98]
(PBMC) which could be significantly
enhanced with heterologous boosting
M.V. Tullius and M.A. Horwitz
Gag (HIV) BALB/c mice 40-fold higher expression using codon [99]
optimization resulted in stronger
immune response; T-cell proliferation
and IFNg ELISPOT (SP); serum IgG
Gag (HIV) BALB/c mice Heterologous prime-boost resulted in [100]
prolonged CTL (SP)
Gag (HIV) BALB/c mice CTL and T-cell proliferation (SP); no [101]
serum Ab
Gag (SIV) Rhesus macaques CD8þ CTL (PBL) [87]
Gag (SIV) Rhesus macaques Heterologous prime-boost; good CTL No protection against cell- [102]
(PBL); no Ab free intravenous SIV

challenge
Gag (SIV) Cynomolgus macaques Heterologous prime-boost; IFNg Protection against mucosal [103]
ELISPOT (PBMC) challenge with SHIV
Gag (SIV) Guinea pigs, Hartley strain Long lasting, very high titer serum IgG; [104]
IFNg mRNA elevated (PBMC)
Gag (SIV) Guinea pigs, Hartley strain DTH; T-cell proliferation (PBMC); IFNg [105]
mRNA elevated (PBMC, SP);
very high titer serum IgG
Env (HIV) BALB/c mice CD8+ CTL (SP) [106]
[15 aa V3 CTL epitope fused to
M kansasii a-antigen]
Env (HIV) BALB/c mice and Guinea DTH; CTL (SP); moderate serum Ab; [107]
[19 aa V3 CTL epitope fused to pigs, Hartley strain Neutralizing Ab
Env (HIV) C57BL/6J mice serum IgG; Neutralizing Ab; IFNg and [108]
[19 aa V3 CTL epitope fused to IL-2 secretion by CD4þ T-cells
Env (HIV) Guinea pigs, Hartley strain DTH; T-cell proliferation (PBMC, [109]
[19 aa V3 CTL epitope fused to SP, I-IEL)
(continued)
143
Table 2 (continued)
144

Antigen
Env (HIV) Guinea pigs, Hartley strain DTH; serum IgG and IgA; [110]
[19 aa V3 CTL epitope fused to Neutralizing Ab
Env (HIV) Rhesus macaques Serum Ab; Neutralizing Ab; weak IFNg No protection against [111]
[19 aa V3 CTL epitope fused to ELISPOT (PBMC) pathogenic SHIV
Env (HIV) BALB/c mice T-cell proliferation (SP); serum IgG [112]
[15 aa V3 CTL epitope (PND) fused
to M tuberculosis chaperonin-10]
Env (HIV) Guinea pigs Moderate serum IgG; Neutralizing Ab [113]
[11 or 12 aa V3 CTL epitope fused
to M kansasii a-antigen]
Env (HIV) BALB/c mice IFNg ELISPOT (SP, lung, FRT); No [114]
serum Ab although could be elicited
with heterologous boost
Env (SIV) BALB/c mice and Guinea CD8+ CTL (LN); serum IgG; fecal IgA; [115]
pigs Neutralizing Ab
Nef (HIV) BALB/c mice T-cell proliferation (LN) [9]
Nef (HIV) BALB/c mice T-cell proliferation (LN); No serum Ab [116]
Nef (SIV) BALB/c mice T-cell proliferation and CD8þ CTL (LN) [117]
Nef (SIV) BALB/c mice T-cell proliferation (PGLN, MLN, CLN, [118]
SP); IFNg and TNFa ELISPOT (PP,
I-IEL, PGLN, MLN, SP); CD8þ CTL
(I-IEL, MLN, SP)
Measles virus
Nucleocapsid (N) protein (measles virus) C3H/He mice Moderate serum Ab but only when rBCG Partial protection [119]
given twice; T-cell proliferation (SP);
low levels of Neutralizing Ab after
infection
Nucleocapsid (N) protein (measles virus) Rhesus macaques CTL and T-cell proliferation (PBMC); no Partial protection [120]
serum IgG
Human papillomavirus (HPV)
HPV type 6b late protein L1, HPV type C57BL/6J or BALB/c mice DTH; serum Ab (weak but could be No protection against [121]
16 early protein E7 boosted); T-cell proliferation and tumor challenge
low CTL (SP)
Cottontail rabbit papillomavirus (CRPV)
L1 (major capsid protein) Outbred New Zealand Serum Ab; neutralizing Ab Good protection [122]
white rabbits
L2, E2, E7 or L2E7E2 fusion Outbred New Zealand Therapeutic vaccine: 71% [123]
white rabbits of papilloma sites had
complete regression
with rBCG expressing

L2E7E2 fusion protein
Porcine reproductive and respiratory
syndrome virus (PRRSV)
GP5, M BALB/c mice Serum Ab; neutralizing Ab; IFNg [124]
(cocktail) secretion (SP)
GP5, M Crossbreed F1 (Landrace Serum Ab, neutralizing Ab in 3 of 5 pigs; Partial protection [125]
(cocktail) Large White) pigs negative IFNg ELISPOT (PBMC)
Hepatitis B virus (HBV)
HBsAg-Middle S, HBsAg-Large S BALB/c mice Serum Ab [126]
Hepatitis C virus (HCV)
NS5a (nonstructure protein 5a) BALB/c C3H/HeN CD8þ CTL (SP); increased IFNg, IL-2, Good protection against [127]
[12 aa CTL epitope of HCV-nonstructure (CC3HF1) mice and IL-12 mRNA (LN) surrogate recombinant
protein 5a (NS5a) as a chimeric protein vaccinia virus
with a-antigen of M kansasii] challenge
CtEm HHD-2 mice Serum Ab; T-cell proliferation, IFNg Good protection against [128]
[multi-epitope antigen, including the (transgenic for HLA-A2.1) and IL-2 secretion, and CTL (SP) surrogate recombinant
major epitope domains of the truncated vaccinia virus
core, mimotopes from E2, and six challenge
HLA-A2–restricted CTL epitopes
from NS3, NS4 and NS5]
145
(continued)
146
Table 2 (continued)
Antigen
Rotavirus
VP6 BALB/c mice, Guinea No serum Ab Partial protection [129]
pigs, Rabbits
Respiratory syncytial virus (RSV)
N, M2 BALB/c mice No serum IgG; IFNg and IL-2 secretion Very good protection [130]
and increased CD69þ T-cells (SP)
Encephalomyocarditis virus (EMCV)
D-variant (Human type I diabetes
model)
VP1 (major outer capsid protein) SJL/J mice, inbred Serum IgG; Neutralizing Ab; DTH; Long-lasting (>10 months) [131]
Guinea pigs T-cell proliferation (SP) protective immunity,
very good protection
(required at least 6
weeks for full
immunity)
Rabies virus
N (nucleoprotein) BALB/c mice Serum IgG [132]
[B-cell and T-cell epitopes fused to
M leprae 18-kDa protein]
Abbreviations: aa amino acid, Ab antibody, CLN cervical lymph nodes, CTL cytotoxic T-lymphocytes, DTH delayed-type hypersensitivity, ELISPOT
enzyme-linked immunosorbent spot, FRT female reproductive tract, I-IEL intestinal intraepithelial lymphocytes, LN lymph nodes, MLN mesenteric lymph
nodes, PBL peripheral blood lymphocytes, PBMC peripheral blood mononuclear cells, PGLN periglandular lymph nodes, PP Peyer’s patches, SP splenocytes
Table 3 Parasitic diseases targeted by recombinant BCG vaccines
Antigen
Malaria (Plasmodium spp.)
CSP (circumsporozoite protein BALB/c and No serum Ab; no IFNg secretion or T-cell [89]
of Plasmodium falciparum) BALB/k proliferation (SP) in response to CSP
mice Th2R peptide (CD4 þ T-lymphocyte
epitope)
CSP (circumsporozoite protein BALB/c mice Serum IgG; T-cell proliferation and IFNg [133]
of Plasmodium falciparum) and IL-2 secretion (SP)
CSP (circumsporozoite protein BALB/c mice Serum Ab in only one of seven mice [134]
of Plasmodium yoelii)
[B-cell epitope of CSP fused to
MSP-1 (merozoite surface protein 1 C3H/He mice IFNg secretion (SP); no serum Ab Very good protection against challenge [135]
from Plasmodium yoelii) with 104 P. yoelii 17XL-parasitized
[15 kDa C-terminal region of MSP-1 erythrocytes
fused to M kansasii a-antigen]
MSP-1 (merozoite surface protein 1 C3H/He mice Long-lasting (9 month) protection [136]
from Plasmodium yoelii) against intraperitoneal challenge
[15 kDa C-terminal region of MSP-1 with 104 P yoelii 17XL-parasitized
fused to M kansasii a-antigen] erythrocytes but protection was
substantially less than at 1 month
postvaccination
MSA2 (merozoite surface antigen BALB/c mice Serum IgG; T-cell proliferation and IFNg [137]
2 from Plasmodium falciparum) and IL-2 secretion (SP)
F2R(II)EBA (fragment 2 region II BALB/c mice No expression data presented; serum IgG; [138]
of EBA-175), (NANP)3, as well T-cell proliferation (SP); increased
as two T-cell epitopes of the splenocyte CD4þ IFNgþ, CD4þ
M tuberculosis ESAT-6 antigen IL-2þ, CD4þ IL-4þ cells in response
to (NANP)3; increased splenocyte
CD4þ IL-4þ cells in response to F2R
(II)EBA; immune responses also
147
obtained against M tuberculosis

ESAT-6 epitopes
(continued)
Table 3 (continued)
148

Antigen
Leishmaniasis (Leishmania spp.)
gp63 (Leishmania major surface BALB/c and Good protection against L mexicana [88]
proteinase) CBA/J mice promastigotes and amastigotes, poor
protection against L major
promastigotes in BALB/c mice
gp63 (Leishmania major surface BALB/c and Partial protection against a challenge with [139]
proteinase) C57BL/6 L major amastigotes in BALB/c-mice
mice
LCR1 (antigen cloned from BALB/c mice Weak protective immunity against L [140]
amastigote L chagasi library) chagasi infection
Schistosomiasis (Schistosoma spp.)
Glutathione S-transferase BALB/c, T-cell proliferation (LN but not SP) [141]
(Schistosoma mansoni, C57BL/6, [BALB/c and C57BL/6 mice]
Sm28GST) and C3H/HeJ
mice
Glutathione S-transferase BALB/c mice High titer and long-lasting (1 year) serum [142]
(Schistosoma mansoni, IgG; Neutralizing Ab
Sm28GST)
Glutathione S-transferase BALB/c mice High titer serum IgG; serum and BALF [143]
(Schistosoma haematobium, IgA; Neutralizing Ab
Sh28GST)
Glutathione S-transferase BALB/c mice T-cell proliferation and IFNg secretion [144]
(Schistosoma japonicum, (SP)
Sj26GST)
Sm14 (from Schistosoma mansoni) BALB/c and No serum Ab; IFNg secretion (SP) Partial protection against subcutaneous [145]
outbred challenge with 100 S mansoni
Swiss mice cercaria; not able to boost protection
with rSm14 protein or rBCG
Sm14 (from Schistosoma mansoni, BALB/c and No serum Ab; IFNg secretion (SP); codon Partial protection against subcutaneous [146]
codon optimized) outbred optimization increased expression challenge with 100 S mansoni
Swiss mice level but did not improve immune cercaria; not able to boost protection
response with rSm14 protein
Toxoplasmosis (Toxoplasma gondii)
GRA1 (a major secreted antigen) OF1 outbred Mice: no serum Ab; no DTH Mice: very weak protection against oral [147]
mice, Suffolk Sheep: no serum Ab; T-cell proliferation challenge
crossbred and IFNg secretion (PBMC) Sheep: shorter duration of pyrexia
sheep following oral challenge
ROP2 (rhoptry protein 2) BALB/c mice Serum Ab; IFNg and IL-2 secretion (SP) Slightly increased survival after [148]
intraperitoneal challenge
Coccidiosis (Eimeria tenella)
Rho (rhomboid gene) Chickens Serum IgG Partial protection [149]
Abbreviations: Ab antibody, BALF bronchial alveolar lavage fluid, DTH delayed-type hypersensitivity, LN lymph nodes, PBMC peripheral blood mononuclear
cells, SP splenocytes
149
Table 4 Bacterial diseases targeted by recombinant BCG vaccines
150

Antigen
Lyme disease (Borrelia burgdorferi)
OspA (outer surface protein A) BALB/c, C3H/HeJ, and High titer serum IgG Complete protection [5]
outbred Swiss Webster
mice
OspA (outer surface protein A) BALB/c mice Prolonged systemic IgG and Complete protection [150]
mucosal IgA
OspA (outer surface protein A) Human phase I clinical trial None of the 24 volunteers [151]
developed anti-OspA Ab
OspA (outer surface protein A) White-tailed deer Serum Ab [152]
Bacterial pneumonia, otitis media,
meningitis (Streptococcus
pneumoniae)
PspA (Pneumococcal surface protein A) BALB/c, C3H/HeJ, and High titer serum Ab 50–100% protection against [153]
CBA/N (Xid) mice intraperitoneal challenge
Leptospirosis (Leptospira spp.)

LipL32 (Leptospira interrogans external Golden Syrian hamsters Serum IgG Partial protection against [154]
membrane protein) intraperitoneal challenge
with L interrogans
LipL32 BALB/c mice Serum Ab [155]
Listeriosis (Listeria monocytogenes)
p60 (major secreted antigen) BALB/c mice CD8þ T-cells (SP) Complete protection with rBCG [156]
expressing membrane-
anchored p60
DPT (Diphtheria–Pertussis–Tetanus)
S1-TTC (hybrid protein of S1 subunit BALB/c mice High titer serum anti-TTC IgG [157]
of pertussis toxin fused to fragment but no anti-S1 IgG; toxin
C of tetanus toxin) neutralizing Ab; IL-2
secretion (SP)
S1 subunit of pertussis toxin BALB/c mice Long-term serum IgG (up to [158]
8 months) and memory
response (15 months)
S1 subunit of pertussis toxin (genetically BALB/c and outbred Swiss Weak serum Ab; IFNg secretion Good protection against [159]
detoxified) mice and T-cell proliferation (SP) intracerebral Bordetella
pertussis challenge
S1 subunit of pertussis toxin (genetically Outbred Swiss mice No serum Ab; IFNg secretion Good protection against [160]
detoxified) (neonates) (SP) intracerebral Bordetella
pertussis challenge
CRM197 (mutated nontoxic derivative BALB/c mice Weak serum Ab [161]
of diphtheria toxin) (nonneutralizing); rBCG

capable of priming a humoral
response to DT vaccine
FC (tetanus toxin fragment C), CRM197 BALB/c and outbred NIH Mice: weak serum anti-FC IgG; 75% protection against a [162]
(mutated nontoxic derivative of mice, Guinea pigs rBCG capable of priming a challenge with 100 minimum
diphtheria toxin) (cocktail) humoral response to DT lethal doses of tetanus toxin
vaccine guinea pigs:
Neutralizing Ab against
tetanus toxin and diphtheria
toxin
FC (tetanus toxin fragment C, ToxC) Outbred NIH Swiss mice Serum Ab [4]
FC (tetanus toxin fragment C, ToxC) Outbred NIH Swiss mice Serum Ab Partial to complete protection [7]
against a challenge with 100
minimum lethal doses of
tetanus toxin
Bovine anaplasmosis (Anaplasma
marginale)
MSP1a BALB/c mice Weak serum Ab; IFNg secretion [163]
(SP)
Abbreviations: Ab antibody, SP splenocytes
151
Table 5 Bacterial proteins used to enhance the adjuvant effect of recombinant BCG vaccines
Antigen Animal model Immunogenicity References
CTB (cholera toxin B subunit) BALB/c mice Increased IgA and TGF b1in [164]
bronchial alveolar lavage
fluid
LTB (B subunit of E. coli heat BALB/c mice Serum IgG and IgA; oral rBCG [165]
labile enterotoxin) also induced mucosal IgA
LTB (B subunit of E. coli heat BALB/c mice LTB used for adjuvant effect; [166]
labile enterotoxin) fused to serum anti R1 IgG and
R1 repeat region of P97 IgA (greater response to
adhesion from Mycoplasma LTB R1 fusion than to
hyopneumoniae R1 alone)
immunological properties primarily in mice and guinea pigs, although several of

these vaccines have been tested in nonhuman primate models as well (Table 2).
Developing a vaccine against HIV has proven extremely challenging [177, 178] and
rBCG HIV vaccines are no exception. It is generally agreed that both broadly
neutralizing antibodies and antiviral cytotoxic T-lymphocytes are needed for a
highly effective vaccine, although CD4þ T cells also have a role in mediating
these effects. Due to the great difficulty in generating broadly neutralizing anti-
bodies, recent HIV vaccine research has focused more on T-cell vaccines. As
rBCG HIV vaccines have been reviewed several years ago [173], this section
will focus on some of the most recent studies.
11.1.1 Recent Studies
Cayabyab et al. constructed rBCG strains expressing SIV Gag, Pol, and Env
localized to the mycobacterial cell wall with the 19 kDa lipoprotein signal under
the control of the a-antigen promoter [96]. Rhesus macaques were vaccinated
intradermally or intravenously with 106 109 CFU rBCG (given as a cocktail of
all three strains) and boosted with an identical dose 23 weeks later. Twenty weeks
after the second immunization, all the monkeys were boosted with 1010 virus
particles of recombinant adenovirus 5 expressing the same SIV antigens as the
rBCG strains. The monkeys developed very weak CD8þ T-cell responses to the
SIV antigens even with two doses of rBCG. However, after the heterologous prime
boost with adenovirus, the monkeys developed strong responses to all three SIV
antigens, as measured by PBMC IFNg ELISPOT responses to Gag, Pol, and Env
peptide pools. The responses to Gag and Pol were greater for the rBCG immunized
animals compared with naı̈ve animals, but a similar response was obtained for Env
in rBCG immunized and naı̈ve animals, which was attributed to instability of
expression of Env by the rBCG vaccine.
Promkhatkaew et al. constructed an rBCG expressing HIV Gag intracellularly
from the strong hsp60 promoter (0.26 0.45 mg/L of culture) [101]. Expression was
reported to be stable. BALB/c mice were vaccinated subcutaneously with 0.1 mg
(2 106 CFU) and cell-mediated immune responses were measured 2 weeks to
Table 6 Cancer and allergic disease targeted by recombinant BCG vaccines
Antigen Animal model Immunogenicity Protective efficacy References
OVA C57BL/6 mice IFNg-secreting CD8+ T cells and Significant protection against [167]
CTL (SP) challenge with OVA-
expressing tumor cells
(B16-OVA)
OVA (SIINFEKL epitope) C57BL/6 and TAP1-/- mice Weak CTL Partial protection against [168]
challenge with
OVA-expressing tumor
cells (B16-OVA)
MUC1 (22 variable-number tandem SCID mice reconstituted IFNg secretion; low-level serum [169]
repeats) þ mIL-2 with 107 human PBL IgG and IgM; rBCG secretes
functional IL-2 but effect of
cytokine is unclear as rBCG
expressing MUC1 alone was
not tested
MUC1 (1, 4, or 8 variable-number SCID mice reconstituted Increased IFNg ELISPOTand CTL; Partial protection against [170]
tandem repeats) þ hGM-CSF with 5 107 human no data on whether coexpressed tumor challenge
PBL hGM-CSF was functional
S1 subunit of pertussis toxin C57BL/6 mice Increased TNFa mRNA (qPCR) Reduction of bladder [171]
(genetically detoxified) tumor volume
Der p I (a major allergen from house C57BL/6J and BALB/b IFNg secretion (SP) [172]
dust mites, immunodominant mice
peptide containing T- and B-cell
epitopes)
Abbreviations: CTL cytotoxic T-lymphocytes, ELISPOT enzyme-linked immunosorbent spot, PBL peripheral blood lymphocytes, SP splenocytes
153
2 months later. Gag-specific CTL to multiple epitopes as well as lymphocyte

proliferation was induced in response to vaccination, but no anti-Gag antibodies
were detected in sera. In a follow-up study, mice were boosted 1 month or 6 months
after subcutaneous or intradermal rBCG vaccination with a replication-deficient
vaccinia virus strain also expressing full-length Gag and cell-mediated immune
responses were measured 1 month later [100]. The prime-boost regimen resulted in
a more persistent CTL response than the single vaccination with rBCG.
Chege et al. tested two rBCG vaccines expressing HIV Gag localized to the
mycobacterial cell wall with the 19 kDa lipoprotein signal for immunogenicity in
a baboon model using a prime-boost regimen [98]. Both humoral and cellular
Gag-specific immune responses to rBCG alone were very weak, but rBCG succeeded
in priming the immune system for a Gag VLP boost (assessed by assaying the IFNg
ELISPOT response to Gag peptides and Gag-specific antibody). rBCG was adminis-
tered at very high doses (108 CFU at 0, 14, 24, and 40 weeks) and boosted twice with
Gag VLPs at 92 and 104 weeks. The exact expression level of Gag by the rBCG
strains was not reported, but the strain with higher expression, which produced better
results, was reported to be less stable. Low expression and/or instability of the rBCG
vaccines may have resulted in a poor immune response in this study.
Kawahara and colleagues examined the long-term immune response of rBCG
expressing full-length SIV Gag in guinea pigs [104, 105]. Gag was expressed intra-
cellularly under the control of a strong promoter (Phsp60) at 0.5 ng Gag/mg of rBCG.
Guinea pigs were immunized intradermally (0.1 mg) or orally (80 mg 2) using
typical human doses. A strong immune response was achieved from both routes of
immunization for up to 3 years as evidenced by: Gag-specific serum IgG 106-fold
greater than control (IgG2 > IgG1), DTH, proliferation of PBMC and splenocytes
in response to Gag, and increased IFNg mRNA in PBMC and splenocytes in response
to Gag, mediated largely through CD4þ T-cells. The high levels of antibody
produced contrasted with the authors’ previous work with an rBCG secreting a 19
amino acid CTL epitope from Env fused to M. kansasii a-antigen [110]. Intrader-
mal vaccination of guinea pigs with 0.1 mg of this strain resulted in no antibody
production, although other routes of immunization did elicit an antibody response.
Im et al. constructed an rBCG expressing the HIVA immunogen localized to the
mycobacterial cell wall with the 19 kDa lipoprotein signal under the control of the
a-antigen promoter [91]. HIVA is a synthetic gene containing ~73% of gag fused to
a multi-CTL epitope from the Gag, Pol, Nef, and Env proteins [179]. The investi-
gators obtained greater expression with an extrachromosomal plasmid compared
with an integrated plasmid and used the higher expressing strain for all of their
animal studies. A balanced lethal system was used to apply selective pressure on
the rBCG strain to maintain the plasmid (a BCG DlysA lysine auxotroph was
complemented with a functional copy of lysA on the plasmid). This strategy was
quite successful in maintaining the plasmid as 10 of 10 colonies isolated from the
spleens of mice 15 weeks after immunization still maintained the kanamycin
resistance marker on the plasmid and 2 of 2 were positive for the HIVA gene by
PCR. Unfortunately, the authors did not go one step further and check the isolated
colonies for continued expression of HIVA. BALB/c mice were vaccinated
intraperitoneally with 106 CFU of rBCG for most experiments and 103 107 CFU for
a dose-titration study. BCG HIVA induced little or no CD8þ T-cell responses
alone, but enabled enhanced responses when used to prime a subsequent boost with
MVA HIVA (given at 102 d). In a dose response experiment, a high priming dose
of BCG HIVA was determined to be important for eliciting a broader
T-cell response. Mice that were primed with a DNA HIVA vaccine, boosted with
BCG HIVA, and challenged with a surrogate replication-competent vaccinia virus
expressing HIVA had significantly increased levels of bifunctional CD4þ T-cells.
Yu et al. constructed rBCG vaccines expressing HIV-1 Env as a surface,
intracellular, or secreted protein [114]. For surface expression, the 19 kDa lipopro-
tein signal was used and for secretion, the a-antigen signal was used. All constructs
were under the control of the a-antigen promoter, a moderately strong mycobacte-
rial promoter. In an attempt to address the very difficult challenge of HIV genetic
diversity, the authors used two artificial consensus env genes (CON6 gp120 or
CON6 gp140CF). BALB/c mice were vaccinated twice (at 0 and 8 weeks) intra-
peritoneally with 106, 107, or 108 CFU of the different rBCG strains, and antigen-
specific T-cell responses were measured by IFNg ELISPOT assays on lymphocytes
isolated from spleens. Vaccines secreting Env yielded the strongest response.
Interestingly, little or no response was obtained with a single dose of rBCG
demonstrating a clear boosting effect of rBCG in this assay. Elevated Env-specific
T-cell responses were also obtained with lymphocytes isolated from the lung and
female reproductive tract after two doses of rBCG. The antigen-specific T-cell
response was primarily due to CD4þ T-cells. rBCG did not elicit anti-Env anti-
bodies on its own, but did prime an antibody response when followed by a boost of
recombinant HIV-1 Env oligomer in RiBi adjuvant.
11.2 Other Viral Diseases
Many other viruses besides HIV have been targeted by recombinant BCG vaccines
(Table 2). Very good protective efficacy has been achieved against respiratory
syncytial virus (RSV) and encephalomyocarditis virus (EMCV) [130, 131]. Good
protection against cottontail rabbit papillomavirus (CRPV) has been achieved
[122, 123], and immune responses to hepatitis C virus (HCV) have provided
good protection against a surrogate challenge with recombinant vaccinia virus
expressing an HCV antigen [127, 128].
11.3 Parasitic Diseases
Nearly 20 studies on recombinant BCG vaccines targeting malaria, leishmaniasis,

schistosomiasis, and toxoplasmosis are cataloged in Table 3. Several studies that
examined protective efficacy of the rBCG vaccines will be highlighted here.
Matsumoto et al. obtained very good protection against rodent malaria (P. yoelii)
with a recombinant BCG secreting the 15 kDa C-terminal region of merozoite

surface protein 1 (MSP1) fused to the M. kansasii a-antigen (six of seven rBCG
vaccinated mice survived versus 0% survival for controls) [135]. Protection was
significantly better than that achieved with recombinant MSP1 protein with adju-
vant. C3H/He mice were immunized intravenously with 106 CFU rBCG, boosted
1 month later intraperitoneally with 106 CFU rBCG, and challenged 1 month after
the second immunization with P. yoelii. As neither route can be used to immunize
humans and the route of immunization is known to affect the immune response to
rBCG in small animals, the results should be interpreted cautiously. In a follow-up
study, the authors found that protective immunity had waned significantly by 4 and
9 months postimmunization (only 4 of 9 and 3 of 9 mice survived at 4 and 9 months,
respectively), but still was better than after immunization with recombinant MSP1
protein with adjuvant [136].
Connell et al. constructed an rBCG expressing the Leishmania major gp63 surface
proteinase intracellularly under the control of a strong promoter (Phsp60) [88]. BALB/
c and CBA/J mice were vaccinated intravenously (104 or 105 CFU) or subcutaneously
(106 CFU) and challenged 10 weeks later. Good protection from cutaneous leishman-
iasis was obtained when the mice were challenged with L. mexicana (promastigotes
and amastigotes) but not against L. major promastigotes. Abdelhak et al. also
developed rBCG strains expressing L. major gp63 [139]. Mice were vaccinated
twice at a 1-month interval (106 CFU intravenously; 107 CFU subcutaneously) and
challenged with L. major amastigotes 1 month after the boost. Partial protection was
observed with the rBCG expressing gp63 fused to the N-terminal portion of b-
lactamase (a secreted protein) but not with the rBCG expressing gp63 intracellularly.
rBCG expressing the Sm14 antigen from Schistosoma mansoni have delivered
partial protection from challenge with S. mansoni cercaria in two studies [145, 146].
In the first study, Varaldo et al. expressed Sm14 fused with b-lactamase under the
control of a strong promoter (pBlaF*) with the fusion protein localized to the cell
wall [145]. A single dose of rBCG (106 CFU subcutaneously) was as effective as a
three-dose regimen of rSm14 protein in alum, with ~50% reduction in worm burden
in outbred Swiss mice. Protective efficacy could not be boosted by a second dose of
rBCG or by rSm14 protein. In a follow-up study, Varaldo et al. constructed a
mycobacterial codon-optimized Sm14 gene and obtained fourfold greater expres-
sion with rBCG expressing the codon-optimized gene compared with their earlier
construct [146]. However, this did not translate into an increased immune response
(IFNg secretion by splenocytes stimulated in vitro with rSm14) or protective
efficacy. This contrasts with an earlier report in which an rBCG strain expressing
codon-optimized HIV Gag was more immunogenic than an rBCG expressing wild-
type Gag (~40-fold increase in expression for the codon-optimized Gag) [99].
11.4 Bacterial Diseases
Some of the earliest rBCG vaccines targeting bacterial pathogens produced very
promising results [5, 150, 153]. Stover et al. developed an rBCG vaccine against
Lyme borreliosis by expressing the outer surface protein A (OspA) of Borrelia

burgdorferi as a membrane-anchored lipoprotein [5]. Using this construct, they
obtained protective antibody responses in inbred and outbred mouse strains that
were 100 1,000 times greater than those obtained with rBCG strains expressing
OspA intracellularly or as a secreted protein. In protection experiments, inbred and
outbred mouse strains were immunized intraperitoneally with 106 CFU rBCG,
boosted 17 weeks later with an identical dose, and challenged intraperitoneally or
intradermally 5 weeks after the booster dose. Excellent protective efficacy was
obtained against both challenge routes. Likewise, a single intranasal dose of this
rBCG strain provided complete protection against intradermal challenge 13 weeks
postvaccination [150]. This group also obtained very good humoral immune
responses in mice immunized with rBCG strains expressing pneumococcal surface
protein A (PspA) [153]. Interestingly, protective immunity was only induced in
mice vaccinated with rBCG secreting PspA or expressing PspA as a membrane-
anchored lipoprotein. Despite inducing a good humoral immune response, no
protective efficacy was obtained with rBCG expressing intracellular PspA. The
rBCG vaccine against Lyme disease was eventually tested for safety and immuno-
genicity in the first phase I clinical trial of an rBCG vaccine (and still the only
human trial of an rBCG vaccine expressing a foreign antigen) [151]. Unfortunately,
in stark contrast to the results obtained in mice, none of the 24 human volunteers
vaccinated intradermally with 2 104 2 107 CFU rBCG developed a humoral
immune response.
Similar to the studies with OspA and PspA above, Grode et al. constructed rBCG
vaccines expressing secreted, membrane anchored, and intracellular Listeria mono-
cytogenes p60 (a major secreted antigen) under the control of a strong promoter
(Phsp60) [156]. BALB/c mice were vaccinated intravenously with 106 CFU of rBCG
and challenged 120 days later. Excellent protective efficacy (80 100% survival at
10 days postchallenge) was obtained with the rBCG strains expressing membrane
anchored or secreted p60, but not with the rBCG strain expressing intracellular p60.
Interestingly, only CD4þ T-cells were needed for protective efficacy in mice
immunized with rBCG secreting p60, but both CD4þ and CD8þ T-cells were
required in mice immunized with rBCG expressing membrane-anchored p60. The
authors suggest that this could be due to decreased access of membrane-anchored
p60 to the MHC class II loading compartment leading to less stimulation of CD4þ
T-cells.
Recombinant BCG vaccines expressing bacterial toxins have produced good
protection in several studies as well [7, 159, 160, 162].
12 Conclusions
As a vaccine vector for a recombinant TB vaccine, BCG is an obvious choice since

it shares so many antigens with M. tuberculosis, and it has efficacy by itself. The
emphases of current efforts aimed at an improved TB vaccine are on expanding the
repertoire of overexpressed immunoprotective M. tuberculosis antigens and

improving the processing and presentation of both vector and recombinant antigens
by altering the intracellular lifestyle of the vector, endowing the vector with
immunomodulatory cytokines, enhancing apoptosis to promote cross-presentation
of antigens, etc. Some of these modifications also show promise for improving
BCG as a therapeutic against bladder cancer, the only other approved use of BCG
vaccine aside from the prevention of TB.
Increasingly, primarily because of its safety record and high immunogenicity,
BCG has been chosen as a vaccine vector to express foreign antigens, particularly
where no convenient or safe alternative vector homologous to the target exists, e.g.,
in the case of parasites, cancer, and allergic disease. Recombinant BCG expressing
HIV antigens are being intensely studied including in nonhuman primates. Some of
these vaccines have induced strong immune responses against key HIV antigens,
particularly when used as part of a heterologous prime-boost vaccination strategy.
Acknowledgments Work on TB vaccines in this laboratory is supported by Grants AI068413 and

AI031338 from the National Institutes of Health. The Phase 1 trial of rBCG30 was sponsored
by the Aeras Global TB Vaccine Foundation with funding from the Bill and Melinda Gates
Foundation.
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Part III
Manipulating Host-Pathogen Interactions
to Make Vaccines
Basic Science Paves the Way to Novel Safe
and Effective Pestivirus Vaccines
Norbert Tautz and Gregor Meyers
Abstract Pestiviruses are among the economically most important pathogens of

livestock. Except for culling, vaccination represents the only feasible way to control
pestiviruses. Therefore, a considerable number of pestivirus vaccines have been
developed and put on the market. However, these vaccines still have disadvantages
that should be eliminated in future approaches, some of which are based on recent
findings and will be outlined in this chapter. One of the most important features of
ruminant pestiviruses is their extraordinary tendency to establish lifelong persis-
tence as the outcome of intrauterine infection. As a result, 1 2% of cattle worldwide
are persistently infected with bovine viral diarrhea virus. The constant dissemina-
tion of the virus by these animals is central for maintenance of this pathogen in
its host population; therefore, future vaccines must address this highly relevant
problem. Elucidation of the molecular features of pestiviruses that are required for
the establishment and maintenance of persistent infection has made significant
progress, and the present knowledge on this topic is summarized in this chapter.
These features include a unique strategy to restrict virus genome replication by a
limiting host factor and viral virulence factors Npro and Erns interfering with the
innate immune response of the host. Accordingly, a framework of viral functions is
involved in the establishment and maintenance of persistence. On the basis of this
knowledge, specific mutations in the recently identified virulence factors have
resulted in the generation of attenuated viruses, building a perfect basis for future
vaccine design.
N. Tautz
Institute for Virology and Cell Biology, University of L€
ubeck, Ratzeburger Allee 160, 23562
L€ubeck, Germany
G. Meyers (*)
Institut f€ur Immunologie, Friedrich Loeffler Institut, Institute for Animal Health, Paul Ehrlich Str.
28, 72076 T€ubingen, Germany

174 N. Tautz and G. Meyers
1 Introduction
Pestiviruses cause economically important diseases of livestock [1]. Moreover,

because they are closely related to hepatitis C virus (HCV), pestiviruses are a
widely used surrogate system for this human pathogen. Classical swine fever
virus (CSFV), two types of bovine viral diarrhea virus (BVDV-1 and BVDV-2),
and border disease virus of sheep (BDV) belong to this group of viruses, and are
classified as one genus within the virus family Flaviviridae [2].
For pestiviruses, both cytopathic and noncytopathic viruses can be discriminated
in cell culture [3]. The biotype of the virus is very relevant, because only noncyto-
pathic viruses are able to establish persistent infections in their natural host.
Persistence is a key feature for maintenance of pestiviruses in their host population.
Persistent infections are achieved by diaplacental infection of fetuses in pregnant
animals. Fetal infection, depending on the time of gestation at which infection
occurs, can also lead to abortion or fetal malformation [4].
Killed and live attenuated vaccines against BVDV and CSFV are currently
commercially available. Both types of vaccines have distinctive disadvantages.
The established live virus vaccines were obtained via repeated cell culture passages
of field isolates. This leads to an unpredictable risk of reversion to virulence,
because the basis of attenuation of these viruses is undefined. Attempts to identify
the molecular markers for attenuation in the viral genomes have not provided final
conclusions [5 7]. Even more important than possible reversion to virulence is the
fact that currently available live pestivirus vaccines are not safe in pregnant animals
because they can be transmitted to the fetus and induce damage, trigger abortion, or
establish persistent infection, depending on the vaccine virus [8]. Therefore, despite
the outstanding efficacy of live pestivirus vaccines, their use is hampered by safety
concerns. As an alternative, a variety of nonlive vaccines have been developed
that contain killed viruses or heterologously expressed structural components of the
viruses (several such vaccines are commercially available). These vaccine formula-
tions are safe but considerably less efficient in inducing protective immunity in host
animals, especially when efficacy is measured with respect to prevention of dia-
placental infection of fetuses in pregnant animals. Sometimes a booster vaccination
is necessary to achieve protective immunity. In addition to standard two-step
immunization schemes using the same vaccine for both steps, prime boost regimes
utilizing a killed virus vaccine as the prime vaccination and a live virus vaccine for
booster immunization have been developed to achieve improved protection.
Based on this background, there is an obvious need for improved pestivirus
vaccines that are safe in young, adult, and, especially, pregnant animals. Vaccina-
tion should efficiently block transplacental transmission of field viruses to the fetus
in pregnant animals and should prevent development of clinical symptoms as well
as virus shedding in challenged animals. Recently obtained data on the biological
functions of pestiviruses have significantly improved our understanding of the
prerequisites for virus persistence and can be used for a rational design of attenu-
ated recombinant pestivirus vaccines.
Basic Science Paves the Way to Novel Safe and Effective Pestivirus Vaccines 175
2 Pestivirus Molecular Biology
Pestivirus particles are enveloped and have a diameter of 40 60 nm. The virions
contain four structural proteins. Within the virion, the viral positive-sense single-
stranded RNA genome is found. The genomic RNA, with a length of about 12.3 kb,
contains one long open reading frame (ORF), coding for all known viral proteins [9,
10]. In the genomic RNA the ORF is flanked by untranslated regions (UTRs), which
contain RNA sequences and structures with crucial importance for viral RNA
replication. The 50 UTR also contains a complex RNA structure, termed an “inter-
nal ribosome entry site” (IRES). The IRES mediates initiation of protein translation
in the absence of a 50 cap structure [11]. Protein expression occurs via translation of
a polyprotein that is co- and posttranslationally processed by viral and cellular
proteases into the mature virus proteins. Within the polyprotein, the individual gene
products are arranged in the order NH2 Npro/C/Erns/E1/E2/p7/NS2/NS3/NS4A/
NS4B/NS5A/NS5B COOH [1] (Fig. 1).
Capsid protein C and the glycoproteins Erns, E1, and E2 are structural compo-
nents of the enveloped pestivirus virion [12]. E2 and, to a lesser extent, Erns are
targets for antibody neutralization [13 17]. E2 interacts specifically with the sur-
face protein CD46, which functions as a pestivirus receptor. CD46 is necessary but
not sufficient to mediate infection [18, 19].
Erns is the second pestivirus protein that is accessible on the surface of virus
particles [14]. It interacts with carbohydrate structures on the surface of target cells
[20 22]. Erns lacks a typical transmembrane sequence or another known type of
membrane anchor and is secreted in considerable amounts from infected cells [23].
Recent analyses have shown that the C-terminal part of the protein functions as
a novel type of membrane anchor [24, 25]. Notably, Erns exhibits RNase activity
[26 28].
The nonstructural proteins include the autoprotease Npro (the first protein
encoded by the long ORF) and the seven proteins located in the polyprotein
Fig. 1 Genome organization of a pestivirus. The 50 and 30 untranslated regions are indicated by
black lines, and the single long open reading frame (ORF) is indicated by a box. The location of the
regions of the ORF coding for the individual viral proteins is indicated, together with the basic
organization of the genome into regions coding for structural and nonstructural proteins. Below the
ORF, the currently known functions of the encoded proteins are given. The processing scheme of
the polyprotein encoded by the ORF is indicated by arrows
downstream of E2. The small hydrophobic p7 is required for virion formation. In

the related HCV system it has been shown that the ortholog of p7 is a so-called
viroporin and forms an ion channel [29 32]. Host cell proteases resident in the
endoplasmic reticulum catalyze all cleavages required for the release of the viral
glycoproteins and p7, and virus encoded enzymes mediate processing of the
nonstructural proteins NS3 to NS5B. The generation of the N terminus of NS3 by
an autoprotease in NS2 is essential for viral RNA replication because NS3, but not
uncleaved NS2-3, is an essential component of the viral replicase [33, 34]. In
contrast, uncleaved NS2-3 is required for virion morphogenesis [34, 35]. NS3 is a
multifunctional protein with protease, helicase, and NTPase activities. To gain full
activity, the NS3 serine protease requires NS4A as a cofactor. This NS3/NS4A
protease complex generates the C terminus of NS3 and catalyzes all downstream
cleavages of the polyprotein [36 38]. No specific functions have been attributed to
NS4B and the Zn-binding phosphoprotein NS5A so far [39]. NS5B is an RNA-
dependent RNA polymerase, which catalyzes the replication of viral RNA, in
concert with viral proteins NS3, NS4A, NS4B, and NS5A and an unknown number
of host factors [40].
Cloning a complementary DNA copy of the entire viral genome into bacterial
plasmids allows the manipulation of the viral sequence and the generation of viral
genome copies in vitro. Upon transfection of these RNAs into cultured cells,
autonomous replication of the viral genome occurs, and infectious progeny are
released into the culture supernatant [41, 42]. This technique is the basis for the
establishment of recombinant pestiviruses with properties desired for a vaccine.
3 Prerequisites for Pestivirus Persistence
Establishment of persistent infection is the strategy mainly responsible for the

maintenance of pestiviruses in their host population. Persistence is best studied
for BVDV, which achieves lifelong constant spread by persistently infected animals
[4]. Any successful vaccination campaign must break this vicious cycle.
Viral persistence requires a delicate balance between the host immune system
and viral factors and strategies that protect the virus against elimination. Intrauter-
ine infection of a fetus in the first trimester (between days 40 and 120 of pregnancy)
by noncytopathic BVDV strains may lead to viral persistence, which is accompa-
nied by an acquired immunotolerance with strict specificity for the infecting virus
strain [43, 44] (Fig. 2). It is generally believed that self-reactive elements of the
adaptive immune system, including those directed against the persisting virus, are
inactivated during this developmental stage of the infected fetus. Thus, the devel-
oping animals do not mount an adaptive immune response against the persisting
virus strain during their lifetime. Furthermore, the virus is protected against the
immune response of the mother cow by the fact that antibodies cannot cross the
bovine placenta. Consequently, viral clearance in pregnant cows with preexisting
antibodies against BVDV must occur prior to infection of the fetus. Otherwise,
depending on the transmitted virus, persistently infected animals will develop, or
Fig. 2 The principal pathway to virus specific acquired immunotolerance and establishment of
persistent infection by bovine viral diarrhea virus (BVDV)
abortion of the fetus will occur. Accordingly, fetal safety requires a stringent level
of immunoprotection against BVDV. Such protection is further hampered by a
high level of antigenic variability within and between the subtypes BVDV-1 and
BVDV-2.
To maintain a persistent infection for years, BVDV must also evade the host’s
innate immune system [45]. Because innate immunity is crucially important to
control viral infections, all viruses seem to encode antagonists counteracting this
system [46]. For BVDV at least three strategies or factors have been identified that
work together to counteract the host’s innate immune response (see below).
Although persistence is somewhat different in other pestiviruses, transplacental
infection of fetuses is a general theme. On the basis of the facts described above,
effective novel live pestivirus vaccine strains (1) must protect a pregnant vaccinee
from even transient viral replication and (2) must not infect the fetus itself or at least
must not establish a persistent fetal infection.
Taking these considerations into account, we should design improved live
pestivirus vaccines on the basis of recently obtained knowledge about virulence
factors and requirements for viral persistence. The description that follows will
focus mainly on BVDV because the available data provide a rather complete
picture of the processes leading to persistence.
3.1 Time Point of Infection, Biotype, and Beyond
As outlined already, infection of pregnant cattle with noncytopathic BVDV in the

first trimester of gestation may lead to infection of the not yet immunocompetent
fetus and the development of persistently infected offspring with an acquired

immunotolerance against the infecting BVDV strain [47]. Infections in the earlier
time phases of pregnancy may have severe consequences for the developing animal,
such as abortion, stillbirth, or malformations. When the fetus is infected in the late
phase of pregnancy, the development of the immune system has already proceeded
to the point where the virus infection may be cleared, but abortion can still occur
[48]. Importantly, the capacity to establish persistent infections is restricted to the
noncytopathic biotype of BVDV. Persistently infected animals can be retarded in
growth or might appear completely healthy. However, all these animals eventually
come down with the so-called mucosal disease. This disease is characterized by
severe ulcerations of the mucosa throughout the gastrointestinal tract and the
destruction of Peyer’s patches, resulting in bloody diarrhea and the death of the
animal. Onset of mucosal disease is correlated with the appearance of a cytopathic
BVDV strain in the animal already persistently infected with a noncytopathic
BVDV strain [43]. These cytopathic BVDV strains mostly arise from the persisting
noncytopathic virus by mutation, often through RNA recombination [49]. The
genomic changes associated with the switch in the viral biotype result in an
uncontrolled release of NS3 from the viral polyprotein, which in turn causes a
large increase in viral RNA replication. Mutations found in those cytopathic
genomes are deletions, accumulated point mutations, or insertions of viral or cell-
derived sequences, sometimes together with large sequence duplications [50]. In
the recombinant genomes, fragments of cellular messenger RNAs are sometimes
found upstream of the NS3-coding region. The cell-derived sequences encode
substrates of cellular proteases such as ubiquitin, ubiquitin-like proteins, or proteins
with ubiquitin-like folds [51]. Because the cellular proteases can recognize their
cognate substrates in the context of the viral polyproteins, they generate the
authentic N terminus of NS3 and thereby release this protein [52]. Moreover,
duplicated versions of Npro located upstream of NS3 in the polyproteins of cyto-
pathic BVDV can also mediate processing at the N terminus of NS3 [53]. Other
cytopathic BVDV strains are characterized by highly efficient NS2-3 processing via
a deregulated NS2 autoprotease [33] (also see below). As a result, a large amount of
free NS3 is present in cells infected with cytopathic BVDV and correlates with
highly efficient viral RNA replication as well as viral cytopathogenicity and
interferon induction [33, 54 56]. Studies of the different viral biotypes have
revealed a unique control mechanism for viral RNA replication in cells infected
with noncytopathic BVDV-1: the autoprotease in NS2 requires a cellular protein as
a cofactor for catalysis of NS2-3 cleavage [57]. This cellular chaperone protein,
termed “Jiv” (for J-domain protein interacting with viral protein), which stably
interacts with two domains in NS2, is required in stoichiometric amounts for the
activation of the NS2 protease and thus for NS3 release [58]. Because the endoge-
nous level and turnover rate of Jiv seem to be very low, the number of Jiv molecules
present in the cell at the time of infection only allows efficient cleavage of the NS2-
3 molecules translated in the first few hours after infection (Fig. 3). After this time,
mainly uncleaved NS2-3 is produced because, for most protease molecules, no
cofactor is available [57]. Since NS3 is an essential component of the viral RNA
early phase
high level
NS2 NS3
RNA
Jiv replication
late phase virion

NS2 NS3 morpho-
genesis
Fig. 3 The Jiv dependent control mechanism of noncytopathic BVDV genome replication and its
connection with different stages of virus replication. NS3 is an essential factor for RNA replica
tion, and uncleaved NS2 3 is required for virion morphogenesis. A switch between these stages is
mediated by the consumption of the cellular Jiv pool
replication complex and cannot be functionally replaced by NS2-3, the efficiency of

viral RNA replication drops with the consumption of the endogenous Jiv pool and
the concomitant decrease in NS3 release. Thus, low endogenous amounts of Jiv
restrict viral replication. This restriction is crucial for the maintenance of the
noncytopathic biotype. Accordingly, Jiv insertions in BVDV and CSFV strains
render those viruses cytopathogenic [55, 59, 60]. Similarly, the regulated over-
expression of Jiv induces a switch of the viral biotype from noncytopathic to
cytopathic [61]. Most interestingly, whereas noncytopathic BVDV seems to be
capable of suppressing the interferon response upon persistent infection via the
action of Npro and Erns, cytopathic BVDV has obviously lost this capacity [62]. For
CSFV, the change of the viral biotype from noncytopathic to cytopathic induced by
a Jiv insertion correlated with an attenuation of the virus in its natural host and the
induction of an interferon-induced gene [59]. This again emphasizes the importance
of the Jiv-mediated regulation of viral RNA replication for the control of the innate
immune system (also see later).
It is tempting to speculate that triggering of the interferon response together with
the induction of apoptosis in the infected cell is causative for the failure of
cytopathic BVDV to establish persistence.
3.2 Mutation of Viral Factors Interfering with the Innate

Immune Response as a Strategy for Pestivirus Attenuation
3.2.1 How Can Innate Immune Reactions Be Reduced to a Tolerable Level?
Despite the control of RNA replication in noncytopathic BVDV, a minimal amount

of viral RNA must be present within the infected cell, inevitably leading to the
presence of double-stranded RNA, a very potent trigger of the type 1 interferon
response. Viral persistence in an organism with huge numbers of infected cells
could hardly be imagined if infection resulted in a constantly high level of type 1

interferon production, with all the consequences for cellular translation, RNA
degradation, induction of apoptosis, and inflammatory responses. In fact, persis-
tently infected calves do not have increased serum levels of type 1 interferon (Bryan
Charleston, personal communication). To avoid triggering the interferon response,
with all its downstream antiviral effects, pestiviruses not only restrict viral RNA
replication, but also express two proteins involved in controlling the type 1 inter-
feron response.
3.2.2 Npro
For both BVDV and CSFV, Npro, the first protein encoded by the long ORF, is
responsible for blocking the type 1 interferon response in pestivirus-infected cells.
Npro is an unusual cysteine protease [63], which cleaves at its own C terminus and
thus generates the N terminus of the capsid protein. There is no other substrate
known for this protease. Moreover, the Npro-coding region can be deleted from the
viral genome without dramatically influencing the growth characteristics of the
virus [64]. Nevertheless, the Npro deletion attenuates the virus [65, 66]. It was first
reported for CSFV that deleting the Npro-coding region results in a mutant virus that
induces an interferon response in infected cells; therefore, Npro was proposed to
interfere with the innate immune system in CSFV-infected cells [67 69]. Loss of
repression of interferon induction was also reported for BVDV Npro deletion
mutants [70, 71].
The observed repression of an interferon response by noncytopathic BVDV was
independent of the proteolytic activity of Npro [70] and correlated with the absence
of activation of gene expression by interferon regulatory factor 3 (IRF3) [72]. It was
reported recently that Npro induces degradation of IRF3 via the proteasome [71,
73 75]. For this degradation the Zn2þ-binding site of Npro is essential, indicating
that the structural integrity of the protein must be maintained [76].
It has been a matter of debate whether Npro deletion results in considerable
attenuation of CSFV in piglets or adult animals. The first published data indicated
that the deletion of Npro in different CSFV isolates leads to attenuation [66]. In
contrast, results obtained in the laboratory of one of the authors gave no indication
that Npro deletion causes considerable attenuation (G.M., unpublished data). More
recent findings from Szymanski et al. showed that the observed attenuating effect is
not due to loss of the Npro-induced repression of the type 1 interferon response, but
most likely results from reduced growth rates probably due to less translation of the
mutated genomic RNAs [77]. This finding indicates that the Npro type 1 interferon
repressing function is not a virulence factor in piglets or adult animals.
3.2.3 Erns
Erns is another fascinating pestiviral protein. Not only is it an essential structural

component of the virus particle, but it also exhibits enzymatic activity [26, 27, 78, 79].
Erns contains sequence motifs typical of the T2 superfamily of RNases and hydro-
lyzes single- and double-stranded RNA upstream of U residues [28, 80 82]. The
enzymatic function of the protein is not necessary for virus growth in tissue culture;
RNase-negative virus mutants replicate in cultured cells as rapidly as wild-type
viruses [83 85]. However, abrogation of the RNase activity by mutation of the
active-site histidines in the conserved T2 RNase motifs results in considerable
attenuation of both CSFV and BVDV [84 86].
The function of the RNase is still obscure, but involvement in pestivirus immune
evasion seems most plausible. In fact, like Npro, the Erns RNase also can prevent
induction of a type 1 interferon response [81, 82, 87]. Since the RNase efficiently
degrades viral RNA [28], a function of the active RNase in the cytoplasm of an
infected cell seems to be highly unlikely. Addition of purified Erns or secretion of
Erns from cells expressing the protein is sufficient to prevent type 1 interferon
induction when extracellular double-stranded and single-stranded synthetic or
viral RNAs are used as triggers [81, 82, 87]. This effect is dependent on RNase
activity and correlates with a double-stranded RNA binding feature of the protein
[81]. Erns cannot prevent a type 1 interferon response when the triggering RNA is
introduced into the cells by transfection. Therefore, it can be concluded that the
RNase does not repress the type 1 interferon response in the cytoplasm of infected
cells and thus has a role that clearly differs from that of Npro.
As already mentioned, Erns is secreted in substantial amounts from infected or
transfected cells. This feature of the protein plays a central role in hypotheses on the
function of its intrinsic RNase activity. Npro seems to function as an intracellular
repressor of type 1 interferon induction in the cytoplasm; Erns probably finds its
target outside the infected cell. Therefore, secretion of Erns could be directly
connected to its function as a virulence factor. The amount of Erns present in the
serum of infected animals has been measured as approximately 50 ng/ml, an
amount sufficient to exert a biological effect [82]. The mechanism leading to
release of Erns from the cell in which it is synthesized is not clear. Erns is a
membrane-bound protein [24] that is not usually found on the surface of the
infected cell [88]. Accordingly, both the membrane anchoring and the intracellular
retention signal of the protein must be circumvented to allow secretion. It was
shown recently that Erns is bound to membranes via a long amphipathic helix,
composed of approximately 40 C-terminal amino acids [24, 25]. Erns is the first
surface protein shown to be anchored in this way. It seems likely that this unusual
membrane anchor plays an important role in Erns secretion and the tuning of the
equilibrium between release and retention. On the basis of the considerations
outlined above, this membrane anchor would also be important for the function
of the RNase.
Erns contains eight cysteines that form intramolecular disulfide bonds [89] and
are conserved in all pestiviruses analyzed to date. A ninth cysteine is found rather
close to the C terminus of the protein in the overwhelming majority of pestivirus
isolates. This cysteine, Erns residue 171, is engaged in forming Erns dimers via
disulfide bonds between two monomers. These homodimers are found in both
infected cells and virus particles [12]. There are very few pestivirus strains, most
notably a biologically cloned virus of the BVDV-1 prototype strain NADL, that
lack C171. The existence of such naturally occurring viruses and of several
engineered virus mutants that lack C171 [90, 91] proves that formation of Erns
homodimers linked via C171 is not essential for pestivirus viability. Since Erns
proteins lacking C171 do no establish dimers stable enough to allow purification
and analysis under mild conditions, the formation of stable dimers does not appear
necessary for pestivirus viability [91]. Nevertheless, Erns dimerization must have a
distinct advantage, because C171 is conserved among the overwhelming majority
of pestiviruses. Indeed, the growth in tissue culture of CSFV and BVDV lacking
C171 is retarded by approximately 1 order of magnitude. More important, two
different CSFV mutants with exchange or deletion of C171 are very significantly
attenuated in animals. Therefore, dimer formation seems to be connected with
virulence [91].
Taken together, the data suggest more than the ability to hydrolyze RNA is
necessary for Erns virulence factor activity. There is evidence for the involvement of
both secretion and dimer formation in this interesting biological function.
3.2.4 Npro, Erns, and Persistence
The function of Npro during infection of adult host animals is not clear. Only mild or
even no attenuation has been observed for Npro-negative mutants [77]. Defined
mutants abrogating the type 1 interferon repressing function of the protein do not
necessarily lead to significant attenuation, so a connection between Npro-induced
repression of the innate immune system and general pestivirus virulence is rather
unlikely [77]. Attenuation due to Npro deletion seems, therefore, to be a secondary
effect, presumably a consequence of reduced viral protein translation. Similarly, the
detailed function of the Erns RNase remains obscure. A clear disadvantage of
RNase-negative viruses was detected when the natural host was inoculated with
them: the virus load in the infected animals was much lower, as indicated by the
absence of virus transmission to contact animals. Despite the demonstration that
secreted Erns can block type 1 interferon induction by extracellular RNA, the
mechanism underlying the RNase effect is not yet known. The presence of signifi-
cantly increased amounts of extracellular RNA within infected animals is not
established, so the mechanism and target of the Erns RNase activity remain obscure.
Interestingly, an adequate interferon response was observed in immunocompe-
tent cattle after acute infection with noncytopathic BVDV [45]. In contrast, absence
of a type 1 interferon response is typical in animals persistently infected with
noncytopathic BVDV and was proposed to play an important role in establishment
and maintenance of persistent infection [92]. Therefore, the real functions of Npro
and the Erns RNase may be to assist in the latter processes in the fetus. If so, these
functions cannot be investigated in the adult animal. In fact, Npro and the Erns RNase
activity are connected with the establishment of persistent BVDV infections [93].
When fetuses in pregnant heifers were directly infected with different BVDV
mutants, as reported previously [92], a noncytopathic wild-type BVDV did not
induce a type 1 interferon response, but a corresponding cytopathic virus induced

type 1 interferon synthesis. Both Erns-RNase-negative and Npro-deletion mutants of
the same noncytopathic virus also induced the expression of type 1 interferon in the
fetus, at levels similar to those induced by the cytopathic BVDV variant. The
combination of these two changes in a double mutant resulted in an extremely
elevated type 1 interferon response 7 days after infection [93].
Although induction of an interferon response in the infected fetus is not directly
connected to prevention of virus persistence, there is at least some correlation. The
RNase-negative or Npro-deletion single mutants of BVDV could establish persistent
infection, as documented by isolation of infectious virus from fetuses 2 months
after infection of pregnant heifers [93]. In contrast, double mutants, with a combi-
nation of Npro deletion and Erns RNase inactivation, were never found in the fetus in
these experiments. When such mutants were introduced directly into the fetus,
abortion within the first 3 7 weeks after infection always occurred in all animals.
Therefore, it can be concluded that the double mutant cannot establish persistence,
presumably because it induces a strong type 1 interferon response, leading to severe
innate immune reactions. The ongoing secretion of type 1 interferon and the
resulting downstream effects should lead to apoptosis of cells, ultimately causing
massive damage to the fetus.
3.3 Textbook of BVDV Persistence and Lessons

for Vaccine Approaches
From currently available data, establishment of BVDV persistence relies on a set of

prerequisites. The first prerequisite is the correct time of infection of a pregnant
cow, so that antigens of the transplacentally transmitted virus are accepted by the
fetus as “self,” preventing adaptive immune responses. The second prerequisite is
the need for a noncytopathic biotype that prevents cell damage by strict control of
viral RNA replication to limit expression of danger signals. Npro and the Erns RNase
are needed to repress innate immune reactions triggered by the presence of viral
double-stranded RNA within the infected cell and at currently unknown additional
sites.
One might expect that loss of either Npro or the Erns RNase function would
prevent establishment of persistence, because both are hypothesized to exert quite
different activities that block the innate immune response. However, because both
seem to counteract the induction of type 1 interferon expression, one could also
imagine a certain degree of redundancy. Alternatively, it may be that the type 1
interferon response must only be reduced below a threshold level, with just one of
the two viral counteracting activities being sufficient to reach this level. In fact,
experiments have shown that the presence of only one of the two functions is
sufficient to allow establishment of persistence. However, it can be hypothesized
that a reduction of the incidence and duration of persistence would be observed if a
sufficiently high number of pregnant animals could be challenged with viruses

lacking an active RNase or Npro. It is also important to note that efficient control of
viral replication is absolutely necessary, because cytopathic viruses cannot prevent
a type 1 interferon response, even in the presence of normal amounts of functional
Npro and active Erns RNase.
Modern BVDV live vaccines should be designed to induce protective immunity
against transplacental infection of the fetus in pregnant animals and to be safe in
pregnant animals. For safety, a live vaccine virus should not establish persistent
infection itself and should not cause any other adverse effects in the heifer or fetus.
Available data indicate that this goal can be achieved by interfering with the viral
repressors of the innate immune response. We have demonstrated that a noncyto-
pathic double mutant, with deletion of the Npro-coding sequence and elimination of
a histidine residue in the active center of the Erns RNase, is a promising vaccine
candidate that meets the above-described criteria. When a pregnant heifer was
inoculated with this mutant, the mutant did not detectably cross the placenta but
did induce protective immunity that prevented fetal infection upon challenge.
Further efforts will be necessary to elucidate the function of the Erns RNase in
detail and define the mechanism underlying the attenuation of RNase-negative
viruses.
As mentioned already, two species of BVDV are found in the host population,
designated BVDV-1 and BVDV-2. All vaccines currently available are derived
from BVDV-1. Published data prove that these vaccines do not protect sufficiently
against a BVDV-2 challenge. Therefore, full protection of cows against BVDV
requires vaccines that most likely must include both BVDV-1 and BVDV-2 or at
least structural proteins from both species.
4 Pestiviruses Other Than BVDV
The development of vaccines against pestiviruses other than BVDV is less

advanced. The authors are not aware of ongoing efforts to produce BDV vaccines,
even though border disease also has a significant economic impact. In contrast,
discussions about novel CSFV vaccines have been reactivated because classical
swine fever outbreaks in the last few decades have required culling campaigns,
which are associated with serious ethical concerns and enormous costs. As for
BVDV, safe and effective modified live vaccines with defined attenuating muta-
tions would be reasonable for CSFV. Recently, a cytopathic strain of CSFV was
established by the insertion of Jiv-coding sequences into the viral genome [59].
This virus showed more efficient NS2-3 processing and upregulation of viral RNA
synthesis, as observed previously for cytopathic BVDV strains with Jiv insertions.
In cell culture experiments the cytopathic CSFV mutant induced the expression of
the interferon-regulated gene Mx, but the parental noncytopathic CSFV strain did
not. Accordingly, increased replication of the cytopathic virus correlated with
induction of an innate immune response. Interestingly, the cytopathic virus was
greatly attenuated in its natural host. Because the cytopathic CSFV mutant induced
high levels of neutralizing antibodies, it is a potential vaccine candidate.
Not only must the vaccine prevent disease, horizontal virus spread, and vertical
virus spread, but the vaccine virus itself must also be safe in pregnant animals.
Because fetal infection and persistence is somewhat different for CSFV, it cannot
be foreseen whether the same mutations used in the live attenuated BVDV vaccine
will be appropriate. This point must be analyzed soon.
5 Alternative Approaches to Virus Attenuation
The attenuation of pestiviruses for vaccine approaches can, of course, also be

achieved by mutations that do not affect the functions involved in controlling the
innate immune response and establishing persistent infection. As a general rule,
reduction of virus fitness by mutations reducing the efficiency of viral replication
will most likely result in attenuation. In this context, mutations affecting genome
replication, viral gene expression, the efficiency of target cell infection, or the
tropism of the virus in its natural host can lead to live attenuated vaccines.
Different mutations reducing the efficacy of pestivirus gene expression
have been identified. One of these changes is directly connected with deletion of
Npro. As mentioned already, the deletion of this nonessential protein usually results
in a certain degree of virus growth retardation. This effect does not seem to be
connected with the loss of interference with the type 1 interferon response, but has
been hypothesized to result from less efficient translation of the viral RNA [77].
Pestiviruses recruit ribosomes for translation of their genome via the IRES located
in the 50 UTR. Mapping of the IRES revealed that, at least in several pestiviruses,
the sequence relevant for efficient translation initiation extends into the region
downstream of the IRES. The importance of the downstream sequences has been
proposed to correlate with secondary structure constraints [94 97]. Thus, deletion
of Npro not only eliminates part of the ability of pestiviruses to interfere with the
innate immunity of the host, but it also impairs IRES function and thereby reduces
the efficiency of virus propagation.
IRES function can also be affected by other mutations. When different altera-
tions were introduced into the UTRs of BVDV to reduce virus replication, a variety
of different mutants were recovered that were viable and stable. These viruses
showed reduced fitness in cell culture and were found also to be attenuated in their
animal host. Such mutants with alterations in the IRES also represent potential
vaccine candidates [98, 99].
Similarly, alterations in the 30 UTR, which contains important cis-acting ele-
ments for virus replication, can attenuate pestiviruses. A 12-nucleotide insertion,
first identified in a lapinized CSFV vaccine strain recovered after consecutive
passage of pathogenic virus in rabbit cells, conferred attenuation when introduced
into the highly pathogenic strain Shimen [100]. The alteration retarded the growth
of the mutant virus in tissue culture by nearly 2 logs. This considerable growth
retardation is the most probable explanation for the observed attenuation in vivo.
The stability of attenuation resulting from such inserted sequences is an important
unanswered question. In theory, an insertion that hampers virus growth can easily
be lost by recombination, restoring virulence.
Interference with virus fitness at the level of infection of a target cell has
been achieved by introducing mutations into structural protein-coding regions. In
one approach, a linear epitope in E2 used to differentiate CSFV from ruminant
pestiviruses was changed in highly virulent CSFV Brescia in different ways to
resemble the corresponding sequence of BVDV strain NADL. Two of the result-
ing viruses showed considerable growth retardation and proved to be attenuated in
pigs [5, 6]. In addition, a 19 amino acid insertion introduced into the C-terminal
region of E1 via transposon linker insertion mutagenesis resulted in an attenuated
virus that had a growth rate equivalent to that of the wild type in tissue culture
[101].
Other approaches to attenuation of CSFV by mutations in structural protein-
coding regions rely on elimination of N-glycosylation sites. This was done for E2,
Erns, and E1 [6, 102, 103]. In all cases, the mechanism underlying the observed
attenuation is not fully understood and the long-term stability of the mutations has
not yet been demonstrated.
Virus fitness can also be impaired in chimeras that contain sequences from two
different strains of one pestivirus species or even from members of two different
species. Several chimeras in which sequences from the CSFV vaccine viruses
“C-strain” and “CS-strain” were introduced into the background of the highly
pathogenic wild-type virus Brescia are attenuated in pigs [5 7].
Similarly, chimeras that combine sequences from CSFV and BVDV have been
established. In several cases, a CSFV background was used, and specific fragments,
such as the Erns-coding region or part of the E2-coding sequence, were replaced by
the corresponding BVDV sequences [104, 105]. These chimeras have acceptable or
even high growth rates in porcine cells. The chimeric viruses are not pathogenic in
pigs, as is expected because the mutants have a CSFV vaccine strain background.
Importantly, vaccination with the chimera protects against a stringent wild-type
CSFV challenge.
A different approach was chosen to construct the chimeric virus CP7 E2alf.
In this case, a BVDV background (stain CP7) was used, and the E2-coding region
was replaced by the corresponding sequence from CSFV strain Alfort 187. After
passage, this virus replicated in porcine tissue culture cells to rather high
titers. Importantly, the chimera propagated in bovine cells to only very low titers.
Vaccination of pigs with this BVDV-based recombinant virus induced protective
immunity against a stringent challenge. A theoretical disadvantage of this approach
is the lower complexity of the CSFV-specific immune response owing to the
absence of CSFV Erns as a second target for neutralizing antibodies [14] and the
absence of a complete set of T-cell epitopes in viral structural and nonstructural
proteins [106]. Owing to these considerations, a less effective cross-protection
would be anticipated after vaccination with this mutant. The same disadvantage,
though less relevant owing to the much smaller size of the exchanged fragments,
would also be intrinsic to other interspecies chimeras. It remains to be determined

whether this theoretical flaw is of practical relevance.
In general, vaccination with interspecies chimeras bears an additional risk,
because new types of viruses are created and spread in the field. The combination
of sequences from different virus species might result in unforeseeable changes in
tropism or virulence in “nontarget species.” Risk assessment is difficult, because
only a limited number of experiments can be conducted. If the most likely target
species have tested negative for adverse affects from a chimeric virus infection, the
risk can be hypothesized to be low. In the end, society must decide whether or not
this risk is necessary and justified.
Additional alternative approaches to new pestivirus vaccines cross the border
between live attenuated viruses and killed virus vaccines. These approaches are
based on establishing autonomous replicons by deleting sequences from viral
genomes. Several such putative replicon vaccines have been established for
BVDV and CSFV [78, 107 110]. In all of these recombinant viruses, essential
sequences were deleted from the genomes so that the vaccine candidates can only
be propagated when the proteins encoded by the deleted sequences are provided in
trans. An advantage of such replicons is certainly their safety, because infection of
a cell is a dead-end, with no infectious virus released. In contrast to killed virus
vaccines, the replicons express genes and viral proteins are synthesized de novo
within the infected cells. Intracellular production of viral proteins allows presenta-
tion of viral peptides on MHC, resulting in a T-cell immune response in addition to
a humoral response. Care must be taken during propagation of the deletion mutants
in complementing cell lines so that fully replication competent viruses are not
restored by recombination between the replicon genome and RNA coding for the
complementing proteins.
Vaccination efficiency is a general question with the replicon approach. Because,
even with a high vaccine dose, only a very limited number of cells are infected and
produce viral proteins, the trigger for the immune system is not very prominent.
Nevertheless, induction of protective immunity has been successfully demon-
strated in stringent challenge models in several cases. It is not yet clear whether a
single vaccination with a replicon can consistently prevent fetal infection in a
pregnant animal.
6 Marker Vaccines
Modern approaches to eradication of viruses often rely on a combination of

vaccination and culling of (persistently) infected animals. Marker vaccines that
allow the differentiation of vaccinated from field-virus-infected animals by sero-
logical techniques have been successfully used for such projects. In the case of
pestiviruses, the benefit of a marker in the vaccine virus is a matter of debate. The
main reason for this debate is the absence of antibodies in persistently infected
animals because of the special way in which these viruses establish persistence. It is
generally accepted in the field that the most important step to control pestiviruses
is eliminating persistently infected animals. In contrast to, for example, animals
infected with herpesvirus, animals persistently infected with pestiviruses cannot be
identified by serological means. A vaccine would have to be designed so that,
although the presence of vaccine virus in persistently infected animals could not be
detected serologically, it could be detected by other means, such as a simple reverse
transcriptase PCR test. In any case, such immunized but nevertheless persistently
infected animals would have to be eliminated.
Despite the problems outlined above, pestivirus marker vaccines could certainly
have advantages for control strategies; therefore, this aim is pursued by different
groups (recently reviewed for CSFV in [111]). Because of the high degree of
mutation and recombination, a positive marker is likely to be lost rather quickly
if it is not combined with a selective marker. A negative marker introduced by a
genomic deletion would be preferable. As a nonessential protein with immune
evasion function, Npro would be a perfect marker; however, Npro does not seem to
be immunogenic enough to induce the required levels of specific antibodies. Other
ideas for establishing marker vaccines are based on the chimeric pestiviruses
(described earlier) that have genomes combined, for example, from BVDV and
CSFV sequences. Other approaches rely on the already mentioned replicons.
After vaccination with such chimeras or deletion mutants, the animals could be
serologically differentiated from animals that had been infected with the corres-
ponding field viruses. As mentioned already, a general problem of such vaccines
could be a lower degree of acceptance owing to difficult risk assessment or lower
vaccination efficiency.
In summary, the search for a feasible marker for pestivirus vaccines remains an
ongoing process waiting for novel ideas.
7 Conclusion
The importance of pestiviruses as livestock pathogens with enormous economic

impact and a significant effect on food production demands the development of
modern, safe, and effective vaccines. Detailed investigation of the molecular fea-
tures of these viruses and their interactions with host animals and the estab-
lishment of infectious complementary DNA constructs for several pestiviruses
provide a solid basis for approaches toward novel vaccines. Because of the need
to protect fetuses in pregnant animals, live attenuated viruses, with their high
immunogenic potential, seem to be most appropriate. The near future will show
which types of putative vaccine viruses are promising enough for development
of commercial products. Because the elucidation of pestivirus molecular bio-
logy is still in progress and frequently provides new surprises, there is also a
good chance that there will be further interesting vaccine approaches in the
future.
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Vaccine 25:205 230
Live Attenuated Influenza Virus Vaccines: NS1
Truncation as an Approach to Virus Attenuation
Natalie Pica, Peter Palese, and John Steel
Abstract Influenza virus causes significant morbidity and mortality worldwide.

Vaccination, usually involving the inactivated type of vaccine, is the primary
mechanism of influenza virus prevention. Live attenuated influenza viruses
(LAIV), however, are also available for the prevention of disease. These vaccines
have been shown to stimulate a robust cellular response, induce IgA and IgG
antibodies, and can provide heterosubtypic protection. Cold-adaptation and tem-
perature sensitivity are two mechanisms of influenza virus attenuation, yielding
viruses that are both safe and immunogenic. At present, novel attenuation strate-
gies, including the manipulation of viral gene sequences and proteins, are being
developed in the hopes of providing new LAIV vaccines. One promising strategy
involves the truncation of the NS1 protein of influenza virus, limiting the interferon
antagonist capabilities of the influenza pathogen. Experimental vaccines that
exploit this mode of attenuation have been tested in several animal models; as
summarized herein, high efficacy in reducing mortality, morbidity, and transmis-
sion of influenza viruses has been observed.
1 Influenza Virus Vaccines: The Current Standard
Vaccination has proven to be the most cost-effective medical intervention targeting

seasonal influenza [1]. Nonetheless, the very young, the elderly, and the immuno-
compromised population groups at high risk of suffering severe complications of
influenza [2 4] do not respond optimally to vaccination. Thus, there is an
identified need for more effective vaccination strategies eliciting greater protective
N. Pica (*) and J. Steel

Department of Microbiology, Mount Sinai School of Medicine, New York, NY 10029, USA
P. Palese
Department of Microbiology, Mount Sinai School of Medicine, New York, NY 10029, USA
Department of Medicine, Mount Sinai School of Medicine, New York, NY 10029, USA

196 N. Pica et al.
immunity in at-risk populations [5, 6], as well as vaccines to better protect the
general population.
1.1 Inactivated Influenza Vaccines
The most widely available human influenza vaccines are of the inactivated (killed)
type. Typically, these vaccine preparations derive from reassortants possessing at
least the hemagglutinin (HA) and neuraminidase (NA) genes from the presently
circulating strains and the remaining genes from the master donor strain A/Puerto
Rico/8/34 (A/PR/8/34) virus. This gene constellation confers high-yield viral
growth of a vaccine preparation [7], while retaining the antigenic characteristics
of the circulating strains. Influenza B virus vaccine preparation does not involve the
use of a high-growth strain to improve titers in egg-based systems. Instead, bulk
vaccine stocks derived from circulating influenza B vaccines are serially passaged
and amplified in embryonated chicken eggs, followed by inactivation [8]. Trivalent
inactivated vaccines (TIVs), however, are of limited efficacy in elderly and pediat-
ric populations [3, 4]. In addition, it is possible that large or multiple doses of
inactivated vaccines would be needed to provide protection against certain strains,
including antigenically novel influenza viruses [9].
2 Live Attenuated Cold-Adapted Influenza Vaccine
Early attempts to create live attenuated influenza virus vaccines aimed to exploit
physiological differences in the human respiratory tract; the temperature of the upper
respiratory tract is approximately 32 34 C, while that of the lower respiratory tract is
37 C [10]. Cold-adapted viruses would theoretically replicate to levels capable of
triggering an immune response in the upper respiratory tract, while keeping virus titers
in the lower respiratory tract low enough to not cause adverse effects in the patient.
Early strategies to exploit these temperature differences included the selection
of temperature-sensitive mutants that were grown in the presence of a mutagen, 5-
fluorouracil. Temperature-sensitive mutants were attenuated in animal models [10,
11], but were overattenuated in human subjects [12]. In addition, the temperature
sensitivity was a result of only one or two genetic mutations [13], making these
viruses less genetically stable and more prone to reversion.
Via serial passage at low temperatures, A/Ann Arbor/6/60 (H2N2) (A/AA/6/60)
[14 16] and B/Ann Arbor/1/66 (B/AA/1/66) viruses [16] were cold-adapted by the
Maassab laboratory in the Department of Epidemiology at the University of
Michigan School of Public Health, United States. These attenuated viruses dis-
played markers of cold-adaptation [17], thought to be a result of multiple genetic
mutations [18, 19]. Specifically, five amino acid changes in polymerase basic 1
protein, polymerase basic 2 protein, and nucleoprotein (NP) have been implicated
in the cold-adapted phenotype of A/AA/6/60 [20]. These mutations are thought to
Live Attenuated Influenza Virus Vaccines 197
reduce levels of M1 protein and vRNA expression and inhibit the export of vRNAs
to the cytoplasm [20]. Mutations in NP, polymerase acid, and matrix (M) proteins
are thought to cause attenuation of B/AA/1/66 virus, affecting polymerase function
as well as efficient virus assembly and budding [21]. The multiple mutations found
in these viruses are thought to contribute to their genetic stability, making them
ideal for vaccine use.
Prior to the advent of reverse genetics, reassortants were created via coinfection
to create vaccine viruses that contained six internal genes from the influenza A or B
Ann Arbor strains and the HA and NA of influenza virus strains circulating in the
community [22]. This coinfection technique for the creation of reassortants,
whereby the master donor strains would be A/AA/6/60 virus or B/AA/1/66 virus,
was therefore an attractive method of creating LAIV vaccines for newly circulating
viruses in a timely fashion.
The advent of reverse genetics allowed for the creation of temperature-sensitive,
cold-adapted reassortants to be used as vaccine viruses, creating “6 þ 2” viruses
(HA and NA of circulating strain and the remaining 6 segments from either A/AA/
6/60 virus or B/AA/1/66 virus) via a plasmid rescue system. This method is of
particular value when creating vaccine viruses for highly pathogenic avian influ-
enza (HPAI) viruses that are normally lethal to eggs due to of the presence of a
polybasic cleavage site.
It is possible that avian influenza viruses could contribute to an antigenically
novel reassortant pandemic virus, as was seen in 1957 and 1968 [23]. HPAI viruses
that could cause systemic infections are of particular concern. HPAI viruses contain
a polybasic cleavage site in the HA molecule that contributes to the pathogenicity
seen in these strains [24]. Ubiquitous proteases can cleave the HA molecule at this
site, activating the HA molecule in a variety of tissue types [25, 26]. Because these
highly pathogenic avian viruses are lethal in eggs, the multibasic cleavage site of
these high pathogenic strains must first be modified by reverse genetics techniques
so that stocks can be grown to high titer in this substrate [27, 28].
Inactivated HPAI virus vaccines are weakly immunogenic in human volunteers
or require adjuvant to have an immunogenic effect [29 31]. “6 þ 2” reassortants in
the cold-adapted Ann Arbor background, however, show promise in animal studies.
Suguitan et al. engineered vaccines on the cold-adapted background containing HA
molecules from 1997, 2003, and 2004, with the multibasic cleavage site deleted.
These vaccines also possessed an avian NA. A single dose of 106 TCID50 of
vaccines with HA from 1997, 2003, and 2004 H5 viruses resulted in 100% survival
in mice following challenge with 50, 500, or 5,000 LD50 of A/Hong Kong/491/1997
and A/Viet Nam/1203/04 H5N1 viruses [28]. Recently, a cold-adapted live attenu-
ated vaccine with a modified HA made against A/Anhui/2/2005 (H5N1) was also
shown to be attenuated in chickens and mice. This vaccine virus also stimulated
neutralizing antibodies and HA-specific CD4þ cells in rhesus macaques. Further-
more, macaques that received two doses of the vaccine did not experience any
weight loss or temperature changes in the 15 days following challenge with 106
EID50 wild-type A/Anhui/2/2005 or A/bar-headed goose/Qinghai/2/05 viruses
(H5N1) [32].
198 N. Pica et al.
Vaccine viruses on the background of A/AA/6/60 are also safe and protective
from wild-type H6 [33] and H9 [34] viruses in mice. However, note that cold-
adapted virus vaccines generated to provide protection against avian influenza
strains appear to be more attenuated than cold-adapted viruses for seasonal influ-
enza in humans [35, 36]. Alternative vaccine strategies against avian influenza
viruses may still be required.
Although the cold-adapted Ann Arbor strains are the present master donor
strains for live attenuated vaccines available in the United States, other countries
have explored the use of alternative virus strains. The former USSR has a long
history in the use of live attenuated vaccine viruses; most influenza vaccination in
Russia today is almost exclusively of the live attenuated type. The main master
donor strains, A/Leningrad/134/57 (H2N2) and B/USSR/60/69, were attenuated
by repeated passage in embryonated eggs at successively lower temperatures [37].
For decades, these viruses have shown to be effective.
2.1 Molecular Mechanisms of Protection
Cold-adapted LAIV vaccines stimulate increased levels of IgA in the upper respi-
ratory tract [38, 39]. Increased IgA levels were seen in young children when
compared with those immunized with TIV. These levels correlate well with
decreased viral shedding in the nasal passageway following challenge with the
parent virus 12 months postvaccination, compared with children vaccinated with
TIV or children who were not vaccinated [39]. Because viral shedding correlates
with severity of illness [40], IgA induction is most likely important to protection
against influenza virus morbidity.
Following vaccination, robust cellular responses are induced [41], and systemic
IgG levels are increased [42]. Interferon-g, an important antiviral cytokine, is
expressed following administration of the cold-adapted virus [43]. It is also possible
that cold-adapted LAIV vaccines could protect patients from infection that occurs
shortly after vaccination by replicative interference [44, 45]. It is thought that the M
protein [46] interferes with vRNA replication of a noncold-adapted strain [47].
2.2 Trivalent Live-Attenuated Influenza Virus Vaccines:

Protection for a Variety of Groups
2.2.1 Protection in Children
The trivalent cold-adapted influenza virus vaccine (CAIV-T) was licensed for use
in healthy children as young as 5 years old in 2003 [48]. Its use has since been
expanded to include children aged 2 4 [49].
In initial trials, cold-adapted influenza virus vaccine administered at a dose of

106 TCID50 was safe and immunogenic in children. Response to the vaccine was
age-dependent, however, with lower antibody titers in children 6 months or younger
[50]. In a large, multicenter, double-blind, placebo-controlled study, administration
of a CAIV-T to 1,602 children aged 15 71 months was safe, immunogenic, and
protective against influenza A (H3N2) and B viruses circulating during the
1996 1997 monitoring season [51]. In this study, the genetic stability of CAIV-T
following vaccination was also demonstrated [52].
Eighty-five percent of children vaccinated in year 1 returned for revaccination in
year 2. This revaccination in 1997 proved to be immunogenic and provided
protection against epidemic A/H3N2 and influenza B viruses in circulation during
the 1997 1998 influenza season, including a variant strain A/Sydney/5/97 (H3N2).
Protection from a virus not contained in the CAIV-T [53] demonstrated the ability
of the vaccine to provide protection from heterologous challenge in children.
Ninety-two percent vaccine efficacy was determined overall (95% CI: 0.89 0.94),
with 89% vaccine efficacy seen in the second year (95% CI: 0.81 0.94), despite the
presence of a strain variant not included in the trivalent vaccine [54]. In later
studies, children 1.5 18 years of age were administered the CAIV-T against
circulating H3N2, H1N1, and B virus strains. Those that received the vaccine in
1999 or 2000 were protected from the H1N1 and B virus strains circulating during
the 2000 2001 influenza monitoring time [55]. Serum HA inhibitory antibody and
IgA levels correlated well with the prevention of shedding [56].
Efficacy was also assessed when children were given CAIV-T annually. While
higher antibody levels were seen after the first vaccination with CAIV-T, antibody
titers were still high, particularly against H3N2 and B strains, in healthy children
vaccinated for 4 consecutive years [57].
2.2.2 Protection in Adults
In early clinical trials, CAIV was well tolerated and had a greater estimated
protective efficacy compared with trivalent, inactivated virus vaccine (85% versus
71%) [58]. In a larger, placebo-controlled, clinical trial, 4,561 working adults were
enrolled. Vaccination caused a statistically significant reduction in severe febrile
illness by 18.8% and febrile upper respiratory illness by 23.6% in those aged 18 49
relative to placebo. This correlated with a reduction in the number of days of illness,
fewer days of lost work, and fewer health care provider visits [59]. A recent study of
1,952 healthy adults has suggested that TIV is more efficacious than CAIV-T in
adults, however, resulting in a 50% reduction (95% confidence interval, 20 69) of
influenza illness in those who received TIV as compared with those who received
CAIV-T prior to the 2008 influenza monitoring time. Vaccine efficacy was also
calculated with respect to influenza A and influenza B viruses. While greater
vaccine efficacy was seen with TIV as compared with CAIV-T with respect to
influenza A viruses, there were not enough culture positive influenza B cases to
draw relative efficacy conclusions [60].
200 N. Pica et al.
2.2.3 Protection in the Elderly
Cold-adapted vaccines enhance IgA levels in the upper respiratory tract in those
65 years and older [61] and cause both systemic and mucosal immune responses
[62]. In many clinical trials, the superiority of CAIV-T over whole inactivated virus
vaccine in inducing serum and secretory antibodies has not been demonstrated in
the elderly [62, 63]. However, the safety of CAIV has been shown [64] and a
randomized, double-blind trial studying nursing home patients over a 3 year period
did confirm that additional protection could be provided if the CAIV (using A/AA/
6/60 as the master donor strain) was administered in conjunction with TIV [65]. In a
double-blind field trial involving nursing home occupants, combining these
vaccines resulted in a 60% decrease of influenza A viral infections when compared
to rates of infection seen in those vaccinated with only TIV [66].
2.2.4 Protection in the Immunocompromised
CAIV-T has not been shown to cause serious adverse effects in HIV positive,
asymptomatic patients. Changes in HIV viral load and CD4þ cell numbers were
not affected in an adult cohort following vaccination [67]. Similar results were seen
in trials involving HIV-positive children [68].
3 Live Attenuated Influenza Virus Vaccines Using

Micro-RNA Technology
Micro-RNAs (miRNA) are endogenous RNA segments that are approximately 21

nucleotides in length and are involved in gene silencing [69]. miRNAs are derived
from RNAs that fold on themselves to create hairpin structures called pri-miRNAs,
primarily in the nucleus [70, 71]. It is believed that in mammalian cells the RNase
III enzyme Drosha converts pri-miRNA into an approximately 70 nucleotide stem-
loop pre-miRNA [72, 73]; this stem-loop is exported to the cytoplasm where it is
cleaved by another RNase III enzyme, Dicer, into a mature, cytoplasmic miRNA
[74 77]. This mature miRNA binds complementary mRNA in the cytoplasm, in
association with members of the RNA-induced silencing complex [78], specifically
with the RNase Argonaute [79 81]. It is by association with this complex that
translational repression or mRNA degradation is executed by miRNAs [69].
Perez et al. sought to attenuate influenza virus by incorporating miRNA response
elements (MREs) into viral genomic segments. To achieve species specificity,
miR-93 was chosen to target viral transcripts. This miRNA is ubiquitously expressed
in mice (Mus musculus) and humans (Homo sapiens), but not in chickens (Gallus
gallus), allowing for robust growth of vaccine stocks in eggs. In order to cause
attenuation, two MREs were introduced into the NP segment of the A/PR/8/34 strain.
The growth of this virus was attenuated in the human embryonic kidney cell line
HEK293 and in mice, but grew to high titer in ovo. Administration of either an A/PR/
8/34 or a H5N1 6:2 reassortant virus (6 segments from A/PR/8/34 virus: modified HA
and unmodified NA from A/Vietnam/1203/04) containing MREs in the NP protein
induced an antibody response and caused less mortality than viruses lacking these
sites [82]. Vaccination with this LAIV vaccine is therefore an exciting new strategy
of attenuation and holds promise as a novel vaccine mechanism.
4 Influenza Virus Immunity Through Other Viral Vectors
The replication deficient vaccinia virus Ankara (MVA) was attenuated via serial
passage in chick embryo fibroblast culture [83]. Attenuation is thought to be a result
of deletions in the virus genome following passage [84]. Although replication is
unhindered in avian cells, MVA is replication deficient in mammalian cells, making
it an attractive mammalian vaccine vector, which can be used to express both viral
and recombinant genes [85, 86]. As a result, recombinant MVA viruses expressing
foreign viral antigens have been developed to protect against a variety of human
pathogens [86 89]. MVA is an ideal viral vector, based not only on its species
specific growth patterns but also due to its safety profile and immunogenicity [87].
MVA viruses expressing the HA of influenza viruses are effective vaccine
viruses [90]. Transfection of virally derived cDNA into MVA-infected cells [91]
allows for the incorporation and expression of recombinant genes under the vac-
cinia virus-specific PsynII promoter [92]. Following two vaccinations of the MVA
virus expressing HA from A/Vietnam/1194/04 (H5N1), mice were fully protected
from signs of morbidity following challenge with 103 TCID50 of the parental virus
and A/Indonesia/5/05 (H5N1). In addition, little to no weight loss was seen follow-
ing challenge with these viruses [90]. Using similar strategies, other groups have
shown safety and antigenicity in mice and chickens [93, 94]. Recently, the efficacy
and safety of a MVA-based vaccine against A/Vietnam/1194/04 (H5N1) [90] was
tested in nonhuman primates [95]. Vaccination with this vaccine was well tolerated.
Following challenge with A/Vietnam/1194/04 or A/Indonesia/5/05 viruses, mock-
vaccinated animals displayed severe necrotizing bronchointerstitial pneumonia.
Animals vaccinated with the MVA-based vaccine were protected, experiencing
only mild bronchointerstitial pneumonia [95].
5 Novel Live Attenuated Virus Vaccines Based

on Modifications of the M2 Ion Channel
The M viral genome segment expresses M1 and M2 proteins by virtue of alterna-

tively spliced mRNA transcripts [96]. M2 acidifies endosomes following virus
binding and cell entry, allowing for the release of viral ribonucleoprotein into the
cytoplasm [96]. When the transmembrane and cytoplasmic domains of the M2
202 N. Pica et al.
protein are ablated by the insertion of stop codons, the mutant virus (M2 knockout
virus M2KO) displays deficiencies in replication and lack of growth in mice [97],
probably due to interruptions in the normal virus life cycle [98]. Because the M2KO
virus displays an attenuated phenotype in cell culture and in vivo, its use as a
vaccine virus holds promise. When an M2KO virus made in an A/PR/8/34 back-
ground was generated by reverse genetics, low levels of virus were detected in the
lungs following vaccination with 3 106 and 3 105 pfu. No virus was recovered
from the lungs, however, when lower doses were administered, except for one
animal vaccinated with 3 104 pfu. Virus-specific antibodies correlated well with
survival rates following lethal challenge with wild-type A/PR/8/34 virus [99].
Although attenuated, virus growth deficiencies can be overcome when grown in
a stable cell line expressing M2 protein [99]. This could therefore be a strategy for
growing large vaccine stocks. This method of attenuation creates an attenuated and
safe live virus vaccine, though further studies with this vaccine construct are
warranted.
6 Novel Live Attenuated Virus Vaccines Based

on Modification of Viral Interferon Antagonists
Research in recent years on influenza virus, as well as many other viral pathogens,
has led to the identification of viral gene products that antagonize mammalian
antiviral responses by inhibiting the type I interferon (IFN) system [100]. The
widespread presence of IFN antagonists in diverse virus families provides a ratio-
nale for the generation of a novel class of live attenuated vaccines [101]. By
engineering viruses that have an impaired ability to inhibit the type I IFN system,
it should be possible to generate vaccine strains that can grow in vitro in IFN-
deficient substrates but will be attenuated in vivo by inducing the host IFN antiviral
response (Fig. 1). As added value, the induction of type I IFN can result in increased
adjuvancy and enhanced B and T cell responses [39, 102 104], so that mutant
viruses may be intrinsically more immunogenic than wild-type viruses. In support
of this concept, it has been demonstrated that NS1 mutant influenza viruses are
potent activators of dendritic cells [105, 106] and potent immunostimulators [107].
6.1 The NS1 Protein of Influenza Viruses
Segment 8 of influenza A virus encodes two proteins through alternative splicing of

its mRNA: the NS1 and NEP proteins [108]. NS1 is a polypeptide of 215 238 amino
acids, depending on the viral strain, and is the most abundant viral nonstructural
protein expressed in influenza virus-infected cells. The NS1 proteins of both influ-
enza A and B viruses inhibit the type I IFN response [109, 110]. This inhibition is
achieved through a block in the transcriptional induction of type I IFN. Antagonism
of this innate cellular response contributes to the virulence of influenza viruses.
Fig. 1 Proposed rationale for live attenuated influenza virus vaccines based on NS1 modification.
(a) Wild type influenza viruses express the NS1 protein, which reduces induction of type I IFN and
other related cytokines. This suppresses the innate and adaptive response of the infected cell, and
virus is able to multiply unhindered. (b) Deletions in the NS1 gene interrupt the protein’s
interferon antagonist capability causing attenuation as a result of an enhancement of host innate
and adaptive immunity. As a result, viral replication is hindered
Indeed, the growth of a mutant influenza virus based on the A/PR/8/34 strain but
lacking the NS1 protein (delNS1) is highly restricted in interferon-competent sub-
strates [109]. Poor replication and lack of disease following delNS1 virus infection
was furthermore correlated to increased levels of IFN production by the host [111].
Thus, NS1 mutant influenza viruses induce higher levels of type I IFN than wild-type
viruses. The induced type I IFN in turn limits further viral replication [111]. In
contrast, NS1-deleted viruses replicate efficiently in IFN incompetent systems such
as STAT1 knockout mice [109]. These initial findings supported the concept that
NS1-mutated influenza viruses have potential as live attenuated vaccine candidates.
However, it remained problematic that viruses carrying the delNS1 mutation may be
too attenuated in animal hosts to constitute a viable live attenuated vaccine. Because
of this potential limitation, mutations in NS1 that partially disrupt NS1 function were
sought, with the aim of generating mutant viruses with intermediate attenuation
characteristics between delNS1 and wild-type virus.
6.2 Mechanisms of NS1 Function
Early studies had demonstrated that the core region of the NS1 protein responsible
for inhibition of type I IFN production lies within its N-terminal dsRNA-binding
204 N. Pica et al.
domain [112]. This domain is a dimer of 73 amino acids, with exposed basic
residues responsible for interaction with dsRNA [113]. These data suggested that
NS1 inhibits the induction of IFN, at least in part, by sequestering dsRNA generated
during viral infection, thereby preventing its interaction with the cellular sensor
involved in triggering the IFN response, RIG-I. Although deletion of portions of the
C-terminal region of the NS1 protein also decrease NS1 IFN-antagonistic functions,
this is, in part, a result of destabilizing the dimer required for efficient dsRNA
binding [112].
Subsequent studies have revealed that NS1 inhibits the production of type I IFN
by inhibiting the activation of IRF-3, NF-kB, and AP-1 three key transcription
factors that coordinate the induction of IFN-b gene expression [111, 114, 115].
Considerable detail of the mechanism underlying NS1-mediated inhibition of type I
IFN production has now been elucidated. A significant milestone involved the
recognition of an interaction between NS1 and RIG-I, which leads to a block of
downstream signaling from RIG-I to the MAVS/Cardif/IPS-1/VISA adaptor mole-
cule and thereby prevents activation and nuclear translocation of the IFN-b enhan-
ceosome [116]. The mechanism of NS1-mediated inhibition has since been further
refined, with the finding that interaction of NS1 with the cellular ubiquitin ligase
TRIM-25 blocks dimerization of TRIM-25 and subsequent ubiquitination of RIG-I.
The lack of ubiquitination of RIG-I results in inhibition of signaling to MAVS and
therefore to a downstream block in transcriptional activation of type I IFN [117].
This in-depth characterization of NS1 function suggested that viruses with
impaired, but not entirely abrogated, type I IFN antagonistic properties were
obtainable, and might prove to be ideal live attenuated vaccine strains. Toward
the realization of this concept, several studies aimed at developing LAIV vaccines
based on modification of the NS1 protein by reverse genetics have been performed
in recent years and key findings are summarized here.
7 Testing the Concept: Vaccination Studies with

NS1-Truncated Viruses
7.1 Studies in Mice
Initial proof of principle experiments were conducted using the mouse-adapted

strain of influenza virus, A/PR/8/34. Thus, modification of NS1 as an attenuation
strategy was tested in mice using the A/PR/8/34 NS1-99 virus. This virus encodes a
truncated NS1 protein that contains only the N-terminal 99 amino acids and
possesses reduced, but not completely absent, IFN antagonist activity, intermediate
between that of wt A/PR/8/34 virus and the A/PR/8/34 delNS1 virus.
The results [101] are summarized in Table 1. Eighty percent of mice receiving
2 103 pfu of wild-type A/PR/8/34 virus died from infection. By contrast, intra-
nasal infection with 1 106 pfu of NS1-99 virus led to the death of only two of ten
Table 1 Survival of mice immunized with NS1 attenuated influenza A viruses and challenged
with wild type influenza A/PR/8/34 virus
Group Vaccine Dose Survivors Survivors Survivors
virus1 (pfu) postvaccination postchallenge2 postchallenge2
1.0 105 pfu 5.0 106 pfu
A A/delNS1 1.0 106 9/9 5/5 4/4
B A/delNS1 3.3 104 5/5 0/5 ND
C A/NS1 99 1.0 106 8/10 5/5 3/3
D A/NS1 99 3.3 104 9/10 3/5 4/4
E A/PR8 2.0 103 1/5 ND3 1/1
F PBS 0 6/6 0/6 ND
Adapted from [118]
1
Refers to the abbreviated description of the genotype used to identify each virus.
2
Mice were either challenged with 1.0 105 pfu or 5.0 106 pfu of wild type A/PR/8/34 virus
3
ND not determined
animals. When vaccinated animals were challenged, most mice immunized with
either high or intermediate doses of NS1-99 virus were protected, indicating that the
virus is immunogenic in vivo despite its significant attenuation. It was observed that
the delNS1 virus required high-dose vaccination in order to provide protection
against challenge. Protection was correlated with the induction of efficient humoral
and cellular immune responses [101]. Although these results were encouraging,
they were obtained using a mouse-adapted influenza A virus strain (A/PR/8/34),
and it remained to be determined whether they would extend to wild-type isolates
of influenza A virus infecting their natural hosts. An initial indication that observa-
tions with A/PR/8/34 could be generalized to other strains of influenza virus came
with experiments by Talon et al., who showed that vaccination with an NS1-
truncated variant of B/Yamagata/1/73 conferred sterilizing immunity against chal-
lenge of mice with human influenza B virus, as assayed by viral growth in the lungs
[118]. In this instance, viral growth in the lungs was used as a correlate of
protection, due to the lack of disease signs normally seen in mice infected with
human influenza B virus strains.
Studies of NS1-truncated influenza B virus vaccines were recently extended by
Hai et al. [119], who exploited a PKR knockout mouse model to study protection
from disease. Three variants of B/Yamagata/16/88 virus were generated; Yam/88/
NS1-80, Yam/88/NS1-110, and Yam/88/del-NS1. In vitro, each NS1-mutated virus
generated an increased type I IFN response relative to wild-type and, in vivo,
none of the mutants induced signs of disease in either PKR-/- or wt C57BL/6
mice. Furthermore, the NS1-truncated viruses grew to less than 104 FFU/ml in
lungs of PKR-/- mice, a greater than 100-fold reduction from wild-type virus levels.
Mice vaccinated with Yam/88/NS1-80, Yam/88/NS1-110, and Yam/88/del-NS1
were protected from disease or weight loss upon challenge from 5 105 pfu of
the homologous strain. BALB/c mice vaccinated with either Yam/88/NS1-80 or
Yam/88/NS1-110 virus were furthermore protected from death and weight loss
upon challenge with the significantly drifted heterologous influenza virus B/Lee/40,
highlighting the breadth of the immune response induced by the NS1-truncated
LAIV vaccines.
206 N. Pica et al.
7.2 Studies in Pigs
To address whether NS1-truncated virus vaccines could be effective in natural

hosts, a study was conducted using a swine influenza virus isolate, A/swine/
Texas/4199-2/98 (H3N2) (TX/98). Through reverse genetics, three TX/98-based
mutant viruses expressing truncated NS1 proteins of 73, 99, and 126 amino acids in
place of the 219 amino acid protein of the wild-type virus were generated [120].
Growth properties, induction of IFN in cell culture, and virulence-attenuation in
pigs of the NS1 mutant TX/98 viruses were analyzed and compared to those of the
recombinant wild-type TX/98 virus. All mutant viruses were impaired in their
ability to prevent induction of type I IFN in swine epithelial cells. Perhaps surpris-
ingly, the NS1-126 virus induced more type I IFN than the shorter NS1 mutant
viruses. Examination of NS1 levels revealed that the 1 126 NS1 protein levels
expressed in virus-infected cells were very low compared to levels of the wild-type
or the 1 73 and 1 99 mutant proteins. Thus, it appeared that different truncations
affect NS1 expression to differing degrees, resulting in viruses with varying
abilities to block induction of type I IFN and, consequently, varying levels of
attenuation.
In intratracheally inoculated pigs, both NS1 mutant viruses were attenuated
relative to the wild-type, with the TX/98/NS1-126 virus exhibiting the greatest
degree of attenuation [120]. All infected animals developed specific humoral
responses characterized by the presence of HA neutralizing (HI) antibodies in
sera by day 8 postinoculation. Sera from animals infected with wild-type viruses
had neutralizing titers approximately twofold higher than sera from those immu-
nized with mutant viruses.
The protective efficacy of the attenuated TX/98/NS1-126 mutant was tested in a
swine vaccination study. The TX/98/NS1-126 virus was administered in two doses,
separated by a 3-week interval, using an inoculum of 2 105 pfu per pig. This
regimen conferred protective immunity against challenge with 2 105 pfu of a
homologous wild-type virus isolate (H3N2 A/swine/Texas/4199-2/98). Further-
more, and remarkably, when vaccinated pigs were challenged with 2 105 pfu
of an H1N1 subtype swine virus (A/swine/MN/37866/99), significantly fewer
lesions in lung tissues and a lower viral load in lung lavage compared to the
nonvaccinated controls at day 5 postinoculation were observed [121]. These data
demonstrate that the attenuated TX/98/NS1-126 mutant has significant potential as
a live attenuated virus vaccine, inducing immunity against homologous challenge,
as well as generating considerable protection against heterosubtypic challenge.
However, as intratracheal inoculation is not a practical route for commercial
vaccination, a further vaccination challenge study involving the TX/98/NS1-126
virus was performed in which intramuscular and intranasal routes of inoculation
were compared [122]. Intranasal inoculation was found to result in a superior
immune priming of the local mucosa, based on the detection of swine influenza
virus-specific antibody in the respiratory tract by ELISA.
For the challenge experiments, the vaccine regimen consisted of two doses of
2 106 TCID50 per pig. Three challenge viruses were employed, H3N2 A/swine/
Texas/4199-2/98, H3N2 A/swine/CO/23619/99, and H1N1 A/swine/IA/00239/04.
Similar results to the intratracheal vaccination were obtained using intranasal
vaccination. Complete, sterilizing protection from challenge with the homologous
virus was observed, while the titers of the drifted A/swine/CO/23619/99 virus in
nasal swabs and lung lavage samples were reduced by around 100,000-fold, to
approximately 0.5 log10 pfu. Animals challenged with the H1N1 virus strain also
had statistically significant reduction in fever and virus titers.
Thus, in the vaccination challenge study, live virus vaccination through the
intranasal route produced an immune response that provided effective and broad
protection, including limited heterosubtypic protection, similar to the results seen
with intratracheal vaccination.
7.3 Studies in Horses
Studies characterizing the growth of equine influenza viruses containing truncated

NS1 proteins in equine hosts have also been performed, to assess the suitability of
NS1-based vaccines in horses. The results from experiments using the reverse
genetics derived influenza A/eq/KY/5/02 strains KY/02/NS1-126, KY/02/NS1-99,
and KY/02/NS1-73 broadly mirror results obtained with the swine influenza viruses
in cell culture and in a mouse model, with attenuated growth of NS1-truncated
viruses observed relative to the wild-type virus [123]. In mouse lung, titers of the
NS1-truncated viruses were reduced by 50-fold (KY/02/NS1-73) to greater than
100,000-fold (KY/02/NS1-126), relative to wild-type virus. Each of the three NS1-
truncated viruses was administered intranasally to horses to ascertain safety and
immunogenicity. No animals developed any clinical signs of infection, while KY/
02/NS1-126 and KY/02/NS1-73 virus-infected animals seroconverted against
influenza virus by 2 weeks postvaccination, as assayed by single radial hemolysis.
Subsequently, a vaccine challenge study was performed in horses. The study
demonstrated that, following homologous wild-type challenge, the KY/02/NS1-126
vaccine strain significantly reduced clinical signs of infection, conferred protection
from febrile response, reduced peak virus shedding by at least 100-fold, and
reduced duration of shedding [124]. These results confirmed the findings of the
vaccine studies performed in pigs and extended the results to a second natural host
of influenza virus.
7.4 Studies in Birds
Because of their extreme virulence, highly pathogenic H5N1 influenza viruses have
caused significant problems for the poultry industry, and have occasionally caused
severe disease in mammals [125, 126]. The potential of NS1-truncated LAIV to
208 N. Pica et al.
Fig. 2 vRNAs of purified A/Viet Nam/1203/04 recombinants. RNA was extracted from A/PR/8/
34 and A/VN/1203/04 recombinant viruses and then separated on a polyacrylamide gel, followed
by silver staining. The viral RNA gene segments are labeled: polymerase (Ps), HA (PR8), HALo
(A/VN/1203/04), NP, NA, M, and NS (of varying lengths). Recombinant viruses contained either
lysine or glutamic acid at amino acid position 627 in PB2. Adapted from [127]
mitigate this problem was examined by testing a panel of eight candidate live
attenuated influenza vaccine viruses for poultry. The viruses were based on the
strain A/VN/1203/04 and had truncated NS1 proteins. In addition to modification of
the NS1, two further alterations to the viral genome were introduced: removal of the
polybasic cleavage site in the HA protein of each candidate vaccine and alteration
of the amino acid at position 627 of polymerase basic 2 protein from lysine to
glutamic acid (to attenuate viruses in mammalian systems). The viruses were
generated by reverse genetics, and their genotypes were initially confirmed through
in vitro characterization (Fig. 2) [127]. Similar to results of previous studies with
NS1-truncated viruses, growth was attenuated and the viruses induced high levels
of interferon in mammalian substrates; nevertheless, each recombinant virus grew
to high titer in embryonated chicken eggs [127]. All eight vaccine candidates were
found to be markedly attenuated in a mouse model and to provide protection against
lethal challenge following a single vaccination dose (Table 2).
A single vaccine that was selected for testing in chickens also protected the
chickens against stringent challenge (100 CLD50) with HPAI (H5N1) viruses. One
hundred percent protection from death was observed with homologous challenge,
and 88% protection was conferred against challenge with a heterologous H5N1
strain (Table 3).
In a separate study using an avian influenza virus with a naturally truncated NS1
protein (A/turkey/Oregon/71-delNS1 (H7N3)), Wang et al. [128], observed that
chickens inoculated with the NS1-truncated virus showed no signs of disease and
evidence of only extremely limited virus replication. Nevertheless, the birds were
protected against a high-dose challenge (106 EID50) with a heterologous H7N2
Table 2 Summary of the genotype and phenotype in mice of candidate vaccine viruses
Virus name1 Genotype2 MDT3 MLD504 Maximum Lowest
(h) weight loss5 protective dose
(EID50)6
VN HALo/ PB2 627K, PB1, PA, HALo, 64, 61 >10 6
18% (4) 103
627K/NS NP, NA, M, NS
FL
VN HALo/ PB2 627K, PB1, PA, HALo, 45, 54 >106 15% (3) 104
627K/NS NP, NA, M, NS 1 126
1 126
VN HALo/ PB2 627K, PB1, PA, HALo, 54, 49 >106 nd 104
627K/NS NP, NA, M, NS 1 99
1 99
VN HALo/ PB2 627K, PB1, PA, HALo, 62, 36 >106 19% (8) 104
627K/NS NP, NA, M, NS 1 73
1 73
VN HALo/ PB2 627E, PB1, PA, HALo, 58, 61 >106 2% (9) 104
627E/NS NP, NA, M, NS
FL
VN HALo/ PB2 627E, PB1, PA, HALo, 51, 54 >106 nd 106
627E/NS NP, NA, M, NS 1 126
1 126
627E/NS NP, NA, M, NS 1 99
1 99
627E/NS NP, NA, M, NS 1 73
1 73
Adapted from [127]
1
Refers to the abbreviated description of the genotype used to identify each virus. Numbers
following “NS” refer to the number of amino acids present in the NS1 protein starting from the
amino terminal methionine. “FL” indicates full length
2
All segments are derived from the A/Viet Nam/1203/04 (H5N1) virus. HALo refers to the HA
segment of A/Viet Nam/1203/04 with the polybasic cleavage site removed, as described in Fig. 2.
Numbering following the PB2 segment refers to the identity of the amino acid residue 627 in the
PB2 protein
3
Mean time to death of eggs infected with VN1203 viruses. The results of two independent
experiments are shown
4
The number of EID50 units required to kill 50% of groups of 6 to 8 week old C57BL/6 mice
(n 4)
5
The maximum average weight loss of groups of mice (n 4) upon vaccination with 106 EID50 of
virus. nd no weight loss detected. Values in brackets represent standard deviation from the mean
6
The lowest dose of vaccination virus that subsequently conferred 100% protection from death
following inoculation with 1,000 MLD50 of challenge virus
virus. Viral load and duration of shedding were significantly reduced relative to
mock vaccinated control animals. The results obtained in this study, as well as those
of Steel et al. [127], support the idea that NS1-truncated virus vaccines are broadly
applicable against avian influenza.
In addition to high protective efficacy, NS1-modified LAIV offers the advantage
of allowing differentiation between vaccinated and naturally infected chickens
210
Table 3 Serological, clinical, and virological outcomes of chicken vaccination and challenge
Group Shedding of vaccine virus in HI/SN titer1 Protection Virus shedding after challenge
trachea (EID50/ml) (day 3, from death (Maximum TCID50/mL)
day 5) Homologous Heterologous Tracheal Cloacal
VN HALo/627E/NS 1- <10–4.8103, <10 20–640/<40–320 <20–320/ND–320 6/6 <50 <50
99/VN1203 challenge
VN HALo/627E/NS 1- <10–480, <10–48 20–640/<40–320 80–640/ND–160 5/62 <50 <50
99/Egret06 challenge
Mock/VN1203 challenge <10, <10 <20/<40 <20/<40–40 0/4 2.32104–1.08105 5.00105–5.00106
Mock/Egret06 challenge <10–20, <10 <20–20/<50 <20/<40 2/4 2–1.08106 1.08104–1.58106
1
Hemagglutination inhibition (HI) and serum neutralizing (SN) titers are given as the reciprocal of the highest dilution of serum that showed activity. Homologous
refers to activity against A/Viet Nam/1203/04 virus, and heterologous refers to activity against A/egret/Egypt/01/06 virus. ND ¼ not determined
2
One bird was alert only when personnel were nearby by 7 days postchallenge (dpc). Disease progressed thereafter, culminating in the development of
neurological signs by 11 dpc when the animal was euthanized
N. Pica et al.
using serological analyses. DIVA (differentiate infected from vaccinated animals)

compatibility is a highly desirable feature of vaccines to be used in agricultural
species because many countries restrict the import of livestock testing positive for
HPAI. Although this concept remains to be rigorously tested, the humoral response
to NS1-modified LAIV should differ from that induced by wild-type virus in that
the former would not include antibodies to the C-terminus of the NS1 protein.
7.5 Studies in Macaques
To gain a better understanding of the cellular mechanisms underpinning protection

conferred by live attenuated virus vaccination, Baskin et al. [129] compared the
protective immune response provoked by inactivated influenza vaccine and an
NS1-truncated LAIV in a macaque model. The authors analyzed both the serologi-
cal and functional genomic immune responses following (1) vaccination and (2)
challenge with the human influenza virus strain, A/Texas/36/91 (H1N1). Three
treatment groups were used in the study: (1) intranasally vaccinated with a single
dose of live vaccine virus, (2) intraperitoneally vaccinated with a single dose of
inactivated vaccine, or (3) unvaccinated [129].
Initial analyses demonstrated that no clinical signs of disease were observed
following vaccination with 6 107 pfu of the live virus vaccine TX/91/NS1-126
(H1N1), and no significant lung pathology was observed (on day 4) postvaccina-
tion, suggesting that the NS1-truncated virus is safe and suitable as a live attenuated
vaccine. Interestingly, global analysis of gene transcription 2 days after vaccina-
tion, using a macaque oligonucleotide array, demonstrated a stronger induction of
interferon-related genes in the lungs of the LAIV group, relative to the killed-
vaccine and unvaccinated groups. This result indicated that a qualitatively different
immune response was induced with the LAIV, most likely due to the growth of
LAIV and interferon stimulation by live virus in the lung [129].
At 21 days postvaccination, all animals were challenged with 6 107 pfu of the
human influenza virus A/Texas/36/91 (H1N1), and tissues were collected 4 days
postchallenge. The frequency of lung lesions, the levels of viral mRNA present in
those lesions, and the severity of inflammation in the upper respiratory tract were all
lower in animals that received the LAIV than in control, unvaccinated, animals and
in animals that received the inactivated virus vaccine. Analysis of postchallenge
blood samples revealed that animals vaccinated with the LAIV had a sevenfold
increase in the number of influenza-specific CD4þ T-cells, higher IgG levels, and
higher HI titers in serum relative to the other groups [129].
Examination of global expression profiles revealed that the LAIV group had a
less pronounced activation of innate immune genes at 4 days postchallenge than did
the killed vaccine and control groups, most likely due to lower levels of replication
of the challenge virus in the lungs at that time. Conversely, activation of cellular
gene pathways associated with the induction of B and T cells was higher in the
lungs of animals that received the LAIV. Thus, there appears to be a relationship
between LAIV, a reduced innate immune response on challenge, and the presence
212 N. Pica et al.
a c
24 h 24 h
Mock vaccinated
b Vaccinated
Naive contact
d Inoculated contact
Inoculated Inoculated
Exposed Exposed
7 7
Nasal wash titer (log10PFU/ml)

6 6
5 5
4 4
3 3
2 2
1 1
0 0
0 2 4 6 8 10 0 2 4 6 8 10
Day post-inoculation Day post-inoculation
Transmission: 0/4 Transmission: 2/2
e g
24 h 24 h
Vaccinated Mock vaccinated
f Inoculated contact h Inoculated contact
Inoculated Inoculated
Exposed Exposed
8 7
7 6
6 5
5
4
4
3
3
2 2
1 1
0 0
0 2 4 6 8 10 0 2 4 6 8 10
Day post-inoculation Day post-inoculation
Transmission: 0/4 Transmission: 2/2
Fig. 3 Vaccination with an NS1 truncated LAIV provides 100% protection against transmission
of the homologous strain and sterilizing immunity against homologous challenge. (a) Schematic
representation of challenge by the intranasal route. Four guinea pigs previously vaccinated with
LAIV (red) were challenged intranasally with 1,000 pfu of Pan/99 virus. At 24 h postinoculation, a
naive contact animal (blue) was cocaged with each of the inoculated guinea pigs. (b) Results of
homologous challenge by the intranasal route. No virus was detected in the nasal washings of
challenged guinea pigs (red squares with dashed lines) or of the naive contact animals (blue
triangles with solid lines). (c) Schematic representation of challenge of mock vaccinated guinea
pigs by the intranasal route. Two previously mock vaccinated control animals (black) were
inoculated intranasally with Pan/99 virus. At 24 h postinoculation, a naive contact guinea pig
(white) was cocaged with each of the inoculated guinea pigs. (d) Results of Pan/99 challenge of
of a stronger, more mature adaptive immune response [129]. Whether this response
is specifically stimulated by the high interferon-inducing NS1-truncated vaccine or
is a property of LAIV in general is of great interest.
8 Blocking Influenza Transmission by Vaccination

with NS1-Modified LAIV
The Centers for Disease Control and public health organizations in many countries
recommend that household contacts of at-risk persons receive annual influenza
vaccinations. This advice is based on the concept of vaccinating to block transmis-
sion and highlights the utility of the indirect effectiveness of immunization [1].
Clinical trials have confirmed the benefit of vaccination with the aim of preventing
transmission [130, 131]. Nonetheless, efficacy in preventing disease is the primary
measure in the evaluation of new influenza vaccines; the potential of a vaccine to
disrupt the chain of transmission is seldom considered.
Using the guinea pig model, Lowen et al. evaluated immunization with an NS1-
truncated LAIV in terms of its potential to reduce interhost transmission of influ-
enza viruses [132]. Immunity from the NS1-truncated LAIV was compared with
that obtained by natural infection and by vaccination with an inactivated influenza
virus preparation. Both vaccines were applied in two doses, spaced 3 weeks apart.
In each case, immunized animals acted as either donors or recipients in transmis-
sion; in this way, the efficacy of vaccination in blocking transmission from and
to treated guinea pigs was tested. All three modes of immunization were found
to reduce transmission to and/or from vaccinated animals. Natural infection was the
most effective, providing sterilizing immunity against homologous challenge and
heterologous challenge with a drift variant virus. The NS1-truncated LAIV also
provided sterilizing immunity against homologous challenge and very good protec-
tion from transmission of the heterologous strain (Fig. 3). Although vaccination
◂
Fig. 3 (continued) mock vaccinated guinea pigs by the intranasal route. Mock vaccinated guinea
pigs were productively infected through inoculation (black squares with dashed lines) and trans
mitted efficiently to naive contact animals (white triangles with solid lines). (e) Schematic
rep‘resentation of challenge through exposure to an infected guinea pig. Four naive guinea pigs
were inoculated intranasally with Pan/99 virus. At 24 h postinoculation, each acutely infected
animal (blue) was placed into the same cage with one previously vaccinated guinea pig (red). (f)
Results of homologous challenge through exposure to an infected guinea pig. Intranasally infected
contact animals shed high titers of virus into nasal washes (blue squares with dashed lines); no
virus was detected in nasal washes of the four vaccinated animals (red triangles with solid lines).
(g) Schematic representation of challenge of mock vaccinated animals through exposure to an
infected guinea pig. Two naive contact animals were inoculated intranasally with Pan/99 virus. At
24 h postinoculation, two previously mock vaccinated guinea pigs were each placed into the same
cage with one infected animal. (h) Results of control challenge through contact with an infected
guinea pig. Intranasally infected contact animals shed high titers of virus into nasal washes (white
squares with dashed lines), and both mock vaccinated guinea pigs (black triangles with solid
lines) became infected through contact with the infected animals. Adapted from [132]
214 N. Pica et al.
with the inactivated virus was found to induce high titers of HI antibodies
(comparable to those obtained with natural infection and LAIV) and to reduce
viral load in vaccinated guinea pigs, protection against transmission was moderate.
Upon homologous challenge, transmission from guinea pigs that had received the
inactivated vaccine was reduced only by 50%, and transmission to guinea pigs
vaccinated with the killed virus was not reduced. Thus, similar to the situation
following natural infection, intranasal vaccination with an NS1-truncated LAIV
was found to be highly effective in blocking secondary transmission from and
to animals that had received the vaccine. These findings support the use of
live vaccines for influenza, and if the results extend to humans point to a simple
and effective way to protect individuals who are less responsive to direct vaccina-
tion from contracting influenza virus infection.
9 Conclusions
LAIV vaccines confer effective protection against influenza virus infection. Despite
the effectiveness of CAIV-T, other attenuation techniques could provide enhance-
ments to the immunization options that are commercially available at present. Over
the last 10 years, a considerable number of in vitro and in vivo studies have been
conducted with NS1-truncated influenza viruses. The results have consistently
indicated that viruses with C-terminal truncations in the NS1 protein are attenuated
in growth in vitro and in vivo (in a number of animal model systems), do not
generate signs of disease in animals, and stimulate the production of IFN in systems
competent to do so. Increasing evidence from these studies suggests that one dose
of vaccine may be sufficient to generate a strong, protective immune response in a
number of host species, and that this response is most likely broader in terms of
cross-protection amongst strains than conventional inactivated vaccines. The effi-
cacy of NS1-truncated LAIV has been demonstrated to extend not only to disease
prevention but also to prevent transmission between animals. It is therefore likely
that NS1-truncated LAIV vaccines are safe and protective in humans.
Acknowledgments This work was supported by the National Institutes of Health Grants PO1
AI058113, PO1 AI59443, U19 AI062623 023, T32 AI07647, HHSN266200700010C, RC1
AI086061, U54 AI057158, U01 AI070469.
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An Attenuated HSV-1 Live Virus Vaccine
Candidate that is Replication Competent
but Defective in Epithelial Cell-to-Cell
and Neuronal Spread
Elizabeth E. Zumbrun and Harvey M. Friedman
Abstract Live attenuated vaccines represent the most successful approach for the
prevention of alphaherpesvirus infections, including varicella zoster virus, pseu-
dorabies virus, and equine herpes virus 1. It is reasonable to consider that live virus
vaccines may also be effective for the prevention of other alphaherpesviruses, such
as herpes simplex virus 1 (HSV-1) and 2 (HSV-2). An HSV-1 mutant strain that is
deleted in glycoprotein E (gE), NS-gEnull, is replication competent but is defective
in spread from one epithelial cell to another, from epithelial cells to axons, and from
the neuron cell body into axons. The defect in spread likely accounts for the
favorable safety profile of the live virus vaccine candidate in mice. The NS-gEnull
mutant is also defective in immunoglobulin G (IgG) Fc receptor binding, which is a
process used by the virus to escape antibody attack. NS-gEnull when used as an
immunogen is highly effective in providing protection against epidermal and
vaginal challenge by wild-type (WT) HSV-1 and HSV-2. NS-gEnull represents a
novel HSV-1 vaccine approach that retains replication competency while impairing
virus spread at the inoculation site and in neurons. Only gE is deleted from the
vaccine strain, ensuring that most viral antigens are presented to the host. This
strategy is worth considering for prevention of HSV-1 and possibly HSV-2.
1 Introduction
Attenuated live virus vaccines are safe and effective for several members of the
alphaherpesvirus family, including varicella zoster virus (VZV), pseudorabies virus
(PRV), and equine herpes virus 1 (EHV-1) [1 4]. Nevertheless, no effective vaccine
E.E. Zumbrun
Center for Aerobiological Sciences, US Army Medical Research Institute of Infectious Diseases,
Fort Detrick, Frederick, MD 21702, USA
H.M. Friedman (*)
Infectious Disease Division, Department of Medicine, University of Pennsylvania School of
Medicine, 502 Johnson Pavilion, Philadelphia, PA 19104 6073, USA

224 E.E. Zumbrun and H.M. Friedman
is available to protect humans against herpes simplex virus 1 (HSV-1) or 2 (HSV-2)

infection. HSV-1 and HSV-2 cause significant morbidity and occasional mortality in
humans with 58% of people in the United States ages 14 49 testing seropositive for
HSV-1 and 17% for HSV-2 [5]. HSV-1 generally causes lesions on the vermillion
border of the lip and HSV-2 typically causes genital ulcers.
An attenuated live virus vaccine that is replication defective is currently under
development for prevention of HSV-2 infection (see chapter “Replication-defective
Herpes Simplex Virus mutant strains as genital herpes vaccines and vaccine
vectors” by D.M. Knipe) [6]. Another novel approach uses a replication competent,
neuronal spread defective HSV-1 that is also defective in evasion of host antibody
responses [7]. This attenuated HSV-1 strain has a large deletion within the unique
short 8 (Us8) gene encoding glycoprotein E (gE) and is the focus of this chapter.
The characterization of this virus revealed its potential utility as an attenuated
vaccine [8].
2 Lifecycle of Alphaherpesviruses
A common feature shared by alphaherpesviruses is that they infect neurons of the

peripheral nervous system (PNS). The neurons of the PNS are also the site where
alphaherpesviruses establish life-long latent infection. In their natural host, these
viruses periodically reactivate, replicate in the cell body of PNS neurons, and
spread along axons to the mucosa or epithelial cells innervated by the neurons.
Reactivation of VZV, HSV-1, and HSV-2 frequently causes itching and discomfort
and produces virus-filled vesicles in the skin or at mucocutaneous borders.
3 Varicella Zoster Virus
VZV causes chickenpox and shingles. Chickenpox is no longer a common child-

hood illness in developed countries due to an attenuated vaccine, VZV Oka, derived
from serial passage of a Japanese clinical isolate. VZV Oka contains a number of
mutations throughout the genome and although lesions may develop at the inocula-
tion site, the vaccine strain rarely causes serious sequelae [9]. VZV Oka is effective
in preventing chickenpox in children, while vaccination of older individuals
reduces the incidence of shingles. In those individuals who develop shingles despite
immunization, the vaccine modifies the severity of postherpetic neuralgia, which is
prolonged and often debilitating pain associated with shingles [10, 11]. The VZV
vaccine thus represents an effective live attenuated virus vaccine against an alpha-
herpesvirus that is safe and effective in humans. This success supports efforts for a
similar vaccine approach against two other human alphaherpesviruses, HSV-1 and
HSV-2.
An Attenuated HSV 1 Live Virus Vaccine Candidate 225
4 Pseudorabies Virus and Equine Herpes Virus 1
PRV is an alphaherpesvirus for which the natural host is the adult pig. Infection of
adult pigs causes poor weight gain and abortion in pregnant sows. Infection of
newborn pigs less than 1 month of age is virtually 100% lethal [12]. PRV infection
of other secondary hosts such as cows, cats, dogs, rats, and mice is uniformly lethal,
causing death within 2 3 days [12]. In many secondary hosts, clinical signs include
a “mad itch” followed by neurological signs that precede death. Humans are not
susceptible to PRV infection. Disease caused by PRV can be economically devas-
tating to the pork industry; therefore, a vaccine was developed in 1961 by serial
passage in tissue culture. The resulting attenuated vaccine, PRV-Bartha, has a large
deletion within the Us coding region, encompassing several genes that are neces-
sary for anterograde spread (defined as movement of virus from one cell to another
that includes movement in axons away from the neuron cell body) [13]. These
genes include Us7, Us8, and Us9 that encode the gI, gE, and Us9 proteins,
respectively. PRV-Bartha is defective in anterograde spread, but retrograde spread
(defined as movement of virus from one cell to another that includes movement in
axons toward the neuron cell body) is intact. Therefore, this vaccine strain has been
used experimentally as a retrograde neuronal tracing virus [14]. PRV-Bartha is
highly effective in protecting swine from infection [15].
Attenuated vaccine approaches have also been successful in preventing infection
of horses with the alphaherpesvirus EHV-1. One attenuated EHV vaccine candi-
date, Kentucky A (KyA), is notable for its similarity to the attenuated PRV vaccine
[16]. KyA was produced by serial propagation in Syrian hamsters followed by
passage in a murine cell line. KyA contains a large deletion in the Us region of the
genome encompassing the genes encoding gE and gI. Another attenuated EHV-1
candidate vaccine strain is defective in gE alone [3]. These vaccine strains are
highly attenuated in horses but only partially protect from respiratory symptoms
after challenge [2, 3, 17, 18].
The attenuation of the PRV-Bartha and EHV-1 KyA strains was done by serial
passage and did not specifically target particular genes; however, it is notable that
both viruses contain deletions of genes required for efficient anterograde spread in
neurons. Extensive similarities exist in genome organization and sequences of
PRV, EHV-1, HSV-1, and HSV-2, and in requirements for neuronal spread as
part of the virus lifecycle. Therefore, an approach of targeted attenuation of HSV-1
or HSV-2 by deletion of a gene or multiple genes required for neuronal spread
represents a potentially effective means for vaccination of humans.
5 Characterization of HSV-1 gE
HSV-1 gE is a type-1 membrane glycoprotein that is incorporated into the virion

envelope and is also expressed on the surface of infected cells. An HSV-1 gE
deletion strain was constructed from a low passage clinical isolate (NS). The gE
deletion strain retains a portion of the Us8 sequence encoding the first 123 amino
acids, but deletes amino acids 124 510, including the transmembrane domain that
is replaced by a LacZ reporter gene [7]. The 30 region of Us8 encoding amino acids
511 552 is not deleted but is not in frame, and therefore should not be expressed.
This HSV-1 gE deletion virus, referred to as NS-gEnull, was evaluated as an
attenuated HSV-1 vaccine candidate. A number of linker scanning mutants within
the Us8 gene were also constructed within the HSV-1 NS background to further
characterize specific gE functions, as discussed later in this chapter.
NS-gEnull does not produce a gE protein, while adjacent genes Us7 and Us9 are
not affected, as assessed by Western blot [19]. NS-gEnull has intact single-step
growth kinetics in Vero cells. When evaluated for cell-to-cell spread in human
epidermal (HaCaT) cells, NS-gEnull forms plaques that are approximately fourfold
smaller than WT virus at 48 h postinfection (hpi) [20, 21]. Repair of the Us8
deletion in NS-gEnull restores the WT plaque phenotype, indicating that gE is
required for efficient cell-to-cell spread. As a vaccine candidate, the ability of
NS-gEnull to replicate normally may provide an advantage in priming the host
immune system compared with replication defective strains. The cell-to-cell spread
defect of NS-gEnull is an important safety feature of the live virus vaccine.
6 Defining the Role of HSV-1 gE in Anterograde

and Retrograde Spread
The mouse flank scarification model is useful for HSV pathogenesis studies. Five-
to six-week-old female BALB/c mice were anesthetized and the hair removed from
their right flanks by shaving followed by application of depilatory cream that is then
rinsed off with water. The next day, mice were again anesthetized and a 10-ml
droplet containing virus at the desired titer was applied to the surface of the
denuded flank skin. Thirty to forty gentle scratches of approximately 0.5 cm in
length were made in different directions through the droplet using a 26 5/8-gauge
needle [7].
Following flank scarification, HSV-1 replicated at the inoculation site forming a
lesion by 3 days postinfection (dpi). During that time, HSV-1 spreads cell to cell in
the epithelial cell layer of the skin and enters local sensory nerves that innervate the
skin. Virus enters the axon terminus and spreads to the cell body of the neuron
located in the dorsal root ganglia (DRG). HSV-1 replicates in neuron cell bodies,
infects adjacent neurons in the DRG, and spreads to the axon terminus and then
to epidermal cells in the skin. Replication occurs in the skin resulting in lesions by
4 5 dpi that are confined to the dermatome innervated by the DRG (zosteriform
lesions) [22]. Therefore, zosteriform disease requires both retrograde and antero-
grade spread.
Mice infected with WT HSV-1 typically die 8 10 dpi; however, infection with
NS-gEnull results in no death or zosteriform disease, while inoculation site disease
is no different than after mock scarification [23]. Therefore, the lack of virulence
of NS-gEnull as a potential vaccine candidate is notable. Virus titers performed at
the inoculation site demonstrate 5log10 less virus in mice infected with NS-gEnull
than WT virus by 3 dpi. Additionally, less viral antigen is observed by immuno-
histochemistry of sectioned skin. These in vivo results can be explained by the cell-
to-cell spread defect observed in cultured cells. NS-gEnull titers in DRG are
negative at 1, 3, 6, and 8 dpi compared with peak WT virus titers of 4log10 at
3 dpi. The negative NS-gEnull DRG titers can also be explained by a cell-to-cell
spread defect in the epidermal cells or by a defect in spread from epidermal cells to
innervating neurons [23]. The failure of NS-gEnull to infect DRG adds an important
safety feature to the candidate vaccine, since DRG are the site of latency for the
virus. Since no virus reaches the DRG, the flank model cannot be used to assess the
spread of NS-gEnull from DRG to skin (anterograde).
We chose the mouse retina infection model to assess NS-gEnull anterograde
spread. Five- to eight-week-old BALB/c mice were anesthetized and a small cut
was made in the sclera with a 30-gauge needle [8]. This needle was then used to
puncture the vitreous body of the eye. A Hamilton syringe was used to inject virus
into the vitreous body through the same puncture hole. The ganglion cell neurons
comprise the innermost layer of the retina and are the first neurons to become
infected. The axons from these neurons form the optic nerve. Three to five dpi
with WT virus (NS) or NS-gEnull, eyes, optic nerves, and brains were removed,
sectioned, and stained for HSV-1 antigen. This staining revealed a robust infection
of the retina with WT and NS-gEnull strains, although more antigen was detected in
retina infected with WT virus, which is consistent with in vitro results demonstrat-
ing a cell-to-cell spread defect for NS-gEnull.
Optic nerve sections from mice injected with NS-gEnull showed no HSV-1
antigen when stained with an anti-HSV-1 polyclonal antibody, compared with
abundant antigen seen after infection with WT virus or the rescue strain, rNS-
gEnull [8]. These results indicate that HSV-1 gE is required for spread of virus from
the retina ganglion cell neurons to the optic nerve. Antigen staining for HSV-1
envelope glycoproteins gB, gC, and gD revealed that none of these envelope
proteins was present in the optic nerves of NS-gEnull-infected mice. Additionally,
the tegument protein, VP22, and capsid protein, VP5, were not detected in the optic
nerve after retina infection with NS-gEnull. Therefore, HSV-1 gE is required for
spread of HSV-1 envelope, tegument, and capsid proteins from the cell bodies of
retina ganglion cell neurons into their axon fibers in the optic nerve. This spread
defect is another safety feature of NS-gEnull as a vaccine candidate.
Further support for the requirement of HSV-1 gE for spread in vivo comes from
an analysis of brain sections from WT or NS-gEnull-infected mice [8]. After retina
infection with WT virus, brains contained antigens in the optic tract, dorsal, and
ventral lateral geniculate nuclei and superior colliculus, which represent regions of
the brain reached by anterograde spread. Additionally, antigens were detected in
brain nuclei reached by retrograde spread of virus from structures in the eye
or orbit, including the intergeniculate leaflet of the lateral geniculate nucleus,
Edinger Westphal, and oculomotor nuclei. No viral antigens were detected in
any region of the brain following NS-gEnull infection, indicating defects in both
anterograde and retrograde spread. The failure of NS-gEnull to reach nuclei in the
brain by routes involving retrograde spread is consistent with the observation in the
flank model that the virus failed to infect DRG.
An in vitro neuronal culture system was employed to further assess the neuronal
spread properties of NS-gEnull [24]. In this system, 17-day embryos were removed
from pregnant Sprague-Dawley rats and sympathetic motor neurons were estab-
lished in Campenot chamber cultures by dissecting the superior cervical ganglia
(SCG) [20]. Campenot cultures contain three separate chambers, the Soma (S),
Middle (M), and Neurite (N) compartments (Fig. 1a). Dissociated SCG neurons are
plated in the S chamber and allowed to differentiate for approximately 2 weeks.
Axons (neurites) sprout from these neuronal cell bodies and grow through a silicone
grease barrier that separates the S and M chambers. The M chamber is filled with
1% methylcellulose to prevent the diffusion of virus between chambers. By 2 3
weeks, neurites grow into the N chamber through a silicone grease barrier that
separates the M and N chambers.
NS-gEnull was added to the S chamber neurons and epithelial cells were added
on top of neurites in the N chamber to evaluate virus spread from neuron cell bodies
to epithelial cells (Fig. 1c). NS-gEnull replicated to levels comparable to WT or
rescue (rNS-gEnull) virus in the S chamber, as determined by titering the contents
of the S chamber at 24 and 48 hpi. In contrast, no NS-gEnull was detected in the N
chamber, compared with 4-5log10 WT or rescue virus at 48 hpi, indicating an
impressive spread defect of NS-gEnull from the neurons in the S chamber to
epithelial cells in the N chamber [20]. Immunofluorescent studies of SCG neurons
Fig. 1 (a). Campenot chamber consists of a Teflon ring that divides the culture dish into three
compartments. SCG neurons are placed in the Soma (S) chamber. Over time, the neurons extend
their axons (neurites) along pin rake groves that are made in the culture dish to guide the growth of
the neurites, which penetrate into the Middle (M) chamber and then Neurite (N) chamber. Prior to
infection of S chamber neurons, the M chamber is filled with methylcellulose to prevent virus
leaking between chambers. (b). For some experiments, virus is added to the N chamber and
neurons are harvested in the S chamber to determine virus transport from axons to neuron cell
bodies. (c). For some experiments, epithelial cells are added to the N chamber prior to infection of
neurons in the S chamber. Contents of the N chamber are harvested to determine spread of virus
from epithelial cells in the N chamber to neurons in the S chamber. Figure is reprinted with
permission of the editor of the Journal of Virology
infected with NS-gEnull demonstrated viral antigens in the cell body but not in the
axons, supporting a role for gE in targeting viral antigens from the neuron cell body
into axons [8]. This in vitro result is consistent with the in vivo observation in the
mouse retina infection model that failed to detect NS-gEnull antigens in the
optic nerve. The mechanism by which HSV-1 gE mediates axonal localization is
unknown and currently under evaluation.
Further experiments were performed to evaluate the contribution of gE to
retrograde spread. WT virus or NS-gEnull was added directly to axons in the N
chamber and virus transport was assessed by titering the contents of the S chamber
(Fig. 1b) [20]. No differences were detected comparing WT and NS-gEnull virus
titers in the S chamber. Therefore, gE is dispensable for virus transport from the
axon terminus to the neuron cell body. The Campenot chamber system was then
modified to test the ability of WT virus or NS-gEnull to spread from epithelial cells
to neurites by seeding HaCaT cells over the neurites in the N chamber. Virus was
added to the HaCaT cells in the N chamber and assayed for spread to the S chamber.
One-hundred-fold less NS-gEnull than WT virus was detected in the S chamber.
Therefore, the defect in NS-gEnull spread stems from the contribution of gE to
virus spread from epithelial cells to axons, which is consistent with a cell-to-cell
spread defect noted in epithelial cells [25]. Table 1 summarizes replication and
spread properties of the HSV-1 gE deletion mutant, NS-gEnull.
The mechanism by which gE contributes to spread from epithelial cells to axons
may relate to the observation that gE is required to target virus to the basolateral
surface of polarized epithelial cells [26]. HSV-1 gE mutant virus is transported
Table 1 Replication and spread phenotypes of NS gEnull

Replication that requires a single growth cycle in Vero cells or Similar to WT virus
SCG neurons
Replication that requires virus spread from cell to cell as Greatly reduced relative
measured by plaque size or skin titers in the mouse flank to WT virus
Spread from one cell to another that requires virus transport Greatly reduced relative
away from the neuron cell body (anterograde) in Campenot to WT virus
chambers (from S to N chamber) or in the mouse retina
infection model (from retina to specific nuclei in the brain such
as the superior colliculus nucleus)
Spread from one cell to another that requires virus transport in Greatly reduced relative
axons toward the neuron cell body (retrograde) in Campenot to WT virus
cultures (from epithelial cells in the N chamber to SCG
neurons in the S chamber), mouse flank (from skin to DRG), or
mouse retina model (from eye to specific nuclei in the brain
such as the Edinger Westphal or oculomotor nuclei)
Targeting of viral antigens from the neuron cell body into axons in Greatly reduced relative
SCG cultures (infect neurons and observe for viral antigens in to WT virus
axons) or mouse retina model (infect retina and observe for
viral antigens in the optic nerve)
Targeting of viral antigens from the axon terminus to the neuron Similar to WT virus
cell body in SCG cultures (infect N chamber axons and titer
virus in S chamber neurons)
primarily to the apical surface of polarized epithelial cells, which may explain the
cell-to-cell spread defect of gE defective virus. An elucidation of the cellular
binding partners of gE in epithelial and neuronal cells may help clarify mechanisms
of gE-mediated virus spread.
7 Defining the Role of HSV-1 gE in IgG Fc Binding Activity
Another important function of HSV-1 gE is its role as an IgG Fc receptor (FcgR) in

the immune evasion of host IgG antibody responses [27]. Earlier work identified gE
and gI as heterodimers that bind the IgG Fc domain [28, 29]. HSV-1 gE forms a
lower affinity receptor for Fc, whereas the gE gI complex constitutes a higher
affinity receptor [30]. The FcgR activity of gE was evaluated using a rosetting assay
in which sheep erythrocytes were coated with anti-sheep erythrocyte IgG and then
incubated with HSV-1-infected cells. WT virus-infected cells express gE at the cell
surface and formed rosettes (defined as 4 erythrocytes per infected cell), whereas
uninfected cells or those infected with NS-gEnull failed to form rosettes, indicating
that WT virus expresses a receptor for IgG Fc [27].
Antibody bipolar bridging is a mechanism that explains the immune evasion
activity mediated by the HSV-1 FcgR [27]. The term “antibody bipolar bridging”
stems from studies indicating the HSV-1 FcgR preferentially binds the Fc domain of
anti-HSV IgG compared with nonimmune IgG. This observation led to the hypothesis
that as the Fab domain of an anti-HSV IgG antibody binds to an antigen on the
infected cell surface, the Fc portion of the same antibody molecule binds to the HSV-1
FcgR to form an “antibody bridge”. By binding the IgG Fc domain, the FcgR blocks
downstream effector functions mediated by this domain, including complement-
enhanced antibody neutralization, antibody-dependent cellular cytotoxicity, and
attachment of granulocytes [31, 32]. The hypothesis of antibody bipolar bridging is
supported by the crystal structure of the HSV-1 FcgR bound to IgG Fc [33].
Interestingly, the HSV-1 FcgR binds the Fc domain of human IgG, but not murine
or guinea pig IgG [34]. The failure of the HSV-1 FcgR to bind murine IgG makes
murine models useful for studying its activity. Mice were passively immunized with
human anti-HSV IgG and then infected with either WT or NS-gE339, which is an
HSV-1 gE mutant strain that has an insertion of four amino acids after gE residue 339
[7]. NS-gE339 is defective in FcgR activity but is much less impaired in cell-to-cell
spread than NS-gEnull, making NS-gE339 a useful mutant strain to evaluate FcgR
function independent of spread activity [25]. Mice infected with WT virus developed
severe inoculation site disease when passively immunized with human anti-HSV
IgG; however, the disease was greatly reduced in mice infected with NS-gE339.
Nonimmune human IgG had no effect on disease caused by WT virus or NS-gE339,
while murine anti-HSV IgG modified both viruses to a comparable degree. These
results indicate that the HSV-1 FcgR mediates evasion from human anti-HSV anti-
bodies, and suggest that another safety feature of NS-gEnull as a vaccine candidate
is its inability to evade host IgG antibody responses.
The gE domains that mediate FcgR activity, cell-to-cell spread and neuronal
spread have been partially characterized through linker scanning mutagenesis of the
Us8 gene [8, 25, 35, 36]. Some mutant strains are defective in spread and others in
FcgR activity, suggesting that these functions are mediated by different domains on
the protein. Of note is the fact that all regions responsible for these activities are
absent in NS-gEnull.
8 NS-gEnull as an Attenuated Vaccine Candidate
NS-gEnull has intact single-step replication kinetics but impaired spread from one
epithelial cell to another, from epithelial cells to axons, and from neuron cell bodies
to axons. NS-gEnull is greatly attenuated in the mouse flank infection model
causing no inoculation site disease and no zosteriform site disease or death.
These features make NS-gEnull a novel candidate for a safe live-virus vaccine.
To address immunogenicity and efficacy of the vaccine candidate, the mouse
flank model was again employed. Mice were immunized by flank scarification with
NS-gEnull, or mock immunized, and 28 days later challenged by flank infection on
the opposite flank with 105 PFU of WT virus, HSV-1 NS [23]. One hundred percent
of mock-immunized mice died, while 100% of NS-gEnull-immunized mice sur-
vived. The NS-gEnull-immunized mice had only mild inoculation site disease and
no zosteriform lesions, in contrast to the severe inoculation and zosteriform disease
that developed in mock-immunized mice. Titers of skin at the inoculation site and
DRG following challenge were striking in that no WT virus was recovered from
NS-gEnull-immunized mice, while 4 5log10 PFU were detected in the mock group.
By real-time quantitative PCR, low levels of viral DNA were detected in DRG of
vaccinated mice 1, 3, 6, and 8 dpi, while DNA copy number was 3 4log10 higher in
mock-immunized mice; however, no determinations were performed to distinguish
vaccine from WT virus DNA in the DRG. The results indicate that immunization
with NS-gEnull protects mice from moderate or severe inoculation site disease,
entirely prevents zosteriform disease and death, and results in greatly reduced titers
of challenge virus reaching the DRG.
DRG explant cocultures were performed to assess the efficacy of NS-gEnull
vaccination in preventing the establishment of latency by WT virus challenge [23].
Mice were either mock vaccinated or vaccinated with NS-gEnull by flank scarifi-
cation and challenged by infection of the opposite flank with HSV-1 KOS. The
advantage of KOS over some other HSV-1 strains is that KOS causes severe
zosteriform disease but rarely leads to death when inoculated by the flank route.
Zosteriform disease indicates that the virus has reached the DRG (site of latency)
and returned to the skin. Survival of the animal enables DRG to be harvested after
infection once latency is established. One-hundred percent of mock-vaccinated
mice challenged with KOS developed severe inoculation site and zosteriform site
disease. In contrast, NS-gEnull-vaccinated mice had only mild inoculation site
disease and no zosteriform lesions. Twenty-eight days after KOS challenge, all
mock-immunized mice had recovered and several weeks had passed since the last
signs of disease. DRG were removed and explant cocultures were performed with
Vero cells. The explant cocultures were observed for 20 days for cytopathic effects
as an indication of virus reactivation from latency. Importantly, 100% of DRG from
mock-vaccinated mice reactivated KOS virus compared with 10% of DRG from
NS-gEnull-vaccinated mice. Therefore, NS-gEnull vaccination was highly effec-
tive in protecting the DRG.
To evaluate whether NS-gEnull-vaccinated mice were protected against differ-
ent HSV-1 isolates, mice were challenged by flank scarification with HSV-1 strains
F and 17 [23]. NS-gEnull-vaccinated mice were protected against death and
moderate or severe inoculation site disease. One mouse challenged with F strain
developed mild zosteriform disease, which indicates that the virus reached the
DRG. These challenge experiments support the KOS explant coculture results in
that DRG were greatly, but not totally protected against challenge virus.
Prior HSV-1 infection appears to provide some degree of cross-protection
against HSV-2 [37]. Therefore, an attenuated vaccine for HSV-1 may provide
protection against HSV-2; however, cross-protection may also be problematic if
prior HSV-2 infection reduces the immunogenicity of an HSV-1 vaccine. Note that
a clinical trial of an HSV-2 gD subunit vaccine showed protection of HSV-1
seronegative women but not HSV-1 seropositive women [37]. The explanation
for this result is either that the vaccine failed to improve the cross-protection
already provided by HSV-1 infection or that prior HSV-1 infection reduced the
ability of the HSV-2 gD vaccine to elicit a robust immune response.
NS-gEnull-or mock-vaccinated mice were challenged by flank scarification with
HSV-2 strain 2.12, a low-passage clinical isolate [23, 38]. One-hundred percent of
mock-vaccinated mice developed severe inoculation site and zosteriform site dis-
ease, and 80% died. In contrast, none of the mice vaccinated with NS-gEnull died.
These mice developed mild inoculation site disease and no zosteriform lesions.
DRG explant cocultures were performed on survivors 1 year after challenge and
revealed no reactivation of the challenge virus. Therefore, NS-gEnull provides
robust protection against HSV-2 infection and the establishment of latency.
The finding that NS-gEnull cross-protects against HSV-2 has important implica-
tions. An attenuated HSV-1 vaccine would likely be given at a young age, since
HSV-1 infection is typically acquired early in life. An HSV-2 vaccine would likely
be given in early adolescence. Therefore, robust cross-protection by an attenuated
HSV-1 vaccine against HSV-2 infection may protect young children against HSV-1
while also providing early protection against HSV-2.
VZV immunity wanes after vaccination, which led to a recommendation to
revaccinate children during adolescence. Immunity to natural infection with VZV
also wanes over time, which is the rationale behind vaccinating adults over the age of
60 years to prevent shingles [10]. The duration of protection after immunization with
NS-gEnull was evaluated by flank challenge 1 year after vaccination. Mice were
protected against death, zosteriform disease, and severe inoculation site disease [23].
NS-gEnull protected against WT virus challenge when immunization was given
by skin scarification, intramuscular (IM), or subcutaneous (SubQ) routes [23].
Table 2 Neutralizing antibody responses to NS gEnull immunization and efficacy against chal
lenge by WT virus
Neutralizing antibody responses Highest when NS gEnull given IM compared
with SubQ or flank scarification
Protection against death after flank challenge by No death at 105 PFU challenge
WT (parental) strain
Protection against inoculation site disease by Almost total protection
WT (parental) strain
Protection against zosteriform disease by WT No disease detected
(parental) strain
Protection of DRG against WT (parental) strain No WT virus isolated
at 1, 3, 6, and 8 dpi
Protection against death after flank challenge by No death at 105 PFU challenge
WT isolates other than parental virus
Protection against inoculation site disease by Mild disease detected
WT isolates other than parental virus
Protection against zosteriform disease by WT Mild disease detected
isolates other than parental virus
Protection of DRG against WT isolates other Substantial, but not total protection
than parental virus assessed by attempts to
reactivate latent virus (explant coculture)
Protection against HSV 1 vaginal challenge No death, no disease, and very low vaginal titers
compared with WT virus
Protection against HSV 2 flank challenge No death, mild inoculation site disease, no
zosteriform disease, and no reactivation of
latent virus from DRG
Neutralizing antibody titers were highest after IM and lowest after SubQ immuni-
zation. Both scarification and IM vaccination resulted in complete protection
against death, severe inoculation site disease, and zosteriform disease when
challenged by the WT strain that was used to derive the vaccine virus. IM is a
more acceptable immunization route than skin scarification, since IM inoculation is
used for many vaccines.
Genital infection caused by HSV-1 rather than HSV-2 comprises 30 40% of first
time genital herpes virus infections [39]. Therefore, NS-gEnull immunization was
evaluated for protection against WT HSV-1 vaginal challenge [23]. All mock-
vaccinated mice developed vaginal disease and had high virus titers in vaginal
swabs until 8 dpi. Mortality was 60%. In contrast, NS-gEnull-vaccinated mice all
survived and had no observable vaginal disease. Vaginal swab titers were 2 4log10
lower than in mock-vaccinated mice and were undetectable by 3 dpi. Table 2
summarizes neutralizing antibody responses and the efficacy of protection provided
by NS-gEnull immunization.
9 Conclusions
Studies using NS-gEnull, an HSV-1 gE deletion mutant, demonstrate that it is

replication competent, but defective in epithelial cell-to-cell spread, epithelial cell
to axon spread, and spread from neuron cell bodies into their axons. The vaccine
candidate is also deficient in IgG Fc-mediated immune evasion. These character-

istics represent important safety features for this attenuated live virus vaccine
candidate. In mouse models, the vaccine provides protection against challenge by
skin and mucosal routes and offers cross-protection against HSV-2 challenge. In
addition, the protection provided by NS-gEnull immunization is long-lasting. A
similar approach may be effective for HSV-2 since the HSV-2 gE protein shares
72% amino acid homology with HSV-1 gE.
Additional safety features may be considered for this live virus vaccine
approach. Along with gE, Us9 and gI are involved in anterograde spread in PRV
and HSV-1 [19, 40 42]. PRV-Bartha has a deletion encompassing the genes
encoding gI, gE, and Us9. A similar deletion in HSV-1 or HSV-2 may provide
added safety by further impairing the spread features of the vaccine strain; however,
excessive attenuation may hamper growth kinetics and impair antigen presentation
to the immune system.
Acknowledgment This work was supported by NIH grant RO1 AI033063.
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Live Attenuated Vaccines for Respiratory
Syncytial Virus
Michael N. Teng
Abstract In the five decades since the identification of respiratory syncytial virus
(RSV) as an important pediatric pathogen, no effective vaccine has been developed.
Previous attempts to develop inactivated RSV vaccines resulted in vaccine-
enhanced disease, resulting in a greater focus on the generation of live attenuated
RSV vaccines. However, identifying a live attenuated vaccine candidate that is
appropriately attenuated and sufficiently immunogenic has proven to be difficult.
Recently, reverse genetics systems have been developed for RSV, allowing
researchers to introduce specific mutations into the genomes of recombinant vac-
cine candidates. These systems provide a means of determining the effects of
known attenuating mutations and identifying novel methods of attenuating the
virus without decreasing immunogenicity. In addition, different mutations can be
combined in a single genome to fine-tune the level of attenuation and immunoge-
nicity to achieve the proper balance in a viable vaccine candidate. Current research
into RSV attenuation includes investigation of point mutations responsible for
temperature sensitivity, nontemperature-sensitive attenuating mutations, and dele-
tion of nonessential viral genes that play roles in viral RNA synthesis and/or
inhibition of innate immune responses. Development of an effective RSV vaccine
will likely rely on using reverse genetics systems to optimize the attenuation and
immunogenicity of a live vaccine candidate, while preserving viral replication
in vitro.
Keywords Live attenuated vaccines Paramyxoviruses Pediatric vaccines

Recombinant vaccines Respiratory infections Vaccine development
M.N. Teng
Department of Biochemistry and Molecular Biology, Pennsylvania State University, University
Park, PA 16802, USA
and
Nanomedicine Research Center, Divisions of Translational Medicine and Allergy & Immunology,
Department of Internal Medicine, University of South Florida College of Medicine, 12901 Bruce
B. Downs Blvd., Tampa, FL 33612, USA

238 M.N. Teng
1 Respiratory Syncytial Virus
Respiratory syncytial virus (RSV) is the most important etiologic agent of pediatric
viral respiratory infection and remains a major cause of morbidity and mortality
among infants. Infection rates for RSV in infants have been found to be 68.8 per 100
children for the first year of life, reaching 82.6 per 100 children for the second year
[1]. Lower respiratory tract illness (LRTI) is more common during year 1, though
LRTI occurs frequently during year 2. Approximately half of all children are
reinfected by age 2, but most children experience only 1 LRTI [1]. RSV infection
accounts for between 70,000 and 120,000 hospitalizations in the United States of
infants under 6 months of age and ~70% of hospitalizations due to bronchiolitis
[2 5]. Severe RSV infection has been associated with long-term effects such as
asthma and wheezing and can cause significant mortality in high-risk groups, such
as premature infants or children with immunodeficiency, chronic pulmonary dis-
ease, or cardiovascular disease [6 9]. In addition, RSV infection is a serious
complication in immunocompromised subjects, particularly bone marrow trans-
plant patients, and the elderly [10].
Previously, RSV bronchiolitis was thought to be caused by an overactive anti-
viral immune response, similar to allergic asthma [11 13]. However, recent evi-
dence indicates that severe RSV disease is likely due to virus-induced cell death and
sloughing of apoptotic cells into the lumen of the bronchioles [14]. Examination of
autopsy specimens from fatal cases of RSV bronchiolitis showed the presence of
overwhelming RSV antigen and massive apoptotic sloughing of epithelial cells, but
a relative dearth of infiltrating T cells. In addition, infants who suffered nonfatal
cases of RSV showed decreased expression levels of cytokines, particularly IFN-g,
IL-17, IL-4, and IL-6, compared to infants infected by influenza [14, 15]. Cytokine
expression levels in RSV-infected infants did not appear to correlate with the
severity of RSV infection. However, viral replication levels directly correlated
with the severity of RSV disease [14, 16]. Thus, severe RSV LRTI is likely due
to high levels of RSV replication in ciliated and nonciliated airway cells, resulting
in cell death and a large influx of neutrophils and macrophages. This hypothesis
also fits with the time course of RSV infection and the observation that corticoster-
oids are ineffective in treating RSV bronchiolitis [17]. These results suggest that
reducing viral replication levels by the induction of protective immune responses
via vaccination is likely to reduce the morbidity and mortality due to RSV infection.
Infection by RSV causes severe disease in the very young (infants under 6
months of age) and the elderly [18]. One distinctive characteristic of RSV infection
is that it does not induce long-lived immunity upon exposure, resulting in recurrent
infection throughout life. Reinfections occur frequently throughout life, though
the symptoms of subsequent infection are generally milder [18]. Thus, the target
populations for RSV vaccines would be individuals at the extremes of age. In both
populations, lung function is suboptimal due to relatively inelastic lungs, either due
to developmental immaturity or loss of elasticity. Premature infants are particularly
susceptible to severe RSV disease due to interrupted lung development, leading to
Live Attenuated Vaccines for Respiratory Syncytial Virus 239
decreased lung function with reduced airway diameter and increased smooth
muscle. In addition, both populations present challenges to vaccination because
of deficiencies in their immune responses. For infants, there are two major hurdles
to effective immunization: (1) developmental immaturity of the immune system
and (2) presence of maternal antibodies. Neonatal immune responses are both
quantitatively and qualitatively different from those in adults, and these differ-
ences persist throughout the first year of life. The neonatal immune system
appears to be biased toward Th2-like responses, although Th1 responses can be
induced in neonates with certain stimuli including certain microbes [19 21]. This
effect is likely due in part to immaturity of dendritic and other accessory cell
populations. Serum antibodies derived from the mother pose a challenge for
vaccine take, as seen in the experience with the measles virus vaccine. In contrast,
premature infants born before 28 weeks of gestation, when maternal antibody
transfer occurs, have increased susceptibility to RSV. Premature infants born
closer to full term are likely better protected, as maternal antibody levels are
proportional to gestational age.
At the other end of the age spectrum, immunosenescence is a hurdle for RSV
vaccination in the elderly population. Not only are adaptive immune responses
blunted in the elderly, but innate immune function appears to be decreased as well
[22 24]. Protection from RSV by vaccination will likely require the induction of
both B- and T-cell responses in the elderly, similar to influenza vaccination [19, 25,
26]. Thus, a more complete understanding of the mechanisms responsible for
immunosenescence is required to improve the efficacy of RSV vaccines in the
elderly.
Immunologic protection from RSV infection requires induction of high-affinity
neutralizing antibody responses. Both infants and the elderly show decreased B-cell
responses compared with healthy adults [27 29]. Moreover, these two populations
display a limited ability to generate diversity in their antibody responses to anti-
genic stimulation [27, 30]. The exact mechanisms for these defects are not well
understood. However, increasing the diversity and affinity of the immunoglobulin
response in vaccinees is essential for efficient protection.
2 Agent
RSV is an enveloped virus classified in the family Paramyxoviridae in the order

Mononegavirales, and is the prototype member of the Pneumovirus genus. The
nonsegmented, negative-sense RNA genome of RSV is 15,222 nucleotides long and
contains 10 genes from which 11 proteins are translated (Fig. 1). The genome is
encapsidated by the viral nucleocapsid (N) protein, and this ribonucleocapsid
complex serves as the template for viral transcription and RNA replication. RSV
enters cells by direct fusion of its envelope with the plasma membrane and
replicates solely in the cytoplasm. RSV packages its own viral RNA-dependent
RNA polymerase (RdRP), which is essential for the initial transcription of its
240 M.N. Teng
Fig. 1 RSV genome and virion structure. The M2 gene overlaps with the L gene. Photograph by
Anthony Kalica (courtesy of Peter Collins, NIAID)
genome upon infection. The RdRP for RSV transcription is minimally composed of
P, M2-1, and L. L encodes the large enzymatic subunit of the polymerase, and P is
an essential cofactor for RNA synthesis. M2-1 is specific for the viral transcriptase
and is an antitermination/processivity factor. The polymerase complex accesses the
genome at a single promoter at the 30 end of the genome and initiates transcription
at the first gene (NS1). Each gene is bounded by conserved transcription initiation
and termination signals and is separated from the adjacent genes by a variable
length of intergenic sequence. The linear array of viral genes is transcribed sequen-
tially in a start/stop fashion, resulting in a polar gradient of mRNA production,
whereby genes proximal to the 30 promoter are transcribed more efficiently than
those that are promoter-distal. At a low frequency, the RdRP will fail to terminate,
resulting in an oligocistronic or “readthrough” mRNA that is terminated at a
subsequent transcription termination signal, or will fail to reinitiate, resulting in
transcription attenuation and a gradient of expression inversely proportional to the
distance from the 30 end of the genome. After primary transcription has occurred,
the polymerase complex begins replicating the viral genome, synthesizing a full-
length copy of the vRNA called the antigenome (cRNA). The regulation of the
switch from transcription to replication by RdRP is not clear; however, the M2-2 protein
is thought to be involved in this process. The antigenome is also encapsidated by N
protein and serves as a template for synthesis of more vRNA. In infected cells, there
is more vRNA than cRNA [10]. Encapsidated vRNA interacts with the matrix (M)
protein and traffics to the plasma membrane where the viral N interacts with the
cytoplasmic tails of the attachment (G) and fusion (F) proteins. Virion morphogenesis
occurs at lipid raft domains in the membrane where F is localized. In addition to G
and F, the RSV viral envelope contains a small hydrophobic (SH) protein of unknown
function. Importantly, G and F are the major neutralizing antigens for RSV. The two
remaining RSV proteins, NS1 and NS2, are nonstructural proteins that have been
shown to inhibit IFN-b induction and signaling but are otherwise dispensable for viral
replication in vitro [31, 32].
3 Treatment
Currently, there are no effective antiviral drugs to treat RSV infection. Ribavirin
has been previously used to treat severe RSV disease, but the efficacy of this treat-
ment is questionable and the cost is high [33 35]. Supportive care with sup-
plemental oxygen is the most common treatment option, although treatment
with corticosteroids and/or b-agonists has been tried with limited success [35].
Nebulized hypertonic saline with or without epinephrine has been found to decrease
length of stay in infants hospitalized with viral bronchiolitis [36, 37]. Immunopro-
phylaxis has been the mainstay for the prevention of RSV infection in high-risk
infants. Synagis (palivizumab), a recombinant humanized monoclonal antibody to
the RSV F protein, has been shown to be effective in preventing infection in
premature infants and children with underlying risk factors for severe RSV disease
[38 40]. The recent development of a higher affinity monoclonal antibody to F has
improved the efficacy profile of RSV immunoprophylaxis [41, 42]. However,
Synagis treatment is not cost-effective in normal populations due to the need to
administer the drug monthly during RSV season and the lower incidence of
hospitalization for severe RSV bronchiolitis.
4 RSV Vaccines
Although RSV is the most important cause of viral lower respiratory tract disease in
infants, initial attempts to develop an RSV vaccine by using inactivated virus met
with failure. In the early 1960s, vaccination of infants with a formalin-inactivated
(FI)-RSV vaccine not only failed to protect against RSV disease during the follow-
ing RSV season but some vaccinees developed enhanced disease upon infection
with RSV, resulting in increased rates of severe pneumonia and two deaths [43 45].
Studies on autopsy samples as well as in the mouse model suggested that the
enhanced disease due to FI-RSV vaccination was due to an imbalanced T helper
cell response, predisposing the vaccinees to a response resembling allergic asthma
upon subsequent infection by RSV (reviewed in [46]). More recently, it has been
determined that the FI-RSV vaccine has reduced the capacity for inducing high
avidity antibodies, due to reduced TLR stimulation, likely resulting in the deposi-
tion of complement in the lungs [47, 48].
In the intervening years, a number of different approaches have been evaluated
including subunit vaccines, vectored vaccines, and live attenuated vaccines; however,
as of the writing of this chapter there remains no licensed RSV vaccine. Currently,
the most promising vaccine candidates for RSV are live attenuated viruses. These
242 M.N. Teng
viruses have several benefits: (1) enhanced RSV disease has not been observed
either after natural infection or vaccination with live attenuated viruses [49 53]; (2)
administration of live attenuated RSV vaccines induces balanced immune
responses that more closely match natural immunity compared with parenterally
administered subunit (or inactivated) vaccines [54, 55]. Also, vaccination with live
attenuated viruses intranasally would likely induce better local immunity compared
with intramuscular injection of subunit or killed vaccines [56]. Live attenuated RSV
vaccines have been in development for several decades, using a combination of
cold passage (cp) and chemical mutagenesis to induce temperature sensitivity (ts)
(reviewed in [57, 58]). The initial RSV vaccine candidates were either under- or
over-attenuated, with reversion of one of the ts mutants in vaccinated children [50,
59 61]. However, children vaccinated with these live attenuated viruses did not
show enhanced disease upon subsequent infection with RSV [62]. Therefore,
further development of live attenuated vaccine candidates was performed, combin-
ing cold passage and chemical mutagenesis to generate temperature-sensitive RSV.
A spectrum of cptsRSV vaccine candidates were produced by this method, with a
range of temperature sensitivity in culture and attenuation in animal models
(Fig. 2a) [53, 63 66]. Candidate vaccines from this method were immunogenic
and protected against RSV challenge in both rodent and nonhuman primate models.
Two candidate vaccines (cpts248/955 and cpts530/1009) were chosen for testing in
the clinic [53]. These candidates induced protective immune responses in seroneg-
ative children; however, both candidates were underattenuated in this age group,
precluding further analysis in infants (Table 1). One additional candidate, cpts248/
404, was found to be sufficiently attenuated and immunogenic in seronegative
children and was tested in 1- to 3-month-old infants [49]. However, cpts248/404
caused nasal congestion in these infants, an unacceptable adverse effect in this
population [49].
Production of live attenuated RSV vaccine candidates by mutagenesis and
screening for temperature sensitivity is a laborious and inefficient process. There-
fore, it is essential to develop a method of systematically deriving tsRSV and
identifying additional attenuating mutations that can be incorporated into RSV
vaccine candidates. The recent advent of reverse genetics systems for RSV has
allowed the development of live attenuated RSV vaccine candidates encoding
specific attenuating mutations, rather than relying on random mutagenesis. The
ability to generate recombinant RSV (rRSV) from cDNAs also allows the identifi-
cation of novel viral targets for attenuation through the investigation of the vir-
us host interactions important for viral pathogenesis. Reverse genetics systems for
RSV rely on the coexpression of the viral polymerase components (N, P, M2-1, and
L) with a complete copy of the viral genome [67, 68]. Coexpression is achieved by
transfection of plasmids encoding each of the viral polymerase genes and a plasmid
encoding the full-length cDNA of the viral genome into cultured cells. Expression
from the plasmids is driven by the bacteriophage T7 RNA polymerase, which is
supplied exogenously. For the purposes of vaccine development, T7 RNA poly-
merase is expressed by cotransfection of an expression plasmid with the other
plasmids into qualified Vero cells [69]. Upon expression of viral components,
Fig. 2 RSV vaccine candidates. (a). Genomic organization of biologically derived, temperature
sensitive RSV vaccine candidates. Arrows indicate relative position of the attenuating mutations
corresponding to the mutant, indicated on the left. (b). Recombinant RSV vaccine candidates. ts
point mutations are identified as in (a). Deletions are indicated with dashed lines. (c). Potential
recombinant RSV vaccine candidates. ts point mutations are identified as in (a). Deletions are
indicated with dashed lines
transcription and replication of the viral genome initiates the RSV infectious cycle,
resulting in the production of infectious rRSV. The cDNA copy of the viral genome
can be mutated by standard molecular biology techniques in order to attenuate the
resultant rRSV.
Initial studies using rRSV focused on two different means of attenuating RSV.
The first method involved combining the known mutations from the cptsRSV
isolates in rRSV strain A2 (rA2) to increase attenuation of the vaccine candidates.
This resulted in the generation of rA2cpts248/404/1009 and rA2cpts248/404/1030,
combining the cpts248/404 mutations with those of 530/1009 and 530/1030 [70].
These new mutants were more attenuated than the cpts248/404 parental virus,
indicating that some mutations have additive effects in attenuation. However,
these studies also showed that certain mutations are incompatible with others, as
the rA2cpts248/404/530 could not be recovered, due to incompatibility of the 530
244 M.N. Teng
Table 1 Clinical trials on live attenuated RSV vaccine candidates

Vaccine candidate Attenuation phenotype Immunogenicity References
Biologically derived
cpRSV Underattenuated in Mild (adults) [53]
seropositive children
cpts248/955 Underattenuated in Good (seronegative [53]
seronegative children children)
cpts530/1009 Underattenuated in Good (seronegative [53]
cpts248/404 Underattenuated in infants Good (seronegative [49]
(partial reversion) children)
Mild (infants)
Recombinant
rA2cpts248/404DSH Underattenuated in Good (seronegative [52]
rA2cpts248/404/1030DSH Sufficiently attenuated in Good (seronegative [52]
Ongoing trials infants (partial reversion) children)
Poor (infants)
rA2cpDNS2 Underattenuated in Mild (seropositive [79]
seropositive children children)
rA2cp248/404DNS2 Underattenuated in Moderate (seronegative [79]
rA2cp530/1009DNS2 Sufficiently attenuated in Poor (seronegative [79]
Vectored
MEDI 534 (rB/HPIV3 Attenuated in seropositive Poor (seropositive [117]
RSV F) children children)
Ongoing trials
mutation with, particularly, the 248 mutation [70]. Therefore, it would be desirable
to have a panel of attenuating mutations from which to select to incorporate into
rRSV vaccine candidates, so that the level of attenuation can be properly tuned. In
order to increase the number of attenuating mutations that could potentially be
combined in a vaccine candidate, specific viral proteins have been mutagenized to
replace charged amino acids with a noncharged amino acid (e.g., alanine). This
procedure has been employed to identify a number of mutations in both P and L that
result in attenuation of RSV, both in culture and in rodents [71 73]. These mutations
thus add to the panel of mutations available for inclusion in future vaccine candi-
dates, either alone or in combination with the previously identified cpts L mutations.
Another avenue of attenuation for RSV has been the deletion of nonessential
genes. Gene deletion should be more stable than the point mutations responsible for
temperature sensitivity, reducing the risk of reversion to virulence of the vaccine
candidate. rRSVs (rA2) lacking one or a combination of NS1, NS2, M2-2, and SH
were generated and shown to be attenuated in preclinical trials [31, 74 76]. RSV
lacking SH (rA2DSH) replicated similarly to wild-type (wt) RSV (rA2) in culture
but showed a low level of attenuation in the respiratory tracts of rodents and
nonhuman primates [77]. Because clinical trials indicated that rA2cpts248/404
was only slightly underattenuated, the SH gene deletion was incorporated into this
vaccine candidate to increase the level of attenuation (Fig. 2b). However, this
vaccine candidate (rA2cpts248/404DSH) was not further attenuated in adults,
seropositive or seronegative children (Table 1) [52]. It was not possible to deter-
mine from these observations whether the SH deletion mutation confers attenuation
to RSV in humans, even though rA2DSH was attenuated in mice and chimpanzees.
These results indicate that attenuation of RSV by combining different mutations is
not necessarily additive. However, subsequent addition of the 1030 mutation to
rA2cpts248/404DSH resulted in a virus that was more ts and more attenuated in
seronegative children [52]. Further trials in seronegative infants showed that
rA2cpts248/404/1030DSH was well tolerated and appropriately attenuated
(Table 1) [52]. Only a minority of vaccinees produced increased neutralizing
antibody responses, even after a second dose of the vaccine virus. However,
replication of the second dose of vaccine was significantly reduced, indicating
that some protective immunity had been induced by the initial dose [52].
Preclinical testing of RSV lacking NS1 or NS2 (rA2DNS1 and rA2DNS2,
respectively) showed that these viruses were deficient in replication in culture
and also attenuated in rodents and nonhuman primates [31, 32, 76, 78]. In chim-
panzees, rA2DNS2 displayed an attenuation phenotype similar to rA2cpts248/404,
and rA2DNS1 was significantly more attenuated in both the upper and lower
respiratory tracts [74, 75]. However, both deletion mutants induced levels of
serum-neutralizing antibodies against RSV to levels comparable or slightly lower
than wt RSV. In addition, chimpanzees immunized with rA2DNS2 were protected
against subsequent challenge with RSV. Therefore, an NS2-deletion rA2 derivative
was then tested in clinical trials as a vaccine for the elderly because it was less
attenuated in chimpanzees than the cpts248/404 vaccine candidate (Fig. 2b) [79].
rA2cpDNS2 was shown to be overattenuated in adults; however, it was also under-
attenuated in children, a contraindication for testing in infants (Table 1). The NS2
deletion virus was further attenuated by inclusion of the ts mutations 248/404 or
530/1009. These vaccine candidates were more attenuated than their parental
strains and modestly immunogenic when tested in seronegative children [79].
5 Live Vectored RSV Vaccines
An alternative means of delivering RSV antigens in attenuated virus vaccines has

been the use of heterologous viral vectors expressing RSV F and/or G. Early efforts
focused on vaccinia viruses (VV) expressing RSV proteins. VV-F and VV-G
together were immunogenic and protective in the mouse model of RSV; however,
these VV recombinants did not induce protective immunity in chimpanzees
[80 83]. In addition, VV is likely too virulent to use as a vector for current vaccine
development. More recently, use of the attenuated modified vaccinia Ankara as a
vector for RSV antigens has shown some efficacy, though a prime-boost strategy
may be required to elicit sufficiently protective immunity [84, 85].
246 M.N. Teng
Adenovirus vectors were initially used to immunize against RSV F and G over
15 years ago and, with the advent of replication-deficient adenovirus vectors, have
been further investigated more recently [86 90]. Adenovirus-vectored F and/or G
have been shown to provide protection to RSV in mice and ferrets; however, this
vaccine modality does not immunize chimpanzees against RSV, indicating that this
strategy will likely not be clinically useful [88, 89]. Alphavirus replicons have also
been tested for their ability to vaccinate against RSV [91 94]. Immunization via either
the intranasal or intramuscular route with Venezuelan equine encephalitis virus
replicons expressing RSV F induces balanced Th1/Th2 immunity, protects mice and
cotton rats against RSV challenge, and induces serum antibodies in macaques [91, 92].
The recent proliferation of reverse genetics systems for the paramyxovirus
family has provided the possibility that RSV antigens can be expressed in the
context of a number of different paramyxoviruses, including Sendai virus, New-
castle disease virus (NDV), and human parainfluenza viruses (HPIV) 1, 2, and 3
(reviewed in [95 97]). Sendai virus and NDV are murine and avian viruses,
respectively, and thus are naturally attenuated in humans due to host range restric-
tion. NDV is a strong inducer of IFN-b and may therefore provide better stimulation
of dendritic cell (DC) maturation and T-cell responses than RSV infection [98].
Both of these vector systems have been shown to be immunogenic and protective
against RSV challenge in animal model systems [98 102].
An additional consideration is the possibility of combining vaccines against
multiple pediatric viral pathogens into a single recombinant virus. Infection of
children by HPIV1 and HPIV2 generally occurs later in life (approximately 6
months of age), so immunization would occur in older infants. Thus, an HPIV1-
or HPIV2-vectored RSV vaccine may be useful as a booster to prevent secondary
disease or as a vaccine in the elderly. In addition, attenuated HPIV1 and HPIV2 are
being developed for use as vaccine candidates [103 109].
Because HPIV3 is also an important cause of pediatric respiratory tract disease,
significant effort has been put into developing a live attenuated HPIV3 vaccine that
could also be used as a vector for an RSV vaccine (Table 1). One candidate vaccine
utilizes the bovine PIV3 (BPIV3) backbone, which has been shown to be safe and
immunogenic in infants [110, 111]. In order to generate a bivalent HPIV3/RSV
vaccine, the BPIV3 F and HN genes were replaced by their HPIV3 counterparts and
RSV F was inserted into the B/HPIV3 chimera; thus, the resulting virus expresses
both RSV and HPIV3 surface antigens. Recombinant B/HPIV3-RSV-F was slightly
more attenuated than the parent virus, but remained immunogenic and was protec-
tive against both RSV and HPIV3 in animal model systems [112 115]. This vaccine
candidate (MEDI-534) has recently been tested in clinical trials. Although the
vaccine was attenuated and safe, it was minimally immunogenic in both adults
and children, indicating that further modification may be required [116, 117].
However, the major advantage of this approach is that the viral vector is also a
vaccine, thus providing protection against multiple pathogens. Because the RSV F
protein is likely not incorporated into its viral envelope, RSV-specific antibodies
were ineffective at neutralizing the chimeric virus [112], suggesting it could be also
used as to boost anti-RSV immune responses.
6 Future Directions
There remain a number of challenges to the development of an efficacious RSV

vaccine. First, it will be important to develop additional animal models for RSV
challenge that more faithfully represent the target populations of infants and the
elderly. Although nonhuman primate models have yielded important information
on both vaccine safety and immunogenicity, these models also have not recapi-
tulated some aspects of the replication of vaccine candidates in humans. For
example, DNS2 was immunogenic in chimpanzees but not in seropositive chil-
dren [75, 79]. In addition, the partial reversion of the ts phenotype seen with the
248/404/1030 mutations in infants was not detected in animal experiments [49,
52, 70, 118]. Defining the correlates of protection and attenuation in animal
models will aid in the selection of vaccine candidates for clinical trials. In
addition, a model that recapitulates stimulation of the immature immune system
in the presence of maternal antibodies will be important for the development of a
pediatric RSV vaccine.
Perhaps the most important challenge in the development of an effective RSV
vaccine has been achieving the proper balance between immunogenicity and
attenuation. The rA2cpts248/404/1030DSH vaccine candidate, which was appro-
priately attenuated in infants, was only mildly immunogenic [52]. It is possible to
enhance immunogenicity of vaccines by increasing the dose or boosting with
multiple inoculations. However, the target population of a pediatric RSV vaccine
would be infants who are entering their first RSV season, thus shortening the
window in which immunization would be effective. Therefore, a better understand-
ing of the induction of immune responses in the target populations for RSV
vaccines will be essential. Identifying signals (e.g., TLR agonists, cytokines) that
can induce DC maturation and/or activate other antigen-presenting cell populations
stimulate Th1 responses that can augment the immunogenicity of an RSV vaccine.
For example, studies in mice suggest that deletion of NS1 results in a virus that has
enhanced capacity to induce DC maturation, likely due to increased production of
IFN-b [119]. In addition, NS1 appears to play a role in viral replication beyond IFN
antagonism, indicating that deletion of this gene might be both attenuating and
immunomodulatory [31].
An alternative method to enhancing immune responses that has been explored is
the expression of cytokine genes as an additional transcription unit in rRSV
[120 122]. Stable expression of additional gene products in the rRSV genome
has been shown for a variety of genes [123]. rRSV encoding GM-CSF as an
additional gene shows reduced replication in the respiratory tracts of mice with a
concomitant increase in the number of pulmonary DCs and in the expression of
IFN-g and IL-12 [121]. By contrast, insertion of genes for the cytokines IL-4
and IFN-g into rRSV results in viruses that caused increased pathogenesis after
immunization and/or challenge [120]. Skewing of the T helper response can have
adverse effects on secondary exposure and even to unrelated viruses [124]. Thus,
significant care must be taken in identifying specific immunomodulators that will
248 M.N. Teng
increase the immunogenicity of an RSV vaccine candidate without causing

enhanced disease.
One potential mechanism of improving B-cell responses to RSV is increasing
the expression of the RSV F and G proteins, which serve as the major protection
antigens [18]. Because of the linear nature of the RSV genome, the promoter-
proximal genes are expressed to a greater extent than the promoter-distal genes
[18]. Rearrangement of the gene order in the related vesicular stomatitis virus
(VSV) has been shown to result in genome site-specific levels of expression for
the viral genes [125]. These rearranged viruses displayed an attenuated phenotype
both in vitro and in vivo and were able to vaccinate pigs against subsequent VSV
infection [126, 127]. For RSV, rearrangement of the gene order in a recombinant
virus, such that the F and/or G genes are the promoter-proximal, resulted in an
approximately twofold increase in protein expression [128]. Unlike VSV, these
viruses replicated slightly better than wt virus in culture and similarly to wt in the
respiratory tracts of mice [128]. Thus, gene rearrangement alone in the context of
RSV is not attenuating. In addition, shifting F to a promoter-proximal position
resulted in an increase in anti-F serum antibody responses in mice, suggesting that
increased F expression may be desirable in a vaccine candidate [128]. Expression
of F and G might be further increased by optimizing the codon usage of these
genes for translation [129]. Combining these relatively small increases in antigen
expression might allow for an additive effect for vaccination. Studies with anti-
RSV F antibody prophylaxis show significant increases in efficacy with even
minor increases in antibody titer [130, 131]. Thus, increasing the amount of
antigen available for presentation to the immune system may allow for a more
robust anti-RSV response.
RSV G is unique among paramyxovirus attachment proteins in that it is
produced in both a membrane-bound and a secreted form. Secreted G (sG) is
produced from the G mRNA by alternative initiation from a downstream AUG
[132, 133]. Ablation of this translation initiation codon in rRSV results in RSV
that produces only membrane-bound G [134]. Studies have shown that the sG
can act both as an antigenic decoy in vitro and as an immunomodulatory factor
in mice [135]. Importantly, sG appeared to affect restriction of RSV replication
in vivo by both anti-G and anti-F antibodies through a mechanism involving
FcgR-bearing immune cells [135]. Thus, a vaccine candidate that does not
express sG may have increased immunogenicity and may be more efficiently
controlled by the immunity induced. In addition, sG showed proinflammatory
functions in the lungs of mice, likely via its CX3C (fractalkine) motif [135].
Because pulmonary inflammation is associated with increased pathogenicity of
RSV, removal of this factor may result in decreased reactogenicity. However,
sG may be necessary for vaccine take in infants in the presence of maternal
antibody. Further studies will clarify these disparate effects of sG on RSV
pathogenesis and immunity. An alternative to ablating the expression of sG
might be removal of the CX3C motif from G; studies have shown that mutagen-
esis or deletion of this sequence does not affect viral replication in vitro or in
mice [136].
One important characteristic of vaccine candidates is genotypic and phenotypic

stability. Genomic stability is important during the scaling up of production for
the vaccine viruses, which undergo multiple rounds of replication and thus have a
greater chance for mutation. In addition, phenotypic stability is essential during
vaccination, during which reversion to virulence can cause increased pathogenicity
and shedding. In this case, the attenuated phenotype is more important than specific
genotype provided that immunity to the major protective antigens is achieved.
Deletion of nonessential viral genes should provide the most stable attenuating
mutations because genetic recombination of RSV is extremely rare and has only
been observed in the laboratory under optimal conditions [137]. In addition to the
NS2 and SH deletion viruses, RSVs lacking NS1 or M2-2 (Fig. 2c) are significantly
attenuated and protective in animal models and are potentially good vaccine
candidates [31, 74, 76, 78].
All of the ts mutations identified in the RSV vaccine candidates that have
undergone clinical trials are point mutants. ts248, ts530, ts1009, and ts1030 are
all missense mutations within the viral polymerase (or L protein), and ts404 is a
point mutation in the M2 gene start sequence [65, 66, 70]. Characterization of virus
shed from vaccinees has shown that these point mutations can readily revert,
resulting in less ts RSV, in some cases despite the “stabilization” of these mutations
in rRSV by changing two residues of the specific codon encoding the ts mutant. For
example, analysis of nasal wash specimens from seronegative infants vaccinated
with rA2cpts248/404/1030DSH showed that approximately one-third of the sam-
ples had lost a measure of their ts phenotype, displaying a 1 3 C increase in
permissive temperature [52]. Sequencing of these clinical specimens identified
reversion mutations at either the ts248 or the ts1030 mutation [52]. Although
these partial revertants retained four of the five attenuating mutations and a measure
of attenuation, these results demonstrate the difficulty of using point mutations to
attenuate RNA viruses, which encode an error-prone viral polymerase. To counter-
act this problem, there are a number of possibilities to generate genotypically and
phenotypically stable ts RSV vaccine candidates.
It is possible to generate phenotypically stable attenuated RSV viruses by
introducing several ts point mutations in a variety of places in the RSV genome.
The difficulty with this approach is that some combinations of mutations might
increase the attenuation of the vaccine virus beyond the level required for inducing
protective immunity. In addition, some ts mutations are not compatible with each
other, resulting in a nonviable virus [70]. Thus, the spectrum of mutations that can
be combined would have to be empirically defined. The benefit to this strategy
is that reversion at any one site should be compensated by the presence of
the additional attenuating mutations. However, as seen with rA2cpts248/404/
1030DSH, particular mutations have a more prominent effect on attenuation of
the vaccine virus and reversion at these sites may result in a significant loss in
attenuation.
One method of preventing reversion is to “stabilize” existing ts mutations by
altering the codon usage to require two mutation events in order for the mutant to
revert to the wt phenotype. Theoretically, the viral polymerase would not be likely
250 M.N. Teng
to introduce two mutations at the same site. Recently, Luongo et al. have con-
structed rRSV that have mutations at position 831 of L (ts248) encoding every
possible amino acid residue. Although most mutants could be recovered, only two
mutants were found to confer temperature sensitivity (831I and 831F) to the rRSV
in addition to the 831L mutation [138]. Furthermore, neither 831I nor 831F was as
attenuated as 831L in the respiratory tracts of mice, suggesting that 831L has an
attenuating function beyond temperature sensitivity. Interestingly, using the differ-
ent codons for Leu resulted in different frequencies of reversion (to wt genotype) or
pseudoreversion (to wt phenotype) [138]. These data suggest that careful selection
of mutant codons may offer a strategy for increasing genotypic stability of attenu-
ating point mutations. However, the genetic code precludes certain mutations from
being “stabilized” by this method, as not all mutations can be made with two
nucleotide differences from the wt assignment.
A novel potential mechanism of providing genotypic stability for point muta-
tions is increasing the fidelity of the viral polymerase. Recent studies with poliovi-
rus (PV) have shown that mutations that alter replication fidelity and/or replication
speed of the PV RdRp produce attenuated viruses that protect mice transgenic
for the PV receptor from a lethal challenge with wt PV [139 141]. Furthermore,
mutation of a single amino acid residue that is conserved in all viral RdRps appears
to control both replication speed and replication fidelity. This amino acid residue is
a lysine that is present in conserved structural motif D of the RdRp [142, 143]. In
the PV model, changes to this residue produce slow, high-fidelity RdRps [143].
Biochemical analysis shows that mutation of the homologous lysine in HIV RT and
T7 RNA polymerase results in similar effects on polymerase speed and fidelity
[143]. Thus, application of this technology to RSV could allow the identification of
an additional attenuating mutation and could prevent or delay the emergence of
more virulent variants of the vaccine candidates. Combinations of L mutations that
increase polymerase fidelity and known attenuating mutations could allow for even
finer tuning of vaccine efficacy and prevent outgrowth of more virulent viruses,
which could then be spread to naive individuals.
7 Summary
Much progress has been made recently toward the development of an effective, live
attenuated RSV vaccine; however, a number of hurdles remain. Most importantly,
achieving the proper balance of attenuation and immunogenicity has been difficult
because of the lack of animal models and immune correlates to investigate induc-
tion of immune responses in infants, a target population for RSV vaccines. Future
studies into the molecular biology of the virus may lead to novel ways to address
current difficulties in RSV vaccine development.
Acknowledgment The author would like to gratefully acknowledge the contribution of Kim C.
Tran for producing the figures and table for this chapter.
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Live Attenuated Cholera Vaccines: Flagella
and Reactogenicity
D. Ewen Cameron and John J. Mekalanos
Abstract The rational design of attenuated Vibrio cholerae strains has been an
attractive method for live cholera vaccine development because the major mechan-
isms of V. cholerae virulence are well defined and convalescence from cholera, the
disease it causes, is a strongly immunizing process. After decades of effort to
develop safe live attenuated cholera vaccines, however, the appearance of reacto-
genicity, defined as adverse symptoms in immunized volunteers, has precluded
further development of most live vaccine candidates. We now know that V. cholerae
flagellar motility is associated with human and animal reactogenicity in early live
attenuated cholera vaccines, and recently developed nonflagellated V. cholerae
mutant strains have shown great promise as live attenuated vaccines in volunteer
studies. This chapter briefly summarizes our current understanding of V. cholerae
pathogenesis and describes efforts to use this knowledge to design immunogenical
and nonreactogenic live cholera vaccines.
1 Vibrio cholerae and Disease
Vibrio cholerae, a highly motile Gram-negative bacterium, is the etiological agent

of cholera, a human diarrheal disease that kills an estimated 100,000 200,000
people annually [1]. Besides its traditional home in countries of the Ganges delta
(i.e., India and Bangladesh), cholera in the last several years has extracted particu-
larly high fatality rates in Africa including countries like Zimbabwe where the
reported case fatality rate was nearly 5% in 2008 [2, 3]. One of the most rapidly
fatal diseases known, cholera can kill its victims in as little as 10 18 h following
initial symptoms due to severe dehydration and hypovolemic shock [4]. In endemic
D.E. Cameron and J.J. Mekalanos (*)

Department of Microbiology and Molecular Genetics, Harvard Medical School, 200 Longwood
Avenue, Boston, MA 02115, USA

262 D.E. Cameron and J.J. Mekalanos
regions, cholera largely targets young children not previously exposed to the
disease, but people of all ages are equally at risk in newly invaded areas during
epidemic spread [5]. V. cholerae was originally identified as the cause of cholera by
Filippo Pacini in 1854, but his observations were largely ignored until Robert Koch
independently discovered the causal connection between the comma-shaped bacte-
rium and voluminous diarrhea in 1884 [6]. Since then, great strides have been made
in understanding the virulence mechanisms of V. cholerae, its ecology, and the
nature of host immunity following convalescence.
2 Ecology of V. cholerae
V. cholerae is found in marine and brackish water and has historically caused
epidemic disease throughout the world [6, 7], but improved sanitation and health-
care facilities in the developed world have largely confined cholera outbreaks to
the coastal regions of southern Asia, Africa, and central America. Classified by
the immunogenicity of lipopolysaccharide (LPS) O-antigen, over 200 serogroups
of V. cholerae have been identified in the environment, but only specific “bio-
type” strains within the O1 and O139 serogroups are known to cause widespread
disease [8]. O1 “classical” strains were likely responsible for at least six pan-
demics that spread throughout Asia, Europe, and the Americas in the nineteenth
century [9], and O1 “El Tor” strains are responsible for the current seventh
pandemic that began in Indonesia in 1961 [10]. In the early 1990s, seroconversion
of an O1 El Tor strain to the O139 serogroup allowed it to quickly overtake the O1
El Tor strain as the primary cause of cholera in India and Bangladesh [11 13],
likely due to its ability to circumvent acquired immunity in endemic communities
against the O1 antigen [14]. In recent years, O1 El Tor strains have reemerged,
and presently both the O1 and O139 strains cause recurrent disease. While these
“toxigenic” O1 and O139 strains cause a vast majority of cholera disease, several
non-O1, non-O139 strains are known to cause sporadic human disease [15, 16],
often using alternative virulence mechanisms including type III and type VI
secretion [17 19].
3 Virulence Mechanisms of V. cholerae
As a waterborne disease, cholera is acquired by ingestion of water or food con-

taminated with V. cholerae. A high-infectious dose of at least 108 bacteria is
required to cause human disease due in large part to V. cholerae sensitivity to the
stomach’s gastric acid barrier [20]. Once in the small intestine, the bacterium uses
its single polar flagellum and an extensive chemotaxis sensory network to target and
then penetrate the mucus layer, a thick glycocalyx gel covering the small intestine
epithelium [21 23]. Recent work suggests that V. cholerae loses its flagellum
Live Attenuated Cholera Vaccines: Flagella and Reactogenicity 263
shortly after entering the mucus layer but is still able to actively transit through the
layer, possibly using an additional as-yet-unidentified motility system [24].
Upon reaching the epithelial layer, V. cholerae uses an intricate regulatory
network known as the toxR regulon to induce expression of virulence genes
including cholera toxin (CT), the AB5 enterotoxin responsible for the bulk of the
diarrheal response seen in the disease, and the toxin coregulated pilus (TCP), a type
IV bundle forming pilus that is essential for V. cholerae intestinal colonization
[25 27]. In the regulatory cascade, the inner membrane protein ToxR acts with its
membrane partner ToxS and a second pair of membrane proteins TcpPH to induce
expression of ToxT, an AraC family transcription factor that then activates tran-
scription of the toxin genes ctxAB and the tcp pilus biosynthetic operon [28].
Further regulatory control is provided by the V. cholerae quorum-sensing system,
which uses expression of the transcriptional regulators AphA and AphB to control
TcpPH levels [29 31].
The host intestinal environment also plays an intricate role in this virulence gene
cascade as both ToxT and ToxR are posttranscriptionally controlled by the compo-
nents of bile, a heterogeneous mixture found at high concentration in the small
intestine where it aids in digestion. Oleic acid and other unsaturated fatty acids
(UFAs) in bile directly inhibit ToxT activity by inducing a “closed” ToxT confor-
mation that is unable to activate tcp and ctx transcription [32, 33]. Since UFAs in
bile exist at high concentration in the intestinal lumen but are readily absorbed by
the small intestine epithelium, the resulting UFA concentration gradient provides V.
cholerae with an ideal measuring stick to ensure that virulence gene expression
occurs only at or near the intestine epithelial surface. Interestingly, the bile acids
cholate and deoxycholate seem to function in an opposing manner: they activate
ToxR to cause increased CT expression independent of ToxT activity [34]. Other
intestinal stimuli including changes in pH and temperature are known to regulate
virulence gene expression, but their specific modes of action remain unknown [35].
4 Cholera Disease Dynamics
In endemic regions, V. cholerae infection rates follow a distinct biannual seasonal-

ity in which a large outbreak of disease occurs following monsoon rains in the fall
and a smaller outbreak occurs in the spring. This pattern of disease correlates
strongly with the presence of culturable toxigenic V. cholerae in the environment,
and several studies have linked this increase in V. cholerae abundance and the
epidemic spread of disease to changes in water salinity, temperature, and zooplank-
ton and phytoplankton blooms [6, 36]. During a cholera outbreak, as many as
several trillion V. cholerae may be shed into the environment by a single symptom-
atic person [37], and these bacteria exist in a transient hyperinfectious state that
reduces the bacterial load required to infect other people in the community [38].
These features of V. cholerae pathogenicity help to explain the explosive nature
of cholera outbreaks, but equally interesting is the self-limiting nature of these
epidemics, which often end as suddenly as they begin. Recent work suggests that
this may be due to lytic bacteriophage in the environment that specifically target O1
and O139 strains of V. cholerae [39]. In this phage-based model of cholera disease
dynamics [40 42], the large number of V. cholerae in the environment (and even
inside patients) during a cholera outbreak provide ample targets for lytic-phage
infection and growth. The resulting high-phage predation rate serves to reduce the
concentration of toxigenic V. cholerae in the environment, and the concomitant
drop in new human infections reinforces the decline since fewer hyperinfectious
bacteria are shed from cholera patients into the environment. In the subsequent
interepidemic months when O1 and O139 strains are in low abundance, the lytic-
phage population in the environment is reduced by dispersion and dilution, allow-
ing for the cyclic reemergence of toxigenic V. cholerae strains.
Recent mechanistic modeling suggests that cholera epidemic dynamics are also
heavily influenced by the high rate of asymptomatic cholera infections in humans
[43, 44]. As discussed below, the resulting spike in transient protection from
reinfection in endemic communities may play an important role in the cyclic
decline and reemergence of cholera disease in these regions.
5 Immunity to Cholera
Convalescence from symptomatic cholera disease induces a durable immunity that

is years in duration and perhaps life-long in some individuals [5, 45]. Because
cholera is a mucosal, noninvasive disease, it has long been thought that elements of
the mucosal immune system play the predominant role in protective immunity, with
secretory immunoglobulin A (IgA) serving to aggregate V. cholerae in the intestinal
lumen and neutralize its toxins and colonization factors [46]. Since direct measure-
ment of IgA levels in the intestinal lumen and mucosal layer is cumbersome, serum
vibriocidal antibody levels have traditionally been used as a correlate for immunity
at the gut mucosal surface [47]. Serum vibriocidal antibody levels in patients
typically rise rapidly following V. cholerae infection but usually drop down to
near baseline levels within 6 months [48]. Serum vibriocidal levels in patients
correlate with immunity to cholera [48], but are at best an incomplete predictor of
protection because some patients with very high vibriocidal titers are still suscepti-
ble to disease [49]. In fact, serum IgG antibodies specific to V. cholerae LPS and the
B subunit of CT (CTB) are abundantly found in convalescent patients, but serum
IgG titers against either of these antigens do not correlate well with protection from
reinfection [50].
Serum IgA levels, on the other hand, do correlate well with protection from V.
cholerae. IgA titer against V. cholerae LPS, CTB, and the TCP pilin TcpA are all
predictive of protection from the disease [51]. Intestinal lavage studies have shown
that V. cholerae-specific IgA levels in the intestinal lumen are elevated shortly after
infection but drop significantly within 4 weeks of convalescence [52]. Since the
resulting resident mucosal IgA levels appear to be too low to prevent V. cholerae
infection in immune individuals, it is likely that a rapid anamnestic response by

memory B and T cells in the gut-associated lymphoid tissue (GALT) is responsible
for the observed protection [53]. Memory T cells specific to V. cholerae antigens
are observed in patients as soon as 7 days after infection [54], and circulating
memory B cells specific to V. cholerae LPS, CTB, and TcpA can be found in
patients at least 1 year after infection [55]. It is still unclear, however, if these
particular memory cells represent the gut lymphocyte population that is actually
protective.
6 Killed Whole-Cell Cholera Vaccines: Parenteral and Oral

Inoculation
The first cholera vaccines contained killed, whole-cell V. cholerae lysates that were
parenterally administered, but broad field trials showed that they failed to elicit an
adequate level of long-term immunity [56 58]. These vaccines elicited high serum
IgG levels against V. cholerae antigens, but they induced only low levels of serum
IgA compared to oral administration of the vaccine. It was eventually recognized
that immune stimulation at the intestinal mucosa was required to induce strong
immunological memory toward V. cholerae [52], and since then the field has
largely focused on developing oral cholera vaccines that trigger an immune
response in the small intestine mucosa.
In the 1980s, oral vaccination against cholera was explored in several volunteer
studies using killed whole-cell V. cholerae strains [59], and Holmgren and collea-
gues were the first to test these vaccines for efficacy in a cholera-endemic country
[52]. In the vaccine, they included a mixture of formalin-treated and heat-killed V.
cholerae strains belonging to both the O1 El Tor and classical biotypes and added
purified CTB to induce additional antitoxin immunity. Three doses of this vaccine
produced 85% efficacy at 6 months and 50% efficacy lasting at least 3 years in
a large-scale field trial in Bangladesh [60, 61], but long-term immunity was much
more predominant in older age groups and tended to fall off in children under
5 years of age, the very group that is most susceptible to infection in endemic
regions. The vaccine was subsequently approved for sale as Dukoral [62], and
similar vaccines that include an O139 strain but are not dosed with CTB have been
developed in Vietnam [63 65] and India [66].
Results from these field trials confirm that cholera vaccines can prevent disease
in endemic countries, but it remains to be determined if killed oral vaccines are the
most effective public health tool to control and ideally eliminate cholera in endemic
settings. Important drawbacks of killed oral vaccines include the following: (1)
multiple doses are required to induce significant immunity, (2) they are less
effective in infants and children who carry a disproportionate share of the disease
burden, and (3) their manufacture may be cumbersome compared to alternatives
such as live attenuated vaccines.
7 Live Attenuated Cholera Vaccines
The concept of using live attenuated microbes as vaccines dates back to work on
viruses such as polio, measles, mumps, and rubella where laboratory propagation of
virulent strains led to attenuation. These attenuated strains serve as good vaccines
because they retain the ability to infect people and induce an adaptive immune
response targeted at actively replicating organisms, but their attenuation allows the
host-immune response to overwhelm the virus before progression to symptomatic
disease can occur. Over half a century ago, naturally attenuated strains of
V. cholerae were also explored for cholera vaccination until molecular genetic
techniques were developed to combine attenuating traits like auxotrophy and
streptomycin-dependence into a single strain [67, 68].
An important shift in cholera vaccine design occurred in the early 1970s when it
was recognized that virulence factors might be particularly good targets for attenu-
ating V. cholerae vaccine strains. In particular, it was hoped that disruption of CT in
a toxigenic V. cholerae strain would stop it from causing diarrheal disease but
would not affect its ability to colonize the human intestine and elicit an adaptive
immune response that normally leads to long-term immunity. Howard reported
isolating CT mutants after chemical mutagenesis in 1971 [69] but these mutants
were not further characterized or tested in volunteer studies. In 1979, Honda
and Finkelstein reported the isolation of Texas Star, a chemically induced mutant
of a V. cholerae O1 El Tor strain that did not produce the CT A subunit (CTA) but
continued to produce the nontoxic B subunit (CTB) [70]. When tested in volunteer
studies, Texas Star did not induce voluminous diarrhea and remained fully immu-
nogenic as had been hoped, but the strain also elicited adverse side-effects in
recipients including cramps, fever, malaise, and mild diarrhea [71]. These symp-
toms are not normally seen in clinical cholera patients, and their induction by
V. cholerae strains has been termed “reactogenicity.”
Advances in the molecular genetics of CT led to the development of attenuated
V. cholerae mutants that had deletions in the CT genes ctxAB created by mutagenic
phage or recombinant DNA techniques [72 74]. It was hoped that the precision
associated with genetic engineering would lead to defined, stable, attenuated
live vaccines that were free of reactogenicity; however, volunteer studies quickly
established that while these strains were highly immunogenic and protective
in experimental human challenge studies, they also remained significantly reacto-
genic [75].
The field as a whole entertained several theories that could explain this reacto-
genicity, including the possibility that it was caused by an additional undefined
accessory toxin [76] or that it resulted from a local inflammatory response caused
by colonization per se of the relatively bacteria-free upper small intestine. Indeed,
volunteer experiments revealed that V. cholerae strains with defined deletions that
caused a defect in intestinal colonization also caused less reactogenicity and
immunogenicity in patients [27], suggesting that V. cholerae colonization, immu-
nogenicity, and reactogenicity are tightly linked.
Eventually one mutant (CVD-103-HgR), derived from the poorly colonizing

V. cholerae classical strain 569B, was found in volunteer studies to induce very
little reactogenicity while eliciting enough immunogenicity to justify significant
development efforts [77]. However, for reasons that to this day remain unknown,
other mutants (CVD101 and CVD110) with a similar constellation of defects as
CVD103-HgR but derived from different parental strains were found to be reacto-
genic [75]. Despite the poor understanding of the underlying mechanism of immu-
nity and reactogenicity in CVD103-HgR, clinical development continued over the
next decade [78, 79] and the vaccine was eventually licensed in Europe as Orochol,
a single-dose oral travelers vaccine for cholera [80]. In 2000, however, the results of
a large, placebo-controlled field trial of Orochol in Indonesia indicated that this
vaccine provided protection from cholera in only 14% of subjects despite inducing
adaptive immune responses in nearly 70% of vaccine recipients [81]. These and
other considerations resulted in suspended manufacture of Orochol.
8 New Insights and New Approaches toward Stable

Attenuation of V. cholerae
With the outbreak of cholera in Peru in 1991 after nearly a century-long hiatus in
the Western hemisphere, new urgency was placed on developing highly immuno-
genic, live attenuated cholera vaccines based on the El Tor biotype strain that
caused the outbreak. This was based in part on the recognition that El Tor strains
expressed an antigenically distinct TcpA that was presumed to be a protective
mucosal immunogen [82] and in part on the assumption that these strains would
be more immunogenic because of their increased colonization capacity compared
to classical strains. Mekalanos and colleagues at Harvard Medical School soon
created V. cholerae O1 El Tor strains with deletions in ctxAB as well as several
genes involved in the integration of the CTX genetic element, a large DNA segment
believed necessary for the acquisition of ctxAB by nontoxigenic V. cholerae strains
[83]. The CTX genetic element was also suggested to encode accessory toxins, but
vaccine strains with deletions in these genes remained reactogenic [84, 85], and the
putative toxin genes (named zot and ace) were later shown to be morphogenesis
genes of the filamentous phage that encoded CT (see below), providing strong
evidence that these putative toxin genes had no role in reactogenicity [86].
The whole concept of a genetically stable live attenuated cholera vaccine was
brought into question by the work of Waldor and Mekalanos who reported in 1996
that the CTX genetic element corresponded to the genome of a filamentous bacte-
riophage termed CTXF [86]. This phage could efficiently transduce nontoxigenic
V. cholerae strains using TCP as a receptor, and the specter of genetically engi-
neered V. cholerae vaccine strains reverting to toxicity by simple phage transduc-
tion immobilized many vaccine developers during this period. It was subsequently
recognized that CTXF requires a 17 bp site called attRS1 to integrate into the
V. cholerae large chromosome; V. cholerae strains deleted for attRS1 can be

transduced by CTXF but the episomal CTXF genome is unstable and is easily
lost because it cannot integrate into the chromosome [86]. Deletion of recA in
vaccine strains was also explored as a stabilizing mutation because it was predicted
to block reacquisition of the CTX phage through other pathways that require
homologous recombination. Deletions in both recA and attRS1 have been
incorporated into newly designed vaccine strains, and live cholera vaccine devel-
opment has continued. While this approach has solved, in theory, the issue of
CTXF-mediated reversion to toxigenicity, the deeper issue of reactogenicity,
remained unsolved.
9 Development of the Concept: Evaluation of Motility

Defective Vaccine Candidates
A breakthrough was achieved when the live vaccine strain Peru-14 was found to be
highly immunogenic but also very well tolerated in volunteer studies [87]. Derived
from an O1 El Tor clinical strain isolated in Peru in 1991, Peru-14 contained
deletions in the entire CTXF genome and attRS1 site, and recA was replaced
with a high-expression construct for ctxB, the gene that encodes the immunogenic
but nontoxic cholera toxin B subunit CTB (Fig. 1). Importantly, Peru-14 also
carried an undefined mutation that gave it a filamentous morphology; Peru-14
was motile when observed by light microscopy but did not penetrate soft agar in
motility assays, so it was thought that Peru-14’s low reactogenicity resulted from its
filamentous morphology and not its motility defect per se. To further address this
possibility, Peru-14’s parental strain Peru-3 was screened for spontaneous muta-
tions that rendered it nonmotile but left its cell morphology intact. One stable
nonmotile mutant, Peru-15, was tested in initial volunteer studies and found to
also be highly immunogenic and completely devoid of reactogenicity [88]. Peru-15
does not form a flagellum and as such represents the first aflagellar bacterial mutant
to be evaluated in human volunteer studies as a live attenuated vaccine.
10 Peru-15: an Aflagellar, Nonreactogenic Cholera Vaccine
The initial positive results with Peru-15 prompted expanded study of its safety and
immunogenicity in various buffers and in a lyophilized form. As expected, Peru-15
was least immunogenic in saline, which cannot neutralize stomach acid, but an oral
dose of 1 108 bacteria in a buffer called CeraVacx induced seroconversion in all
ten volunteers immunized [89]. A lyophilized version of Peru-15 was evaluated in a
larger double-blind, placebo-controlled volunteer immunization/challenge trail by
Cohen and colleagues [90]. In this study, 59 volunteers were randomly selected to
receive either 2 108 colony-forming units (CFU) of lyophilized Peru-15 vaccine
CT-AB5 (toxic)
C6709 (01 El Tor) CTX M010 (0139)

//
Peru 1991 clinical isolate India 1993 clinical isolate
rec
A
CT-B5 (non-toxic)
Peru-3 (deleted) Bengal-3

//
immunogenic ctx immunogenic
reactogenic reactogenic
B
CT-B5 (non-toxic)
Peru-14 Peru-15 (deleted) Bengal-15

//
immunogenic immunogenic immunogenic
non-reactogenic
ctx
non-reactogenic non-reactogenic
B
filamentous aflagellar aflagellar

reduced motility
Fig. 1 Live cholera vaccine design. Peru 3 and Bengal 3 were created from V. cholerae clinical
isolate strains C6709 and MO10 by deleting the CTXF genome and replacing recA with a ctxB
expression construct. Peru 15 and Bengal 15 contain spontaneous mutations that produce aflagel
lar cells that retain immunogenicity but strongly reduce reactogenicity in vaccine recipients
reconstituted in CeraVacx buffer or a placebo containing only CeraVacx buffer.

After unblinding, it was found that Peru-15 was well tolerated compared to buffer
alone and impressively 98% of the Peru-15-immunized volunteers showed at least a
fourfold rise in serum vibriocidal antibody. Three months after immunization, 36
volunteers were challenged with V. cholerae O1 El Tor strain N16961, a toxigenic
clinical isolate from the current seventh pandemic. Remarkably, none of the 24
volunteers who received the vaccine developed even moderate diarrhea while 5 of
the 12 placebo recipients (42%) developed either moderate or severe diarrhea.
11 Bengal-15 and More Evidence for the Link between

Motility and Reactogenicity
In order to produce a live attenuated vaccine protective against O139 serogroup

strains of V. cholerae and obtain more evidence for the role of flagellar motility in
vaccine reactogenicity, another vaccine derivative called Bengal-15 was developed
and tested in North American volunteers [91, 92]. Starting with the toxigenic O139
strain MO10, Bengal-15 was constructed to contain the same genetic “blueprint” as
Peru-15 including its aflagellar phenotype (Fig. 1). Bengal-15 and its motile
parental strain Bengal-3 were evaluated in a small volunteer study for their immu-
nogenicity and reactogenicity after oral inoculation. At a dose of 1 108 bacteria,
one of the four Bengal-3 recipients experienced diarrhea but none of the ten Bengal-
15 recipients did, suggesting that the loss of flagella formation in Bengal-15
reduced its reactogenicity. Upon challenge with the toxigenic MO10 strain 1
month after vaccination, five of six patients who had received only the placebo
vaccine developed severe diarrhea while only one of seven patients vaccinated with
Bengal-15 developed mild diarrhea (protective efficacy 83%). It was concluded that
the aflagellar strain Bengal-15 was a nonreactogenic vaccine candidate that was
highly immunogenic and protective against cholera caused by O139 serogroup
strains of V. cholerae.
12 Field Trials of Peru-15 in a Cholera-Endemic Region
Building on the impressive results obtained in North American volunteers immunized

with the aflagellar Peru-15 strain, Qadri and colleagues sought to evaluate the vaccine
in adults in Bangladesh, a region where cholera is endemic [93]. These studies
were carried out by a team at the International Center for Diarrhea Disease Research,
Bangladesh (ICDDR,B) in collaboration with the investigators at the International
Vaccine Institute (IVI, Seoul, Korea), Avant Immunotherapeutics (now Celldex
Therapeutics, Needham, Massachusetts), and Harvard Medical School, Boston,
USA. In the double-blind, placebo-controlled study, no major adverse events
were associated with the vaccine in adult volunteers, and despite the fact that
Peru-15 was detected in stools of only one volunteer, 30 of the 40 vaccine recipients
(75%) seroconverted for serum vibriocidal antibody and 35 (88%) displayed elevated
levels of serum IgA antibodies directed against V. cholerae LPS O-antigen. Detectable
immunogenicity against CTB, however, was lower in the Bangladeshi volunteers than
had been previously observed in North Americans.
Demonstration of the safety and immunogenicity of Peru-15 in adults from
endemic communities set the stage for further vaccine trials in Bangladeshi children
in an age-descending study [94]. In a double-blind, randomized, placebo-controlled
trial, 140 children aged 9 months to 5 years were given the vaccine, with a further
100 children receiving placebo. Two different doses were examined and children
were monitored for adverse reactions, excretion of the vaccine strain, and serocon-
version by vibriocidal assay and IgA anti-LPS responses. Peru-15 was not associated
with adverse events in this study and was isolated from only 8 of 140 recipients.
However, 84% of toddlers (age 2 5 years old) and 70% of infants (9 23 months
old) showed a serum vibriocidal response after receiving the higher dose of Peru-15
(2 108 CFU). Sixty percent of the vaccinated toddlers and 34% of the vaccinated
infants also showed IgA responses against V. cholerae LPS-O antigen compared
to 15% of toddlers and 12.5% of infants who received a placebo. Responses to
CTB subunit were lower but also significant (46% of toddlers and 36% of infants).
Thus, Peru-15 was clearly safe and immunogenical in Bangladeshi children when
administered in a single dose. Further development of Peru-15, known commer-
cially as CholeraGarde, awaits creation of a formulation suitable for large-scale
manufacture followed by efficacy trials in a cholera-endemic setting [95].
13 Host-Innate Immunity and Flagellin Signaling
Development of the aflagellar vaccines Peru-15 and Bengal-15 in the mid 1990s
actually predated the recognition that bacterial flagellins were key signaling mole-
cules of the innate immune system and were capable of inducing proinflammatory
responses. Early reports by Mizel and colleagues identified flagellin as a bacterial
protein that could be recognized by high-affinity receptors on human monocytes in
a process that mysteriously activated cytokine production [96, 97]. Eventually,
Hayashi and colleagues reported that Toll-like receptor 5 (TLR5) recognizes a
conserved amino acid sequence in the flagellin protein [98]. We now know that
flagellin is one of many highly conserved components of bacterial cells such as
lipopolysaccharide, peptidoglycan, lipoproteins, and DNA that contain pathogen-
associated molecular patterns (PAMPs) that are recognized by host pattern recog-
nition receptors (PRRs) [99]. Upon binding their cognate PAMP, PRRs like TLR5
then activate signal transduction pathways leading to proinflammatory cytokine
production. In brief, TLR5 responds to flagellin binding by signaling through
MyD88 and the serine/threonine kinase IRAK-4 to ultimately activate the mitogen-
activated protein kinase p38 (p38 MAPK) and the transcription factor NF-kB,
which go on to induce expression and secretion of the cytokines TNF-a, IL-8,
and IL-1b (Fig. 2) [100]. These cytokines promote an influx of neutrophils and
other inflammatory leukocytes and induce further cytokine expression and release
by surrounding cells. IL-1b must be activated by caspase 1, found in an innate
immune complex known as the inflammasome that can independently recognize
flagellin [101]. Such inflammation in the gut could cause symptoms such as fever,
cramps, and nausea that closely resemble the reactogenicity induced by flagellated
live cholera vaccines.
TLR5 is expressed in mucosal tissues and can recognize flagellins produced by
invasive pathogens [102] as well as extracellular organisms like Pseudomonas
aeruginosa [103]. Intestinal epithelial cells (IECs) were originally thought to
intestinal lumen V. cholerae
mucus
villi crypt
epithelium surface
epithelial
cells
lumen
TLR5
flagellin
p38 MAPK
NF- B
IL-1
IL-8
TNF
inflammation
+
leukocyte dendritic cell
recruitment lamina propria
Fig. 2 V. cholerae flagellin induced inflammation model. During infection, V. cholerae pene
trates the small intestine mucus layer and colonizes the epithelium where membrane bound TLR5
receptors of epithelial or dendritic cells recognize V. cholerae flagellin. TLR5 mediated innate
immune signaling causes activation of NF kB and p38 MAPK and subsequent expression and
secretion of proinflammatory cytokines including IL 1b, IL 8, and TNF a into the lamina propria
express TLR5 only on the basolateral membrane [104], but more recent work has
shown that TLR5 is found on the apical surface as well and readily detects flagellin
produced by noninvasive pathogens like V. cholerae [105]. To avoid detection by
TLR5, some enteric pathogens such as Campylobacter jejuni and Helicobacter
pylori have altered their flagellin peptide sequence [106]. Salmonella, on the other
hand, may actually use flagellin to activate TLR5 signaling and induce inflamma-
tion as part of its infection process before downregulating flagellin expression
after gaining intracellular access to intestinal enterocytes [107, 108]. V. cholerae
produces a membranous sheath that covers the length of its flagellum and helps to
hide its flagellin components from TLR5 recognition [109]. Nevertheless, all five of
the flagellins that V. cholerae produces are abundantly found in culture supernatants
and all five are recognized by TLR5 [110].
14 Motility and Reactogenicity: The Infant Rabbit Model
The Peru-15 and Bengal-15 vaccine trials detailed above provided strong correlative
evidence that flagellar motility plays an important role in live cholera vaccine
reactogenicity, but the precise cause of these patient symptoms remained undefined
because of the pleiotropic role that flagellar motility plays in V. cholerae pathogen-
esis. Flagellar motility is required for V. cholerae to efficiently colonize the small
intestine in the infant mouse model of infection [111], and flagellar motility is
thought to be necessary for the bacterium to traverse the thick mucus layer protect-
ing the intestinal epithelium [23]. At a genetic level, the flagellar biosynthesis
regulon in V. cholerae directly regulates the quorum sensing and virulence cascades
in vitro [24, 112] and may serve as a mechanical sensor to induce virulence gene
expression in response to mucus-induced flagellar shearing. Finally, the flagella
itself could be the principle cause of reactogenicity since TLR5 recognition of its
flagellin components may cause a local inflammatory response at the intestine
epithelial surface.
As a result, the aflagellar vaccine strains may not induce reactogenic diarrhea
because it mislocalizes in the intestine, is unable to initiate virulence gene expres-
sion, or fails to induce an inflammatory innate immune response. Finally, it is
important to point out that Peru-15 and Bengal-15 were isolated in screens for
spontaneous nonmotile mutants and may contain secondary mutations, so it is
formally possible that the reduced reactogenicity of these strains is independent
of their aflagellar morphology.
All these theories provide clear testable hypotheses, but until recently there was
no adequate animal model of diarrheal disease for cholera. The infant mouse has
long served as an accurate model for V. cholerae intestinal colonization and
virulence gene induction, but the lack of a robust diarrheal phenotype negates its
use in any comprehensive examination of diarrheal disease [113]. Rabbit models
of cholera infection, including the ligated ileal loop and removable intestinal
tie adult rabbit diarrhea (RITARD) models, have been developed to measure
diarrheal disease following V. cholerae inoculation [114, 115], but these models
rely on surgical intervention to block peristaltic flow through the intestine during
V. cholerae infection, a severe nonphysiologic condition that limits their use in
modeling natural cholera disease in people.
To address many unanswered questions about cholera pathophysiology, Richie
et al. [116] have recently developed an infant rabbit model of cholera infection that
closely resembles the diarrheal disease seen in human victims. When infant rabbits
are pretreated with cimetidine to inhibit acid production in the stomach, orogastric
inoculation of V. cholerae causes a voluminous watery diarrhea that resembles
cholera disease in people, and a large majority of the infected rabbits die 24 30 h
after infection. Importantly, deletion of the ctxAB toxin genes in this strain strongly
reduces the occurrence of watery diarrhea and enables the rabbits to survive
infection, but most of the animals go on to exhibit a self-limiting noncholeric
fecal diarrhea that resembles the reactogenic diarrhea observed in people inoculated
with motile live cholera vaccines.
To directly determine the cause of reduced reactogenicity in the Peru-15 vaccine
strain, Rui et al. [117] introduced defined mutations into flagellar genes of an O1 El
Tor strain of V. cholerae that is deleted for ctxAB and is closely related to the
parental strain of Peru-15. To separate any reactogenic phenotype associated with
flagellar biosynthesis from that associated with swimming motility, they measured
reactogenicity in rabbits infected with either a flagellar motor mutant that is
flagellated but nonmotile or a flagellar filament mutant that lacks all five flagellin
genes and is both aflagellate and nonmotile. The flagellated nonmotile mutant
caused reactogenicity at nearly the same level as the parental strain but the
aflagellar mutant caused very little reactogenicity in the animals. Disruption of all
five flagellins was necessary to achieve the lowest reactogenicity levels, suggesting
that all five of the flagellins are able to induce reactogenic diarrhea.
An intriguing observation in these rabbit studies was that the aflagellar mutant
was able to penetrate the intestinal mucus layer and travel into the deep intestinal
crypts, an area thought to be inaccessible to nonmotile strains [21, 23, 118]. The fact
that motile and nonmotile V. cholerae strains show the same intestinal localization
in these experiments may help to explain why the nonmotile Peru-15 strain is able
to induce the same immunogenicity in vaccine recipients as its motile Peru-3 parent
strain [88, 94], and it lends further weight to the recent suggestion that V. cholerae
uses flagellar-independent motility to travel through the intestinal mucus layer [24].
In tandem with data showing that V. cholerae flagellins directly activate TLR5,
these infant rabbit experiments strongly suggest that the reduced reactogenicity
seen in Peru-15 is specifically due to reduced flagellin production.
15 Perspective on other Live Attenuated Vaccines vis-à-vis

Flagellins and Reactogenicity
In conclusion, studies of flagellar defective V. cholerae vaccine candidates in

volunteer subjects and new animal models for reactogenicity have now provided
strong evidence that flagellin is a significant reactogenic factor elaborated by the
organism. V. cholerae produces other potentially reactogenic factors including the
MARTX toxin [119] and effectors of the newly described type VI secretion system
[120], but in the context of human infection with CTXF-deleted vaccine candi-
dates, it seems likely that flagellin production is the most important proinflam-
matory signal and is directly responsible for the reactogenicity seen in vaccine
recipients. It is worth noting that other live attenuated bacterial vaccine candidates
have suffered from the problem of reactogenicity in human subjects, and one might
speculate that these organisms produce flagellin in vivo that might elicit reacto-
genicity as well. For example, live attenuated Shigella vaccines are often reacto-
genic in volunteer studies [121, 122], and although Shigella are notoriously
nonmotile, some strains have been reported to contain intact flagellar operons and
to produce flagella [123]. Even the flagellin produced by commensal organisms
such as Escherichia coli may be involved in pathology associated with inflamma-
tory disease. In human disease, flagellin has been implicated as an elicitor in
inflammatory bowel diseases such as Crohn’s [124, 125] and mutations in TLR5
are associated with enhanced susceptibility to Legionella disease [126].
Clearly, recognition of flagellin by the innate immune system is an early host
response that may affect the outcome of infection through induction of locally
protective inflammatory responses. However, in the context of a live bacterial
vaccine such a local immune response may cause adverse symptoms and could
even block the development of long-term immunity in the vaccine recipient by
controlling the infection before it can induce a strong adaptive immune response.
Continued exploration of flagellin as a reactogenic factor in natural infection and
experimental immunization seems warranted.
Acknowledgments We thank Shannon Humphreys for help in designing the figures.
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Part IV
New Types of Replicating Vaccines
Replication-Defective Herpes Simplex Virus
Mutant Strains as Genital Herpes Vaccines
and Vaccine Vectors
David M. Knipe
Abstract Viral vaccines have traditionally been live, attenuated viruses, or inacti-
vated virus/subunits. Herpes simplex virus (HSV) vaccine candidates based on
inactivated viruses or subunits have not been effective thus far. In addition, attenu-
ation of HSV to make a safe vaccine candidate has not allowed good immuno-
genicity to be retained. Therefore, novel vaccine strategies have been initiated,
including replication-defective and single-cycle HSV strains. In this chapter, I will
review the design and properties of these replication-defective virus vaccine can-
didates and the preclinical and clinical results that have been obtained using them.
1 Introduction
The herpes simplex viruses cause significant morbidity and mortality, including
encephalitis, neonatal herpes, and keratitis [1]. Despite the existence of some good
antiviral drugs, there is enormous need for a herpes vaccine, in particular for a
genital herpes vaccine. Herpes simplex virus 1 (HSV-1) causes orofacial lesions
including the common cold sores or fever blisters, but causes more serious enceph-
alitis and keratitis in a limited number of cases. Herpes simplex virus 2 (HSV-2)
causes most of the genital herpes infections but becomes life-threatening in neo-
nates and immunocompromised individuals. Importantly, in terms of global public
health, HSV-2 infection increases the susceptibility to human immunodeficiency
virus (HIV) by 3 4-fold [2, 3]. This effect is possibly exerted through the herpetic
lesions providing breaks in the epithelial mucosa that allow HIV to enter the
epithelium and which also contain elevated numbers of CD4+ T lymphocytes, the
host cell for HIV, and dendritic cells, which transport HIV to lymph nodes where it
D.M. Knipe
Department of Microbiology and Molecular Genetics, Harvard Medical School, 200 Longwood
Avenue, Boston, MA 02115, USA
e mail: david [email protected]

286 D.M. Knipe
can infect CD4+ T-lymphocytes. Herpes suppressive drug therapy of individuals

doubly infected with HSV-2 and HIV reduces viral loads of HIV [4, 5]. However,
herpes drug treatment of HSV-2 seropositive individuals did not reduce the risk for
HIV infection [6, 7]. Therefore, novel strategies, such as a genital herpes vaccine,
are needed to reduce HSV-2 infection and thereby reduce the risk of HIV infection.
For these reasons, an effective genital herpes vaccine would be a major advance in
global public health.
2 History of HSV Vaccines
Traditionally, viral vaccines were either live, attenuated viruses, or inactivated

viruses. Newer forms of inactivated viral vaccines consist of viral protein subunits.
There are several examples of successful vaccines of each type. Live, attenuated
vaccines have included the smallpox vaccine, the yellow fever vaccine, the Sabin
polio vaccine, and the measles vaccine. Killed viral vaccines have included the Salk
polio vaccine. Viral protein vaccines have included hepatitis B vaccine and human
papilloma virus vaccine.
Several of the early herpes vaccines were tested in trials that were not placebo-
controlled, so their efficacy could not be determined (reviewed in [8]). An
attenuated HSV recombinant virus, R7020, constructed by Bernard Roizman’s
laboratory was safe in phase I trials but was not immunogenic [9]. A replication-
competent HSV-2 strain constructed in Aurelian’s laboratory [10, 11] was tested
in a phase I therapeutic clinical trial in Mexico and reported to reduce recurrences
[12]. The safety profile of this replication-competent HSV-2 strain has not been
described, but it is capable of establishing latent infection albeit at a reduced
level [11].
Biovex Inc. has a “novel replication-competent HSV-2 virus,” based on inacti-
vation of viral immune response genes, which it is testing as a prophylactic genital
herpes vaccine (http://www.biovex.com/immunovex.html), but the precise geno-
type and properties of this virus have not been reported in the scientific literature.
Their ImmunoVEXHSV-2 vaccine product was approved for a phase I clinical trial in
the United Kingdom.
Recently, Friedman’s group has used an HSV-1 glycoprotein E (gE)-null mutant
virus to immunize mice, and they observed protection against flank challenge
infection by HSV-1 [13]. This approach is discussed in another chapter of this book.
Thus far, it has been difficult to attenuate HSV to make it safe enough to give as a
prophylactic vaccine. Therefore, subunit vaccines have been tested as herpes
vaccines. The Chiron HSV-2 glycoprotein B and D (gB-2 and gD-2) subunit
vaccine in MF-59 muramyl dipeptide adjuvant provided no clinical protection in
prophylactic [14] and therapeutic trials [15, 16]. The GlaxoSmithKline gD-2 subunit
vaccine in alum and monophosphoryl lipid A (Herpevac vaccine) showed no
efficacy in men or in women who were HSV-1 seropositive but showed partial
Replication‐Defective Herpes Simplex Virus Mutant Strains 287
protection against HSV-2 infection in women who were seronegative for HSV-1
and HSV-2.
Thus, subunit vaccines have not provided broad protection against HSV-2 infec-
tion. As a result, novel vaccine strategies including DNA vaccines, peptide vac-
cines, replication-defective mutant viral strains, and single-cycle mutant viral
strains have been tested as potential HSV-2 vaccines (reviewed in [18]). In this
chapter, we will consider the replication-impaired viruses as herpes vaccines and
vaccine vectors.
3 Replication-Impaired HSV Mutants
Two types of replication-impaired HSV mutant viral strains have been tested as
HSV vaccines, replication-defective mutant strains, and single-cycle mutant
strains. HSV-1 replication-defective mutant viruses were among the first replica-
tion-impaired viruses used as vaccines [19]. An HSV-2 replication-defective
mutant induced protection against genital HSV-2 infection in guinea pigs [20].
These replication-defective mutants can infect cells and express immediate-early
and early viral gene products and even many late gene products but contain defects
in viral DNA replication, so the replication cycle is absolutely blocked. Viral late
gene expression is observed with mutant viruses defective for ICP8 even though
there is no viral DNA replication, likely because ICP8 and/or a complex of viral
DNA replication proteins exerts an inhibitory effect on viral late gene expression in
the absence of viral DNA synthesis [21].
To generate a safe, potential vaccine strain for clinical use, we deleted two
essential HSV-2 genes, UL5 and UL29, from the HSV-2 186 strain virus to generate
the dl5-29 vaccine candidate virus [22, 23]. The UL5 gene encodes one of the
subunits of the viral helicase primase complex, while UL29 encodes ICP8, the viral
single-stranded DNA-binding protein. Both are essential for viral DNA synthesis
and viral growth. Two deletions separated by a large distance on the viral genome,
both unable to recombine with the genes in the complementing cell line, were
engineered into the vaccine strain to reduce the likelihood that the vaccine virus
could recombine with HSV in the immunized individual to generate a replication-
competent virus. Recombination of the HSV-2 mutant virus with wild HSV-
2 would generate a replication-competent HSV-2 strain, likely no different from
the virus with which the individual was already infected. Therefore, only recombi-
nation of the HSV-2 mutant with a wild HSV-1 would generate a new infection with
a new replication-competent HSV-2-like virus. The dl5-29 mutant virus also has a
latency defect in that the small amount of viral DNA that reaches sensory ganglia
are not maintained stably [22].
Immunization of mice with dl5-29 virus protects them against genital challenge
with virulent HSV-2 in that virus shedding from the genital tract was reduced,
lesions were reduced, and lethal encephalitis was prevented [22]. Studies in guinea
pigs have compared dl5-29 immunization with gD-2 protein and plasmid pgD-2
288 D.M. Knipe
immunization. Prophylactic immunization of guinea pigs with dl5-29 virus reduced

virus shedding and disease during primary infection and latent viral load similarly
to immunization with gD2 in complete Freund’s adjuvant and to a greater extent
than plasmid immunization [24]. Furthermore, therapeutic immunization of guinea
pigs with dl5-29 virus was effective in reducing recurrent infection [24]. Surpris-
ingly, immunization with dl5-29 induced higher neutralizing antibody titers than
immunization with gD-2, a major target for neutralizing antibodies. Immunization
with dl5-29 virus induced anamnestic T cell responses that were recruited rapidly to
sites of viral infection. These studies demonstrated that dl5-29 virus induces strong
humoral and cellular responses and that it is superior to previously tested vaccine
candidates.
The dl5-29 virus was licensed by Acambis (now Sanofi Pasteur Biologicals) and
is undergoing preclinical studies as a vaccine candidate known as ACAM-529.
4 Durability
One concern about a replication-defective HSV strain that does not establish latent
infection is that the immune responses may not be durable. We have observed that
the protective immunity induced by HSV-1 d301 UL29 gene mutant persisted for at
least 7 months [25]. Similarly, immunization with HSV-2 dl5-29 virus induced
protective immunity that persisted for at least 7 months in mice [26]. Therefore,
immunity induced by replication-defective mutant viruses lasts for a significant
portion of a Balb/c mouse’s lifetime [27]. Durable immune responses in the mouse
may involve (1) persistence of viral genomes that can express viral antigens in
various cells and/or (2) trapping of antigen with complement on complement
receptors on bone marrow-derived cells in local lymph nodes [28]. In terms of
the mechanisms of protection, Morrison [29] has found that CD4+ T cells are
required but CD8+ T cells are not essential for protective immunity induced by
replication-defective HSV-2 viruses. Therefore, dl5-29 induces durable T-cell
responses in the murine system.
5 Safety
A prophylactic genital herpes vaccine would ideally be used to immunize children

before they became sexually active; therefore, safety is of the utmost importance
for this application. Replication-deficient strains are ideal for this because the
virus cannot spread significantly beyond the injection site. In mice injected intrace-
rebrally with virus, dl5-29 was at least 250,000-fold less virulent than wildtype
HSV-2, and dl5-29 did not cause any disease in the immunodeficient SCID mice
[30]. Therefore, dl5-29 has a very desirable safety profile from these preclinical
studies.
6 Pre-Existing Immunity
Because the GSK gD2 vaccine in alum and MF59 was not effective in HSV-1
seropositive women, it was conceivable that other vaccines might not be effective
in HSV-1 seropositive individuals. To investigate this question, Hoshino et al. [31]
tested dl5-29 and gD-2 in guinea pigs that were HSV-1-seronegative or -seroposi-
tive. In HSV 1-seronegative animals, dl5 29 induced the highest titers of neutraliz-
ing antibody, and after vaginal challenge with wild-type virus, dl5 29 resulted in
lower rates of vaginal shedding, lower levels of latent viral DNA in sensory ganglia,
and less acute and recurrent genital herpes, compared with the gD2 vaccines. In
HSV-1-seropositive animals, both vaccines induced similar titers of neutralizing
antibodies and showed similar levels of protection against acute and recurrent
genital herpes after vaginal challenge with wild-type virus, but dl5 29 reduced
vaginal shedding after challenge more than did the gD2 vaccine. Therefore, dl5-29
appeared to be efficacious in HSV-1-seropositive guinea pigs.
7 Route of Immunization
The level and type of immunity induced by replication-defective virus immuniza-

tion depends on the route of delivery. Subcutaneous immunization with HSV-1
induced protective immunity against HSV-1 ocular challenge in mice [25]. Simi-
larly, subcutaneous immunization with HSV-2 5BlacZ UL29 mutant virus induced
protective immunity against HSV-2 genital challenge in mice [32]. Immunization
of mice by the intranasal route with HSV-2 5BlacZ also induced protective immu-
nity, but the best protection was observed with combined intranasal and subcutane-
ous immunization [32]. Intravaginal immunization of guinea pigs with dl5-29 was
only partially protective against later vaginal challenge [24].
8 Cross Protection Against HSV-1
There is growing epidemiological evidence suggesting an increase in the incidence

of genital herpes caused by HSV-1 [33, 34]; therefore, a herpes vaccine should
ideally protect against both HSV-1 and HSV-2. Van Lint et al. [35] tested the ability
of HSV-2 dl5-29 to protect against ocular infection with HSV-1 and observed that
dl5-29 immunization induced protective immunity that reduced viral shedding from
the cornea, ocular disease and reduced latent infection by the challenge virus.
Further studies are needed to determine if dl5-29 can protect against genital
challenge by HSV-1, but these results support the idea that HSV-2 dl5-29 might
be broadly effective against HSV-1 infection.
290 D.M. Knipe
9 Single-Cycle Mutant Viral Vaccine
HSV-1 and HSV-2 glycoprotein H mutant viruses were tested as a single-cycle

mutant virus vaccine. HSV gH mutant viruses complete the replication cycle in
normal cells and form progeny virus particles, but because they lack gH, the
progeny virus particles are noninfectious or at least have reduced infectivity [36].
The gH mutant viruses are grown in a complementing cell line that expresses gH,
and immunization with these pseudotyped viruses gives immune protection against
HSV-1 challenge in the ear pinna [37] and genital HSV-2 challenge in mice and
guinea pigs [38]. These viruses may not be completely replication-defective in vivo
because latent infection by an HSV-1 gH mutant virus, as detected by latency-
associated transcript expression in sensory neurons, was observed [39]. These viruses
were named defective infectious single-cycle (DISC) viruses by Cantab Pharmaceu-
ticals. An HSV-2 gH mutant virus was tested in clinical trials by Cantab and was safe
but showed no clinical or virological benefit in a therapeutic phase II trial against
genital HSV-2 disease [40]. On the basis of available information, the development of
the DISC vaccines by Xenova, which acquired Cantab, has been stopped.
10 Future Improvements
10.1 Immune Evasion
HSV encodes a number of gene products that act to reduce the host immune
response. These include: (1) ICP47, which binds to TAP and prevents peptide
transport into the lumen of the ER for loading on MHC class I molecules [41];
(2) virion host shutoff (vhs), a virion tegument protein that, upon entry into the
cytoplasm, becomes a ribonuclease that digests host mRNA and causes shutoff of
host protein synthesis [42, 43]; (3) ICP34.5, which activates a phosphatase to
reverse phosphorylation of eIF2a by PKR [44]; (4) ICP0, which blocks IRF-3
[45] and Toll-like receptor 2 signaling [68]; and (5) US3, which blocks interferon
g responses [46].
Inactivation of the viral genes that inhibit host immune responses could lead to
better immune responses to a vaccine strain if the mutation does not reduce viral
gene expression. As described above, BioVex Inc. has engineered an HSV-2 strain
with mutations in a number of viral immune evasion genes, but the complete
description of the viral strain is not available. HSV ICP47 is an obvious gene to
target to increase immunogenicity; however, ICP47 affects TAP function only in
higher mammals, and there is not a good nonhuman primate model for HSV
because rhesus monkey cells are poor host cells for HSV [47] and other nonhuman
primates are less available.
The effect of vhs inactivation has been tested in several HSV strains. Inactiva-
tion of the vhs/UL41 gene has been shown to increase the immunogenicity of
replication-competent [48] and replication-defective HSV-1 viruses [49]. Inactiva-

tion of the vhs gene in dl5-29 virus generated the dl5-29-41L virus, which was more
immunogenic and induced greater protection than dl5-29 virus in some but not all
situations [26, 30]. Replacement of the HSV-2 vhs gene with the HSV-1 vhs gene,
which encodes a less active form of vhs, to give the dl5-29-41.1 virus, gave a virus
that replicated better than dl5-29, expressed slightly lower levels of viral proteins,
and induced immune responses and protective immunity that approximated those
induced by dl5-29 [50]. These results argue that the HSV-1 vhs protein enhances
growth of HSV-2 but that the RNase activity may have to be inactivated to give
optimal immunogenicity. In total, these studies show the potential of increasing the
immunogenicity and/or replicative ability of dl5-29 by the incorporation of addi-
tional genetic changes in the viral genome.
11 Coexpression of Immune Stimulatory Molecules
An additional potential approach for enhancement of immune responses induced by

the replication-defective mutant strains is coexpression of immune stimulatory
molecules by the virus. To test this hypothesis, Vagvala et al. [51] constructed
a UL29 mutant that expressed the B7-2 costimulatory molecule and found that
B7-2 expressed by the recombinant virus enhanced its immunogenicity and protec-
tive immunity. Therefore, this general approach represents a very promising way to
improve the replication-defective virus vaccine candidates.
12 Genetic Diversity of HSV-2 Strains
One of the remaining questions about HSV-2 is the extent to which genetic
diversity exists in strains around the world. Certain geographical areas, such as
Sub-Saharan Africa, show very high HSV-2 seroprevalence, greater than 40%
among antenatal attendees in one clinic in Africa, [52] and as high as 60 95%
among female sex workers in Sub-Saharan Africa [53, 54]. Therefore, it is impor-
tant that a potential genital herpes strain be efficacious against HSV-2 strains found
in Sub-Saharan Africa. While little is known about the genetic diversity of HSV-2,
one paper has reported that phylogenetic analysis of three genes, US4 (encoding
glycoprotein G or gG), US7 (gI), and US8 (gE), of the genomes of 47 HSV-2 isolates
from Tanzania, Norway and Sweden show at least two genogroups with genogroup
A and B being represented in the Tanzanian isolates and group B in the Scandana-
vian isolates [55]. Because there is more genetic diversity in the Tanzanian HSV-
2 isolates, we reasoned that the Sub-Saharan isolates might also diverge antigeni-
cally. We tested the ability of HSV-2 dl5-29 mutant virus, which is based on a US
isolate, to induce protective immunity against the US isolate, HSV-2 G, or a South
African isolate SD-90 (Dudek and Knipe, personal communication). We observed
292 D.M. Knipe
that dl5-29 did induce protective immunity against both viruses but that higher
doses of dl5-29 virus were needed to protect against SD-90 infection as compared
to that needed for protection against G virus infection. Similarly, we observed that
a United States UL29 virus protected better against three US HSV-2 challenge
strains than against three South African HSV-2 challenge strains, and vice versa, a
South African UL29 virus protected better against South African HSV-2 challenge
strains than against the three US HSV-2 challenge strains. Further studies are
needed, but these results suggest that optimal protection in South Africa may require
a replication-defective virus based on the genomic backbone of an HSV-2 isolate
from that region.
13 HSV as a Vaccine Vector
HSV replication-defective mutant virus strains can also serve as recombinant

vaccine vectors. There are several advantages to HSV as a vaccine vector. First,
HSV infects a wide variety of human cell types, likely because it has multiple virion
glycoproteins that facilitate entry through any of several receptor molecules [1].
Second, the HSV genome can be expanded by at least 15 kbp [56], and with
deletion of nonessential viral genes, the size of the inserted sequences could be
increased further. Third, HSV activates innate responses so an adjuvant is not
needed. Fourth, HSV induces Th1 helper T cell responses against antigens
expressed by the vector [57]. Fifth, as discussed above, replication-defective HSV
strains can be used to immunize systemically or mucosally.
Replication-defective and replication-competent HSV-1 strains expressing sim-
ian immunodeficiency virus (SIV) envelope (env) and nef were constructed by
insertion of the SIV gene expression cassette into the viral thymidine kinase gene
[58]. The HSV recombinants induced antienvelope antibody responses that per-
sisted at relatively stable levels for months after the last administration. Two of
seven rhesus monkeys vaccinated with recombinant HSV were solidly protected,
and another showed a sustained reduction in viral load following rectal challenge
with pathogenic SIVmac239 at 22 weeks following the last vaccine administration.
These results provided proof-of-concept for the use of HSV recombinants as AIDS
vaccine vectors.
To improve the HSV replication-defective vector, we evaluated the properties of
a second-generation HSV vaccine vector, an HSV-1 multiple immediate-early (IE)
gene deletion mutant virus, d106, which contains deletions in the ICP4, ICP27,
ICP22, and ICP47 genes [59]. Because several of the HSV IE genes have been
implicated in immune evasion, we hypothesized that inactivation of the genes
encoding these proteins would result in enhanced immunogenicity. The d106
virus expresses few HSV gene products and shows minimal cytopathic effect in
cultured cells. When we inoculated d106 virus into mice, we observed that viral
DNA accumulated at high levels in draining lymph nodes, consistent with an ability
to transduce dendritic cells and activate their maturation and movement to lymph
nodes. A d106 recombinant expressing E. coli b-galactosidase induced durable

b-gal-specific IgG and CD8þ T cell responses in naive and HSV-immune mice.
Finally, d106-based recombinants were constructed that express simian immuno-
deficiency virus (SIV) gag, env, or a rev tat nef fusion protein for several days
in cultured cells. Thus, d106 shows many of the properties desirable in a vaccine
vector: limited expression of HSV gene products and cytopathogenicity, high level
expression of transgenes, ability to induce durable immune responses, and an
ability to transduce dendritic cells and induce their maturation and migration to
lymph nodes.
The d106 vectors expressing SIV proteins were used to immunize rhesus maca-
ques [60]. Three macaques were inoculated with recombinant HSV vectors expres-
sing Gag, Env, and a Tat Rev Nef fusion protein of simian immunodeficiency
virus (SIV). Three other macaques were primed with recombinant DNA vectors
expressing Gag, Env, and a Pol Tat Nef Vif fusion protein prior to boosting with
the HSV vectors. Robust anti-Gag and anti-Env cellular responses were detected in
all six macaques. Following intravenous challenge with wildtype, cloned SIV-
mac239, peak and 12-week plasma viremia levels were significantly lower in
vaccinated compared to control macaques. Plasma SIV RNA in vaccinated maca-
ques was inversely correlated with anti-Rev ELISPOT responses on the day of
challenge (P value < 0.05), anti-Tat ELISPOT responses at 2 weeks post challenge
(P value < 0.05) and peak neutralizing antibody titers prechallenge (P value 0.06).
Therefore, the d106 vectors were capable of inducing efficacious humoral and
cellular immune responses specific for SIV proteins.
HSV-1 d106 recombinants expressing HIV clade A env, clade C env, HIV
clade B gag, and HIV have been constructed ([61]; Sen et al. personal communi-
cation). The recombinant vector expressing clade A env is genetically stable for
up to ten passages in cell culture, gives burst sizes of 200 250 PFU/cell, and
shows a specific infectivity of less than 100 particles/PFU (Sen and Knipe,
personal communication). Therefore, the d106 vectors are genetically stable,
grow well for production purposes, and show high infectivity. The d106 vectors
show low infectivity and/or expression of the transgene in rhesus fibroblasts
relative to human cells; therefore, the tests of the HSV-1 vectors in rhesus
macaques may underestimate the immunogenicity of these viral vectors. Clinical
tests of these recombinant vectors in humans are highly justified based on these
preclinical results.
14 HSV Amplicons as Vaccine Vectors
HSV amplicons have also been tested as vaccine vectors. HSV amplicons are
replication-defective viruses that are deleted for all genomic sequences except for
an origin of DNA replication and DNA packaging sequences [62]. The viral
proteins needed for replication of the genome and for assembly of the virion are
provided by a helper virus [62] or by a set of five cosmids that contain an entire
294 D.M. Knipe
genome of HSV-1 deleted for DNA packaging signals [63]. An HSV-1 amplicon
expressing HIV-1 gp120 induced durable cellular and humoral responses in mice
[64]. An HSV-1 amplicon that expressed HIV-1 env elicited polyfunctional T cell
responses specific for env and strongly boosted responses to an adenovirus vector
primed mice [65]. However, production of these vectors has been a challenge, and
expression of the transgene seems to be limited, possibly due to the absence of
expression of the HSV IE ICP0 protein, which prevents host chromatin silencing of
the viral genome [66, 67]. Immunogenicity and protection studies in nonhuman
primates have not been reported for the amplicon vectors.
15 Perspectives
Replication-defective viruses have been very promising as genital herpes vaccines

and vaccine vectors in animal model systems. Their real potential needs to be tested
in clinical trials in that the animal model systems may overestimate or underesti-
mate the immunogenicity of these vaccine candidates. Testing of these vaccine
candidates in humans is a high priority, which would allow an assessment of their
efficacy in humans followed by the construction and testing of further generations
of recombinant viruses that are designed with the types of improvements described
in this article.
Acknowledgments Research in the author’s laboratory on HSV vaccines is supported by NIH

grant AI057552 and on HSV vaccine vectors is supported by NIH grant HIVRAD grant AI46006.
DMK is co inventor on a patent on HSV replication defective mutant vaccine technology that has
been licensed by Harvard University to Sanofi Pasteur Biologicals. DMK is a consultant to Sanofi
Pasteur Biologicals.
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Nucleic Acid-Based Infectious and
Pseudo-Infectious Flavivirus Vaccines
Justin A. Roby, Roy A. Hall, and Alexander A. Khromykh
Abstract The genus Flavivirus contains a number of important pathogens of

humans including yellow fever virus (YFV), dengue virus (DENV), Japanese
encephalitis virus (JEV), tick-borne encephalitis virus (TBEV), and West Nile
virus (WNV). Despite causing significant morbidity and mortality worldwide,
commercially available vaccines only exist for YFV (live-attenuated), TBEV, and
JEV (inactivated). Flavivirus vaccine research has been driven by the need for
cheap, safe, thermally stable, and efficacious preparations amenable to use in
developing nations. The creation of infectious cDNA clones of various flaviviruses
has led to the development of genetically engineered, nucleic acid-delivered,
attenuated live vaccine candidates. These provide effective immunity from a single
immunisation, however share the same safety concerns as traditional live-attenuated
vaccines. The generation of large internal deletions in the capsid gene of flavivirus
genomes creates a vaccine that secretes large amounts of immunogenic prM/E
particles from self-replicating RNA but does not form a spreading infection.
Packaging of these capsid-deleted RNAs into virus-like particles (VLPs) using a
cell line that produces capsid gene from another expression vector creates a pseudo-
infectious vaccine that elicits a highly efficient immune response from a single dose
and is safer than infectious virus. However, production of these VLPs is cumber-
some and the resulting product is heat labile. Providing the capsid gene in trans
from another promoter but within the same plasmid DNA as the capsid-deleted viral
genome creates a DNA vaccine capable of producing VLPs in vivo. Uptake of this
plasmid DNA results in the generation of self-replicating, capsid-deleted RNA and
the capsid protein in the same cell, leading to production of secreted single-round
infectious particles (SRIPs). These SRIPs then deliver capsid-deleted RNA to
adjacent cells where it replicates to produce more prM/E particles. As functional
J.A. Roby, R.A. Hall, and A.A. Khromykh (*)

Centre for Infectious Disease Research, School of Chemistry and Molecular Biosciences,
University of Queensland, Brisbane, QLD 4072, Australia
e mail: [email protected], [email protected], [email protected]

300 J.A. Roby et al.
capsid cannot be produced in SRIP-infected cells, further spread does not occur.
SRIP-producing DNA was shown to be highly effective in mice and horses and
provides an easier to manufacture and thermally stable alternative to other vaccine
candidates currently being developed.
1 Introduction
The genus Flavivirus is composed of a number of arthropod-borne disease agents

within the Flaviviridae family of positive-strand RNA viruses [1]. The viral genome
encodes a single polyprotein that undergoes post-translational cleavage to form
three structural proteins; the capsid (C), precursor membrane/membrane protein
(prM/M), and envelope protein (E), as well as seven non-structural proteins (NS1,
NS2A, NS2B, NS3, NS4A, NS4B, and NS5) [1].
The most notable human pathogens among the flaviviruses are the mosquito-
borne yellow fever virus (YFV), Japanese encephalitis virus (JEV), the four ser-
otypes of dengue virus (DENV), and West Nile virus (WNV), as well as tick-borne
encephalitis virus (TBEV) [1, 2]. All of these viruses cause significant morbidity
and mortality within their areas of endemicity. Substantial increases in the freedom
of trade and travel in the post-World War II era have given these viruses an
unprecedented potential to expand their natural geographical range. Perhaps the
most demonstrative example of this is the introduction of WNV into New York City
in 1999 and the subsequent rapid invasion of the virus into most areas of the United
States, parts of Canada, Central America, the West Indies, and substantial areas of
South America [3 7].
There are no specific therapies for the diseases of humans or livestock caused by
these flaviviruses, thus the development and application of safe and efficacious
vaccines is of paramount importance for the management of outbreaks. To date the
only commercially licensed vaccines against flavivirus diseases in humans are the
live-attenuated 17D YFV vaccine developed in 1936 [8 10], formalin-inactivated
whole-virus vaccines against TBEV [11, 12] and JEV (both mouse brain- and cell
culture-derived) [13 16], and a live-attenuated SA14-14-2 strain JEV vaccine
produced in primary hamster kidney cells in China [17, 18]. These live-attenuated
and inactivated whole-virus vaccines have been highly successful in providing
effective prophylaxis for communities at risk. However, there are specific chal-
lenges that must be overcome in the light of stringent demands on the safety,
efficacy, and manufacturing cost of such approaches [19].
Despite their success, recent concerns have been raised in the medical commu-
nity in regards to adverse reactions observed after administering the currently
licensed 17D YFV and mouse brain-derived JEV vaccines. Severe and often fatal
yellow fever vaccine-associated neurotropic disease and yellow fever vaccine-
associated viscerotropic disease have been estimated to affect 1.3 16 individuals
per million vaccinated and 2.13 2.5 individuals per million vaccinated, respec-
tively [8, 20, 21]. The inactivated, mouse brain-derived JEV vaccine has also been
Nucleic Acid Based Infectious and Pseudo Infectious Flavivirus Vaccines 301
associated with allergic reactions and neurological adverse events (estimated to

affect 0.1 1% and 1 2 patients per million vaccinated, respectively) [8, 22 24].
Although these instances of adverse reactions are relatively low, vaccine technol-
ogy has drastically improved since the development of the currently used vaccines.
Modern incarnations of flavivirus vaccines should thus strive to abrogate these
negative outcomes.
When administered in compliance with the recommended dosage regimen, con-
temporary flavivirus vaccine preparations are characterised as having high levels of
seroconversion and rates of protection in vaccinees [10]. Any innovations in vaccine
research need to demonstrate comparable efficacy. Live-attenuated vaccines hold an
advantage in this regard as inactivated and subunit vaccines require booster immu-
nisations to generate sufficient immune response to be protective.
Flavivirus-mediated diseases primarily affect developing nations with compara-
tively poor healthcare systems and limited budgets in regards to generating and
maintaining vaccine stockpiles [2]. Passages of live-attenuated virus in chicken
eggs are relatively expensive compared to cell culture, and both live and inactivated
viruses require low temperature storage to maintain vaccine integrity. Constructing
a cheap and stable alternative to the current vaccine strategies remains an important
research goal.
Recently, a range of novel flavivirus vaccine candidates exploiting nucleic acid
technologies that overcome the obstacles of cost and safety (Table 1) have been
developed. The establishment of infectious cDNA clones has allowed the delivery
of live-attenuated virus to the vaccinee as highly stable nucleic acids that are
relatively cheap to produce en masse. Further manipulation of this technology has
engendered the concept of capsid-deletion mutants, the genomes of which are able
to replicate in cells; however, virus dissemination is disabled. Such mutants provide
an extra degree of safety to vaccine recipients. Capsid-deleted vaccines may be
delivered as RNA, DNA, or virus-like particles (VLPs). The latest development of
this concept involves the delivery of DNA encoding both the capsid-deleted
flavivirus genome and the intact capsid gene from different promoters but on the
same plasmid DNA. Replicating capsid-deleted genomes are thus packaged into
VLPs in initially transfected cells and undergo one cycle of infection before losing
their ability to spread. These single-round infectious particles (SRIPs) produced
in vivo (otherwise functionally and structurally analogous to VLPs produced
in vitro) provide a bridge between competing strategies combining the safety,
thermal stability, and ease of manufacture of capsid-deleted DNAs with the efficacy
close to that of live-attenuated viruses.
2 Infectious cDNA Clones
The generation of infectious cDNA clones of RNA viruses has allowed direct
manipulation of their genomes revealing information about their replication and
gene expression [36]. In the context of vaccines, cDNA clones have allowed the
302
Table 1 Replicating nucleic acid-based candidate flavivirus vaccines under development

Class Vaccine Flavivirus Animal model Delivery Minimal protective Challenge References
targeted (mode/route) dose (route/dose)
Infectious cDNA pTNd/cD10847 TBEV Mouse RNA/GGa Single, 0.6 ng NDb/100 LD50c [25]
clones pKUN1 WNV Mouse DNA/IMd Single, 0.1 mg IPe/20 IUf [26, 27]
DNA/GGa Single, 1 mg ICg/20 IUf
Single, 0.02 mg IPe/100 IUf
pCMVWN WNV Mouse DNA/IMd Single, 0.01 mg IMd/10 LD50c [28]
Capsid-deleted C(D28–89)-S TBEV Mouse RNA/GGa Two, 1 mg each IPe/>1,000 LD50c [29, 30]
genomes pCMVWN(DC) WNV Mouse DNA/IMd Two, 0.1 mg each IMd/10 LD50c [28]
VLP-delivered RepliVAX WN WNV Mouse, hamster VLP/IPe Single, 4 104 IPe/10 LD50c (mouse) [31–33]
capsid-deleted or SCh VLPs or 10f FFUi
genomes (hamster)
RepliVAX JE JEV Mouse VLP/IPe Single, 4 104 IPe/5 106 FFUi [34]
VLPs
RepliVAX D2 DENV-2 Immunocompromised VLP/IPe Single, IPe/1,000 FFUi [35]
mouse 5 105 VLPs
DNA-delivered pKUNdC/C WNV Mouse DNA/GGa Single, 0.02 mg IPe/100 IUf [27]
SRIPs vaccine
a
GG ¼ gene gun; bND ¼ not described; cLD50 ¼ 50% lethal dose; dIM ¼ intra-muscular; eIP ¼ intra-peritoneal; fIU ¼ infectious units; gIC ¼ intra-cranial;
h
SC ¼ subcutaneous; iFFU ¼ focus forming units
J.A. Roby et al.
investigation into defined targets of virus attenuation and provide a novel means of
delivery of live-attenuated viruses. In contrast to the traditional, empirical means of
live-virus attenuation (i.e. serial passage of the virus to select for growth in chicken
eggs, cell culture, or laboratory animals), cDNA clones allow the introduction of
targeted mutations at specific sites and a subsequent determination of their pheno-
typic importance in attenuation [37].
Thus far infectious clones have been constructed for most of the major patho-
genic flaviviruses: DENV-1 [38, 39], DENV-2 [40 44], DENV-3 [45, 46], DENV-
4 [47], JEV [48, 49], WNV [50], Kunjin [51], TBEV [52, 53], Langat [54], Murray
Valley encephalitis virus [55], wild-type YFV [56], and 17D YFV [57]. Mutagene-
sis and manipulation of these cDNA clones has led to the development of several
new vaccine candidates.
Replacing the prM and E genes of a cDNA clone with those of a heter-
ologous flavivirus allows the generation of chimeric vaccines. This technique
has been applied to the attenuated 17D YFV vaccine backbone to generate
the very promising ChimeriVax series of flavivirus vaccines that have been
reviewed extensively elsewhere [58, 59]. This concept has also been applied to
a backbone of attenuated DENV-4 [45, 60 65] and of DENV-2 PDK-53 [61,
66, 67].
In addition to using the infectious clones as a platform to generate attenuated
viruses, the nucleic acid itself may be utilised for the delivery of the vaccine,
creating a more stable preparation that is cheaper to manufacture (Table 1). Mandl
et al. [25] were the first group to explore this approach with flaviviruses, using
in vitro transcribed RNA corresponding to the genome of the Neudoerfl strain of
TBEV with a 470 nucleotide deletion in the 30 untranslated region (Fig. 1a). RNA
was coated onto gold micro-particles for delivery via gene gun with as little as
0.6 ng conferring protective immunity in outbred Swiss-albino mice [25]. The
authors showed that coating onto gold micro-carriers improved the stability of
RNA when stored at 4 C. However, if a vaccine based upon infectious RNA-
coated gold micro-particles is to be used in clinics, further comprehensive studies
on its cost-effectiveness, long-term durability, and efficacy in humans are
required.
The addition of a eukaryotic promoter upstream of the 50 untranslated region of
an infectious clone allows the plasmid itself to be delivered as a DNA-based
vaccine (Fig. 1b), an approach that has been previously utilised for developing
alphavirus replicon-based vaccines [69]. DNA delivery has an advantage over RNA
vaccines as it is more stable and easier to manufacture. The first demonstrated use
of this approach for developing a flavivirus vaccine employed a cytomegalovirus
(CMV) immediate early promoter upstream of an infectious cDNA of an attenu-
ated Kunjin subtype of WNV, creating the plasmid pKUN1 (Table 1) [70]. Later
challenge studies indicated that intra-muscular injection of 0.1 mg of the pKUN1
DNA vaccine generated sufficient immune response to protect BALB/c mice from
20 infectious units (IU) of the virulent NY99 strain of WNV, whereas 1 mg of
DNA was required for protection when the same challenge dose was administered
intra-cerebrally [26]. A later investigation demonstrated that gene gun-mediated
Infectious vaccines:
Infectious nucleic acids:
a Infectious RNA Live virus
C prM-E NS1-5
b Infectious RNA Live virus

P C prM-E NS1-5
Pseudo-infectious vaccines:
Capsid-deleted nucleic acids:
c Replicon prM-E RNA
dC prM-E
prM-E NS1-5 particles
d dC Replicon prM-E RNA

prM-E
P prM-E NS1-5 particles
VLP-delivered capsid-deleted RNAs

dC
prM-E NS1-5
Replicon prM-E RNA
e prM-E
particles
Packaging VLP
cell
SRIP-producing DNAs
C mRNA SRIPs
dC
f C PP prM-E
prM E NS1
NS1-5
5
Replicon
prM-E RNA
prM-E
p No further
particles viral spread
Fig. 1 Schematic representation of various strategies employed for construction, delivery, and
mode of action for nucleic acid based flavivirus vaccines. Infectious vaccines are based on
infectious cDNA clones that may be delivered either as in vitro transcribed RNA (a) or as plasmid
DNA in which the genomic cDNA is placed under the control of a eukaryote promoter (b). This
approach leads to the generation of live virus in vivo. Pseudo infectious vaccines are based on
capsid deleted flavivirus genomes that may also be delivered either as RNA (c) or as DNA (d).
Capsid deleted RNA can also be packaged into virus like particles (VLPs) using a packaging cells
expressing capsid protein (e). Alternatively, both capsid deleted RNA and an mRNA for the intact
capsid gene can be produced from the same plasmid DNA but under the control of different
eukaryotic promoters thus allowing generation of single round infectious particles (SRIPs) in vivo
(f). As functional capsid gene is not encoded within the capsid deleted RNAs, no further produc
tion and spread of infectious virus can occur in vivo in any of the pseudo infectious vaccines based
on capsid deleted genomes. Figure adapted from Khromykh, Chang, and Hall [68]
immunisation of BALB/c mice with as little as 0.02 mg of pKUN1 provided

complete protection against intra-peritoneal challenge with 100 IU of virulent
NY99 strain of WNV [27].
The cDNA of an attenuated lineage 2 WNV strain 956D117B3 has also been
used in the construction of a DNA-delivered live vaccine candidate, pCMVWN
(Table 1; Fig. 1b) [28]. A single intra-muscular injection of as little as 0.01 mg of the
pCMVWN construct was able to protect NIH Swiss outbred mice from intra-
muscular challenge with 10 LD50 of WNV NY99 (Table 1). This appears to be a
very encouraging preliminary result, and further comprehensive efficacy trials in
small and large animal models should reveal full potential of this vaccine candidate.
3 Capsid-Deleted Genomes
Immunisation utilising nucleic acids that encode live-attenuated flaviviruses cer-

tainly resolves problems associated with the stability and expense of vaccine
preparations. However, this method does little to address the rare instances of
vaccine-associated diseases observed with the currently licensed live-attenuated
vaccines [20, 21]. An elegant solution to this problem is to limit the infectivity of
a vaccine strain by disabling its ability to package replicating RNA into virus
particles, an approach that was previously used for developing vaccines against
herpesviruses [71]. The principal strategy used to achieve this goal with flaviviruses
has been the introduction of internal deletions within the capsid gene (Table 1;
Figs. 1c and d and 2) [19]. This concept was first explored using TBEV as a novel
means of live-virus attenuation [73], but was soon expanded leading to the applica-
tion of a non-infectious replicon vaccine [29].
Experimentation with TBEV internal capsid deletions revealed that removal of
62 amino acids (residues 28 89), corresponding to the second, third, and fourth and
part of the first alpha helices (Fig. 2), generated a replication-competent, packag-
ing-deficient genome [29]. Smaller internal deletions either did not abolish packag-
ing (1 16 amino acids primarily between alpha helix 1 and 2; Fig. 2) [73] or
resulted in spontaneous mutations within the capsid gene that restored the packag-
ing phenotype (19 30 amino acids roughly corresponding to alpha helix 2; Fig. 2)
[74]. A later investigation with WNV also demonstrated that removal of all of the
second and third alpha helices could in some instances lead to a viable packaging
phenotype (Fig. 2) [75]. Early investigations indicated that transfected packaging-
deficient replicon RNAs still allowed the secretion of prM/E sub-viral particles
(SVPs) (the primary mediators of anti-TBEV humoral immunity in this vaccine
approach) (Fig. 1c), but only at relatively low levels [29]. Using rationale devised in
previous investigations [76, 77], supplementary point mutations were introduced
into the signal sequence upstream of the prM gene to increase the efficiency of
signalase cleavage. This “idealised” capsid-deletion mutant C(D28 89)-S was thus
able to liberate a significantly greater proportion of SVPs following transfection
[29]. Gene gun-mediated immunisation of BALB/c mice with approximately 1 mg
of C(D28 89)-S RNA followed by a booster with the same dose at 4 weeks was
sufficient to provide protection against intra-peritoneal challenge with more
than 1,000 LD50 of the virulent TBEV strain Hypr [29]. Subsequent research by
1 10 20 30 40 50 60 70 80 90 100 110 120 130
central hydrophobic region signal sequence
N α11 α22 α33 α44 prM

viral protease signalase
a Viable mutants
Δ28-44 TBEV [64]
Δ39-75 WNV [67]
Δ51-87 WNV [67]
b Pseudo-revertants
Δ28-58 TBEV [66]
Δ39-87 WNV [67]
c Nucleic acid- Δ28-89 TBEV [65]

delivered replicons Δ44 59 WNV [62]
d VLP-delivered
Δ30-101 WNV [106, 109]
Δ23-93 YFV [106]
replicons Δ56-95 WNV shrtC for RepliVAX D2 passaging [110]
e DNA-delivered Δ18-100 Kunjin [61]

SRIPs vaccine
Fig. 2 Schematic representation of the flavivirus capsid protein outlining capsid deletion mutants
utilised in the construction of vaccine candidates. The crystal structure of capsid protein of Kunjin
virus was used as a model for the location of the a helices, hydrophobic domains, and protease
cleavage sites in the diagram [72]. (a) Internal deletions that do not completely abrogate packag
ing, leading to generation of a viable viruses. D28 44 in TBEV capsid and D39 75 and D51 87 in
WNV capsid were the largest deletions allowing recovery of viable viruses. (b) Deletions that
initially abrogated recovery of infectious viruses but resulted in compensatory mutations else
where in the capsid gene which restored the ability to produce infectious viruses. D28 58 in TBEV
capsid and D39 87 in WNV capsid represent a series of deletions that have this effect. (c e)
Capsid deletions that completely disabled the ability to produce infectious viruses. Outlined
deletions are further separated by the strategy used for delivery of capsid deleted genomes, i.e.
naked nucleic acid (c), VLP (d), and SRIPs producing DNAs (e)
the same group demonstrated that the aforementioned consecutive gene gun immu-
nisations of mice were able to induce humoral and cellular (Th1 and CD8þ T cell)
immune responses equivalent to those produced by live vaccines and that even a
single 1 mg C(D28 89)-S RNA dose could induce a long-lasting (1 year) neutralis-
ing antibody response [30].
The capsid-deletion approach has also been applied to WNV DNA-delivered
replicon vaccines (Table 1; Fig. 1d) [28]. Residues 44 59 of the attenuated lineage
2 WNV infectious clone pCMVWN were deleted to generate the replicon mutant
pCMVWN(DC) (Fig. 2c). Although incorporating only a relatively small deletion
of 16 amino acids, the packaging-deficient phenotype appears to have been stable
with no infectious virions present in murine sera after 2 weeks of monitoring
following immunisation with up to 10 mg of pCMVWN(DC) DNA, and no virus
was detected in transfected cell culture during the entire period of observation
(48 h) [28]. Serum conversion was demonstrated to be three- to sixfold lower for
capsid-deleted pCMVWN(DC) compared to infectious pCMVWN DNA following
a single intra-muscular injection of mice with comparable amounts of DNA. An
equivalent immune response could be achieved via booster immunisation with
pCMVWN(DC) DNA [28]. Prime and boost immunisations with as little as 0.1 mg
of pCMVWN(DC) DNA provided adequate immune response to completely protect
mice from intra-muscular challenge with 10 LD50 of WNV NY99 [28]. Despite
this impressive protective efficacy and a demonstrated stability of the packaging-
deficient phenotype, concerns still exist in regards to the long-term durability of the
pCMVWN(DC) construct. As described earlier, studies using TBEV mutants have
shown that capsid-deletions of less than 30 amino acids may result in spontaneous
reversion to replicon-packaging competency [74]. A more rigorous investigation
into the construct’s stability and safety is thus warranted.
4 VLPs for Delivery of Capsid-Deleted RNAs
Immunisation with capsid-deleted nucleic acids is a promising line of research,

though this approach is not without shortcomings. Single immunisation with these
vaccines generates an insufficient immune response to provide protection against
virulent challenge [27], thus necessitating the practice of secondary immunisation.
Booster shots are necessary as nucleic acids traditionally have very low transfection
efficiency in vivo [78, 79]. Temporal separation of two small doses of nucleic acid-
based vaccines allows the establishment of a low-level memory immune response
prior to subsequent activation and proliferation (the clonal selection hypothesis)
[80], leading to an exponential increase in humoral immunity. Simply increasing
the initial dose of capsid-deleted DNA or RNA would not reduce the cost and is
unlikely to generate an immune response equivalent to that achieved via booster
immunisation. One laudable alternative to delivering naked nucleic acid is to
deliver the packaging-deficient replicon RNA via VLPs (Table 1; Fig. 1e), a
technique that has been successfully applied to alphaviruses, lentiviruses, and
poliovirus [81 83]. Flavivirus structural genes that have been deleted in replicons
can be provided in trans from a different expression vector [84].
Method for the generation of flavivirus VLPs was first developed using Kunjin
virus replicons with deletions in the structural genes corresponding to the removal
of all of prM and E, and all but the first 20 codons of C (C20DXrep) [84, 85].
C20DXrep RNA was packaged into secreted VLPs by the Kunjin structural proteins
produced from a Semliki Forrest virus (SFV) replicon, designated SFV-prME-C107
[84]. Sequential electroporation of 2 106 BHK21 cells with 10 20 mg of Kunjin
replicon RNA followed by SFV-prME-C107 replicon RNA generated a maximum
titre of approximately 1.3 105 VLPs/ml of culture fluid, a relatively low titre
compared to the wild-type virus (~107 infectious virions/ml) [84]. To improve the
efficiency of packaging, a stable BHK21 cell line, tetKUNCprME, was later
established incorporating the structural gene cassette under the control of a tetracy-
cline-inducible promoter, which facilitated packaging of subgenomic Kunjin repli-
con RNA with much greater efficiency [86]. In this system, electroporation of
3 106 cells with approximately 20 mg of in vitro-transcribed Kunjin replicon
RNA generated up to 1.6 109 VLPs/ml of culture fluid over a 4 day period [86].
The tetKUNCprME cell line also demonstrated an ability to successfully package

replicon RNA from other flaviviruses, though at reduced efficiencies (WNV and
DENV-2 replicons were encapsidated at approximately 70% and 1% of the effi-
ciency of the Kunjin replicon, respectively) [86].
Although none of these Kunjin-based VLPs were tested as flavivirus vaccines,
the establishment of trans-encapsidated replicon technology encouraged investi-
gations by other groups. To date, packaging of flavivirus subgenomic repli-
cons by structural genes provided in trans has been applied to TBEV [87, 88],
WNV [89, 90], YFV [91, 92], JEV [93], and DENV-1 and -2 [94, 95]. Each of
these flavivirus encapsidation systems uses an approach resembling that
described for the Kunjin replicons. For the majority, one or more of the structural
genes sustained large in-frame deletions within the flavivirus replicon, which
were in turn complemented by the expression of these structural genes in trans,
either within a stable cell line [90, 94 96] or via another expression vector (either
replicon RNA or eukaryotic expression plasmid) [88, 94, 97, 98]. Other techni-
ques have employed cell lines stably housing flavivirus replicons which are
packaged upon transfection with trans-complementing constructs expressing
structural genes [89, 91, 94] and a dual replicon system with complimentary
constructs alternately replacing C with the green fluorescent protein gene, and
prM/E with the gene for the red fluorescent protein Cherry [92]. VLP packaging
efficiency was quite high for TBEV, WNV, and YFV, with approximately
108 109 VLPs/ml being produced for each system [89, 92, 96], while DENV
and JEV VLP production was low in comparison with only 1.5 105 and
4.3 105 VLPs/ml produced, respectively [93, 95].
Each of the trans-packaged VLPs thus far described have not been applied
directly to studies concerning vaccines against flaviviruses. Rather their construc-
tion has led to their utilisation as a platform for the transient introduction of foreign
genetic information into eukaryotic cells both in vivo and in vitro. Reporter genes
such as b-galactosidase, green fluorescent proteins, and luciferases may be intro-
duced allowing the development of diagnostic and screening tools as well as
analysing various steps in virus life cycle such as investigations into RNA replica-
tion, visualisation of localised areas of translation of viral proteins within cells,
mechanisms of interferon inhibition, sites of initial infection in feeding mosquitoes,
virus interactions with host cells, generation of antibody neutralisation tests, and
screening for anti-viral drugs [89, 93, 94, 99 109]. Incorporation of genes
corresponding to pathogen or tumour antigens and subsequent immunisation with
packaged VLPs has been used to elicit strong cell-mediated immunity against
specific targets [31 33, 86, 99, 110, 111].
The trans-packaged flavivirus VLP configurations described above have primar-
ily employed replicons with in-frame deletions in all of the structural genes.
Research into TBEV capsid-deleted genomes [19, 29] provoked interest in the
production of replicons that retain the prM/E sequences, requiring complementa-
tion of capsid only. Such an approach is advantageous as it allows the production of
secreted prM/E particles (SVPs) as well as all the non-structural proteins in cells
infected with the VLPs, thus boosting both humoral (anti-E antibody) and cellular
(T-cell responses to non-structural proteins) immunity (Fig. 1e). Hence, this is a

system much more amenable to application as flavivirus vaccines.
Initial investigations concerned the production and characterisation of replica-
tion deficient VLPs of WNV and YFV [34]. Deletions of codons 30 101 of WNV
capsid and 23 93 of YFV capsid were sufficient for stable abrogation of encapsida-
tion without interfering with genome replication (Fig. 2d). Generation of stable
BHK21 cell lines harbouring recombinant Venezuelan equine encephalitis virus
replicons encoding either the YFV C or WNV C/prM/E genes linked to a puromy-
cin acetyltransferase (Pac) gene (VEErep/C2opt/Pac) allowed packaging of capsid-
deleted replicons into VLPs, with titres reaching approximately 108 VLPs/ml [34].
Passage of VLPs in packaging cells allowed further amplification of titres. Single
intra-peritoneal immunisation of outbred Swiss Webster mice with as little as
3 104 WNV VLPs afforded complete protection against challenge with 100 LD50
of the virulent NY99 strain of WNV [34].
Later development of the WNV VLP vaccine (designated RepliVAX WN)
improved the stability and safety of the packaging cell lines by introducing a
ubiquitin gene into the Venezuelan equine encephalitis virus replicon and by
removing the prM/E genes from the construct (hence decreasing likelihood of
recombination with the capsid-deleted replicon) [35, 112]. Serial passage of Repli-
VAX WN in packaging cell lines was unable to generate recombination leading to
recovery of the infective phenotype, but did select for mutations in the proteolytic
cleavage sites flanking the prM signal sequence (Fig. 2) [35]. Mutations of K to M
in the viral protease cleavage site upstream of the prM signal sequence and of S to C
at the signalase cleavage site upstream of prM both led to an increase in the amount
of packaged VLPs; however, only the later mutation was engineered into the
candidate RepliVAX WN vaccine [35]. Single immunisation of Swiss Webster
mice or Syrian hamsters with as little as 4 104 VLPs, either intra-peritoneally or
subcutaneously, engendered sufficient immune response to completely protect them
from intra-peritoneal challenge with either 10 LD50 (mice) or 106 focus forming
units (FFU) (hamsters) of NY99 strain WNV [35, 112]. When challenged 6 months
after this single immunisation, hamsters were still completely protected from
challenge [112].
RepliVAX technology has also been used for the generation of JEV vaccines.
RepliVAX JE was created by replacing the prM and E genes of the RepliVAX WN
replicon with those of JEV strain Nakayama [113]. RepliVAX JE initially presented
with low VLP yields; however, blind passage in packaging cell lines was able to
select for a mutation in the viral protease cleavage site upstream of the prM signal
sequence (K to N) [113]. This mutation was incorporated into the original Repli-
VAX JE sequence, leading to the generation of titres exceeding 107 VLPs/ml [113].
Single intra-peritoneal immunisation of Swiss Webster mice with as little as
4 104 VLPs afforded complete protection against intra-peritoneal challenge
with 5 106 FFU of virulent JEV strain Beijing P3 [113].
DENV-2 vaccines have likewise been generated using the RepliVAX system.
RepliVAX D2 was constructed via replacement of the prM and E genes of
the RepliVAX WN replicon with those of DENV-2 New Guinea C strain [114].
RepliVAX D2 was initially unable to produce encapsidated VLPs upon expression

in packaging cell lines [114]. To overcome this obstacle a new replicon was
constructed containing a smaller deletion within C for use in passaging to select
for compensatory mutations (Fig. 2d). To summarise, several mutations were
found, but only two of them located in the M sequence (G R and E V) and one
in the E sequence (T K) were required for RepliVAX D2 RNA to be packaged with
reasonable improvements in efficiency [114]. Yield was further increased to titres
of approximately 106 VLPs/ml by replacing trans-complemented WNV C with
trans-complemented DENV-2 C via generation of a new cell line stably expressing
the recombinant replicon VEErep/Pac-Ubi-D2C [114]. Single intra-peritoneal
immunisation of severely immunocompromised AG129 mice with 5 105 Repli-
VAX D2 VLPs protected them from intra-peritoneal challenge with wild-type
DENV-2 New Guinea C strain, with only 20% of animals showing illness and no
recorded deaths [114].
The RepliVAX strategy for flavivirus vaccine production appears very
promising. Each construct has demonstrated an impressive capacity to provide
protective immunity in animal models after only a single immunisation; stable
cell lines allow the passage and production of large titres of VLPs; and the
durability of the trans-complementation system has been established with a proven
lack of recombination leading to recovery of the infectious virus. However, this
approach lacks some of the advantages of DNA-based self-replicating vaccines.
Production and purification of VLPs is somewhat cumbersome and relatively
expensive, and the stability of the VLPs at higher ambient temperatures, while
not intensely studied, are unlikely to be as robust as that of DNA.
5 DNA-Based Vaccine Producing SRIPs In Vivo
One of the latest innovations in flavivirus vaccine research combines the advan-
tages of RepliVAX-like technology (efficient delivery and durable immune
response) with those of DNA vaccines (easy manufacture and robust genetic and
thermal stability). SRIP-producing DNA vaccine technology (Table 1) consists of a
capsid-deleted flavivirus cDNA under the control of one eukaryotic promoter
combined with cDNA for the complete capsid gene transcribed from another
eukaryotic promoter in the same plasmid (Fig. 1f). [27]. The vaccine does not
require any additional manipulations, i.e. production of VLPs in a packaging
system/cell line, as the capsid-deleted RNA is both produced and packaged into
SRIPs in vivo (Fig. 1f). As the capsid gene provided in trans is not encoded in the
RNA packaged into SRIPs, further spread of infection is not possible (Fig. 1f). Both
DNA-transfected and SRIP-infected cells contain replicating capsid-deleted RNA
resulting in increased production of immunogenic prM/E particles and CTL-induc-
ing non-structural proteins which in turn leads to an enhanced immune response
(Fig. 1f) [68].
A SRIP-producing DNA vaccine targeting WNV (pKUNdC/C) has recently

been investigated [27]. The vaccine is based on infectious DNA of Kunjin virus,
pKUN1, with the codons corresponding to the amino acid residues 18 100 of the
capsid gene deleted (Fig. 2e). The full-length capsid gene is encoded in reverse
orientation under the control of a second CMV promoter (Fig. 3a) [27]. Character-
isation of pKUNdC/C in vitro demonstrated a production of SRIPs reaching titres of
approximately 105 particles per 1 mg of transfected DNA by day 2 post-transfection,
a yield that was maintained for a 5-day period [27, 68]. Capsid expression has been
demonstrated to be limited to initially transfected Vero cells, whereas envelope
protein expression was observed both in capsid-expressing transfected cells and in
adjacent cells that had been infected with SRIPs released from transfected cells
(Fig. 3b) [27]. Importantly, passage of the secreted SRIPs in Vero cells did not show
any signs of infectious virus (Fig. 3c), thus, implying an inability of the capsid
mRNA to recombine with the genomic replicon RNA [27]. The limited spread of
infection was confirmed in an ex vivo experiment utilising skin from cattle ears that
had been bombarded with gold micro-carriers coated with pKUNdC/C DNA
(Fig. 3d). The expression of E was detected in both cells initially bombarded with
DNA (containing gold particles) and adjacent cells not containing gold particles for
pKUNdC/C and pKUN1 DNAs but not for pKUNdC DNA (Fig. 3d), clearly
demonstrating the release and spread of SRIPs.
The pKUNdC/C construct has performed very well in a small animal model.
Single immunisation with as little as 0.02 mg of gene gun-delivered DNA provided
complete protection to BALB/c mice when challenged intra-peritoneally with
100 IU of virulent WNV NY99 [27]. The construct has also shown promise as a
vaccine for horses that are highly susceptible to WNV infection. Two gene gun
immunisations with 4 mg of pKUNdC/C were sufficient to elicit detectable neu-
tralising antibody response in all horses against both Kunjin and WNV NY99, with
a third immunisation greatly increasing the neutralising antibody titres [27].
SRIP-producing DNA technology thus far appears to be the most comprehensive
approach for designing modern pseudo-infectious flavivirus vaccines. The ease of
production coupled with the robust genetic and thermal stability make these
vaccines highly amenable to use worldwide, including developing nations where
specialised resources may be in short supply. The proven efficacy of the pKUNdC/
C construct in mice and the immunogenic nature of this vaccine in horses [27]
warrant further investigation and should be followed up with a challenge study in
larger animals. Considering the relative ease of construction and the availability of
capsid-deleted flavivirus clones, adaption of this technique to the development of
vaccines against other flaviviruses appears to be straightforward. Given the proven
competence of chimeric flavivirus constructs (i.e. ChimeriVax and RepliVAX),
changing the Kunjin virus replicon backbone may not even be necessary, as merely
swapping the prM and E genes with those of heterologous flaviviruses could in
theory generate effective vaccines against different flaviviruses. Although the
SRIPs-producing DNA-based approach is relatively recent, all indications point
to this strategy being highly attractive as the next-generation of replicating vac-
cines. As such, continuing developments in this technology should be pursued.
b c
Fig. 3 In vitro and ex vivo demonstration of the ability of pKUNdC/C DNA to produce SRIPs. (a)
Schematic diagram of pKUNdC/C DNA. cDNA for capsid deleted Kunjin virus genome is placed
under control of CMV promoter, while cDNA for the intact capsid gene is placed in the reverse
orientation under the control of a second copy of CMV promoter. Upon transfection into cell,
pKUNdC/C is transcribed to produce an mRNA encoding the capsid gene and a capsid deleted
RNA encoding all the remaining Kunjin virus genes. Subsequent translation of these provides all
of the necessary proteins for RNA replication and its packaging into SRIPs. (b) Dual immuno
flourescence analysis of DNA transfected Vero cells with anti E (green) (panels 1, 4, 7) and anti
Capsid (red) (panels 2, 5, 8) antibodies show capsid production in each of the cells transfected or
infected with pKUN1 (panel 9), in none of the cells transfected with pKUNdC (panel 6), and only
in those cells transfected with pKUNdC/C but not in the adjacent cells infected with the released
SRIPs (panel 3). (c) Immunoflourescence analysis with anti E antibodies shows small foci of
positive cells after transfection with pKUNdC/C DNA (panel 1), large foci after transfection with
infectious pKUN1 DNA (panel 7), and only individual positive cells after transfection with
pKUNdC DNA (panel 4). Three days post transfection, culture fluids were collected and used to
infect naive Vero cells (first infection, panels 2, 5, and 8, respectively). Three days after infection,
the culture fluid was again collected and used to infect naive Vero cells (second infection, panels
3, 6, and 9). The pKUNdC DNA did not produce any infective particles, the infectious pKUN1
DNA produced infectious spreading virus as expected, and pKUNdC/C DNA produced infectious
6 Summary
Flaviviruses have been recognised as important agents of human disease for over a
century [1]; however, vaccines are currently licensed for only a handful of flavivi-
rus species (YFV, TBEV, and JEV). Several deficiencies have also been identified
with existing vaccines. They are in general relatively expensive to produce, difficult
to purify, and unstable at ambient temperatures. Considering the global flavivirus
disease burden is predominantly afflicting developing nations [2], such complexity
and expense limit the efficacy of vaccine utilisation. Safety concerns have also been
raised over the currently licensed vaccine preparations. Although they are highly
effective at preventing lethal infections when correctly administered, several
adverse reactions have been observed in patients following immunisation with
commercial flavivirus vaccines [8, 20 24]. Thus, flavivirus research continues to
be driven by the need for cheap, stable, safe, and efficacious vaccines.
The generation of infectious cDNA clones of various flavivirus species has
allowed their specific, directed attenuation and subsequent use as nucleic acid-
delivered, live vaccines. Such vaccines have proven highly effective in animal
models; however, they neglect to address concerns over the safety of live-
attenuated vaccines in regards to observed adverse reactions. Generation of large
in-frame capsid deletions in these cDNA clones has led to the development of
vaccines that are safer but at the same time maintain many of the positive immuno-
logical features of live vaccines. Capsid-deleted clones are unable to package
replicating genomic RNA into virus particles, thus cannot generate a spreading
infection in the host. Immunogenicity of the vaccine preparation suffers however,
as capsid-deleted replicon vaccines must be administered at least twice in order to
generate protective immunity. Single-dose efficacy has been achieved via the trans-
packaging of capsid-deleted replicons into VLPs and delivery of these into the host.
VLP-based vaccines, however, suffer from being relatively expensive to produce,
difficult to purify, and they have low thermal stability. SRIP-producing DNA vac-
cines one of the latest innovations provide the complementary capsid gene in trans
within the same plasmid as the capsid-deleted genome allowing delivery of the
vaccine as naked DNA. Thus, they overcome difficulties with preparation, purifica-
tion, costs, and stability of vaccine. SRIP-producing DNA vaccine has been demon-
strated to not produce any infectious virus and to be highly efficient at generating
protective immune response. The low cost, simple manufacture, safety, efficacy, and
Fig. 3 (continued) particles capable of only one round of infection. (d) Sections of cattle ear
epidermal cells bombarded with DNA coated gold particles and analysed by light microscopy
(panels 1, 3, and 5) and by immunofluorescence with anti E antibodies (panels 2, 4, and 6). Cells
containing gold particles (initially transfected cells) are indicated by solid arrows. Cells that
express E but do not contain gold particles are indicated by open arrowheads. The presence of
E positive but gold particle negative cells indicates infection with virus particles (pKUN1, panels
5 and 6) or SRIPs (pKUNdC/C, panels 1 and 2). The capsid deleted DNA pKUNdC does not
engender a spreading infection (panels 3 and 4). Figure modified from Chang et al. [27]
high genetic and thermal stability of SRIP-producing DNA vaccines make them an
attractive candidate for future flavivirus vaccine development.
Acknowledgements The work was supported by the grants from the National Health and Medical
Research Council of Australia and the National Institute of Health, USA.
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Application of Cleavage Activation Mutants
of Influenza Virus as Live Vaccines
Juergen Stech and Hans-Dieter Klenk
Abstract Influenza viruses are major human and animal pathogens. In man, they are
responsible for annual epidemics and less frequent but more severe pandemics. Avian
influenza viruses cause devastating outbreaks in poultry, and influenza virus infec-
tions in pigs and horses also lead to high economic losses. Vaccination is an effective
instrument to control the disease burden of human and, to some extent, also of animal
influenza. Inactivated human vaccines have been used for more than 60 years.
Furthermore, cold-adapted, live attenuated vaccines have been licensed in some
countries. Attenuated viruses with reduced pathogenicity can also be obtained when
the cleavage site of the hemagglutinin is mutated. Such protease activation mutants
have not only been generated for the production of inactivated vaccines against highly
pathogenic avian influenza viruses, but they have also the potential to be used as live
vaccines. Two types of protease activation mutants have been investigated for use as
live vaccines. In the first group, the polybasic cleavage site of the hemagglutinin, a
prime determinant of pathogenicity, was cut short to a single arginine. These viruses
require additional mutations in other genes for full attenuation. In the second group,
polybasic or monobasic cleavage sites are replaced by an elastase cleavage site. These
viruses are fully attenuated, yet have retained their immunogenicity.
1 Cleavage Activation of the Influenza Virus Hemagglutinin
Influenza viruses belong to the family of Orthomyxoviridae. Among them are the
three genera: influenza A virus, influenza B virus, and influenza C virus. Type A
is a large group of viruses comprising 16 hemagglutinin (HA) and 9 neuraminidase
(NA) subtypes. The genome of influenza A viruses consists of eight single-stranded
RNA molecules of negative polarity embedded in an enveloped virion. The envelope
J. Stech
Friedrich Loeffler Institut, Greifswald Insel Riems, Germany
H.D. Klenk (*)
Institute of Virology, Philipps University, Marburg, Germany
e mail: [email protected] marburg.de

322 J. Stech and H. D. Klenk
contains HA and NA spikes. HA initiates infection by mediating binding

to N-acetyl-neuraminic acid-containing receptors and membrane fusion. Fusion
activity and therefore virus infectivity depends on proteolytic cleavage of HA.
X-ray crystallographic studies have shown that the cleavage site is located in a
circular loop projecting away from the surface of the precursor HA into the solvent
(Fig. 1). The activating enzymes are provided by the host. Avian influenza viruses
show large variations in cleavability, and these differences are prime determinants
of pathogenicity. In the following we will give a brief outline of this concept.
For more detailed information, the reader is referred to previous reviews by us [2]
and others [3].
Mammalian influenza A viruses and low pathogenic avian influenza viruses
(LPAIV) have HAs with a single arginine at the cleavage site. They are activated
by proteases synthesized by epithelial cells that are present only in respiratory or
intestinal tissues. Infection is therefore restricted to these organs (Fig. 1). A number
of trypsin-like proteases, such as plasmin, tryptase Clara and factor X, activate HA
with a monobasic cleavage site in vitro (for references see [2]), but they are unlikely
to activate these viruses in their natural setting. However, recently two serine
proteases (TMPRSS2 and HAT) from the human airway epithelium have been
found to activate human influenza A viruses as well as LPAIVs [4, 5]. Bacterial
proteases may also activate HAs of restricted cleavability and promote the devel-
opment of pneumonia in mice after combined viral-bacterial infection [6].
Highly pathogenic avian influenza viruses (HPAIV) are activated by a different
cleavage mechanism. Their HAs are activated at multibasic cleavage sites by furin,
a member of the proprotein convertase family of eukaryotic subtilisin-like serine
H1-H16 H5, H7
R
X
R K/R
R
trypsin-
like protease furin
apathogenic pathogenic
virus virus
local systemic
infection infection
Fig. 1 The cleavage site of HA determines the pathogenicity of avian influenza viruses. HA is
cleaved into subunits HA1 (blue) and HA2 (red). The cleavage site is located in a loop (yellow)
projecting from the surface of the molecule [1]. LPAI viruses (subtypes H1 H16) have a single
arginine at the cleavage site that is recognized by trypsin like proteases that are present only in
specific tissues, such as intestinal epithelia. These viruses cause therefore local infection. HPAI
viruses (subtypes H5 and H7) are activated at a multibasic cleavage site by the ubiquitous protease
furin. Therefore, these viruses spread throughout the organism. An electron micrograph of a virus
particle with HA and NA spikes protruding from the surface is also shown
Application of Cleavage Activation Mutants of Influenza Virus as Live Vaccines 323
endoproteases [7]. The ubiquity of this enzyme accounts for the systemic infection
typical for these viruses (Fig. 1). Furin is a factor of the constitutive secretory
pathway in almost all cells and accumulates in the trans-Golgi network, which is
also the cellular compartment where this HA type is cleaved (for references see [2]).
Other proprotein convertases, which resemble furin in structure and substrate
specificity, are PACE4, PC5/6, and LPC/PC7. Like furin, PC5/6 activates HAs
with multibasic cleavage sites, whereas PACE and LPC/PC7 do not [8, 9]. The HAs
of most HPAI viruses have the consensus sequences R-X-K/R-R [10] or R-X-X-R
[11] at the cleavage site, motifs that are both recognized by furin. The only
exception to these rules is the HA of A/chicken/Pennsylvania/83 (H5N2) which
contains the unusual tetrapeptide K-K-K-R [12]. Another important determinant is
a carbohydrate side chain close to the cleavage site that interferes with protease
accessibility. Loss of this carbohydrate resulted in enhanced HA cleavability and
pathogenicity [13]. However, masking of the cleavage site by this oligosaccharide
was overcome when the number of basic amino acids was increased [12, 14]. It was
also shown that HA can acquire high cleavability only if the stretch of basic
residues was introduced by insertion, but not by amino acid exchanges in the
carboxy-terminus of HA1 [15].
Increased pathogenicity as a consequence of insertions at the cleavage site has first
been observed in laboratory studies involving sequential cell culture passages of
strains A/seal/Massachusetts/1/80 (H7N7) [16] and A/turkey/Oregon/71 (H7N3). In
the latter case, the acquisition of the furin recognition motif resulted from the
recombination of the HA gene with 28S ribosomal RNA [17, 18]. The HA gene
may recombine not only with cellular RNA but also with other viral gene segments,
as has been observed when new HPAIVs emerging in the field have been analyzed.
Thus, comparison of A/chicken/Chile/02 (H7N3) isolates revealed that the HA genes
of the highly pathogenic strains had an insertion of 30 nucleotides at the cleavage site
that was presumably derived from the nucleoprotein gene of the unrelated A/gull/
Maryland/704/77 (H13N6) virus [19]. Recombination between HA and matrix pro-
tein genes of the same virus generated the highly pathogenic A/chicken/BC/04
(H7N3) viruses [20]. Polymerase slippage has been suggested as an alternative
strategy by which a multibasic cleavage site is generated [21, 22]. However, there
are other examples where the mechanism of insertion is not clear [23].
It is also not clear yet why the acquisition of a multibasic cleavage site and
therefore the generation of HPAI viruses occurs in nature only with subtypes H5
and H7. Interestingly, however, high cleavability was also observed with a subtype
H3 HA after in vitro insertion of a multibasic cleavage site and removal of an
adjacent oligosaccharide by recombinant DNA technology [15] and with recombi-
nant avian H3 viruses with an engineered multibasic cleavage site [24]. Thus it
appears that confinement of HPAIVs to subtypes H5 and H7 cannot be attributed to
structural restrictions of the HA protein, but that the responsible mechanisms are at
the level of RNA replication [2].
In contrast to natural evolution where HPAIVs generally appear to be derived
from LPAIVs, recombinant viruses with reduced pathogenicity can be generated by
in vitro mutation at the cleavage site. Because this attenuation technique does not
affect virus yield, it has been employed for the production of inactivated pandemic
vaccines. Furthermore, cleavage activation mutants of influenza viruses have the
potential to be used as live vaccines.
2 Attenuation by Exchange of a Polybasic for a Monobasic

Cleavage Site
For production of inactivated pandemic vaccines, recombinant H5N1 strains have

been generated containing HA and NA from a human H5N1 virus and the remain-
der of their genes from PR8. In these viruses the polybasic cleavage site of HA was
replaced by a single arginine to allow safe manufacturing [25 30] (Fig. 2).
To obtain pandemic H5N1 live vaccines, introduction of a monobasic HA
cleavage site had to be combined with additional attenuation mechanisms. Thus,
6:2 reassortants have been generated containing a modified HA gene and the NA
gene of H5N1 viruses and the other gene segments of a cold adapted influenza virus.
These reassortant viruses showed low pathogenicity for chickens and were attenu-
ated in mice and ferrets, while growing to high titres in eggs. Inoculation of ferrets
and mice protected against lethality from homologous and heterologous challenge
after one dose and conferred protection against viral replication after two doses
[31, 32]. In another approach a H5N1 recombinant was generated in which a modified
HA cleavage site was combined with an 11-amino acid deletion at the cytoplasmic
tail of M2. This virus replicated efficiently in vitro in M2-expressing cells and was
attenuated in mice. It provided protection from lethal challenge with homologous
Fig. 2 Generation of vaccine strains by genetic manipulation of the HA cleavage site. Attenuation
is accomplished by replacement of (1) a polybasic with a monobasic cleavage site, (2) a monobasic
with an elastase cleavage site, and (3) a polybasic with an elastase cleavage site
and heterologous H5N1 viruses in mice [33]. Finally, protease activation mutants of
H5N1 virus have been used in which increased interferon sensitivity resulting from
a truncated NS1 protein contributed to attenuation [34b].
3 Attenuation by Introduction of an Elastase Cleavage Site
Protease activation mutants of LAIV requiring elastase or another atypical protease

instead of trypsin for HA cleavage have been obtained previously by classical
virological techniques. These mutants proved to be attenuated and immunogenic
[34a]. With the advent of recombinant DNA technology, it is now possible to
specifically design such viruses by site directed mutagenesis. Moreover, it has
been possible to generate live attenuated vaccines against HPAIVs by this
approach.
Using reverse genetics, the HA cleavage site of the strain A/WSN/33 (H1N1)
was changed from a monobasic motif to an elastase motif by exchanging the
arginine at the cleavage site to valine (Fig. 2), resulting in strict dependence of
the generated mutant (WSN-E) on porcine pancreatic elastase. WSN-E replicated in
cell culture with elastase equally well as a wild-type (WSNwt) in the presence of
trypsin. However, replication of WSN-E in vivo was restricted due to poor access to
elastases. Accordingly, in contrast to the lethal wild-type, WSN-E is fully attenu-
ated in mice, confined to the lung and undergoes abbreviated replication, just
reaching levels close to the inoculum dose. One intranasal immunization with 105
or 106 pfu protects mice against lethal challenge with WSNwt four weeks later.
Although some animals from the group vaccinated with 105 pfu WSN-E developed
temporary weight loss and milder disease symptoms, they eventually recovered. All
animals vaccinated with the highest dose of 106 WSN-E survived the challenge
without displaying any weight loss or other visible symptoms of illness. Corre-
spondingly, challenge virus could be detected in lung homogenate from the animals
immunized with 105 pfu, whereas no virus could be recovered from animals with
the highest immunization dose of 106 pfu. One intranasal inoculation of WSN-E
induced a substantial, dose-dependent local and systemic immune response despite
low virus titres in the lung. A dose of 105 or 106 pfu led to remarkable hemaggluti-
nation inhibition titers, serum IgG and mucosal IgA titers. They were lower than
those induced by 103 pfu of WSNwt because its longer replication enables antigenic
stimulation. However, challenged animals showed almost comparable systemic and
mucosal antibody titers if immunized with 106 pfu WSN-E. This indicates that a
notable immunological memory had already been induced in these animals [35].
To check for the emergence of revertants of WSN-E, sequential lung passages in
mice were carried out. After the first passage in mouse lung, the entire amount of
WSN-E was in the range of 105 106 pfu. Such virus populations are too small for
generation of double-point revertants having an equilibrium frequency of approxi-
mately 10–5 to 10–8. Therefore, the elastase-dependent WSN-E was passaged on
MDCK cells in the presence of trypsin, beginning with different inocula of 108, 107,
or 106 pfu each in 10 parallel cell cultures. From all 108 pfu inocula and from six out
of ten 107 pfu inocula, a trypsin-dependent virus with lysine at its cleavage site was
found. No revertants from the lowest inoculum of 106 pfu could be obtained.
Therefore, the reversion frequency within the WSN-E stock is approximately
10–7. This low reversion rate and the small viral loads of WSN-E in the mouse
lung explain the absence of revertants during mouse passages. Another reason for
the genetic stability of WSN-E in vivo is the restriction to one replication cycle due
to the absence of the appropriate protease [35].
A frequent objection against the use of influenza virus live vaccines is the
possibility of reversion to pathogenicity. Because of the double point mutation
(from any arginine to the valine codon) within the cleavage site, two nucleotides
together must be replaced for back-mutation; suppressor mutants outside of the
cleavage region seem to be very unlikely. This explains the low reversion frequency
in cell culture. In hen eggs, a factor X-like protease is present, which should cause a
considerably higher proportion of revertants. Therefore, eggs might not be suitable
for vaccine production. However, in cell culture the substitution of trypsin by
elastase for propagation of WSN-E leads both to positive selection for elastase-
dependent virus and to negative selection against revertants. A reversion frequency
of approximately 10-7, as found for WSN-E, underlines that a live vaccine virus
should carry several independent attenuating mutations; the modified cleavage site
would be useful as one attenuating component of such a vaccine.
The suitability of elastase cleavage site mutants for use as influenza live vaccines
was further investigated with the mouse-adapted highly pathogenic H7N7 strain
SC35MH [16, 37]. This virus has been obtained by repeated passages of the isolate
A/seal/Massachusetts/1/1980 (H7N7) in chicken embryo fibroblasts and afterwards
in mouse lungs [16, 36]. The elastase cleavage site mutant SC35MH-E, which was
generated by replacing the polybasic cleavage site of HA by a single valine, grew in
cell-culture in the presence of elastase as well as the wild-type (Fig. 2). In mice,
SC35MH-E was attenuated. Animals inoculated intranasally with doses of 103 to
106 pfu survived. In contrast, the parent virus had an LD50 of 101.4 pfu. SC35MH-E
could be detected on day 1 at titers close to the inoculum dose. Moreover, the organ
tropism of SC35MH-E was severely restricted compared to that of the wild-type,
which replicates to high titers in lung, brain, and heart from day 1 to 7 post
inoculation. For the immunization studies, two 6:2 reassortants were also gener-
ated: WSN-H7N7-E is composed of the internal protein genes from A/WSN/33
(H1N1) and the surface protein genes from SC35MH-E; and reciprocally, SC35M-
H1N1-E carries the internal protein genes from SC35M and the surface protein
genes from WSN-E (H1N1). Intranasal inoculation with SC35MH-E, SC35-E,
WSN-H7N7-E or SC35M-H1N1-E induced high titers of serum IgG and mucosal
IgA antibodies detectable 4 weeks later. Four weeks after intranasal immunization,
mice that had received a dose of 106 pfu of any of the viruses survived the challenge
with 100 LD50 of SC35MH. The lower immunization doses of 103, 104 or 105 pfu
also ensured survival of all animals with the homosubtypic SC35-E, SC35MH-E
and WSN-H7N7-E (except that three of four mice inoculated with either SC35-E
104 pfu or SC35MH-E 103 pfu survived). In contrast, immunization with the
heterosubtypic SC35MH-H1N1-E led to lower rate of survival (two of four mice).

This lower efficacy corresponds to the absence of antibodies that neutralize H7
virus in the latter group and suggests viral clearance due to cell-mediated immunity.
Taken together, this pilot study demonstrates that HPAIVs are highly attenuated
when the polybasic cleavage site is replaced by an elastase cleavage site and that
these mutants can be used as live vaccines [37].
Recently, elastase-dependent live attenuated viruses have been tested in vacci-
nation trials in pigs, a relevant host for influenza A viruses. Two elastase-dependent
mutants were generated from strain A/swine/Saskatchewan/18789/02 (H1N1):
R345V and R345A. These viruses were administered intratracheally and were
shown to be attenuated in pigs [38]. Both mutants induced IgG and IgA in serum
and bronchoalveolar lavage fluid, high serum hemagglutination inhibition titers,
enhanced levels of lymphocyte proliferation and higher numbers of interferon-
gamma secreting cells at the infection site. After a second immunization, the
animals developed cross-reactive antibodies and cell-mediated immune responses
at higher levels. Accordingly, pigs were vaccinated twice with R345V at a dose of
4 106 pfu and challenged with either the homologous wild-type, the homo-
subtypic H1N1 strain A/swine/Indiana/1726/88 or the heterosubtypic H3N2 strain
A/swine/Texas/4199-2/9/98. No vaccinated animals developed signs of illness after
the challenge. Unvaccinated animals infected with either H1N1 virus developed
fever and mild respiratory symptoms; infection with the less virulent H3N2 virus
did not lead to any apparent clinical signs even in the mock-immunized group. Pigs
challenged with the homologous H1N1 viruses displayed no detectable viral repli-
cation in lungs. The animals challenged with the heterologous H3N2 virus were
protected partially: four of seven vaccinated pigs developed significantly milder
macroscopic and microscopic lung lesions accompanied by significantly lower viral
replication compared to the mock-immunized animals. These data suggest that the
very brief replication of the elastase-dependent mutants in vivo requires two
vaccinations to generate an adequate immune response and that two vaccinations
could mimic natural immunity after infection with wild-type. Taken together, these
results demonstrate that elastase cleavage site mutants are candidate vaccines for
pigs. More practical routes of application, such as intranasal immunization, remain
to be developed [39]
4 Conclusions
Proteolytic activation of the hemagglutinin is essential for multicycle replication of

influenza A and B viruses. The required monobasic and multibasic cleavage sites
are attractive targets for virus attenuation. Conversion of the multibasic cleavage
sites of HPAIVs to a monobasic motif leads to an avirulent phenotype. However, it
should be taken into account that any H5 or H7 strain with a monobasic cleavage
site found in poultry surveillance is still considered a potential HPAIV precursor.
It is therefore common practice to cull the whole flock. Our proof-of-principle
studies in mice demonstrated that elastase cleavage site mutants are attenuated and
immunogenic. Further immunization studies in pigs revealed the suitability of this
approach for relevant hosts; vaccinated animals were protected against homosub-
typic challenge and partly protected against heterosubtypic challenge. For safety in
the field, an influenza live vaccine probably must be attenuated by several mechan-
isms, among which introduction of an elastase cleavage site appears to be particu-
larly attractive. Moreover, such a modified cleavage site prevents reassortment of
the hemagglutinin gene of the vaccine strain into circulating viruses.
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Alphavirus Particle-Based Vaccine Vectors
Scott J. Balsitis, Clayton W. Beard, and Peter W. Mason
Abstract Most of the vaccines in use today are live attenuated, killed, or protein
subunit vaccines. Although these vaccines have saved countless lives, there is still a
need to develop safer and more efficacious ones. These improved new generation
vaccines will enable us to protect more people from a greater number of different
infectious disease threats. One such new vaccine technology is derived from the
alphaviruses, which are single-strand, positive-sense RNA viruses in the family
Togaviridae. By removing the genes that encode for the viral coat proteins, and
replacing them with an antigen-encoding gene, these viruses become a replicon that
can replicate its genome but cannot propagate new virus particles. The resources
that the virus once expended to make new progeny are now diverted to making
vaccine antigen within the host. As a result of this molecular alteration, the
alphavirus particle-based vectors serve as an attractive technology for the develop-
ment of new and improved vaccines.
1 Overview
At a very basic level, the goal of vaccination is to trick the body into believing that it
is under attack from a pathogen. If this deception is successful, the immune system
will mount a robust response to the perceived threat with the end result being the
generation of a long-lived and protective immune state. One of the most effective
ways to carry out this ruse has been through the use of live-attenuated vaccines
(LAV) because they actually do infect the host, but with lowered pathogenicity.
The fact that LAVs are live agents that can potentially regain the ability to cause
disease (or are pathogenic in a subset of the population) results in concerns about
safety. A safer alternative is to use vaccines that deliver a killed pathogen, or pieces
of a pathogen as subunits. These vaccines are quite safe but tend to stimulate weak
S.J. Balsitis, C.W. Beard, and P.W. Mason (*)

Novartis Vaccines and Diagnostics, 350 Mass Ave, Cambridge, MA 02139, USA

332 S.J. Balsitis et al.
immune responses. The alphavirus-based vaccine vectors described in this chapter

are able to fully convince the immune system that the host is infected with a
pathogen such that a full-blown immune response is launched. These vectors
simultaneously produce large amounts of any specified protein antigen that is
then fed into the strong immune response that they have stimulated. The end result
is a LAV-type immune response, but with the safety profile of a killed or subunit
vaccine.
2 Alphavirus Biology
2.1 Classification, Transmission and Epidemiology
Alphaviruses are positive-sense RNA viruses that form the largest genus in the
Family Togaviridae. The Alphavirus genus has approximately 30 members, all of
which are known to be arthropod-borne [1]. A number of alphaviruses are important
human pathogens, including Venezuelan equine encephalitis, eastern equine
encephalitis, and western equine encephalitis (VEEV, EEEV, WEEV). These
three agents are maintained in nature in highly restricted mosquito-warm blooded
vertebrate infection cycles, in which their vertebrate hosts undergo acute self-
limiting infections that produce viremias sufficient to reinfect the appropriate
mosquito vectors. Infections of vertebrates, including man and domestic animals,
can result in severe morbidity and mortality, producing a range of illnesses of
varying severity [1]. The encephalitic alphaviruses can cause lethal infections, with
case fatality rates in human beings ranging from 30 to 50% (EEEV and WEEV) to
less than 5% (VEEV). VEEV, WEEV, and EEEV are restricted to the New World,
while a different group of alphaviruses, whose pathogenesis is limited to arthralgias
and rashes is largely (but not exclusively) found in the Old World [1]. These Old
World alphaviruses include the prototype virus of the family, Sindbis virus (SINV),
which is the best-studied member of the alphavirus genus, as well as Semiliki Forest
virus (SFV), Ross River virus (RRV), and Chikungunya virus (CHIKV) [1]. Among
these, SINV and SFV are not considered to be important human pathogens, whereas
RRV and CHIKV are known to cause debilitating, but rarely fatal, arthralgias.
Despite the global distributions of alphaviruses, infections are low in most human
populations [2].
2.2 Alphavirus Molecular Biology
The alphavirus particle is a 70 nm diameter icosahedron that contains a single-

strand, positive-sense RNA genome that is capped and polyadenylated. The
genome is approximately 11.5 kb in length and is complexed to the capsid protein
(C), producing a nucleocapsid core that is enveloped in a cell-derived lipid bilayer
Alphavirus Particle Based Vaccine Vectors 333
studded with 80 spikes, consisting of three heterodimers of the E1 and E2 glyco-

proteins. Both glycoproteins have transmembrane domains (TMDs) that anchor the
spike to the bilayer, and the internal extension of the E2 protein contacts the
nucleocapsid core of the virion, orienting the glycoprotein spikes on the nucleo-
capsid core [3].
The alphavirus replication cycle begins with cell surface binding. A number of
different cellular receptors have been identified for alphaviruses, consistent with the
broad in vivo and in vitro host ranges of these viruses. E2 appears to mediate cell
binding to cell-surface proteins, and repeated passage of alphaviruses in culture has
been shown to select viruses that have an increased affinity for ubiquitous cell
surface glycosaminoglycans (GAGs). As expected, viruses selected in this manner
display GAG-dependent increases in specific infectivity. Following attachment,
alphavirus particles are internalized into an endocytic compartment in a clathrin-
dependent manner, where a drop in pH triggers the dissociation of E1 and E2,
exposing a fusion peptide on E1 that is responsible for virion fusion to the
endosomal membrane, transferring the nucleocapsid core to the cytoplasm, where
it dissociates and releases its RNA cargo.
The released genomic RNA is translationally competent, and produces two
polyproteins, which encode nonstructural (ns) proteins nsP1-nsP2-nsP3 and nsP1-
snP2-nsP3-nsP4 [3]. The former is more abundant, due to the positioning of an opal
stop-codon between nsP3 and nsP4. Both polyproteins are processed by enzymatic
activity in nsP2 to yield the four functional components of the viral replication
apparatus: nsP1 methyltransferase/guanylyltranferase, nsP2 proteinase/helicase
(responsible for processing of both polyproteins), nsP3 phosphoprotein, and the
nsP4 RNA-dependent RNA polymerase (RdRp). The translated ns proteins form
replication factories on the surface of intracellular membranes that transcribe full-
length negative-strand copies from the infecting genome, and this negative-strand
copy then serves as a template for two positive-strand RNA molecules: the genomic
RNA and a shorter collinear subgenomic RNA that corresponds to the 30 third of the
genomic RNA. This subgenomic RNA, also known as the 26S RNA, encodes the
structural proteins of the virus (C and E1/E2). This RNA is transcribed at extremely
high levels later in the viral replication cycle, permitting the accumulation of the
structural proteins during the later phases of the viral life cycle, facilitating efficient
assembly of the progeny virus. The alphavirus structural proteins are synthesized as
a polyprotein precursor. The C protease is only active in cis, and its activity per se is
not required for its assembly into the virion (this property is critical to certain types
of transpackaging systems, see below).
The C coding region is followed in the 26S RNA by sequences encoding a
glycoprotein precursor, which is co- and post-translationally processed into the
mature viral glycoproteins by cellular proteases [3]. The glycoprotein portion of the
26S coding region encodes four “mature” peptides, in the order E3-E2-6K-E1. The
complex cleavage scenario that produces these mature products requires both early
(signal peptidase) and late (furin) cleavages to allow for the sequential formation of
multiple folding intermediates that are needed to produce the mature virion spikes,
consisting of three E2 E1 heterodimers that accumulate on the plasma membrane
of the infected cell. In the final step of virion assembly, the preformed nucleocapsid
cores bud through spike-containing regions of the plasma membrane to produce
mature virions [3].
2.3 Pathogenesis
Early work on the characterization of a cell culture-adapted, live-attenuated viral

vaccine for VEE (TC-83) important provided insights into the mechanism of
alphavirus pathogenesis. The strain was shown to lack the neuroinvasiveness of
its parental Trinidad donkey (TRD) strain in multiple VEEV-susceptible animals.
Interestingly, this attenuated strain was also shown to be much more quickly
cleared from the circulatory system than the parental strain [4], and this property
was proposed by these same authors to be related to a previous finding, that the
attenuated virus bound more tightly to cells in culture than its parent [5]. The
parental and attenuated viruses differ at a handful of genetic loci, and subsequent
studies identified specific mutations in the 50 UTR and E2 regions as the major
sources of the attenuation of TC-83 [6]. The E2 mutation (AA120 from Thr to
Arg) [7] results in an increase in positive charge of the attenuated glycoprotein,
which increases binding to heparan sulfate (HS), a negatively charged GAG.
Similar mutations have been selected by cell passage of a number of alphaviruses
[5], as well as other RNA viruses that are subjected to cell culture passaging
regimens. In fact, similar E2 mutations have been identified for SINV [8, 9], and
short-term passaging of VEEV in the presence of trypsin, designed to produce
rapidly penetrating variants of the virus, selected variants with attenuated pheno-
types, and a number of HS-binding mutations, including one at precisely the same
position found in E2 of TC-83 [7]. Based on the expected increase in affinity for
ubiquitous cell surface molecules, all of these mutations would be expected to
enhance viral clearance from the circulatory system, that would be expected, in
turn, to reduce a virus’ ability to cross the blood brain barrier, correlating with the
reduced neuroinvasiveness of many cell culture-selected isolates. The second
major attenuating mutation found in TC-83, which was found at nucleotide 3 in
the genome [6], allows the mutant virus to produce less subgenomic (26S) RNA in
infected cells [10]. This property could contribute to its attenuation, since VEEV
virulence is controlled, in part, by the C protein (see below).
Work on adaptation of Old World alphavirus replicons to persist within cells led
to the discovery that their nsP2 protein was a major virulence factor. These studies
showed that wild-type replicons of SINV or SFV were virulent in cells in culture,
but that forcing these replicons to persist in cells produced variants that had
acquired mutations in nsP2 that rendered the replicons less cytopathic [11 14].
The selected mutations were shown to have dramatic effects on the persistence
phenotype that promotes the ability of the mutant replicons to persist by up to a
100,000-fold relative to the WT replicons [11 13]. Interestingly, work by Frolov
and coworkers to adapt replicons from VEEV to persist in cells found that non-
cytopathic VEEV replicons were much easier to obtain, and although the mutations
associated with persistence were found in the same region of nsP2, these mutations
had a less dramatic effect on this New World replicon than on its Old World
counterparts [15]. Additional studies on VEEV led to the conclusion that its ability
to inhibit host gene expression and counteract innate antiviral responses were
associated with its C protein, rather than nsP2 [16]. Further support for the role of
the C protein of New World alphaviruses in inhibition of the host’s innate immune
response came from work on EEEV [17].
2.4 Alphaviruses as Vectors
Several groups have adapted alphaviruses for use as vaccination vectors [18 20]. In
the simplest form, this is accomplished by replacing the structural protein genes of
the alphavirus with a heterologous gene of interest (see Fig. 1). The resulting RNA,
called a replicon, is capable of directing its own replication and heterologous gene
expression when introduced into the cytoplasm of host cells, but is incapable of
forming virions or spreading to adjacent cells because it does not encode the
alphavirus capsid or glycoprotein genes. If these replicons are introduced into a
helper cell, in which the capsid and glycoprotein genes are expressed in trans,
output virions are produced which are structurally identical to wild-type alpha-
viruses, but which encapsidate the replicon RNA in place of a normal alphavirus
genome. These virus replicon particles (VRPs) are capable of infecting cells in vitro
or in vivo, and expressing the encoded gene of interest, but are single-cycle
particles incapable of cell-to-cell spread due to the lack of structural protein
genes in the replicon.
Several features of alphaviruses and alphavirus replicons make them attractive
candidates for use as vaccine vectors. First, VRPs stimulate strong immune responses
against the encoded antigen. The broad tropism of alphaviruses allows VRPs to
deliver antigen to a variety of cell types, including antigen-presenting cells [21 23].
Antigen is expressed at very high levels from the replicon subgenomic RNA,
comprising up to 25% of total cell protein in cultured cells [19]. The production of
antigen in vivo from replicating RNA stimulates innate immune defenses which
recognize RNA virus infection and potentiate adaptive immune responses [24],
while allowing antigen presentation both intracellularly and extracellularly. This
results in a balanced TH1/TH2 profile of cellular and humoral immune responses
similar to those induced by LAVs (see studies reviewed in [25, 26]).
In addition to inducing strong systemic immune responses, alphavirus VRP
vaccination also has further, unexpected benefits. First, subcutaneous or intramus-
cular immunization with VRPs induces potent mucosal immune responses and
efficiently protects against challenge with mucosal pathogens [27 33]. This allows
for development of vaccines against mucosally transmitted pathogens without the
need to vaccinate at a mucosal site. Second, VRPs have an adjuvant effect on
a Alphavirus genomic RNA
7mG 5’ nsP1234 Capsid Glycoproteins 3’ An
Viral subgenomic RNA 7mG Capsid Glycoproteins 3’ An
Alphavirus replicon
7mG 5’ nsP1234 GOI 3’ An
Antigen-expressing sgRNA 7mG GOI 3’ An
b Defective helper RNAs
Single DH 7mG 5’ Capsid Glycoproteins 3’ An
7mG 5’ Capsid 3’ An
Split DH
7mG 5’ Glycoproteins 3’ An
c VRP production by electroporation d VRP production in packaging

Input RNA cell lines
7mG 5 nsP1234 GOI 3

Input VRPs
7mG 5 Capsid 3 7mG 5 Glycoproteins 3
7mG 5 Capsid 3
7mG 5 Glycoproteins 3
Endogenous
helper
RNAs
Output
VRPs
Fig. 1 Alphavirus vector constructs and production methods. (a) Schematics of alphavirus
genomic and subgenomic RNAs as produced by wild type viruses, and an alphavirus replicon
RNA in which the alphavirus capsid and glycoprotein genes are replaced by a gene of interest
(GOI). (b) Schematics of the defective helper RNAs most commonly used for VRP production.
Single DH constructs contain all the alphavirus structural genes in one RNA, while split DH
constructs divide these across two RNAs. (c) VRP production by electroporation. In this method,
the replicon RNA and helper RNAs are introduced to an unmodified susceptible cell, such as BHK
or Vero cell. (d) VRP production in packaging cell lines (PCLs). In this method, a cell line is
modified to express the helper RNAs from DNA cassettes integrated into the host genome.
Infection of a PCL with a replicon then triggers helper activation and VRP production
immune responses to purified protein antigens codelivered with VRPs in the

inoculum [24, 31, 34], likely as a result of immune stimulation by replicating
RNA. This adjuvant effect opens some intriguing possibilities, such as development
of new vaccines that utilize “killed” antigens but elicit a “live” vaccine immune
response [31], or making combination vaccines in which VRPs stimulate immunity
to one antigen, while simultaneously potentiating responses to another, unrelated
antigen, provided that the formulation and delivery are suitable for both the VRP
and protein components.
Importantly, published studies suggest that the excellent immunogenicity
profile of alphavirus VRPs is not compromised by pre-existing immunity to either
the vector or the encoded antigen. Unlike poxvirus or adenovirus vectors, the
immunogenicity of alphavirus VRPs is not subject to widespread pre-existing
antivector immunity in the human population, as seroprevalence of antibodies
against alphaviruses in humans is low [35, 36]. Furthermore, even the antivec-
tor immunity induced by repeated immunization with VRPs, did not impede
subsequent immunization with VRPs encoding a different antigen in mice [20].
Similarly, a boost in immunity to a VRP encoded target antigen was observed in
humans even when two prior immunizations had elicited substantial antivector
neutralizing titers [37]. Additionally, in contrast to LAV, the immunogenicity of
VRP vaccines is not greatly affected by the presence of maternal antibody against
the target antigen [38].
Complementing the potent immunogenicity of alphavirus VRPs are their safety
features. In contrast to the spreading infection caused by LAVs, the single-cycle
replication of VRP infection ensures that vaccination cannot initiate a pathogenic
infection, provided that the VRPs are free of any contaminating replication-compe-
tent viruses. An additional layer of safety is added by the RNA genome of
alphavirus vectors, which only persists for about 7 days postvaccination, does not
disseminate to systemic sites, and has no potential to integrate into the host genome
[39]. This contrasts with plasmid DNA vaccination, where the vector DNA persists
for months postvaccination, both at the injection site and at distal sites, and which
could potentially integrate into host DNA [39].
In light of all these advantages of VRP vaccination, it is perhaps not surprising
that VRPs effectively elicit protective immunity against an extensive list of bacte-
rial, viral, and protozoan pathogens in a variety of small animal and primate models
(reviewed in [25, 26]). While immunizing against one pathogen at a time is the most
straightforward approach, multiple VRPs expressing different antigens can also be
combined into a single immunization to simultaneously induce protective immunity
against multiple pathogens [40]. These studies confirm that VRP vaccination is a
versatile approach to inducing protection against a broad array of important human
and animal pathogens.
3 Alphavirus Vector Vaccine Production
While alphavirus vectors have advantages over other vaccine vector candidates in
several respects, clinical application of alphavirus VRPs has lagged behind other
viral vectors, in part due to the challenges of producing VRPs at large scale. Cost-
effective, scalable systems for manufacturing alphavirus VRPs have not been fully
worked out, although considerable progress has been made. This section will
review the issues inherent in producing alphavirus VRPs at industrial scales, and
review work performed to date to overcome these limitations.
3.1 Electroporation Systems
Alphavirus VRP production occurs whenever permissive cells contain both an

alphavirus replicon RNA and the necessary helper RNAs expressing alphavirus
structural proteins. In most published work, VRPs are produced by coelectropora-
tion of these RNAs into a susceptible cell, typically BHK cells, and harvesting
output VRPs which are present at high titer by the next day. Such an approach has
been used to efficiently produce SINV, SFV, and VEEV VRPs at small scales
[18 20]. However, two problems with electroporation systems have thus far limited
their utility: (1) the ability of alphavirus replicons to recombine with or copackage
with helper RNAs, and (2) the difficulty of performing electroporation at industrial
scale and in GMP-quality cell lines. Nevertheless, several products produced from
electroporated Vero cells have made it into clinical studies [37, 41].
3.2 VRP By-Products
Recombinant viruses. The earliest attempts to produce alphavirus VRPs used an

alphavirus replicon introduced into cells with a single defective helper RNA
(encoding the capsid and glycoproteins as a single polyprotein, as it occurs in
wild-type alphavirus genomes) [18 20]. Although these initial systems produced
high titers of VRPs, this single helper system also produced plaque-forming viruses
(also called replication-competent virus, RCV) at a very high frequency, with
approximately 1 RCV detected for every 104 105 VRPs produced [18 20]. These
RCV were generated by a recombination event (or RdRp strand switch) joining the
helper genome structural protein coding regions to the replicon genome, thus
reforming a genome that encoded all of the proteins necessary for producing
progeny virions and a spreading infection. This outcome was consistent with
studies which found alphavirus replicons to be highly prone to intragenomic
RNA RNA recombination events in electroporation systems [42], provided that
the helper RNA has functional 30 end replication signals [43]. Interestingly, these
and other studies found that alphavirus RNA recombination appears to be predom-
inantly nonhomologous recombination, and thus cannot be prevented simply by
eliminating regions of homology shared by the replicon and helper RNAs [20, 42,
43]. Moreover, SFV or VEEV VRP preparations containing RCV contaminants are
lethal to intracranially inoculated mice, demonstrating that RCV poses a very real
threat to vaccine safety [18, 20].
Segmented genome RCV. Importantly, the frequency of RCV detected in VRP
preparations produced by electroporation of replicon and single-defective helper
RNAs can be higher than the frequency of recombination events, and if the
defective helper RNA contains signals for efficient packaging into virions, RCV
titer can be similar to VRP titer [18, 20]. These observations suggest that these
plaque forming RCVs are the result of efficient packaging of single helpers and
replicons into the same infectious particle. In these scenarios, the alphavirus has, in
effect, been converted into a virus with a segmented genome. Indeed, investigators
have succeeded in designing two or three genome SINV that are efficiently repli-
cated [44, 45], emphasizing that efficient helper RNA packaging could enable
production of unwanted segmented genome RCV in electroporation of replicons
and helper genomes.
Helper expression. The problem of segmented-genome RCV can be alleviated to
a great degree by eliminating packaging signals from helper RNAs to make helper
packaging less efficient [18, 20, 44]. However, even if reduced, expression of helper
RNA in VRP-infected cells in vivo would be expected to elicit immune responses to
vector structural proteins, which may impact the immunogenicity of subsequent
vaccinations with the same vector. Furthermore, efficient helper packaging into
VRPs could provide the opportunity for replicon-helper recombination to occur
in vivo and produce RCV in the vaccinated host, even if RCV was not present in the
initial inoculum.
3.3 Safety Improvements
RCV prevention. Of the three by-products of VRP production listed above, repli-
con-helper recombination has received the most attention because it appears to have
the greatest potential to produce an RCV that compromises vaccine safety, espe-
cially for alphaviruses that are highly pathogenic in humans such as VEEV.
Consequently, considerable effort has been made to reduce the frequency with
which recombination events produce functional infectious virus.
One of the most successful approaches thus far to prevent RCV while retaining
high VRP yields has been the “split-defective helper” approach [46]. In this
system, the single defective helper RNA used in earlier studies is “split” into
two helper RNAs, one encoding the capsid gene, and a second encoding the
glycoprotein genes, so that a replicon RNA must make two independent recombi-
nation events to form a virus genome containing all of the alphavirus genes. This
approach, first published with SINV [46], was also adapted to SFV and VEEV
VRPs [20, 47], and in all cases no RCV was detected in 108 5 109 VRPs
produced using the split-DH system. As an added benefit, the split-DH system
probably also reduces the likelihood of forming segmented-genome RCV, as a
three-component genome would be less likely to form and efficiently passage than
a two-component genome.
In the SFV iteration of this split helper system, a further safety enhancement to
the split-DH system was made by ablating the autoprotease activity of the capsid
protein so that even if two recombination events rejoined the capsid and glycopro-
tein genes into a single polyprotein gene, the resulting protein would not function
[47]. This innovation would certainly prevent regeneration of a wild-type alpha-
virus genome, with a single subgenomic promoter driving a structural protein
polyprotein, but would not prevent the formation of an RCV replicon encoding
the capsid and glycoprotein genes under the control of two separate subgenomic
promoters.
Although the split DH system has eliminated detectable RCV in small research-
scale VRP lots, mathematical considerations suggest that the split-DH approach
may not reduce RCV to levels needed to ensure safe use in humans. Since
recombination between a replicon and one helper RNA occurs at a frequency of
10 4 10 5 [18, 20, 48], the expected frequency of a dual-recombination event
between a replicon and two helper RNAs would be approximately 10 8 10 10.
This is consistent with the published literature which did not find RCV in lots of this
size when split-DH RNAs were used [20, 46, 47], but suggests that an industrial-
scale lot of 1016 VRP could be contaminated with 106 108 RCV. Thus, either
additional safety features may still be needed for alphavirus VRPs to reach com-
mercialization, or the RCV formed must be sufficiently attenuated to ensure that it
does not pose a human health risk.
One design innovation that may be able to reduce the frequency of replicon-
helper recombination events is the removal of the subgenomic promoter from the
helper RNAs [49]. Doing this reduces the theoretical number of possible recombi-
nation events that would still result in functional structural protein expression from
the recombinant replicon, because the helper-replicon recombination must occur in
a precise way that places the structural protein under the control of the subgenomic
promoter that was present in the replicon, as opposed to the structural protein gene
having an attached promoter that can function from a variety of recombination sites.
This approach can be used successfully without a reduction in VRP titer [49], but
the theoretical benefit has yet to be tested experimentally to show that the rate of
RCV is actually reduced in this system.
RCV attenuation. Alphavirus envelope glycoproteins strongly affect both viral
tropism and pathogenicity. Consequently, if VRPs are made with glycoproteins
from alphaviruses with low pathogenicity in humans, then the health risk posed by
RCV is reduced. Two groups have pursued such a strategy to improving VRP
safety, in both cases with the use of VEEV replicons.
In the first approach, attenuating mutations can be introduced into the envelope
glycoproteins of VRPs. For example, mutations that increase virion affinity for
heparan sulfate decrease the neuroinvasiveness of alphaviruses by reducing the
level of viremia that is established during infection [7]. Introducing these mutations
into defective helpers would attenuate any RCV produced, but would also alter
VRP tropism, and therefore could affect immunogenicity, in vivo. However, VEEV
VRPs packaged using envelope glycoproteins with varying heparan sulfate affinity
showed only moderate variation in immunogenicity in vivo [50], demonstrating
that attenuating mutations can be used in VRPs without dramatically altering
immunogenicity. Thus VRP vaccine safety can be improved by packaging repli-
cons in virions derived from attenuated virus strains; however, even attenuated
strains of VEEV retain some pathogenicity by peripheral inoculation in animal
models [7], so it is unclear whether introduction of attenuating mutations into
glycoproteins alone will be sufficient to ensure that RCV by products do not
compromise the safety of VRP vaccines.
A much greater degree of RCV attenuation appears to be possible with a second
approach based on chimeric VRPs. In this case, VRPs were made in which the
replicon is derived from VEEV, but the structural proteins are derived from SINV
[51]. To ensure efficient packaging of VEEV replicons into SINV virions, the well-
defined SINV packaging signal was introduced into the nsp3 region of the VEEV
replicon. The resulting VRPs are similar to VEEV VRPs in production yields from
electroporated BHK cells, RNA replication and antigen expression in infected cells,
interferon resistance, and immunogenicity in vivo [51].
Thus, VEE/SIN chimeric VRPs retain important features of VEE VRPs, but
there are several reasons why any RCV produced from a VEE/SIN VRP system is
likely to be much less pathogenic than RCV produced from a VEEV-only system.
While VEEV is a virulent human pathogen capable of causing fatal encephalitis,
SINV is not neurotropic in humans, causes disease only rarely, and is not life-
threatening [1]. Furthermore, several chimeric alphaviruses have been made in
which the replicon genome of a low-pathogenicity virus (SINV) is combined with
the structural proteins of a more pathogenic virus (VEEV, WEEV, EEEV, or
CHIKV). In all cases, the chimeras were attenuated compared to the pathogenic
parent viruses [52 56], suggesting that chimerization is inherently attenuating to
alphaviruses. This was confirmed in vitro for a chimeric virus constructed
between the VEEV nonstructural proteins and SINV structural proteins [57],
which resembles the RCVs that could occur during VEE/SIN chimeric VRP
production. This chimeric virus is unable to shut down host cell gene expression,
is noncytopathic in vitro, is cleared from cultures of interferon-competent cells,
and was reported to be nonlethal when inoculated intracranially into mice [57].
This degree of attenuation is likely a result of the fact that the VEEV capsid and
glycoprotein genes are both major pathogenicity determinants for VEEV, and
VEE/SIN RCV encodes neither of these proteins. Combined, these data suggest
that VEE/SIN RCV may be severely attenuated and pose little or no human health
risk. Safety could be enhanced even further if attenuating mutations were added to
glycoprotein genes of the chimeric VRP, so that multiple layers of protection
prevent RCV from being pathogenic in vivo. However, more detailed animal
pathogenicity studies are still needed to thoroughly document the attenuation of
chimeric RCV.
3.4 Industrial-Scale Production
Electroporation has been the most common methodology for VRP production
because at small scale it is simple and affordable, and the only specialized equip-
ment required is a commercially available electroporator. However, it is not clear if
electroporation can be performed at industrial scales in a cost-effective manner for
all target populations. A typical VRP electroporation protocol requires trypsiniza-
tion of adherent cells, followed by multiple wash steps and electroporation of cells
in individual cuvettes, followed by cell plating in adherent format and harvest the
next day. While such a multistep protocol is easy to carry out on a bench top with a
limited number of cells, automating electroporation for industrial use is difficult
and will require specialized equipment that does not currently exist. While these
hurdles may be surmountable, they are likely to add considerably to the cost of
developing an alphavirus VRP-based vaccine. Nevertheless, electroporation has
been successfully performed under GMP at scales at least sufficient for phase I
clinical trials [37].
As an alternative, VRP packaging cell lines (PCLs) could be used for VRP
production. One type of PCL that has been developed consists of an alphavirus-
permissive cell line with DNA cassettes expressing the defective helper RNAs
stably integrated into the host genome. In this PCL, the helper RNAs are constitu-
tively expressed, but the alphavirus structural proteins are not, because the genes
are under the control of an alphavirus subgenomic promoter [58]. Upon introduc-
tion of an alphavirus replicon into the PCL by transfection (or VRP infection), the
replicon-encoded replicase enzymes are produced, and trigger replication of
the cell-encoded capsid and glycoprotein genomes and subgenomes, producing the
structural proteins needed to package the replicon genome (Fig. 1). Thus, PCLs
allow VRPs to act as self-propagating viruses. This technology allows VRPs to
be produced in much the same manner, and using the same equipment and meth-
odologies that are in use for the production of traditional or genetically engineered
live-attenuated viral vaccines. Despite these advantages, amplifying VRPs through
multiple passages on PCLs also may provide multiple opportunities for recombina-
tion and RCV formation, although in published work PCLs utilizing the split-DH
system did not produce detectable RCV at small-scale [58].
In summary, while considerable progress has been made in alphavirus VRP
production, issues of safety and scalability must be more fully addressed before the
potential of alphavirus VRPs will be fully realized.
4 Preclinical/Clinical Evaluation
Many alphavirus replicon-based vaccines have been tested at the preclinical level.
Replicons have been used to express antigens from infectious disease agents such as
influenza, HIV, SHIV, SIV, Louping ill, dengue virus, Plasmodium falciparum,
hepatitis C virus, infectious bursal disease virus, human papilloma virus, Lassa
virus, Marburg virus, equine arteritis virus, and Ebola virus, as well as tumor
antigens. Most of these constructs have performed well in small animal models,
and those that have moved further into preclinical development have functioned well
in nonhuman primates. A VEEV-based replicon expressing the G protein from
Marburg virus was able to completely protect nonhuman primates (cynomolgus
macaques) from viremia and disease upon lethal Marburg virus challenge [59]. Both
Semliki Forest virus-based [60] and VEEV-based [61] replicon vaccines expressing
SIV immunogens have been found to confer partial protection from lentiviral disease
as exemplified by lowered peak viral titers or lessened disease signs. A chimeric
replicon system consisting of a VEE based replicon packaged with SINV structural
proteins has been used to develop a prototypic next generation measles vaccine,
expressing the measles virus hemagglutinin (H) or hemagglutinin and fusion (H+F)
proteins. When tested in nonhuman primates, the vaccine was found to induce
neutralizing antibody titers and T cell responses that were similar to those induced
by the current live attenuated measles vaccine [62]. These animals were protected
from measles virus challenge, but did show a reappearance of measles virus RNA in
their PBMCs 4 months after challenge. This recrudescence was not seen in maca-
ques that received the measles LAV, and further experiments will be required to
fully understand the immunological mechanisms that surround measles virus clear-
ance and to determine if additional antigens are required for more effective vaccina-
tion. The chimeric replicon expressing the measles hemagglutinin protein, was
compared to a formalin-inactivated alum-precipitated vaccine in mice. The LAV
measles vaccine is unable to replicate in mice and was therefore not included. The
replicon-based vaccine induced a balanced B and T cell response to the H protein
that produced high affinity neutralizing antibody titers, [63] while the formalin-
inactivated vaccine elicited no neutralizing titers. This work fully demonstrates the
benefits that the alphavirus-based replicon system provides over a formalin-killed
vaccine.
Recently, the results of a phase 1 clinical trial of a VEE alphavirus replicon-
based vaccine against cytomegalovirus (CMV) has been reported [64]. This vaccine
contained two replicon particles, one that expressed CMV gB, and the second that
expressed a CMV pp65/IE1 fusion protein. Four groups of eight individuals each
received either a lower dose (1 107 IU) or higher dose (1 108 IU) by subcuta-
neous or intramuscular injection at 0, 8, and 24 weeks. The vaccine was found to be
safe and produced only mild to moderate reactions at the injection site. All
individuals generated neutralizing antibody titers against CMV following the initial
immunization and these titers increased following each subsequent immunization,
despite the development of antivector immune responses. This alphavirus replicon
vaccine also induced polyfunctional CD4+ and CD8+ antigen-specific T cell
responses to CMV pp65, gB, and IE1 in almost all of the study participants. The
ability of these vaccine vectors to safely stimulate both humoral and cellular
immune responses in healthy adults provides good support for their continued
development for use in human vaccine development.
5 Future Prospects
The future of alphavirus-vectored vaccines is bright. They offer a vehicle that can
produce a desired antigen within the vaccinated individual in such a way that the
immune system reacts with both cellular and humoral arms. This response results in
CD4 and CD8 T cell, systemic antibody, and mucosal antibody responses that can
effectively prevent infection by a vast number of pathogens. The large amount of
data investigating the molecular mechanisms of alphavirus replication and patho-
genesis leaves us with the road maps and tools to fine-tune these vaccine candidates
for optimal antigen production and immune responses.
Acknowledgments We thank Susan Barnett and Christian Mandl for their assistance during the
preparation of this chapter.
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Recombinant, Chimeric, Live, Attenuated
Vaccines Against Flaviviruses and Alphaviruses
Thomas P. Monath
Abstract Many arthropod-borne flaviviruses are important human pathogens respon-

sible for diverse illnesses, including yellow fever (YF), Japanese encephalitis (JE), and
TBE and dengue. Live, attenuated vaccines have afforded the most effective and
economical means of prevention and control, as illustrated by YF 17D and JE SA14-
14-2 vaccines. Recent advances in recombinant DNA technology have made it
possible to explore a novel approach for developing live attenuated flavivirus vaccines
against other flaviviruses. Full-length cDNA clones allow construction of infectious
virus bearing attenuating mutations or deletions incorporated in the viral genome. It is
also possible to create chimeric flaviviruses in which the structural protein genes for
the target antigens of a flavivirus are replaced by the corresponding genes of another
flavivirus. By combining these molecular techniques, the DNA sequences of DEN4
containing a deletion in the 30 NCR, a DEN2 PDK-53 candidate vaccine and YF 17D
vaccine have been used as the genetic backbone to construct chimeric flaviviruses with
the required attenuation phenotype and expression of the target antigens. Encouraging
results from preclinical and clinical studies have shown that several chimeric flavivi-
rus vaccines have the safety profile and satisfactory immunogenicity and protective
efficacy to warrant development as products for human use. The chimeric flavivirus
strategy has led to the rapid development of novel live, attenuated vaccines against
DEN, TBE, JE and WN. This chapter reviews an extensive body of work on the
development of these vaccine candidates, one of which is licensed and others are in
advanced clinical development.
A similar approach is being used to create vaccines against alphaviruses. Here
the experience is less, but some promising data have been developed, particularly
using SIN virus as a vector for structural genes of heterologous alphaviruses. The
principal issues for this technology will be to achieve convincing nonclinical data
on safety, the proper balance of attenuation and immunogenicity, and proof of
concept in large animal models and ultimately humans.
T.P. Monath
Kleiner Perkins Caufield & Byers LLC and Harvard School of Public Health, 295 Townsend Hill
Road, Townsend, MA 01469, USA

350 T.P. Monath
1 Introduction
This chapter focuses principally on live, chimeric vaccines against flaviviruses,

since these vaccines are in clinical development or are licensed. Similar technology
is being used for development of new vaccines against medically important alpha-
viruses, and this topic will be discussed more briefly.
It is assumed that the reader has a general knowledge of flavivirus and alphavirus
structure and replication, and an understanding of the medical need for new
vaccines against flaviviruses and alphaviruses. Chimeric vaccines contain the
structural genes of one flavivirus (alphavirus) against which immunity is desired
and the genes encoding nonstructural (NS) proteins of another flavivirus (alpha-
virus), which serve as the vector or “backbone”. The NS proteins from the vector
provide the replicative machinery of the chimeric vaccine, whereas the immune
response to infection with the vaccine virus is directed principally at the envelope
(E) protein (flaviviruses) or proteins (E1 and E2, alphaviruses) provided by the gene
donor. The E protein(s) contains critical antigens for stimulation of neutralizing
antibodies, which are the principal mediator of protective immunity against flavi-
viruses and alphaviruses. Attenuation of virulence is determined by a combination
of factors, including chimerization itself and mutations in structural and/or vector
sequences resulting from natural evolution, passage in laboratory hosts or cell
culture, or site-directed mutagenesis. Assembly and replication of chimeric virions
are efficient due to the consistency of genome organization and function across
members of the genus Flavivirus (Alphavirus).
The extraordinary immunity provided by natural infection with flaviviruses
(alphaviruses) provides an important benchmark for defining immune correlates
of protection during the development of new vaccines. Fortunately, the evolution-
ary diversity of members of the genus Flavivirus (Alphavirus) defined by neutrali-
zation is very different from influenza, HIV, and enteroviruses, where antigenic
diversity greatly complicates vaccine development. Each medically important
flavivirus represents a single serotype; the same is true of medically important
alphaviruses, with the exception of Venezuelan equine encephalitis (VEE), which
occurs in multiple subtypes. There are few examples of important human or
veterinary flavivirus diseases caused by multiple virus species that cocirculate in
sympatric fashion, requiring a multivalent vaccine. Dengue (caused by four distinct
viruses) is the only important example. Even where a syndrome (encephalitis) is
caused by sympatric flaviviruses [e.g., Japanese encephalitis (JE) and either West
Nile (WN) or tick-borne encephalitis (TBE) viruses in parts of Asia, or WN and St.
Louis encephalitis (SLE) in the Americas], one disease so overshadows the other
that multivalent vaccines have not been a priority for development. The situation is
similar for the medically important alphaviruses, with eastern equine encephalitis
(EEE), western equine encephalitis (WEE), epizootic VEE subtype I, Getah, Ross
River (RRV) and (except for some areas of Africa) chikungunya (CHIK)1 having
1
In parts of Africa, a related virus o’nyong nyong has an overlapping range with CHIK.
Recombinant, Chimeric, Live, Attenuated Vaccines Against Flaviviruses and Alphaviruses 351
geographically distinct distributions. In addition to the lack of antigenic diversity

and relatively simple epidemiological situation, a tremendous advantage to vaccine
development is the quality of the immune response to flaviviruses (alphaviruses)
and the availability of well accepted immunological correlates of protection.
Infection is always followed by natural immunity, and it is strong and highly
durable (essentially life-long). These qualities associated with natural infection
have been embodied in artificial immunization with the live, attenuated vaccines,
especially yellow fever (YF) 17D vaccine. The basis for the remarkable immuno-
genicity of YF 17D virus has been the subject of recent enquiry, which showed, as
had been long suspected, that infection efficiently activates multiple pathways of
the innate immune system, a prerequisite for effective adaptive immunity and long
term memory [1 3]. It is not surprising, therefore, that this property of live flavivi-
rus infection would be harnessed by using live vaccines as vectors for foreign
genes, with the expectation that the vector would impart a similar quality of
immune response on the foreign gene carried by it. Over the last 15 years a wealth
of information has accumulated on this strategy, with the result that there are
multiple chimeric flavivirus vaccines in clinical development, using several vector
species. As with any platform technology, it takes dedicated industrial develop-
ment, years of evidence based science, and regulatory consensus before applica-
tions “take off”. One chimeric live vaccine (against WN) is now approved for
veterinary use, another (against JE) is in registration in several countries, and,
perhaps most importantly, several vaccines against dengue (DEN) are in clinical
development.
2 Principles for Use and General Properties

of Chimeric Vaccines
This review focuses on live, chimeric vaccines against flavivirus, and, more briefly
alphavirus infections. Other chapters address different vaccine technologies,
including single cycle replicons and DNA immunization, that could utilize a
chimeric approach. Chimeric recombinant E proteins [4] represent another yet
approach not covered in this chapter.
Multiple live vector platforms have been developed in addition to flaviviruses
and alphaviruses, poxvirus, adenovirus, cytomegalovirus, vesiculovirus, paramyx-
oviruses (Newcastle’s disease virus), and others. A major problem common to these
technologies is that pre-existing immunity to the vector or immunity generated to
the vector after priming constrains their use. A principal approach to skirting this
problem is to remove protective antigenic determinants from or to switch serotypes
of the vector. This, however, raises a number of issues, since one serotype of the
vector may be significantly less immunogenic than another (a common problem for
adenoviruses). Flavivirus and alphavirus vectors have the advantage that the struc-
tural genes can be substituted across multiple viruses in the genus and that the
352 T.P. Monath
remaining genes (of the vector backbone) do not contribute materially to protective
immunity. In this way novel vaccines can be constructed against multiple members
of each genus without interference from immunity to the vector.
This chapter will describe the use of flaviviruses (and alphaviruses) for con-
structing live vaccines against other flaviviruses (alphaviruses). Use of flavivirus
(alphavirus) vectors for foreign genes will not be considered, but the reader should
be aware that there is interest in this approach. For example, it is possible to insert
relatively small foreign gene coding inserts at one or more sites within the envelope
(E) gene of the flavivirus vector, so that 180 copies of the gene product would be
displayed on each E protein monomer on the virion surface; this approach is
restricted to small inserts, the size of a single epitope, that do not perturb virion
assembly. In another example, larger genes (up to 1 2 kb) may be positioned in a
flavivirus vector downstream of the structural genes. Various insertion points have
been successfully employed, including the intergenic regions between the E and
NS1 genes [5] or the NS2b and NS3 genes [6, 7] , or the placement of an internal
ribosome entry site and foreign sequence between NS5 and the 30 untranslated
region (UTR). Since the structural genes of the vector are preserved, these
approaches are complicated by antivector immunity; it may be necessary to use a
vector for which there is very low immunity in the intended target population and to
construct one or more vectors with different E proteins for use in boosting immu-
nity. In the case of foreign genes inserted in the flavivirus (alphavirus) backbone, it
is possible to avoid anti-vector immunity by exchanging structural gene sequences
for one of up to 70 flaviviruses (29 alphaviruses) comprising the genus.
The terminology used in this chapter to refer to chimeric constructs is to
place the virus donating structural genes and the target for immunization first,
and the vector second. Thus a chimeric virus with structural genes of JE and the
backbone of YF is referred to as a “JE/YF chimera.”
3 Flavivirus Vaccines
3.1 Molecular Construction and Rationale Design
The construction of chimeric Flavivirus vaccines depends on recombinant techni-

ques employing cDNA plasmids derived from flavivirus RNA, site directed muta-
genesis to introduce desired deletions and mutations, an understanding of the
structure and function of the Flavivirus genome, and knowledge of the molecular
basis of virulence and antigenic structure. These aspects are covered in recent
reviews [8]. Briefly, the 10.6 kb single strand, positive sense RNA genome is
organized with a single long open reading frame with three structural genes
encoding the capsid (C), premembrane (prM) and envelope (E) genes at the 50
end and genes for seven NS proteins at the 30 end. The viral RNA is translated as a
polyprotein and is post-translationally processed into the individual viral proteins
by host and virally encoded enzymes. There are short 50 and 30 NCRs at the
respective termini which play important roles in translation and replication of the
viral RNA. Rice et al. [9] first described the generation of full-length infectious
RNA transcripts derived from cDNA of yellow fever 17D (YF 17D) virus. These
transcripts were prepared using a two-plasmid system by cloning the 50 and 30
halves of the genome separately and then ligating them in vitro prior to transcrip-
tion. Instability of full-length cDNA in E. coli proved to be an impediment for
several other flaviviruses. The two-plasmid system was also used to obtain the
infectious RNA transcripts of JE virus strain JaOArS982 [10]. A stable full-length
cDNA copy of wild type DEN4 strain 814669 was first cloned in E. coli strain
BD1528 for transcription of infectious RNA [11]. Full-length cDNA clones of
many other flaviviruses including DEN, WN, and Langat viruses have subsequently
been described.
There are several general approaches to the construction of a flavivirus chimeric
vaccine. The first and most commonly used approach substitutes the prM-E struc-
tural protein genes in the vector for the corresponding genes of the heterologous
virus against which immunization is desired. All three structural genes (C-prM-E)
can be replaced, but in general these constructs are more attenuated, less likely to
replicate efficiently for manufacturing and less immunogenic. Construction of a
chimera requires that the structural genes be sourced from another flavivirus, since
proper assembly of progeny virions requires a high degree of sequence homology.
The use of cDNA clones for introduction of mutations into the vector backbone
or E gene(s) by site-directed mutagenesis allows analysis of their effects on the
phenotype of the recovered viruses. Many studies have shown that the virulence
phenotype can depend on a single mutation that decreases or increases the effi-
ciency of viral replication in cultured cells or in animals. Genetically defined
mutants have been constructed for YF [12], DEN4 [13], DEN2 [14], JEV [15,
16], WN [17] and TBE [18]. Mutations in DEN chimeric viruses have been
engineered to improve viability and increase yields in cell cultures [19]. Mutations
in the NS genes have been introduced to attenuate DEN4 virus [20]. Deletions
engineered into the 30 and 50 NCRs of several flaviviruses produced attenuation in
cell culture and in animals [21 26]. Mutations in the hinge region spanning
domains I and II of the flavivirus E glycoprotein [16, 17, 27] and in the upper
lateral surface of domain III [18] have been shown to reduce virulence; but, in one
case a hinge region mutation increased virulence [28]. Ablation of the glycosylation
site in E or in NS1 has been shown to markedly attenuate viremia and neuroinva-
siveness [29]. Site directed mutagenesis is an important method for rationally
attenuating vaccine candidates and for deciphering the biological effects of muta-
tions that occurred during passage in the empirical development of live vaccine
strains used as gene donors or backbones in constructing chimeras. Wild type and
mutated DEN4, the DEN2 PDK53 candidate vaccine, and YF 17D vaccine strains
have been explored as vectors in constructing chimeric vaccines.
Infectious clone technology has many advantages for manufacture of live,
attenuated vaccines in cell culture. The manufacturing method begins with full
length chimeric RNA transcribed from cDNA, and transfection of the RNA into an
354 T.P. Monath
acceptable cell line for manufacture. This approach is recognized by regulatory

authorities as reducing the likelihood of contamination with adventitious viruses,
and such testing may potentially be avoided. Moreover, the recombinant vaccine
virus is clonal and can be precisely defined by sequencing for quality control and
assessment of genomic stability. However, since normally flaviviruses and alpha-
viruses are genetic swarms, and their biological behavior is determined by the
population of variants, care must be taken to determine that the clonal population
has the desired biological phenotype. A powerful strategy for evaluating clonality
and expression of recombinant inserts is limiting dilution cloning and evaluation of
50 100 clones by sequencing.
A critical aspect of the use of any live vaccine, including chimeric viruses, is
obtaining the correct balance of attenuation and immunogenicity (Fig. 1). This
balance, which also determines the rate of growth and yields in cell cultures used
for manufacturing, is determined by both the gene insert and the vector backbone.
Some confidence can be derived by use of a vector with known properties of
attenuation and immunogenicity, for example an attenuated live vaccine with a
long history like YF 17D. As will be described below, both the structural gene insert
and the NS backbone sequences influence the biological phenotype of the chimera,
so it is sometimes necessary to further attenuate the virus by introducing mutations
or deletions in the structural gene insert or backbone. It is well established that the
process of chimerization is in itself attenuating and that substitution of structural
genes or the nonstructural backbone for sequences from wild-type virulent viruses
usually does not usually confer a virulence phenotype exceeding the parental
viruses [30 32]. This is not always so. When the backbone virus is attenuated,
the insertion of structural genes from a virulent virus can confer a higher degree of
virulence (an example being the increased neurovirulence observed when TBE
structural genes were inserted in a DEN4 backbone [33]). Therefore each construct
must be subjected to empirical study.
Attenuation must be assessed carefully in various model systems before introdu-
cing a chimeric virus into humans. These assessments generally include evaluation
Neurovirulence
Viremia
Adverse events
Strong immune
response
Attenuation
Safety, tolerability
Weak immune
response
Fig. 1 Phenotype of live vaccines requires proper balance of attenuation and immunogenicity that
must be sought through empirical testing
of neuroinvasiveness and neurovirulence in suitable animal models [34], since all

flaviviruses have the capacity to infect and cause damage to brain and spinal cord
tissue. Viscerotropism or special pathogenic features may be more difficult to assess
in animal models, but viremia (mean peak, mean duration, area under the curve),
levels of virus replication in tissues of the host, tissue specific enzymes, and levels of
proinflammatory cytokines may be useful markers [28, 30]. It may be instructive
to employ hosts that are deficient in adaptive or innate immune responses, such
as interferon receptor-deficient mice, to reveal residual virulence of a chimeric
construct or to demonstrate safety in the presence of immune deficiency. Attenuation
of DEN chimeric viruses has been assessed using SCID mice reconstituted with
human hepatoma (HuH-7) cells [20, 25, 26] since liver is a target organ for DEN
viruses in humans.
A theoretical concern about the use of chimeric viruses for immunization is the
reliance on epitopes within the structural (transplanted) proteins to confer “com-
plete” immunity. While neutralizing antibodies to the E glycoprotein are accepted
correlates of protective immunity, and are specific for the pathogen represented by
the foreign genes, NS proteins that may contribute to immunity are derived from the
heterologous vector. Relatively little is known about the contribution of the NS
proteins to protective immunity, but there is evidence that both anti-NS1 antibodies
[35] and T cell responses [36] play a role in protection and immunological memory.
Immunity to shared T cell epitopes in the vector and target virus may account for
some cross-protection. As experience has accumulated, these concerns have not
proven critical to vaccine development. First, multiple T cell epitopes, including
MHC-I restricted epitopes, reside in the flavivirus E protein [37 40]. Second, the
contribution of anti-NS immune responses to protective immunity may not be
great. In one illustrative example, a WN/DEN2 chimera induced high titers of
DEN2 specific NS1 antibodies (and presumably T cell responses to DEN2 NS
proteins) but afforded limited protection against DEN2 virus challenge [41]. More-
over, immunity to chimeric live vaccines has been durable (lasting years) and recall
occurs on boosting. In the specific case of DEN vaccines, it may in fact be desirable
to use a heterologous vector, since T cell responses to DEN (in the absence of
neutralizing antibodies) may play a role in the pathogenesis of dengue hemorrhagic
fever (DHF) [42].
When developing a new vaccine it is important to consider carefully the indica-
tions for use and the target product profile (Table 1). This can be an early guide to
the applicability of a chimeric flavivirus vector.
3.2 Chimeric Flaviviruses Using Yellow Fever 17D Vaccine

as the Vector
Yellow fever 17D vaccine was developed in 1936 by empirical passage as a highly
effective live attenuated vaccine, and has been used in 500 600 million travelers
356 T.P. Monath
Table 1 Target product profile for a live, attenuated vaccine

l Disease indication
l Incidence, severity and lethality of the disease to be prevented (important in assessing risk:
benefit equation)
l Vaccine formulation [virus(es), stabilizers, adjuvant, excipients]
l Vaccine potency (minimum and maximum), test method, units
l Presentation (liquid, lyophilized, vial size and no. doses/vial, prefilled syringe, etc.)
l Storage temperature (before and after reconstitution)
l Thermostability (before and after reconstitution)
l Toxicity (if any) in nonclinical tests (e.g., lethal for infant mice 5 days of age or less when
inoculated IC)
l Age group(s) indication
l Geographic, occupational, vocational indications
l Dose (volume, potency units)
l Route of inoculation
l Safety (incidence of severe and serious adverse events; list specific expected adverse events
if known)
l Tolerability (incidence of common side effects, if any; list expected adverse events, if known)
l Viremia level and incidence
l Precautions
l Contraindications
l Optimal immunogenicity (e.g., seroprotection rate, if level of protective antibodies known; or
non inferiority of GMT to a licensed vaccine)

l Minimal acceptable immunogenicity
l Secondary immune response measures (e.g., T cell assays)
l Specificity (immunity vs. all antigenic variants or subtypes, genotypes of virus targeted?)
l Cross protection against heterologous, related virus species
l Interactions with other vaccines
l Transmissibility by vector mosquitoes (ticks)
l Shedding, secretion, environmental concerns
Each of these factors should be described early in the development process, and refined as work
proceeds
and residents of endemic countries in Africa and South America (reviewed in [43].).
YF 17D is delivered as a subcutaneous (SC) inoculation, but can also be adminis-
tered by the intramuscular, intradermal (ID) or epidermal [44, 45], or intranasal [46]
routes. Wild-type YF virus has been shown to infect monkeys by the oral route
(intragastric inoculation) [47], and there are two recent reports of adverse events
caused by inadvertent YF 17D infection in infants breastfeeding on mothers who
were recently vaccinated (CDC, unpublished), suggesting that use of 17D vectors
for oral or enteric immunization might be possible. Yellow fever 17D is one of the
most, if not the most effective vaccines, rapidly inducing neutralizing antibodies
the mediator and surrogate of protection in 90% of vaccinees within 10 days and
in 99% within 30 days after inoculation [43]. Yellow fever 17D also evokes
robust cytotoxic T cell and memory T cell responses [48, 49]. The vaccine contains
a large excess of virus, about 4.7 log10 plaque-forming units (PFU)/0.5 mL dose,
whereas the 50% immunizing dose is only approximately 50 PFU [43]. This
remarkable efficacy demonstrated by low dose requirements was also observed
for a JE/YF chimeric virus (ChimeriVax™-JE) in humans [50]. The YF 17D
vaccine is indicated for persons 9 months of age or older. Immunity lasts decades
and is probably life-long [51]. However, sterilizing immunity is not complete, since
booster doses result in an increase in antibody levels, albeit typically blunted [52].
Based on these observations, it is likely that a similar negative boosting effect might
occur when a chimeric vaccine with prM-E proteins against a virus for which
preformed neutralizing antibodies were present is used for primary immunization
or boosting. Yellow fever 17D vaccine is well tolerated, and local and systemic side
effects are minimal (reviewed in [43].). The incidence of serious neurotropic and
viscerotropic adverse events is 0.8 and 0.4 per 100,000, respectively [53]. The
occurrence of these adverse events raises the obvious question of whether similar
reactions will be seen with chimeric viruses utilizing the 17D as a vector. However,
data to be presented below indicate that YF chimeric vaccines are more attenuated
than parental 17D with respect to neurovirulence, are attenuated for growth in
human liver cells [54], and are more rapidly cleared from tissues of nonhuman
primates [39]. Finally, the YF 17D vaccine virus and chimeric vaccines with the YF
17D backbone are incapable of infecting mosquitoes by the oral route.
Based on the biological characteristics outlined above, YF 17D is considered an
ideal vector for heterologous genes encoding protective antigens. Multiple groups
are engaged in vaccine development using this strategy, including Sanofi Pasteur
(formerly Acambis), Fiocruz, and the Rockefeller University. An underlying
hypothesis for use of YF 17D as a live vector is that it will impart to the resulting
chimera a phenotype that resembles the parental virus. This is an assumption that
needs to be tested on a case by case basis, but it has held up quite well.
What accounts for the remarkable immunogenicity of YF 17D and for the
durability of the immune response? It is generally understood that the live virus
infection strongly up-regulates innate immunity, including activation of the AIM2/
inflammasome pathway and toll-like receptor pathways, which lead to production
of IL-1b and interferon-a/b, driving the adaptive immune response [3]. The tropism
of flaviviruses for cell receptors is determined by ligands on the E glycoprotein.
An important target cell for YF 17D and other flaviviruses are dendritic (DC) cells
[7, 54] including Langerhans cells in the skin [55]; the receptor is the lectin DC-
specific intercellular adhesion molecule 3-grabbing non-integrin (DC-SIGN) and
the ligands on flaviviruses are glycosylation sites on the E protein. Thus, YF 17D
hijacks DCs for initial replication and movement to regional and systemic lymphoid
tissue and functional maturation. In doing so, multiple activators of innate immu-
nity are triggered, including toll-like receptors (TLR) 2, 7 and 8 [1]. TLR ligands
enhance effector functions of other innate immune cells and synergize with T cell
receptor and B cell signaling to enhance cytotoxic T cell and antibody responses.
Querec et al. [2] and Gaucher et al. [3] studied the early innate immune gene
activation and cytokine profiles in humans vaccinated with YF 17D and found
multiple correlations between specific gene activation signatures and B and T cell
responses. Since many if not all flaviviruses utilize DCs in early stages of replica-
tion, it is likely that chimeras utilizing YF 17D as the backbone but with heterolo-
gous flavivirus prM-E genes will similarly activate innate immune pathways.
358 T.P. Monath
Use of an established commercial vaccine as a vector provides a benchmark

against which the chimeric virus phenotype can be compared. Thus the nonclinical
and clinical behavior of chimeric YF vaccines can be measured against comparator
groups that receive the commercial YF 17D vaccine. The safety of YF 17D vaccine
and chimeric vaccines derived therefrom may be tested preclinically by intracere-
bral (IC) inoculation of mice and monkeys, and neurovirulence compared to
parental YF 17D in dose response experiments (establishing PFU/LD50) or by
semiquantitative scoring of histopathologic lesions [34, 56]. These methods (par-
ticularly dose response neurovirulence tests in infant mice) can detect the biological
effects of single mutations [34]. A chimeric vaccine that is more neurovirulent than
YF 17D in animal models would be expected to be associated with a higher
incidence of neurotropic adverse events in humans, as established many years
ago by Fox et al. [57] In a recent study, a Langat/DEN4 chimera proved more
neurovirulent than YF 17D in monkeys and thus potentially unsuitable for further
development [58]. Similarly, viscerotropism of a chimeric virus can be bench-
marked against YF 17D by determining viremia profiles or other markers such as
liver enzymes or levels of proinflammatory cytokines. See the section on chimeric
WN/YF and JE/YF viruses for specific examples.
3.2.1 Chimeric JE/YF Vaccine (ChimeriVax™-JE, IMOJEV™)
The vaccine candidate ChimeriVaxTM-JE was originally developed by Acambis

Inc. and was acquired by and now branded as IMOJEV™ by Sanofi Pasteur. Since
published work to date refers to the vaccine as ChimeriVax™-JE, this terminology
will be used below.
ChimeriVax™-JE is a live, attenuated, genetically engineered virus, prepared by
replacing the prM-E genes of YF 17D vaccine virus with the corresponding genes
of JE virus [59]. The E protein contains redundant epitopes specifying neutralizing
antibodies and T cell responses [40, 60]. At least four major B cell epitopes have
been identified in the E protein of JE virus at amino acid residues 327 333,
337 345, 373 399 and 397 403 [61 63] and important T cell epitopes at amino
acid residues 60 68 and 436 445 [64, 65]; collectively, these epitopes as well as
others not yet defined confer protective immunity.
The chimeric virus was constructed from a YF 17D infectious clone, substituting
the 17D prM-E genes for the corresponding genes of the SA14-14-2 strain, a live,
attenuated JE vaccine licensed for use in China, South Korea, India and elsewhere
in Asia. The genetic rearrangement was accomplished by standard cloning techni-
ques, employing two bacterial plasmids containing cDNA copies of the prM-E
genes of JE SA14-14-2 virus and the remaining genes of YF 17D [59]. In con-
structing a viable chimera, a critical element was precision of the C-prM junction to
ensure that the yellow fever 17D specific signal sequence (KRRSHDV) for the
NS2b-2 protease was maintained.
The biological properties of the SA14-14-2 strain are well documented and it is a
safe and effective human vaccine [66]. The E protein sequence of SA14-14-2 has
Table 2 Comparison of the amino acid differences in the E protein of chimeric JE/YF, JE SA14
14 2 and wild type JE viruses SA14 and Nakamaya
Virus E E E E E E E E E E
107 138 176 177 227 244 264 279 315 439
JE SA14 14 2 PDKa F K V T S G Q M V R
JE SA14 14 2 PHK F K V A S G H M V R
YF/JE SA14-14-2 F K V A S G H M V R
YF/JE Nakayama L E I T P E Q K A K
JE Nakayamab L E I T P E Q K A K
JE SA14/JAPc L E I T S G Q K A K
JE SA14/CDCd L E I T S G Q K A K
JE SA14/USAe L E I T S E Q K A K
Six residues distinguish the ChimeriVaxTM JE virus (JE SA14 14 2/YF) virus from wild type,
virulent strains SA14 and Nakayama (shown in bold). Residues that are shared between Chimeri
VaxTM JE and SA14 substrains but distinguish ChimeriVaxTM JE from JE Nakayama virus are
shown in italics
a
JE SA14 14 2 vaccine strain sequenced after passage in primary hamster kidney used to manu
facture the vaccine (Aihara et al. [67]) and after additional passages in primary dog kidney (PDK)
cells [68]
b
Wild type (prototype) JE virus (virulent)
c
SA14 strains sequenced by Aihara et al. [67] (virulent)
d
SA14 virus sequenced by Nitayaphan et al. [68] (virulent); sequence corrected by Ni et al. [69]
e
SA14 virus sequenced by Ni et al. [70] (virulent)
been sequenced by a number of laboratories and compared to the virulent parental

SA14 strain (Table 2). The mutations in the E protein of SA14-14-4 underlie the
attenuated phenotype of the vaccine, and it was hypothesized that they would also
contribute to the safety profile of the chimera. The E protein sequence of the JE
(SA14-14-2)/YF chimeric virus (ChimeriVax™-JE) was identical to the JE SA14-
14-2 vaccine strain, sequenced after passage in primary hamster kidney used to
manufacture the vaccine at Chendu (China) [67]. Six amino acid differences at
positions E107, 138, 176, 279, 315 and 439 distinguish all attenuated SA14-14-
2 substrains from both virulent JE viruses, SA14 (the parental strain) and Nakayama
(the JE prototype strain). Four additional mutations at E 177, 227, 244, and 264
distinguish the ChimeriVaxTM-JE virus from the JE prototype strain, Nakayama.
Mutations at positions E177 (A) and 264 (H) seem to be host cell dependent
because they are found in SA14-14-2 primary hamster kidney (PHK) cell grown
virus, (and ChimeriVaxTM-JE derived from PHK passed virus) but not in primary
dog kidney (PDK) cell passaged virus, or parental SA14 virus. Since the PDK
vaccine has wild-type amino acids at these positions, but is attenuated, the E177 and
E264 mutations in ChimeriVax™-JE are unlikely to play a significant role in
attenuation. The putative attenuating amino acid residues map to three subregions
of Domains I and II of the flavivirus E protein model. These include the fusion
peptide (position 107), the hinge cluster (positions 138, 279) and the exposed
surface of Domain I (positions 176).
The complete consensus nucleotide sequence of the ChimeriVax™-JE virus was
determined and the vector backbone sequence compared to published sequences for
360 T.P. Monath
other YF 17D vaccine strains. Genomic heterogenity between YF 17D substrains

has been noted by several authors, reflecting the uncloned nature of these viruses
and differences in passage level [71, 72]. A single nucleotide difference at position
4025 (causing a V!M amino acid mutation in NS2a) was found in the YF sequence
of ChimeriVax™-JE compared to other vaccine substrains.
Plaque-reduction-neutralization tests (PRNT50) were performed on the chimeric
viruses containing SA14-14-2 or Nakayama prM-E genes using YF and JE-specific
hyperimmune polyclonal ascitic fluids and YF-specific purified IgG monoclonal
antibody (2E10). The JE (SA14-14-2)/YF and JE (Nakayama)/YF chimeras and
SA14-14-2 virus were neutralized only by JE hyperimmune ascitic fluid, whereas
YF 17D was neutralized in a specific fashion by YF hyperimmune ascitic fluid and
by 2E10 monoclonal antibody.
To determine whether the SA14-14-2 mutations were important to the attenua-
tion of ChimeriVax™-JE two lines of evidence were followed. In the first, a
chimeric virus was constructed in which the prM-E genes were derived from a
virulent wild-type JE strain (Nakayama). The second approach evaluated the
contribution of each of the SA14-14-2 specific mutations to attenuation. Virulence
was assessed in mice inoculated by the IC route. The JE(Nakayama)/YF virus was
100% lethal for adult mice inoculated by the IC route with 4 log10 PFU, whereas
ChimeriVax™-JE was fully attenuated (0% mortality). Therefore attenuation was
determined by the prM-E genes, and one or more of the SA14-14-2 mutations in
ChimeriVax™-JE caused a dramatic attenuation of neurovirulence. There are ten
amino acid changes in the E protein of ChimeriVax™-JE when compared to the JE
(Nakayama)/YF chimera (and wild-type JE Nakayama virus). Six of these ten
amino acids (E107, 138, 176, 279, 315 and 439) are suspected to be critical
neurovirulence determinants based on: (1) sequence comparison studies between
SA14-14-2 virus substrains, ChimeriVax™-JE and the virulent parent strain SA-14
envelope proteins (Table 3); (2) the observation that these residues map within
putative functional domains of the E protein; (3) published studies showing that
several attenuated vaccine strains of JE (SA14-14-2, SA14-5-3, and SA14-2-8)
differ from virulent parental SA14 virus at amino acids E138, E176, 315 and 439
[69, 73]; and (4) identification of a virulence determinant at E138 by mutagenesis of
a full-length JE infectious clone [15].
A nominal requirement for a genetically stable vaccine is the presence of
multiple mutations independently conferring the attenuation phenotype to avoid
reversion to virulence with a single back mutation. To exclude the possibility of a
single reversion event altering the virulence phenotype of ChimeriVax™-JE, a
series of chimeras with single and multiple changes in amino acid residues was
generated, converting SA14-14-2-specific mutations to the corresponding wild-type
(Nakayama) residues [16]. To define the neurovirulence phenotype of the revertant
viruses, weanling ICR mice were inoculated IC with 4 log10 PFU of each revertant.
Based on these results, revertants were classified as lethal (showing 100% mortal-
ity), partially attenuated (<100% mortality), or fully attenuated (0% mortality)
(Table 3). The analysis showed that reversion of three or four amino acids was
required to restore the mouse neurovirulence typical of the wild-type JE virus. This
finding, as well as the stability of the SA14-14-2 specific residues during sequential
Table 3 Neurovirulence of single site and multisite revertants of SA14 14 2 spe

cific mutations in ChimeriVax™ JE to wild type (Nakayama) amino acid residues,
from Arroyo et al. [16]
Virus or revertanta No. of dead mice/total AST
no. of mice (%)b (days)
YFV/JEV SA14 14 2 0/8 (0)
1 (107; F!L) 0/8 (0)
2 (138; K!E) 0/8 (0)
3 (227; S!P) 0/8 (0)
4 (244; G!E) 0/8 (0)
5 (264; H!Q) 0/8 (0)
6 (279; M!K) 1/8 (13)
7 (315; V!A) 0/8 (0)
8 (439; R!K) 0/8 (0)
9 (176/177; V/A!I/T) 0/8 (0)
10 (107, 176/177) 0/8 (0)
11 (107, 138)c 1/8 (13) 9.0
12 (138, 176/177) 1/8 (13) 13.0
13 (107, 138, 176/177)d 9/9 (100) 9.4
14 (107, 138, 279) 3/8 (38) 11.3
15 (138, 227, 264, 279) 2/9 (22) 11.5
16 (107, 138, 227, 264, 279) 8/9 (89) 9.4
17 (138, 176/177, 227, 264, 279) 9/9 (100) 9.0
18 (107, 176/177, 227, 264, 279) 0/8 (0)
19 (107, 138, 176/177, 227, 264, 279) 8/8 (100) 8.1
YFV/JEV Nakayama 8/8 (100) 9.3
YFV/JEV Nakayama 7/8 (88) 11.6
a
Viruses with neurovirulent phenotypes are indicated by boldface. For viruses 1 9,
the positions of and reversions at the single sites are indicated. For viruses 10 19,
the positions of the multiple reversions are indicated
b
For revertants 1 to 12 compared with YFV/JEV SA14 14 2, there was no signifi
cance. For revertants 14 and 15 compared with YFV/JEV Nakayama, P was 0.01
and 0.002, respectively. For revertants 13, 16, and 17 and YFV/JEV Nakayama
compared with YFV/JEV SA14 14 2, P was 0.0001, 0.0005, 0.0001, and 0.0001,
respectively
c
One of two experiments; the mortality ratio for the second experiment was 0%
d
One of two experiments; mortality was identical for the second experiment (AST
was 9.9 days)
passage (see below) indicated that the risk of reversion to virulence was exceed-
ingly remote.
Since attenuation relied on multiple specific mutations in the E protein, it
was critical to assess genetic stability of the ChimeriVax™-JE vaccine, as RNA
viruses have a high mutation rate due to lack of proof-reading enzymes. To
ascertain genetic stability of the chimeric virus, and to search for “hot spots” in
the genome susceptible to mutation, the virus was serially passaged at high
multiplicity of infection (MOI) in two substrates considered for manufacturing
[diploid fetal rhesus lung (FRhL), Vero] and partial or complete genomic
sequencing and mouse neurovirulence studies performed at low and high pas-
sage levels [74]. None of the SA14-14-2 specific mutations were affected by up
to 18 serial passages in vitro. In each of two different Vero cell passage series
and with passage in FRhL, a single E protein mutation appeared, but these
362 T.P. Monath
mutations appeared at different sites, indicating that there were no amino acid-
specific “hot spots”. However the changes all occurred in the in hinge 4
(bounded by amino acids E266 to E284) of the molecular hinge region of the
E protein responsible for a pH-dependent conformational change during virus
penetration from the endosome into the cytoplasm of the infected cell. The
molecular hinge therefore appeared to represent a region of relative instability.
No changes occurred in the YF 17D backbone. There were no changes in
neurovirulence for mice associated with the hinge region mutations. However
a plaque size change (from large to small in Vero cells) was associated with a
mutation (T!K) at E281 in the FRhL cell passage series. In vivo stability was
also assessed [74]; no changes in brain titer or neurovirulence were found upon
six sequential brain passages in mice. Collectively, these studies led to the
development of specifications for quality control of ChimeriVax™-JE, namely:
(1) each lot would be consensus sequenced across prM-E and all SA14-14-
2 specific mutations were to be retained (other mutations were tolerated); (2) the
passage level would be maintained by a seed lot system; and (3) a statistically
powered test for neurovirulence (in infant mice) would be performed on each lot
in mice, and would meet criteria for complete attenuation.
Interestingly, during the early attempts to manufacture ChimeriVax™-JE in
FRhL cells, a reversion (M!K) at E279 (one of the SA14-14-2 specific sites)
appeared at passage 5 and was associated with a small plaque phenotype. The E279
mutation is located in a beta-sheet in the hinge 4 region of the E protein. The
reversion was not seen in the seed viruses. This mutation caused concern because it
was close to the mutation at E281 observed in the genetic stability studies in FRhL
cells that had also resulted in a small plaque size alteration, indicating genetic
instability at that region during FRhL cell passage. Moreover, Arroyo et al. [16] had
shown that the single-site E279 revertant was the only one with any evidence of a
change in virulence, with 1 of 8 animals succumbing after IC inoculation (Table 3).
These observations triggered a change to Vero cells for manufacturing and a further
investigation of the role of the E279 site in the attenuation phenotype of Chimer-
iVax™-JE. The neurovirulence of the E279 revertant was studied in mice and
monkeys. Outbred mice 4 days of age were inoculated IC with graded doses
of ChimeriVax™-JE FRhL3 (FRHL passage 3, no mutation), ChimeriVax™-JE
FRhL5 (E279 M!K), or a JE(SA14-14-2)/YF chimera in which a single mutation
E279 (M!K was introduced by site-directed mutagenesis [16]. The LD50 values of
the two viruses containing the E279 mutation were >10-fold lower than the FRhL3
construct without the mutation, indicating that the E279 M!K mutation increased
the neurovirulence of the chimeric virus. The FRhL5 virus was 18.5 times more
virulent than FRhL3 (p < 0.0001). This sensitive methodology for detecting differ-
ences in neurovirulence was subsequently employed more widely in developing
chimeric flaviviruses [34].
In the formal monkey neurovirulence test, the FRhL5 virus containing the E279
reversion was significantly more neurovirulent than FRhL3 (no mutation), but less
neurovirulent than commercial YF 17D vaccine. Interestingly, there was an inverse
relationship between neurovirulence and viscerotropism of the E279 revertant as
reflected by viremia, the FRhL5 revertant virus displaying decreased viscerotropism

(statistically lower viremia following IC inoculation of monkeys) compared to FRhL3
virus [28]. Sera from monkeys inoculated with ChimeriVax™-JE FRhL3 and FRhL5
were examined for the presence of plaque size variants. Only large plaques were
observed in sera from monkeys inoculated with the FRhL3, whereas the virus in the
blood of monkeys inoculated with FRhL5 had the E279 revertant small plaque
morphology. These studies reinforced the specification for the vaccine requiring
stability of SA14-14-2 mutations and a phenotypic in vivo release test for attenuation.
During the course of further development of the vaccine, it was decided to
change the cell substrate from the original Vero cell bank grown in medium
containing fetal calf serum to a new cell bank grown in serum-free (SF) medium.
In addition, whereas the original chimeric virus used to prepare seed stocks had not
been biologically cloned, it was decided to plaque purify the new seed virus. The
starting place for developing the new seeds was the RNA transcript used to prepare
the original virus. SF Vero cells were transfected, and a series of expansion and
plaque purification steps were then undertaken, leading to a newly derived pre-
master seed at Passage 10. During this passage series a mutation (R!C) at amino
acid position 60 in the M protein occurred and became the dominant genotype.
Analysis of virus containing the M60 mutation revealed an increased rate of
replication in SF Vero cell culture, with a peak titer approximately 0.5 1 log10
higher when compared to the nonmutant ChimeriVax™-JE. This suggested that the
presence of the M60 mutation could potentially improve genetic stability by pre-
venting accumulation of unwanted mutations associated with restricted growth, and
could also increase virus yields during manufacturing. The genetic stability of this
virus was ascertained by sequencing following three large-scale passages in sta-
tionary culture and stirred tank bioreactors2.
Preclinical studies evaluated the safety profile, immunogenicity and protective
activity of ChimeriVax™-JE in different hosts [16, 74]. ChimeriVax™-JE virus
was fully attenuated for weaned mice inoculated by the IC route, whereas commer-
cial YF 17D vaccine (commercial YF-VAX®) caused lethal encephalitis with an
LD50 of 1.67 log10 PFU. Since the chimeric virus causes no illness in mice after IC
inoculation, it was of interest to determine whether the virus replicated in brain
tissue. Ten groups of 3 4 week old ICR mice were inoculated by the IC route with
3.0 log10 PFU of ChimeriVaxTM-JE virus. The virus replicated to a peak titer of
approximately 6 log10 PFU/g on day 6 after inoculation; titers then decreased over
time. Thus the virus is able to replicate to reasonably high titer in mouse brain
without causing illness.
2
The manufacture of ChimeriVax™ JE is performed by infecting Vero cells grown in serum free
medium on microcarrier beads in a stirred tank bioreactor. The cell culture fluid containing the
virus is harvested and the virus purified by depth filtration, ultrafiltration and diafiltration [early
lots also included a nuclease (Benzonase®) digestion step, but it was later determined that
acceptable levels of residual host cell DNA were achieved without nuclease digestion.] The
final product is lyophilized in a proprietary stabilizer. The vaccine is administered as a 0.5mL
SC injection containing approximately 4.7 log10 PFU.
364 T.P. Monath
Fig. 2 Results of neurovirulence release test (survival curve) used for a typical lot of Chimeri
Vax™JE. Infant mice 8 days of age were inoculated IC with ChimeriVax™ JE 4,5, or 6 log10 PFU
(32 mice/group); YF VAX® 4 or 5 log10 PFU (32 mice/group), or diluent (32 mice). Dose groups
did not differ and were pooled for analysis. The ChimeriVax™ JE survival curve is significantly
different from that of YF VAX® (p < 0.0001, log rank test)
The ChimeriVaxTM-JE virus was neurovirulent after IC inoculation in mice up to

6 days of age, whereas YF 17D is neurovirulent in weaned and adult mice [74].
Partial resistance to IC infection with ChimeriVax™-JE was observed in mice 7 and
9 days of age. The age at which mice become fully resistant to the chimeric virus
occurs between 10 and 28 days of age. Since mice 7 9 days old were partially
susceptible to IC inoculation with ChimeriVax™-JE, this host system was used to
develop a sensitive test for changes in the attenuation phenotype in vaccine lots.
The test was statistically powered to show a difference between a test article and
YF-VAX® in mortality ratio and employed three groups of 32 mice precisely 8 days
of age inoculated with 4, 5 and 6 log10 PFU by the IC route. A reference control
(YF-VAX®) and negative control were included in the test. Results of a typical study
are shown in Fig. 2, with all three dose groups of the ChimeriVax™-JE pooled for
analysis (all had 0% mortality). The new plaque purified vaccine seeds and drug
product developed in serum-free medium and containing the M60 R!C mutation
were also assessed for attenuation in this way. The M60 mutation did not change the
neurovirulence profile when compared to virus lacking the M60 mutation.
The principal control test for safety of YF 17D is the monkey neurovirulence test.
The monkey neurovirulence test was performed according to Good Laboratory
Practice (GLP) regulations using the methods described in the World Health Organi-
zation (WHO) requirements for testing YF 17D vaccine for preclinical safety [75].
The WHO test was modified to include determinations of clinical laboratory tests and
microscopic examination of selected visceral organs in addition to the brain to search
for unexpected extraneural organ dysfunction or pathology. The test involves the
inoculation of 0.25 mL of the Master Virus Seed into the frontal lobe of a minimum of
ten healthy, nonimmune rhesus or cynomolgus monkeys. Another group of ten
monkeys is inoculated with the vaccine virus. A reference control preparation
Table 4 Neurovirulence test, cynomolgus monkeys, ChimeriVax™ JE plaque purified seed

viruses made in SF Vero cells vs. YF VAX®
Parameter ChimeriVax™ JE YF VAX®
Master Virus Production Virus (N 7)
Seed (N 9) Seed (N 10)
Proportion with clinical signs 0% 0% 0%
Proportion viremic (%) 78% 90% 100%
Viremia mean peak (PFU/mL) SD 351 472 272 263 239 188
No. viremic days (Mean SD) 2.89 1.76 2.80 1.40 2.86 0.38
Neuropath score (target areas, 0.160 0.20a 0.223 0.35 0.436 0.19
mean SD)
Neuropath score (discriminator areas, 0.155 0.17a 0.106 0.14a 0.610 0.42
mean SD)
Neuropath score (combined, 0.159 0.16a 0.167 0.23a 0.526 0.19
mean SD)
Test articles (5 log10 PFU in 0.25 mL) inoculated IC on Day 1, observed for 30 days, then
euthanized and necropsied
a
Significantly different from YF VAX® group
(commercial YF-VAX®) is inoculated into a third group of ten monkeys, and the test
article is compared to the control, with respect to specific outcome measures including
clinical observations, viremia and scoring of lesions in defined areas of the brain and
spinal cord 30 days after inoculation. ChimeriVax™-JE (Master Virus Seed and
vaccine lot) were significantly less neurovirulent and produced significantly lower
viremia than YF 17D [76]. The neurovirulence test was repeated on the plaque purified
seed viruses prepared in serum-free Vero cells containing the M60 mutation (Table 4).
An additional safety feature was the finding that ChimeriVax™-JE vaccine was
incapable of infecting Culex and Aedes mosquitoes by oral feeding and had
markedly restricted replication after intrathoracic inoculation [77]. Other work in
Australian and Asian JE virus vectors (Culex annulirostris, Culex gelidus, and
Aedes vigilax) also showed that these mosquitoes failed to become infected feeding
on 6.1 log10 PFU/mL of ChimeriVax™-JE vaccine, which is >10,000 times greater
than the peak viremia in seen in vaccinees [78].
In a pilot study, the susceptibility of rhesus monkeys to challenge by the intrana-
sal (IN) and IC routes with wild-type JE virus was explored. Three young adult
monkeys were challenged by the IC route and five monkeys were challenged IN with
a high dose of JE IC-37 virus (0.25 mL containing 5.4 log10 PFU). None of the
monkeys challenged by the IN route developed signs of illness, whereas all three
monkeys inoculated by the IC route developed encephalitis. The time to onset of
specific neurologic signs of encephalitis was 8 13 days. All monkeys inoculated by
the IN and IC routes developed viremia, but the virus titers in serum were variable.
Based on these results, it was concluded that the IC challenge could be used to model
severe JE in the monkey, and predicted that the long incubation period between
inoculation and illness would provide sufficient time for immunity induced by
prechallenge vaccination to abrogate infection in the central nervous system.
Studies in mice [74] and monkeys [76, 79] showed that a single dose of
ChimeriVax™-JE was highly immunogenic and protected the animals against
lethal IC challenge with wild-type JE virus. Monkeys were inoculated by the SC
route with graded doses of ChimeriVax™-JE virus (Table 5). All monkeys
Table 5 Representative studies in non-human primates of the viremia and antibody response following subcutaneous inoculation of graded doses of
366
ChimeriVax™-JE and protective efficacy following intracranial challenge with wild-type JE virus
Parameter Study 1 (rhesus macaques) [76] Study 2 (rhesus macaque) [79] Study 3 (Cynomolgus macaque) (not published)
Uncloned Not vaccinated Original uncloned Not vaccinated Original uncloned Plaque-purified Diluent
ChimeriVax™-JE ChimeriVax™-JE ChimeriVax™-JE ChimeriVax™-JE
vero passage 2 FRhL passage 5 vero passage 5 (M60 R–>C
(no mutations) GMP vaccine lot mutant) vero
passage 13 GMP
vaccine lot
Number of animals 6 4 12 2 5 4 4
Vaccine dose, route 4 3, 5 3 N/A 2, 3, 4, 5 N/A 4 0 log10 PFU, SC 4 0 log10 PFU, SC N/A, SC
log10 PFU, SC log10 PFU, SC
(N ¼ 3/group) (N ¼ 3/group)
Viremia (% viremic) 100% 0 100% 0% 100% 100% 0%
Viremia duration (mean 40 0 1 7–2 1 0 34 3 75 0
days )
Viremia (mean peak log10 1 2–1 8 0 1 8–2 3 0 24 22 0
PFU/mL
PRNT50 Day 30/31 GMTa 1,016 <10 320–761 0 1,689 761 <10
Seroconversion % 100% 0% 100% 0% 100% 100% 0%
Pre-challenge PRNT50 449b <10b 640–1,280 <10
GMTa
Challenge virusc dose, route 5 4 log10 PFU, IC 5 4 log10 PFU, IC 5 4 log10 PFU, IC 5 4 log10 PFU,
IC
Challenge % ill 33% (17% severe) 100% 0% 100%
Challenge % dead 17% 100% 0% 100% Not challenged
Challenge % viremic (mean 0% 100% (2 8) 0% 100% (1 5)
peak log10 PFU/mL)
PRNT50 Day 30 Post 4,222b <10b (dead) 6,089–10,240 <20 (dead)
challenge GMT
a
To homologous virus (ChimeriVax™-JE ) unless otherwise specified
b
To Nakayama strain
c
T.P. Monath
Challenge virus JE (IC-37)

developed low-level, transient viremias similar to those induced by YF 17D.

Neutralizing antibodies appeared between days 6 and 10, and by day 30, responses
were similar across dose groups, with PRNT50 titers between 320 and 2,560.
Immunized monkeys and sham-immunized controls were challenged by the IC
route with 5.4 log10 PFU of wild-type JE virus. None of the immunized monkeys
developed viremia, few developed signs of illness, and survivors had mild residual
brain lesions 30 days after challenge, while sham-inoculated controls all developed
viremia, lethal encephalitis, and severe histopathologic lesions. Challenge by the IC
route is a severe test of immunity, since natural infection occurs by intradermal
inoculation during mosquito feeding and the virus must undergo extraneural repli-
cation and cross the blood brain barrier.
To evaluate the role of antibodies in protection, hyperimmune ascitic fluids
were prepared in mice to either ChimeriVax™-JE or a commercial inactivated
JE vaccine (JE-VAX®). The antisera neutralized a battery of wild-type JE virus strains
in an equivalent fashion. C57 Bl6 mice were passively immunized and then
challenged IC with a variety of different wild-type JE strains representing different
genotypes [80]. Antisera against ChimeriVax™-JE and JE-VAX® passively protected
strongly against challenge with genotypes II and III JE strains, and less well against
genotypes I and IV. However, overall the antiserum against ChimeriVax™-JE
appeared slightly superior. The study confirmed that protection was mediated by
neutralizing antibody.
Clinical trials with ChimeriVax™-JE are summarized in Table 6. The initial,
randomized, double blind, outpatient Phase I study of ChimeriVax™-JE was
conducted in 36 healthy young volunteers to assess safety, tolerability and
immunogenicity in comparison to YF 17D [81]. This study also addressed the
theoretical concern that prior YF immunization could blunt the efficacy of
ChimeriVax™-JE vaccine by antivector immunity (anti-NS1 antibody or cell-
mediated responses against nonstructural YF proteins). Mild, transient injection
site reactions and flu-like symptoms were noted in all treatment groups, with no
significant difference between the groups. Nearly all subjects inoculated with
ChimeriVax™-JE at both dose levels developed a transient, low-level viremia
similar in magnitude and duration to that following YF-VAX®. There was a
suggestion of slightly higher viremias in subjects with preexisting YF immunity.
Neutralizing antibody seroconversion rates to JE were 100% in the high and low
dose groups in both naı̈ve and YF-immune subjects. Geometric mean neutralizing
antibody responses were higher in the ChimeriVax™-JE high dose groups (naı̈ve
subjects PRNT50 254; YF immune subjects PRNT50 327) than in the low dose
groups (naı̈ve subjects PRNT50 128; YF immune subjects PRNT50 270). Prior
yellow fever vaccination increased the neutralizing antibody response to the
chimeric vaccine, probably due to memory responses to shared antigenic deter-
minants in the E glycoprotein or carrier priming mediated by preexisting immu-
nity to nonstructural proteins.
In a follow-up study, ten adults who had been inoculated 9 months earlier
with ChimeriVax™-JE vaccine and ten control subjects were challenged with
noninfectious JE virus antigen [inactivated mouse-brain JE vaccine (JE-VAX®)],
Table 6 Summary of clinical studies performed with ChimeriVax™-JE
368
Phase Location Age Treatment Dose Adverse events Viremia Neutralizing antbodies to JE
(years) (no. subjects) (log10 Serious, Nonserious Proportion Duration Peak, GMT Seroconversion
PFU) related viremic (range, mean (PRNT50a) (%)a Day
(%) days) log10 Day 28–31 28–31
PFU/mL
1 US 18–49 CV-JEb (N ¼ 12) 5.0 0 AEs were qualitatively 83% 0–3 1.1–1.6c 254–327c 100%
CV-JE (N ¼ 12) 4.0 0 and quantitatively 92% 0–5 1.4–1.5c 128–270c 100%
YF (N ¼ 12) 5.0 0 similar across the 100%d 2–3d 1.6d 1513c 42%
three treatment
groups
1 US Follow-on study in subjects who were immunized in the study above to determine memory response to “challenge” with inactivated JE antigen (see
text)
2 US 18–59 CV-JE (N ¼ 10) 5.8 0 AEs were qualitatively 50% 0–4 0.8 262 100%
CV-JE (N ¼ 44) 4.8 0 and quantitatively 67%e 0–5 1.1 299 100%
CV-JE (N ¼ 11) 3.8 0 similar across all 82% 0–3 1.2 210 100%
CV-JE (N ¼ 11) 2.8 0 treatment groups 100% 1–6 1.6 103 91%
CV-JE (N ¼ 11) 1.8 0 82% 0–5 1.3 285 100%
YF (N ¼ 11) 5.0 0 64% 0–3 1.3 <10 9%
Placebo (N ¼ 11) 0 0% 0 0 <10 0%
2 Australia 18–55 CV-JE (N ¼ 201) 3.8 1f AEs were qualitatively Not determined; a sample for 258–389g 98.5%
and quantitatively viremia taken 14 days after
similar across the vaccination was negative
Placebo (N ¼ 199) 0 treatment groups <10 0%
2 Australia 18–55 CV-JE and YF 3.8 See text and Table 7
(N ¼ 108)
2 Australia 18–49 CV-JE (N ¼ 32) 5.0 0 AEs were qualitatively 28% 0–11h 0.5 2,060 93.5%
CV-JE (N ¼ 32) 4.0 0 and quantitatively 53% 0–7h 0.8 2,152 93.8%
CV-JE (N ¼ 32) 3.0 0 similar across the 44% 0–3 0.6 1,809 100%
Placebo (N ¼ 32) 0 treatment groups 0% 0 0 <10 0%
3 Australia 18 CV-JE [N ¼ 410 ~4.0i 0 Lower incidence of 1,392j 99.1%j
& US (safety); 346 AEs in CV-JE
(efficacy)] group than
JE-VAX® Licensed 0 JE-VAX® group 37.4 74.8%
[N ¼ 410 product (p ¼ 0.031)
T.P. Monath
(safety); 365
(efficacy)]
3 Australia 18 CV-JE (N ¼ 1,600) 4.0 1k Similar incidence of
& US AEs Day 0–30
between active and
placebo treatment
groups (p ¼ 0.203;
higher incidence in
CV-JE group Day
0–7 and 0–14
(p ¼ 0.038).
Headache, myalgia,
inj site pain,
Placebo (N ¼ 404) 0 generally mild
a
Titers are to homologous strain (also see text); PRNT50 ¼ 50% plaque reduction neutralization test
b
ChimeriVax™-JE; YF ¼ YF 17D (YF-VAX® or Stamaril®)
c
YF nonimmune – YF immune
d
YF-nonimmune subjects
e
There were 33 subjects in this group tested for viremia
f
“Acute viral illness” onset 8 days post vaccination with diarrhea, fever, dehydration, hospitalized; recovered w/out sequelae
g
The study was a cross-over design in which half of the subjects received CV-JE first and 30 days later received placebo, and the other half received placebo, then
CV-JE. The range of GMTs represents the 30 day post CV-JE titers for each cross-over subgroup. The seroconversion arte is combined
h
Viremias were intermittent, with 0–4 days positive out of 14 days tested; the latest day positive was taken as the longest duration in the range shown
i
Three consistency lots were tested in 136 subjects each
j
The titer and seroconversion are to homologous JE strains (ChimeriVax™-JE for the CV-JE treatment group and Nakayama for the JE-VAX® treatment group.
The statistical noninferiority and clinical consistency (between lots) endpoint were met
k
Pyrexia, hospitalized, recovered w/out sequelae
370 T.P. Monath
100
80
Seropositive (%)
60 Dose (log10 pfu)
5.8
40 4.8
3.8
2.8
20
1.8
0
0 14 21 30
Day
Fig. 3 Kinetics of the neutralizing antibody response to a single SC inoculation of graded doses of
ChimeriVax™ JE (From Monath et al. [50], with permission)
representing a surrogate for exposure to live virus transmitted by mosquitoes under

conditions of natural exposure [50]. Subjects who had previously received Chimeri-
Vax™-JE responded with a rapid rise in neutralizing antibodies (Fig. 3). Mean
antibody levels on Day 7 were approximately 20-fold higher and on Day 14 100-fold
higher than pre-inoculation levels. In contrast, JE-VAX® control subjects responded
more slowly and had low antibody levels. Differences in mean antibody levels
between the treatment groups were highly significant on all study days. These
results demonstrate that strong immunological memory is induced by the Chimeri-
Vax™-JE vaccine. Vaccinated individuals would be expected to have a rapid
anamnestic response if exposed to wild-type JE virus.
A randomized, double blind, placebo-controlled, out-patient Phase 2 study was
conducted in the US in 99 healthy subjects, 18 59 years, to further evaluate the
safety and immunogenicity of ChimeriVax™-JE vaccine (Table 6). Subjects
received one or two inoculations (30 days apart) across a wide dose range (1.8 5.8
log10 PFU) [82]. Treatment groups included volunteers who received YF 17D 30
days before or after inoculation of ChimeriVax™-JE to investigate vaccine interac-
tions. The vaccine at all dose levels was well tolerated, and there were no differences
in the incidence of adverse events across active vaccine and placebo treatment
groups after the first or second dose. ChimeriVax™-JE vaccine was associated
with a brief viremia of low magnitude. Viremias were somewhat higher in the low
dose than high dose treatment groups. This inverse relationship between dose and
response has also been noted in the case of YF 17D vaccine [43]. The 90% effective
dose was estimated to be 17 PFU, which is not dissimilar to the ED90 of YF 17D
(50 PFU) [43]. Eighty-two (94.3%) of 87 subjects who received a single inoculation
of ChimeriVax™-JE at all dose levels (Groups 1 6, 8 and 9) seroconverted to JE
by neutralization test within 30 days. Seroconversion rates varied between 82 and
100%
90%
P = .0001
Day 0
P = .0001
4.5 90% Day 3
Day 7
P = .0001
4.0 Day 14
3.5 Day 30
3.0 50% 40%

Mean LNI (+SD)
P = .0017
2.5
P = .0024
2.0
20%
1.5
20%
1.0
0.5
0% 0% 0%
0.0
–0.5
Immune Naive
Group
Fig. 4 Seroconversion rate (% above bars) and mean neutralizing antibody levels [log10 neutrali
zation index (LNI, histogram] for subjects in the Phase 1 trial (Table 6) who (left) had been
immunized with ChimeriVax™ JE 9 months earlier or (right) naı̈ve subjects by day after “chal
lenge” with inactivated JE vaccine (JE VAX®). P c values (t tests) compare immune and naı̈ve
treatment groups. From Monath et al. [50], with permission
100% across the range of doses from 1.8 to 5.8 log10 PFU without relationship
to dose. No statistical differences in mean antibody titers were found across dose
groups. Antibody titers increased rapidly over 2 3 weeks after primary inoculation
(Fig. 4), appeared to peak around Day 30, did not increase after boosting, and in fact
tended to decrease slightly by Day 60. Follow up studies have now documented
persistence of antibody for >3 years following a single dose. Neutralization tests
were performed against ChimeriVax™-JE and three wild-type JE virus strains
(Beijing-1, Nakayama and 902/97); seroconversion rates were high to all strains
across all dose groups, but GMTs were higher to the homologous (ChimeriVax™-
JE) virus. The antibody response to ChimeriVax™-JE was not influenced by vacci-
nation against YF performed 30 days previously; however there was a suggestion
that prior inoculation of ChimeriVax™-JE diminished the serological response to
YF 17D. 64% of ChimeriVax-JE-immune subjects seroconverted to YF, compared
to 91% of ChimeriVax™-JE-naı̈ve subjects; the difference was, however, not
statistically significant. The mean antibody titer to YF 30 days after inoculation
was lower in ChimeriVax-JE-immune subjects than in ChimeriVax-JE-naı̈ve
subjects, but again the difference was not statistically significant. It was concluded
from these results that ChimeriVax™-JE has a safety profile and viremia pattern
similar to those of YF 17D vaccine. ChimeriVax™-JE rapidly elicited high titers of
neutralizing antibodies after a single inoculation at very low doses, an advantage
over existing inactivated vaccines that require multiple inoculations.
Another randomized double-blind Phase 2 study conducted in 2004 at a site in
Australia further evaluated the interaction of ChimeriVax™-JE and YF 17D
372 T.P. Monath
vaccine (Stamaril®). The study enrolled 108 subjects 18 55 years of age. Thirty-six
subjects received ChimeriVax™-JE followed 30 days later by Stamaril®; 36 sub-
jects received Stamaril® followed by ChimeriVax™-JE; and 36 subjects received
the two vaccines simultaneously in different arms. Subjects who were Flavivirus
naı̈ve at baseline (JE, YF, MVE, KUN, ALF) were analyzed (Table 7). YF 17D
elicited higher levels of neutralizing antibodies against YF than ChimeriVax™-JE
against JE. Subjects who received ChimeriVax™-JE before Stamaril® had higher
antibody responses than those who received Stamaril® in advance of or concur-
rently with ChimeriVax™-JE. However, the two vaccines evoked strong responses
in nearly all subjects regardless of the schedule of immunization.
Another Phase 2 study in 201 military personnel in Australia included a double-
blind stage during which subjects received two vaccinations, one SC administration
of 3.8 log10 PFU of ChimeriVax™-JE and one 0.5 mL SC dose of placebo (diluent),
28 days apart in a cross-over, parallel group design. The seroconversion rate on
Day 30 to homologous virus was 98.5% and the GMT was 258 389. The serocon-
version rate and GMT to wild-type JE virus strains representing different genotypes
of JE virus were determined (Table 8). The seroconversion rate to genotype IV
virus was lower than to the other genotypes. Genotype IV is the most evolutionarily
divergent subgroup of JE viruses and is also the least likely to be associated with
human disease. The GMTs were lower to genotypes II and IV than to genotype I and
Table 7 Seroconversion rate and geometric mean antibody titer (GMT) by 50% plaque reduction
neutralization test to JE and YF 30 days after administration of two sequential vaccinations or
coadministration with ChimeriVax™ JE or YF 17D (Stamaril®)
Statistic ChimeriVax™ JE Stamaril® then Coadministration
then Stamaril® ChimeriVax™ JE
JE seroconversion 30 days after 17/17 (100%) 21/23 (91%) 22/23 (96%)
both vaccinations
JE GMT 30 days after both 688 426 344
vaccinations
YF seroconversion 30 days after 17/17 (100%) 23/23 (100%) 23/23 (100%)
both vaccinations
YF GMT 30 days after both 3,289 2,175 2,094
vaccinations
Table 8 Seroconversion rates and neutralizing antibody geometric mean titer (GMT) to wild
type JE strains representing different genotypes 28 days after vaccination with ChimeriVax™ JE
ChimeriVax Genotype I Genotype II Genotype III Genotype IV
JE Korea Thailand Beijing Indonesia
TVP 8236 B1034/8 JKT 9092
Seroconversion 194/197 98.5% 194/197 98.5% 181/197 91.9% 194/197 98.5% 175/197 88.8%
ratea
GMT group Ab 388.6 209.2 65.4 211.7 55.7
GMT group Bb 258.4 236.9 72.7 228.0 64.3
a
Seroconversion is presented for the combined groups A and B
b
Group A and B represent subjects in the different arm of this cross over study, subjects in group A
received ChimeriVaxTM JE then placebo, subjects in group B received placebo then Chimeri
VaxTM JE
III. These differences are not believed to be clinically significant since neutralizing
antibody titers 10 are considered to be protective [82].
A randomized placebo controlled Phase 2 study was designed to test safety and
immunogenicity of the plaque-purified, lyophilized formulation of ChimeriVax™-
JE. Groups of 32 healthy subjects received graded doses (3.0, 4.0, 5.0 log10 PFU) of
ChimeriVax™-JE or placebo. The safety profile was good, with no differences in
adverse events across dose groups or placebo. Viremias were very low in all dose
groups. Based on historical data the viremia was lower than that observed following
inoculation of YF 17D. Seroconversion rates in the ChimeriVax™-JE 3.0 log10
PFU, 4.0 log10 PFU and 5.0 log10 PFU groups were 100%, 93.8%, and 93.5%,
respectively. The homologous GMT in the ChimeriVax™-JE 3.0 log10 PFU, 4.0
log10 PFU and 5.0 log10 PFU treatment groups were 1,809, 2,152 and 2,060,
respectively. There were no statistical differences in seroconversion or GMT across
dose groups, confirming earlier dose response data. Tests were conducted with
wild-type JE strains; as in the previous study (Table 8), responses to genotypes II
and IV were lower than to genotypes I and III.
Two Phase 3 (pivotal) trials were conducted by Acambis in 2006. The first
study was a multicenter, randomized, double-blind, study of the comparative
immunogenicity, safety and tolerability ChimeriVax™-JE vs. inactivated mouse
brain JE vaccine ( JE-VAX®) and was conducted in Australia and the US. The
trial enrolled 410 subjects who received placebo on Days 0 and 7 and Chimer-
iVax™-JE on Day 30 and an equal number of subjects who received JE-VAX®
on Days 0, 7 and 30. The primary endpoint was a test for non-inferiority of
ChimeriVax™-JE to JE-VAX®, with the test intended to rule out a 5% difference
in JE neutralizing antibody seroconversion rates to each respective homologous
virus (ChimeriVax™-JE or JE Nakayama) 30 days after immunization. Secondary
endpoints examined GMT at 30 days and early response (Day 14 seroconversion
and GMT). The trial met all of its endpoints; the seroconversion rate and GMT
were statistically higher in the ChimeriVax™-JE group after a single dose
than following three doses of JE-VAX® (Table 6). The GMT following
ChimeriVax™-JE was 37 times higher than after JE-VAX®. In addition, the
study showed that the antibody response was more rapid than that following
JE-VAX®, with 93% seropositive after 14 days.
In parallel, a randomized double blind multicenter Phase 3 study was conducted
in 2006 in 2000 subjects >18 years of age in Australia and the US. The objectives of
the study were to assess safety in 1,600 subjects receiving ChimeriVax™-JE and
400 subjects who received placebo (0.9% saline). The study showed that the new
vaccine was safe and well tolerated.
In 2006, an open label Phase 3 trial of ChimeriVax™-JE was initiated in India,
enrolling children aged 9 months to 5 years. A smaller Phase 2 trial addressed
interactions of ChimeriVax™-JE and measles vaccine in infants.
Following the successful Phase 3 trials, a partnership with Sanofi Pasteur was
formed for commercialization of the vaccine. In 2008, Sanofi Pasteur acquired
Acambis. The ChimeriVax™-JE vaccine, now called IMOJEV™, is in registration
in Australia, Thailand and elsewhere.
374 T.P. Monath
ChimeriVax™-JE vaccine was shown to cross protect against Murray Valley

encephalitis [83] virus (a member of the JE antigenic complex) in mice. If con-
firmed, this strategy could be a useful intervention in the event of outbreaks of
MVE, which occur at infrequent intervals in Australia, but further development
would be needed. It would be of interest to test subjects who receive IMOJEV™ for
cross-neutralizing MVE antibodies.
3.2.2 Chimeric Flavivirus Vaccines Against Dengue
Because of the worldwide importance of dengue fever and severe dengue [previ-
ously called dengue hemorrhagic fever (DHF)], there has been a sustained interest
in the development of vaccines. Before the development of infectious clone tech-
nology allowing rational vaccine design, efforts focused on empirical derivation of
live, attenuated DEN vaccines by serial passage; these efforts have largely been
abandoned due to difficulties in getting the correct balance of attenuation and
immunogenicity (Fig. 1). Major issues that complicate the development of DEN
vaccines include: (1) the role of enhancing antibodies and T cells in sensitizing
the host to severe dengue on exposure to a heterologous dengue virus to which
solid immunity is not induced; (2) the requirement therefore to evoke (preferably
simultaneously) durable protective immune responses against all four dengue sero-
types; (3) interference between the four DEN serotypes when combining them in
a live vaccine formulation; (4) difficulty in establishing immunological surrogates
of protection due to the inability to distinguish between homotypic neutralizing
antibody and cross-reactive heterotypic nonprotective antibody.
With the advent of infectious clone technology, several groups have developed
DEN vaccine candidates based on chimeric flavivirus constructs [84, 85]. Three
different strategies are being deployed currently. The farthest along in development
is the DEN/YF chimeric (ChimeriVax™) DEN vaccine first constructed by Tom
Chambers at St Louis University, subsequently developed by Farshad Guirakhoo,
Konstantin Pugachev, and Thomas Monath at Acambis [86, 87], and acquired
(in 2008) by Sanofi Pasteur (Acambis and Sanofi Pasteur had collaborated on this
project beginning in 1999). Ricardo Galler and colleagues (BioManguinhos/
FioCruz) in Brazil are independently developing DEN/YF chimeric vaccines
[88]. At NIAID, Michael Bray and Ching-Juh Lai pioneered the construction and
testing of intertypic dengue chimeras [89, 90], and a full vaccine development
program ultimately evolved under the leadership of Steven Whitehead and Brian
Murphy [85, 91]. At CDC (Ft. Collins) Richard Kinney and Claire Huang devel-
oped an intertypic dengue vaccine platform [92] that was licensed to and is under
development by Inviragen Inc.
A fundamental question in selection of an appropriate vector backbone is whether
it is preferable to utilize a heterologous backbone virus (e.g., YF 17D) or a homo-
typic vector backbone (DEN) that may contribute to anti-DEN immunity via T cells
and anti-NS1 antibody. The central objective in vaccine immunity is to stimulate
strong neutralizing antibody responses directed against the E glycoprotein. That will
be determined principally by the ability of the vector backbone to replicate in the

host, generating sufficient antigenic mass, and its ability to activate innate immune
signaling pathways. In mouse models, the DEN NS proteins have not contributed
materially to protection [41]. From a safety perspective, in order to prevent sensiti-
zation to severe DEN in the event antibody levels fall, it may be preferable not to
have cytotoxic T cells directed against DEN NS proteins (particularly memory
T cells directed against cross-reactive epitopes, that may have lower avidity for
the infecting virus resulting in altered proinflammatory T-cell functional responses),
especially in the absence of strong neutralizing antibodies [42]; therefore, in the case
of a DEN vaccine, a heterologous (non-dengue) backbone might have some advan-
tages.
3.2.3 Chimeric DEN/YF Vaccines (ChimeriVax™-DEN and Others)
Several groups have independently reported the successful construction of chimeric

DEN/YF viruses [86, 88, 93, 94]. Guirakhoo et al. [86, 95] used donor prM-E genes
from low passage, recent isolates from Asia for construction of all four YF/DEN
chimeras. Various other donor genes have been used, e.g., DEN2 prototype New
Guinea C virus, DEN2 Americas I genotype (PR159) or DEN1 from Venezuela
VeMir95 [88, 93, 94, 96]. In contrast to the JE/YF chimera, wild-type, unmodified
donor genes were used for construction of a vaccine candidate without modification
(mutagenesis), and reliance was placed on the chimerization process itself and the
vector NS genes to confer appropriate attenuation. The rationale for this approach
was the successful empirical development at Mahidol University of a live DEN2
vaccine by 53 serial passages in primary dog kidney cells (PDK53), which was
shown to be attenuated in clinical studies and to contain no mutations in the E gene
[97]. Therefore it was presumed that presentation of the wild-type dengue 2 prM-E
sequence in the background of another vector (YF 17D) with attenuating mutations
in the backbone would yield an acceptable phenotype. The first biological assess-
ments of various chimeric DEN2/YF viruses showed them to be less neurovirulent
than YF 17D after IC inoculation of mice while eliciting antibodies to DEN2.
In contrast, a DEN4/YF chimeric virus constructed by Chambers et al. [94] utilized
a mouse-neuroadapted and neurovirulent DEN4 gene donor strain and the resulting
chimera was neurovirulent, showing [as for JE (Nakayama)/YF] that the structural
gene insert influences the virulence phenotype of chimeras constructed on a highly
attenuated backbone. The fact that wild-type DEN viruses are naturally attenuated
for neurotropism suggested that it would be possible to use unmodified donor
prM-E genes for constructing chimeras, but each serotype and construct would
have to be tested empirically.
Guirakhoo et al. [86] showed that the DEN2/YF chimeric vaccine inoculated SC
over a wide dose range, caused low-titer viremia in rhesus monkeys compared to
wild-type virus, elicited strong neutralizing antibody responses, and protected
monkeys against wild-type DEN2 challenge. Based on these promising results,
manufacture and testing of clinical grade vaccine was undertaken, with the first
376 T.P. Monath
IND for a monovalent DEN2/YF chimera (ChimeriVax™-DEN2) filed to the Food

& Drug Administration (FDA) in early 2002. The method of manufacture was
similar to that described above for ChimeriVax™-JE. Seed virus was tested in
infant mice and by the monkey neurovirulence test and the chimeric virus shown to
be significantly less neurovirulent than parental YF 17D. An interesting aspect of
the preclinical assessments in monkeys was the observation that viremia patterns
following chimeric DEN vaccination differed from those seen after YF 17D in that
virus could be detected (at very low levels or intermittently) for a more prolonged
period, sometimes into the second week.
An important milestone was the clinical proof-of-concept study [98]. Parenthet-
ically, there was considerable internal debate within the development team about
the practical value of conducting clinical studies of each monovalent YF/DEN
serotype independently. It was concluded that human testing of the tetravalent YF/
DEN vaccine would provide more conclusive data on the interaction of viruses in
the formulation than further studies in nonhuman primates or clinical tests of
monovalent components.
The monovalent ChimeriVax™-DEN2 trial was conducted in healthy subjects,
18 49 years of age, at a single center in the US (Table 9) [98]. The study
randomized 42 eligible healthy adult subjects without prior immunity to YF to
receive a single SC vaccination of either ChimeriVax™-DEN2 (5 log10 or 3 log10
PFU dose) or YF 17D (YF-VAX®). In addition, 14 subjects previously vaccinated
to YF received high dose (5 log10) ChimeriVax™-DEN2 as an open-label vaccine.
The vaccines were well tolerated, with no differences in adverse event profiles
between ChimeriVax™-DEN2 and YF-VAX®. More YF-naı̈ve subjects vaccinated
with ChimeriVax™-DEN2 compared to YF-VAX® developed viremia: 8 (57%) in
the ChimeriVax™-DEN2 5.0 log10 group; 9 (64%) in the ChimeriVax™-DEN2 3.0
log10 group compared with 2 (14%) in the YF-VAX® group. The proportion of
subjects who developed viremia following vaccination with ChimeriVax™-DEN2
5.0 log10 PFU was slightly greater in YF-immune than in YF naı̈ve subjects (79% v
57%). The mean peak viremia level following ChimeriVax™-DEN2 5.0 log10 PFU
Table 9 Phase 1 clinical trial of monovalent ChimeriVax™ DEN2; viremia and antibody
responses. (Data from Guirakhoo et al. [98])
Group No. YF Vaccine Dose Viremia (mean) PRNT50a
subjects immune log10 % Duration Peak Seroconversion GMT
PFU in Viremic (days) (PFU/ (%)
0.5 mL mL)
Double-blind, randomized
1 14 No ChimeriVax™- 5.0 57 1.4 12.1 100% 921
DEN2
2 14 No ChimeriVax™- 3.0 64 1.2 11.4 100% 570
DEN2
3 14 No YF-VAX® 5.04 14 0.4 20.0 0% <10
Open label
4 14 Yes ChimeriVax™- 5.0 79 1.9 29.3 100% 975
DEN2
a
antibodies to the homologous ChimeriVax™ DEN2 virus
was greater in YF immune subjects than that in YF naı̈ve subjects. While none of
these values were statistically significant with this small sample, the finding may be
evidence of immune enhancement of ChimeriVax™ replication in persons with
pre-existing YF immunity, an observation similar to that shown for ChimeriVax™-
JE in some studies. ChimeriVax™-DEN2 was highly immunogenic; 100% of
subjects seroconverted; geometric mean neutralizing antibodies were statistically
higher in the higher dose group. The sera were also tested against three different
wild type DEN2 strains as well as heterotypic dengue viruses (types 1, 3 and 4).
Nearly all subjects (93 100%) vaccinated with ChimeriVax™-DEN2 serocon-
verted to the wild-type DEN2 viruses but GMTs were lower than to the homologous
strain; minimal, low titer responses were seen to heterotypic dengue serotypes.
Interestingly, prior YF immunity had a dramatic effect on stimulating broad
heterotypic responses to DEN 1, 3 and 4 following immunization with Chimeri-
Vax™-DEN2. The results suggested that in areas where YF immunity is prevalent,
e.g., South America, DEN immunization might be enhanced. This result was
anticipated by earlier studies of empirically developed live dengue vaccines in
YF immune vs. nonimmune subjects [99]. T cell responses were measured by IFNg
production by PBMC cultured for 7 days in the presence of inactivated Chimeri-
Vax™-DEN2 virus. All four vaccine groups showed a significant increase in IFNg
production at day 31 relative to day 1. A slightly lower IFNg response was seen in
YF-immunized subjects, but it was statistically similar to the response in either
dose group of ChimeriVax™-DEN2 immunized subjects. This suggested that
nonstructural proteins present in the YF backbone of the ChimeriVax™-DEN2
vaccine made a significant contribution to the T cell response. The IFNg response
to ChimeriVax™-DEN2 vaccination was not diminished by prior immunity to YF.
Importantly, neutralizing antibody titers to DEN2 in this trial were substantially
higher than observed in subsequent trials of the tetravalent vaccine (see below),
illustrating the effect of interaction of multiple dengue strains in a combined
(mixed) vaccine.
The first recombinant tetravalent DEN vaccine was developed by Acambis [95].
ChimeriVax™-DEN1 4 viruses were constructed by inserting prME genes of
wild-type DEN viruses into core and nonstructural genes of YF 17D virus. The
origin of the donor wild-type strains used to construct these viruses is shown in
Fig. 5. Different methods for genetic constructions were employed, including
the standard two-plasmid system, and in vitro ligation of an overlap extension
PCR amplicon (DEN1/YF) or multiple DNA fragments (DEN3/YF). The initial
viruses derived by transfection of recombinant RNA were not plaque purified and
accumulated a number of mutations after passages in Vero cells.
Biological characterization of the chimeric candidate viruses was performed in
mice and monkeys [95]. The candidates exhibited a high level of attenuation in
mice. A focus of the preclinical evaluation was whether or not there was interfer-
ence between the four subtypes in the tetravalent vaccine. Monkeys inoculated with
the tetravalent mixture developed satisfactory immune responses to all four sero-
types, although the response to DEN2 appeared to predominate [95]. Signs of
378 T.P. Monath
Fig. 5 The three major approaches to construction of rationally designed live, attenuated DEN
vaccines. (a). Chimeric vaccines in which the prM E genes of YF17D are replaced by the
interference were relatively subtle; the incidence and duration of viremia to DEN 1
and 3 were less than in monovalent controls [86, 95].
To improve the genetic stability, the viruses were rederived and plaque purified in
an attempt to make pre-master seed (PMS) viruses with wild-type consensus
sequences and minimize mutations arising on passage [100]. A principal goal was
to derive DEN 1, 3 and 4 viruses that had wild-type sequences, since it was noted that
the original constructs contained a small number of mutations in the prME genes
[100], whereas the DEN2 chimera did not have mutations. It was possible that this
accounted for the predominant immunogenicity of the DEN2/YF chimera in monkeys
[86, 95]. Plaque-purified pre-master seeds (PMS) at passage 7 (P7) were produced in
Vero cells, and passed three times under cGMP to produce vaccine lots at P10.
Preclinical studies of ChimeriVax™-DEN1-4 viruses demonstrated that the
vaccine candidates had the following product profile:
l Produced high yields in Vero cell culture for vaccine production
l Underwent minimal genetic changes during passage up to P20 in Vero cells
l Were not neurovirulent in 3 4 week old mice inoculated by the IC route
l Were less neurovirulent than YF-VAX® in infant mouse and monkey models
l Did not become more neurovirulent upon extensive in vitro passaging (measured
by a sensitive infant mouse neurovirulence test)
l Had restricted replication in mosquito vectors, similar to the YF 17D virus, and
significantly lower than their parental wild-type DEN viruses
l Did not interfere with each other in terms of replication in host (mouse, monkey
[101] and mosquito models)
l Gave a balanced response (low viremia and high neutralizing) when adminis-
tered at an equal mixture (e.g., 3,3,3,3 or 5,5,5,5 log10 PFU formulation of the
chimeras for DEN 1, 2, 3 and 4 serotypes) and
l Protected monkeys against a severe heterologous challenge after a single mono-
valent or tetravalent dose [100]
Genetic stability was assessed by full genomic sequencing at various passage
levels, including pre-master seed (P7), Master Seed (P8), Working Seed (P9),
vaccine (P10) and P20. The biologically cloned viruses had no mutations from
the parental wild-type sequence at P7. With passage, a small number of mutations
were observed, but these were fewer than seen in the original uncloned preparations
and no mutations arose in prME of DEN3 at P10 (vaccine lot) or in DEN4 at P20
(genetic stability passage) (Fig. 6). Mutations in the YF backbone (NS4B) chimeras
were likely adaptations to growth in SF Vero cells and had been observed previ-
ously when passing uncloned chimeric DEN2. To ensure that the accumulated
Fig. 5 (Continued) corresponding genes from wild type DEN 1 4 strains (Acambis/Sanofi
Pasteur); (b e). Various approaches used at NIAID to develop mutagenized and chimeric DEN
vaccines against DEN1 (Panel B), DEN (Panel C), DEN3 (Panel D) and DEN4 (Panel E);
(f). Chimeric vaccines in which the prM E genes of DEN2 PDK53 vaccine are replaced by the
corresponding genes from wild type DEN 1,3 and 4 strains (CDC/Inviragen)
380 T.P. Monath
Fig. 6 Mutations in plaque purified, reconstructed ChimeriVax™ DEN monovalent seeds and
vaccine lot during GMP production in serum free Vero cells
mutations during cell culture passages had not increased the neurovirulence phe-
notypes of these viruses, 4-day-old suckling mice were inoculated IC with various
doses of P7 (Pre-Master Seed) and P20 viruses. Neurovirulence of the chimeras was
not increased from P7 to P20; in fact, some chimeras lost their neurovirulence upon
in vitro passages. In addition to the in vitro stability studies, virus recovered from
the sera of monkeys were sequenced and if mutations were found, tested in the
4-day mouse test. Point mutations were found in DEN1 and DEN3 chimeras but
neurovirulence for 4-day-old mice remained less than that of YF 17D.
The E protein mutation (K!R) at residue 204 that appeared between P7 and P8
in ChimeriVax™-DEN1 was the subject of investigation to determine the effects on
biological phenotype [102]. Although mutations were found in the other chimeras
(Fig. 6), none affected neurovirulence. However, the E204 (K!R) mutation in
ChimeriVax™-DEN1 increased the plaque size of the virus and reduced neuro-
virulence in the 4-day mouse test. At P7 (no mutations), the LD50 for 4-day-old
mice was <2.0 log10 PFU, whereas for the virus containing the mutation the LD50
was >5.1 log10 PFU. Monkeys inoculated SC with P7 (no mutations) developed
higher and more prolonged viremia than monkeys inoculated with virus containing
E204 (K!R), indicating a linkage between attenuated neurovirulence and viscer-
otropism for monkeys. Fortunately, this mutation and attending attenuation had
minimal effect on immunogenicity of the E204 mutant virus. As predicted from the
SC inoculation experiment, monkeys inoculated IC with P10 vaccine had lower
viremias (mean peak titer 48 PFU/mL than monkeys inoculated with P7 (no
mutation) virus (722 PFU/mL) (p ¼ 0.0432 ANOVA). All monkeys in both groups
developed DEN 1-specific neutralizing antibodies. On Day 31, antibody titers
ranged from 640 to 10,240 and from 2,560 to >20,480 in the ChimeriVaxTM-
DEN1 P7 and P10 treated groups, respectively, indicating that attenuation due to
E204 (K!R) did not impair immune responses.
Fig. 7 Mortality ratios by dose, 4 day old infant mice inoculated IC with plaque purified P10
ChimeriVax™ DEN vaccines and YF VAX®. All chimeric candidates were significantly attenu
ated for neurovirulence compared to YF VAX®
Safety of each final monovalent component and the tetravalent formulation was
assessed by studies of neurovirulence in mice using a sensitive 4-day-old suckling
mouse test and shown to be improved compared to YF 17D (Fig. 7). No interference
for replication in mouse brain tissue between serotypes was found when the mixture
and individual components were inoculated IC [100]. A GLP monkey neuroviru-
lence test was performed on the tetravalent vaccine. As predicted by mouse studies,
the histopathological lesion scores were significantly lower than seen in monkeys
inoculated with YF 17D.
The ability of the chimeric dengue vaccine viruses and tetravalent formulation
(P10 vaccine level) to infect Ae. aegypti mosquitoes by intrathoracic (IT) inocula-
tion or oral feeding was evaluated, and compared to wild type DEN viruses and YF
17D, which is not transmissible by mosquitoes [103]. The replication profile of the
chimeric viruses in mosquito tissue was similar to that of YF 17D virus. In
mosquitoes, the growth rate of each chimeric virus was similar whether it was a
single serotype infection, or part of the tetravalent mix, with no interferences
observed. The chimeric viruses replicated and disseminated to head tissue, but
mean titers of all chimeric viruses were lower than that of IT-inoculated YF 17D
virus. The ChimeriVax™-DEN viruses infected mosquitoes poorly via infectious
blood meals compared to the wt DEN parent viruses, which indicates that the
chimeric viruses are not able to infect and replicate in Ae. aegypti midgut tissue.
Similar studies and conclusions were reported by Vanlandingham et al. [104].
To evaluate efficacy of the plaque-purified chimeras, studies were performed in
monkeys inoculated SC with 5 log10 PFU of P10 monovalent chimeras or tetrava-
lent vaccine [100, 101]. Serotype specific viremias were measured by a validated
monoclonal immunofocus assay that detected the four different serotypes in a
382 T.P. Monath
mixture without interference between serotype viruses. Viremia in monkeys was

similar to that induced by YF 17D and lower than those following inoculation with
the wild-type parental dengue strains. All monkeys inoculated with the tetravalent
vaccine developed neutralizing antibodies to all four serotypes (Tables 10 and 11).
The predominance of DEN2/YF seen in earlier studies with uncloned viruses was
not observed. Neutralizing antibody titers following tetravalent vaccine were
robust, indicating that interference was not a problem (in the monkey model).
There was no clear advantage in safety or immunogenicity in monkeys of formula-
tions with different dose ratios of the four component viruses (Table 11). Low level
viremias were observed to most of the individual components, but in some cases
viremia to DEN1 and DEN2 was not detected. In general DEN3 and 4 chimeras
tended to induce higher and longer viremia than DEN 1 or 2. Despite those
observations, the vaccine elicited antibody responses to all four serotypes. Monkeys
were divided into groups, and challenged with wild-type DEN viruses; all were
protected against viremia following challenge except for one monkey in the 5,5,5,3
group challenged with DEN1 and one animal in the 3,5,5,3 group challenged with
DEN4 [101]. Compared to unimmunized controls, viremias in these animals were
abbreviated and delayed. The two monkeys in question had the lowest prechallenge
neutralization titers (20 and 40, respectively). The formulations containing 5 or 3
log10 PFU of each component appeared to perform best; a formulation containing
3.1 3.7 log10 of each component was selected for the first clinical trial. An IND was
filed in 2004 and a Phase 1 trial initiated.
Despite these encouraging results, there were some subtle signs for interactions
(interference) between chimeric subtype vaccines in the monkey model. Viremias
to all four subtypes were less frequently detected (or in the case of DEN2) and were
of shorter duration when the viruses were inoculated in a tetravalent formulation
Table 10 Viremia and neutralizing antibody responses to monovalent and tetravalent plaque
purified ChimeriVax™ DEN vaccines (P10) in cynomolgus macaques
Virus Dose Viremia Antibody to virus
(log10 inoculated
PFU/ml) No. Mean peak Mean Seroconverted GMT
viremic/ viremia duration
tested (log10 (Days)
PFU/mL)
ChimeriVax™ DEN1 5 1/3 2.7 6 3/3 1016
ChimeriVax™ DEN2 5 1/3 2.0 3 3/3 320
(GMP lot) 5 3/3 1.8 2.3 3/3 127
ChimeriVax™ DEN3 5 3/3 1.8 3 3/3 403
ChimeriVax™ DEN4 5 3/3 2.1 4.3 3/3 50
ChimeriVax™ (5,5,5,5) 3/3 1.9 3.7 DEN1 3/3 254
tetravalent DEN2 3/3 403
DEN3 3/3 806
DEN4 3/3 2032
YF 17D (YF VAX®) 5 3/3 1.7 2.3 NT NT
Table 11 A second experiment in groups of 6 cynomolgus macaques inoculated SC with different

mixtures of chimeric viruses; monkeys were randomized and subsequently challenged with wild
type dengue virus; modified from Guirakhoo et al. [101]
Formulation ChimeriVax™ Proportion PRNT50 GMT by Wild type DEN
DEN 1,2,3,4 DEN serotype viremic (%) serotype Challenge (with
log10 PFU by serotype serotype shown in
column 2)
Day 31 Day 121 Viremic post 4 fold
(prechallenge) challenge rise in
(mean peaka, antibody
durationb)
5,5,5,5 1 5/6 (83%) 452 359 0/1 1/1
2 0/6 (0%) 508 359 0/2 2/2
3 6/6 (100%) 452 1,016 0/2 2/2
4 6/6 (100%) 508 508 0/1 1/1
3,5,5,3 1 0/6 (0%) 90 101 0/1 1/1
2 4/6 (67%) 285 452 0/1 1/1
3 4/6 (67%) 254 160 0/1 1/1
4 2/6 (33%) 10 29 1c/3 (1.7, 2.0) 3/3
5,5,5,3 1 4/6 (67%) 226 142 1d/2 (3.3, 4.0) 2/2
2 5/6 (83%) 452 508 0/3 3/3
3 5/6 (83%) 275 320 0/1 1/1
4 5/6 (83%) 26 718
3,3,3,3 1 4/6 (67%) 254 63 0/2 2/2
2 4/6 (67%) 359 320
3 4/6 (67%) 285 254 0/2 2/2
4 6/6 (100%) 452 34 0/2 2/2
Not 1 <10 4/4 (3.2, 6.0) 4/4
vaccinated 2 <10 4/4 (2.5, 4.5) 4/4
3 <10 4/4 (2.2, 4.2) 4/4
4 <10 4/4 (3.2, 5.2) 4/4
a
log10 PFU/mL
b
Days
c
Prechallenge neutralization titer was 20
d
Prechallenge neutralization titer was 40
compared to monovalent vaccination. These subtle effects in the monkey model did
not predict the more intense interactions observed clinically.
The initial human trial of tetravalent ChimeriVax™-DEN (unpublished) pro-
vided the first insights into the phenomenon of interference between the live virus
components. The randomized, double-blind, placebo controlled Phase I study
evaluated the safety, tolerability and immunogenicity of live attenuated tetravalent
ChimeriVax™-DEN vaccine and YF 17D vaccine (YF-VAX®) in 99 healthy adult
volunteers in the US. In the first stage, 33 subjects received tetravalent Chimer-
iVax™-DEN (Group 1), 33 subjects received YF-VAX® (Group 2) and 33 subjects
(Group 3) received placebo (saline). A second vaccination was performed 6 months
later (open label). Subjects in all three groups received tetravalent ChimeriVax™-
DEN containing 3.1 3.7 log10 of each component . The vaccines were well tolerated,
with no safety signals attributable to the tetravalent chimeric vaccine. A higher
384 T.P. Monath
Fig. 8 Viremia in human subjects following tetravalent ChimeriVax™ DEN (mixture of 4 log10
of each virus) or YF VAX®. (a). Viremia after a single dose of tetravalent ChimeriVax™ DEN
showing total viremia and serotype specific viremia. Subjects in Group 1 after the first dose of
chimeric vaccine and subjects in Group 3 who had previously received placebo and then Chimeri
Vax™ DEN were pooled for analysis. (b). Viremia following a second vaccination of tetravalent
ChimeriVax™ DEN, or a single vaccination in YF immune subjects 6 months after primary
vaccination versus subjects receiving a primary vaccination with ChimeriVax at that time point
incidence of viremia and more prolonged viremia were observed after the first dose
of chimeric vaccine than seen following YF-VAX® (Fig. 8a). As predicted from the
studies of plaque-purified chimeric vaccines in monkeys [100, 101], viremia was
principally caused by the DEN3 and DEN4 components of the vaccine, and rarely
by DEN1 or 2. A biphasic pattern was also observed. When the chimeric vaccine
was used to boost subjects at 6 months, viremia was blunted compared to subjects
receiving primary vaccination (Fig. 8b), demonstrating a degree of protection. Prior
YF vaccination did not affect the early phase of viremia but appeared to modulate
viremia in the second week. All peak viremia levels were less than 2.2 log10 PFU/
mL. These data, together with the viremia data from the Phase I study of monova-
lent ChimeriVax™-DEN2 [98] showing higher DEN2 viremias indicated that there
was interference between the serotypes in the tetravalent mixture principally due to
DEN3 and 4 which caused more active infections.
These observations were borne out by tests for antibody. Neutralizing antibodies
were measured against the homologous (vaccine) virus and at least one different
wild-type strain of each serotype (listed in the footnote to Table 12). Sera taken
after the first dose of ChimeriVax™-DEN were tested against two wild-type strains
Table 12 Phase 1 clinical trial, tetravalent ChimeriVax™ DEN, percent of subjects seronegative
at baseline and developing neutralizing antibodies (titer 10) to the homologous vaccine virus or
at least one wild type strain of each serotype 30 days after one or two doses (6 months apart) or one
dose 6 months after YF 17D vaccination. The GMT is also shown by serotype
Statistic Serotype Group 1 ChimeriVax Group 2 Pooled single
DEN > ChimeriVax YF VAX > dose Chimeri
DEN ChimeriVax DEN Vax™ DEN*
30 days post 30 days post 30 days post 30 days post 30 days post
primary boost primary boost primary
(N 33) (N 29) (N 33) (N 26) (N 54)
Seroconversiona DEN1 21 (64%) 23 (79%) 6 (18%) 26 (100%) 38 (70%)
DEN2 22 (67%) 17 (59%) 1 (3%) 25 (96%) 33 (61%)
DEN3 32 (97%) 27 (93%) 3 (9%) 25 (96%) 47 (87%)
DEN4 26 (79%) 23 (79%) 0 (0%) 20 (77%) 37 (69%)
GMTa DEN1 25 57 9 102 34
DEN2 26 42 5 183 29
DEN3 225 87 5 163 152
DEN4 100 44 5 45 61
a
Sera were tested against the homologous virus (ChimeriVax) and 2 wild type strains: DEN 1 16007
and Western Pacific 74; DEN2 16681 and S16803, DEN3 16562 and CH53489, and DEN4 1036
and TVP 360. Not all 30 day post boost sera were tested against the second wild type dengue virus
of each serotype. This was important because, in contrast with the study of
monovalent ChimeriVax™-DEN2 [98], some subjects seropositive for a wild-
type strain were seronegative for the homologous virus. While respectable sero-
conversion rates were observed, antibody titers (particularly for DEN 1 and 2) were
substantially lower and more variable than observed following monovalent DEN2
immunization [98]. The highest responses were seen against DEN 3 and 4, as
expected from the viremia patterns (Fig. 8). Interestingly, in this study a booster
dose at 6 months provided little increase in antibody titers. However, as noted in
the monovalent vaccine trial, prior YF 17D immunization followed by tetravalent
ChimeriVax™-DEN vaccine increased antibody responses to DEN 1 and 2. Over-
all, the results of the trial were encouraging and guided further vaccine develop-
ment. However, the formulation for further development was changed to increase
the dose of each component to 5 log10 PFU, and emphasis was placed on delivering
multiple doses on an optimal schedule.
The tetravalent vaccine is now in advanced Phase II/III trials in Thailand,
involving 4,000 children who will receive three doses of tetravalent Chimeri-
Vax™-DEN vaccine or a control in a 2:1 ratio. Leading up to these trials, Sanofi
Pasteur conducted Phase II studies in the US, Australia, Mexico and the Philippines
in both adults and children. The results have not been published, but have been
presented at meetings and summarized recently by Guy et al. [105]. Safety in over
880 subjects has been good, with no increase in adverse events over subjects
receiving placebo or control vaccines. It is notable that no dengue-like adverse
reactions (rash, transaminase elevations, neutropenia) have been observed, whereas
these adverse events have been consistently observed following the empirically
derived live, DEN vaccines as well as rationally designed mutagenized and chimeric
386 T.P. Monath
DEN vaccines (see below). Viremia levels have been consistently low and, impor-
tantly, were not enhanced in Mexican and Filipino children who were DEN-
immune prior to vaccination. In a trial conducted in 66 flavivirus-naive adults,
18 40 years of age, in the US, subjects received three injections of vaccine or
placebo at 0, 3 4 and 12 months. 100% seroconversion was noted after the third
dose with GMTs of 67, 538, 122 and 154 against DEN 1 4, respectively. In Mexico,
72 children 2 11 years, 36 adolescents, and 18 adults received the same regimen
outlined above except that a control vaccine (YF 17D) was given in lieu of placebo;
the prevalence of dengue immunity at baseline was 8%. The proportion of subjects
developing neutralizing antibodies increased after each successive injection of
vaccine; after the third vaccine dose, 82% of subjects had developed neutralizing
antibodies to three or more serotypes. The seroconversion rate was lowest to DEN 1
(79%) and highest to DEN 4 (92%). GMTs to the four serotypes (67, 538, 122 and
154) were higher than seen in the initial Phase 1 study in US adults. In the youngest
age group (children 2 5 years) seroconversion was 94 100% for DEN types 2 4
and 88% for DEN 1. A trial in the Philippines had a design similar to that in Mexico,
but there the prevalence of flavivirus (DEN and JE) immunity was 80%. The results
were similar to those in Mexico, except that the background of natural immunity
provided a booster effect and two doses of tetravalent vaccine (in the control group)
evoked a similar response to three doses. These findings reinforce the conclusion
that preexisting flavivirus immunity, whether due to DEN, JE or YF will be an
important potentiating factor in the efficacy of tetravalent DEN vaccines.
The mechanisms underlying interference effects between serotypes in the tetra-
valent vaccine have not been elucidated, but probably involve competitive inhibi-
tion of replication in the skin and draining lymph nodes mediated by rapid innate
immune responses , with monovalent components of the vaccine that replicate more
rapidly shutting off sister components in the mixture. In vitro, absent immune
mechanisms, interference is not observed even though the components of the
tetravalent vaccine grow at different rates. The growth curves of single Chimer-
iVax™-DEN1 4 viruses in vitro have been compared to wild-type DEN viruses.
The titer and growth rate of each chimeric serotype was similar, whether it was a
single infection, or part of the tetravalent mix, (Fig. 9). No interference by one
chimeric virus with a faster growth rate over a slower-growing serotype was
observed. The interference effects in vivo are observed very early, during the
viremic period. To wit, 64% of humans injected with 3 log10 PFU of monovalent
ChimeriVax™-DEN2 developed detectable viremia (Table 9), whereas only 3.5%
of subjects receiving tetravalent vaccine containing the same dose of Chimeri-
Vax™-DEN2 developed viremia (Fig. 8). These observations support a role of
innate immunity in the interference phenomenon.
To avoid the interference effects, several strategies have been investigated,
including favoring the “weaker” viruses by adjusting the ratio of doses in the
tetravalent formulation, or injecting bivalent vaccines at different anatomical
sites, or spaced in time. Compared to simultaneous injection of all four serotypes
in nonhuman primates, an improved response could be achieved by injecting a
bivalent vaccine in different arms or at an interval of 2 months [105]. Priming with
9
8
7
6
DEN-1
5
4 ChimeriVax-DEN1
3 ChimeriVax-DEN1 in
ChimeriVax-DEN1,2,3,4 mix
2
1
0
0 2 4 6 8 10
7
Log10 RNA transcripts/ml
6
5
4 DEN-3
3 ChimeriVax-DEN3
ChimeriVax-DEN1,3,4 mix
1
0
0 2 4 6 8 10
4 DEN-4
3 ChimeriVax-DEN4
ChimeriVax-DEN1,2,3,4 mix
0
0 2 4 6 8 10
Day after infection
Fig. 9 Virus growth of ChimeriVax™ DEN alone or in a tetravalent formulation in C6/36 Ae.
albopictus cells (MOI 0.01 PFU/cell). Virus titer shown is the mean titer SD from triplicate cell
culture assays
a monovalent DEN vaccine (or YF 17D) also had the desired effect of stimulating a
broad response after boosting with tetravalent vaccine. However, it would not be
feasible to prime with a monovalent or bivalent dengue vaccine in human popula-
tions which may be placed at higher risk of developing severe DEN if exposed to
wild-type dengue before the immunization regime could be completed. For practi-
cal reasons other approaches are being pursued, principally the use of multiple
doses, spaced appropriately in time. It is important that booster doses not be given
too early, since adaptive immune responses can interfere with the response to
vaccination. The mechanism underlying short term cross-protection, observed by
388 T.P. Monath
Sabin to last about 6 months in early studies of dengue [106], is unknown, but could
be IgM antibodies [105], antibodies that have not undergone affinity maturation,
antibodies to cross-neutralizing epitopes on domains I and II that wane below
protective titers, or cell mediated immunity. Cell-mediated immune responses to
the tetravalent vaccine have been studied in some depth by Guy et al. [38]. Th1
oriented responses to DEN4 predominate after the first dose, but broaden after a
boost.
A different approach being considered for avoiding interference of primary
immunization is the construction of a virus with an E protein that contains neu-
tralizing epitopes to all four DEN viruses. Such a protein can be generated by gene
shuffling [107], [108] and naked DNA immunization with plasmids encoding the
shuffled E protein has been shown elicit broad responses. Recombinant adenovirus
carrying multiple DEN E genes have shown promise as well. It is uncertain whether
a live chimeric virus with a shuffled E protein can be constructed and elicit strong
tetravalent antibodies, but if so, use of a single virus could obviate interference
phenomena.
3.2.4 Chimeric Dengue/Dengue and Deletion Mutant Vaccine

Candidates Developed at NIAID
The development by several groups of live DEN vaccines using empirical passage
has clearly demonstrated the feasibility of attenuating DEN virus. With the advent
of infectious clone technology, an obvious strategy for rationale design would be to
harness an attenuated DEN strain as a vector for insertion of the structural genes of
other serotypes. One potential advantage of such an approach is that the entire
chimeric virus would be composed of dengue genetic material, which might add
to protective immunity and ensure long term immunological memory. However, a
potential disadvantage of this approach is that heterologous cross-reactive T cell
responses might favor DHF if neutralizing antibody responses were incomplete or
not durable [42].
NIAID scientists developed DEN 4 virus to serve as the backbone for construc-
tion of intertypic chimeras. The first constructions utilized a cDNA clone of the
wild type DEN4 strain 814669. The structural genes of DEN4 were replaced by the
corresponding genes of another DEN serotype virus [11, 89]. Replacement of genes
is facilitated by the fact that there is significant sequence conservation among
the four DEN serotype viruses. Initially, the chimeras were constructed with all
three structural genes (C-prM-E) of wild type DEN1, Western Pacific strain (WP),
or the mouse-adapted DEN2, New Guinea C strain (NGC) and the remaining
sequence from the DEN4 virus. A chimeric DEN3/DEN4 virus with the C-prM-E
genes of the wild type DEN3 strain H53489 was also constructed [109]. The
chimeras were attenuated for replication as the result of chimerization [89]. Con-
struction of a chimeric DEN2/DEN4 virus that contains only the DEN2 prM-E
genes was also constructed as an alternative strategy.
The biological behavior of the DEN4 (wild-type backbone) chimeras was

explored in rhesus macaques inoculated by the SC route with DEN1/DEN4
(C-prM-E, Western Pacific), DEN2/DEN4 (prM-E, mouse adapted NGC), and the
parental DEN4, DEN1, or DEN2 strains used to derive the chimeras [90]. Monkeys
inoculated with the monovalent chimeras or a mixture of two chimeras developed
respectable neutralizing antibody responses and were protected against challenge.
These experiments were encouraging, but did not elucidate whether the chimeras
were sufficiently attenuated for human use.
Mutations were subsequently introduced into the wild type DEN4 backbone to
attenuate the virus, using biomarkers of reduced replication in vitro and reduced
viremia in rhesus monkeys [21]. To avoid potential problems of reversion at point
mutations, deletion mutants were preferred. A panel of mutant DEN 4 viruses with
deletions ranging from 30 to 262 nucleotides (nt) in the 30 NCR were investigated
by Men et al. [23] A DEN4 virus with a 30-nt deletion (D30) in the 30 NCR
(nt 172 143 from the 30 terminus in the TL2 stem-loop structure exhibiting reduced
plaque size in C6/36 cells, reduced replication in LLC-MK2 cells and reduced
viremia in rhesus monkeys elicited a moderate level of neutralizing antibodies. The
virus was not restricted for growth in mosquitoes after intrathoracic inoculation, but
was unable to establish disseminated infection of Ae. aegypti mosquitoes after oral
feeding, and thus could not transmit the vaccine virus [110]. This virus was selected
as a candidate DEN4 vaccine and intertypic chimeric vector.
Clinical grade recombinant DEN4 D30 (rDEN4D30) vaccine was manufactured
in Vero cells and evaluated for safety, tolerability and immunogenicity in a Phase I
clinical trial (Table 13) [111]. A single dose of 5 log10 PFU was administered by the
SC route to 20 seronegative volunteers. A macular rash developed in 50% of the
subjects; a transient mild-moderate elevation in liver enzymes (alanine aminotrans-
ferase, ALT) was observed in 25% of the subjects; and 15% of the subjects had mild
and transient neutropenia. The rash was faint, difficult to discern, nonpruritic, and
was seen only in subjects who had detectable viremia. Viremia occurred in 70% of
subjects, but was low (mean peak 1.6 log10 PFU/mL). These side effects were not
unexpected as similar experiences have been described in studies of empirically
attenuated live dengue vaccines [117]. All 20 volunteers seroconverted to DEN 4
and developed high levels of neutralizing antibodies (GMT 580). The level of
antibodies was similar in subjects who were viremic and those that were not.
A subsequent controlled clinical trial was conducted in which lower doses (1,2
and 3 log10 PFU) were given to groups of 20 subjects (together with placebo in four
subjects) to determine if the rash and transaminase elevations were less frequent at a
lower vaccine doses [114]. While rash and neutropenia were observed at all dose
levels, no ALT elevations were seen at doses of 1 or 2 log10 PFU and only one (5%)
of subjects given 3 log10 PFU had ALT elevation. As with other live vaccines,
including the JE and DEN/YF chimeras, there was no dose response for immunoge-
nicity; 95 100% of subjects seroconverted and developed robust neutralizing anti-
body responses (GMT 139 380). The DEN4 genome with the 30-nt deletion in the
30 NCR was considered an attractive vector for the construction of intertypic dengue
chimeras expressing the antigenic regions of DEN1, DEN2, and DEN3 [118].
Table 13 Summary of clinical studies of DEN vaccines developed by NIH
Phase Location Age Vaccine Dose Adverse events Viremia Neutralizing antbodies to JE Mutations in Reference
(Vaccine) (years) (No. subjects) (log10 Serious, Nonserious Proportion Duration, Peak, GMT Seroconversion D30 region
390
PFU) related viremic (%) days mean (PRNT60b) (%)b Day of virus
log10 Day 28–31 28–31 recovered
PFU/mLa from serum
1 (DEN4) US 18–45 rDEN4D30 5.0 0 Minimal local reactions; 70% 4.4 1.6 567 100% No [111]
(N ¼ 20) no fever-arthraligia- (426–652c)
headache syndrome
(dengue fever); 50%
maculopapular rash;
15% neutropenia;
20% ALT transient
elevation
1 (DEN4) US 18–50 rDEN4D 5.0 85% had min. rash at 0% – – 150 95% [112]
30-4995 injection site; systemic
(N ¼ 20) AEs similar across
Placebo – active and placebo; 0% – – <10 0%
(N ¼ 8) 10% maculopapular
rash; 5% neutropenia;
5% transient ALT
elevation
1 (DEN4) US 18–50 rDEN4D30- 5.0 No signif. Local reactions; 0% – – 100 100% [113]
200,101 systemic AEs similar
(N ¼ 20) across active and
Placebo (N ¼ 8) – placebo; 5% mild 0% – – <10 0%
fever; 20%
maculopapular rash;
10% neutropenia (but
also in 2/8 placebo);
0% ALT elevation
2 (DEN4) US 18–50 rDEN4D30 3.0 0 Minimal local reactions; no 35% 1.6 0.5 139 100% No [114]
(N ¼ 20) differences in AEs
rDEN4D30 2.0 0 between active and 55% 2.6 0.7 189 95% No
(N ¼ 20) placebo; 1.7% low-
rDEN4D30 1.0 0 grade fever; 70% 65% 1.8 0.6 380 100% No
(N ¼ 20) maculopapular rash;
Placebo _ 0 23% neutropenia; 0% 0 0 <10 0%
T.P. Monath
(N ¼ 12) 1.7% ALT transient

elevation
1 (DEN1) US 18–50 rDEN1D30 3.0 0 Minimal local reactions; 45% 2.8 1.0 444 95% No [115]
(N ¼ 20) headache higher in
Placebo (N ¼ 8) _ 3 placebo, no other 0% 0 0 <10 0%
differences in AEs
between active and
placebo; 40%
maculopapular
rash;40%
neutropenia;0% ALT
elevation
1 (DEN2) US 18–50 rDEN2/ 3.0 0 Minimal local reactions; no 55% 3.2 0.6 147 100% No [116]
DEN4D30 differences in AEs
(N ¼ 20) between active and
Placebo (N ¼ 8) – 0 placebo, similar profile 0% – – <10 0%
to other candidates
above; no fever; 45%
maculopapular rash;
35% neutropenia;15%
ALT elevationd
a
Mean peak for viremic subjects only
b
Titers are to homologous strain (also see text); PRNT50 ¼ 50% plaque reduction neutralization test
c
Nonviremic (N ¼ 6) – viremic (N ¼ 14) subjects
d
2/3 subjects with ALT rise had elevated ALT on Day 0 (prevaccination) but had been normal on screening
392 T.P. Monath
It was thought, however, that the elevated liver enzymes associated with
rDEN4D30 could be problematic as vaccine usage scaled up. In an attempt to
abrogate this feature of the virus, two new vaccine candidates were constructed
introducing two mutations at positions 200 and 201 in NS5 or a single mutation at
position 158 of NS3 (rDEN4D30-4995) (Fig. 5). The rDEN4D30-200,201 and
rDEN4D30-4995 candidates were more attenuated than the rDEN4D30 parent in
monkeys and SCID mice reconstituted with human liver cells (HuH 7) [20, 119] but
still elicited neutralizing antibodies. Clinical trials were performed with both
vaccines (Table 13) [112, 113]. These studies showed that the vaccines were
more attenuated than parental rDEN4D30, produced no viremia, little or no hepa-
totoxicity (0% of subjects for rDEN4D30-200,201; 10% for rDEN4D30-4995), and
lower neutralizing antibody levels, and a lower incidence of neutropenia and
generalized rash. The -4995 candidate was associated with a localized rash around
the injection site in a high proportion of subjects. Sera from vaccinees in different
trials who received rDEN4D30-4995, rDEN4D30-200,201 and rDEN4D30 in dif-
ferent trials were compared in a single neutralization test [112]. The rDEN4D30-
200,201 candidate had a GMT only twofold lower than rDEN4D30. The two
vaccine candidates are therefore being evaluated further in the context of tetrava-
lent immunization.
Multiple constructs were evaluated in the development of a chimeric DEN 1
vaccine, in which all structural genes (C-prM-E) or just the prME genes of DEN1
Puerto Rico/94 virus were inserted into either unmodified DEN4 or the DEN 4D30
vector (Fig. 5) [120]. The constructs were evaluated in the SCID-HuH-7 mouse
model, rhesus monkeys and Ae. aegypti mosquitoes. The viruses grew to 6 7
log10 PFU/mL in Vero cells. During passage in Vero cells to produce virus stocks,
1 3 mutations appeared in all viruses, including mutations in NS4B likely to be an
adaptation to growth in Vero (as was seen for ChimeriVax™-DEN). The D30
deletion restricted replication in mosquitoes. The viruses with unmodified DEN4
backbone were not attenuated for rhesus monkeys and the rDEN1 /4(prME) virus
was not attenuated in the SCID-HuH-7 model when compared to wild-type DEN1
virus. The rDEN1/4D30 (C-prM-E) virus was over-attenuated, failed to elicit
antibodies in monkeys or protect against wild-type DEN1 challenge. However,
the rDEN1/DEN4D30 (prME) virus which retained the DEN4 capsid evoked a
modest neutralizing response in 66% of monkeys and protected animals against
challenge. This activity was considered to be insufficient for an effective human
vaccine. An additional vaccine candidate had been developed, based on the clinical
success of the rDEN4D30 candidate and the fact that the 30 NCR sequences are
highly conserved across dengue serotypes the D30 deletion was introduced into
the 30 -NCR of DEN 1 [118]. The rDEN1D30 vaccine candidate was attenuated in the
SCID-HuH7 mouse and showed reduced viremia in rhesus monkeys, while being
immunogenic and protective against DEN1 challenge [121]. However, the virus was
not restricted for growth in mosquitoes following intrathoracic inoculation.
Durbin et al. [115] evaluated rDEN1D30 at a dose of 3 log10 PFU in 20 healthy
adult subjects; eight subjects received placebo in the double-blind study (Table 13).
As observed for the rDEN4D30 candidate, asymptomatic rash (in 40%) and
neutropenia (in 40%) were associated with the rDEN1D30 vaccine, though none of
the subjects developed clinical dengue fever and none had elevated liver enzymes
(ALT). One subject had a transient, mild thrombocytopenia lasting 1 day. The rash
was a faint maculopapular eruption, typically not self reported, rarely pruritic, and
lasting up to a week. The investigators concluded that the vaccine candidate was
well tolerated. Viremia was low (45% viremic; mean peak 1.0 log10 PFU/mL; mean
duration 2.8 days), and antibody responses were robust, with Day 28 GMT of 444
and 95% seroconversion rate.
Attempts were made to develop DEN2 and three candidates were constructed
with D30 mutations in the 30 -NCR analogous to the DEN1 and 4 candidates.
Unfortunately, these constructs did not prove to be satisfactorily attenuated for
monkeys [122, 123]. Development logically next focused on the construction of
chimeric viruses using the DEN4D30 vector backbone [91, 122]. The DEN2
chimeric vaccine candidate was constructed by insertion of the prM-E genes of
prototype New Guinea C (NGC) virus into the attenuated DEN 4D30 vector. The
chimeric virus was attenuated in both SCID-HuH-7 mice and rhesus monkeys
compared to wild-type DEN2 NCG virus. As for other D30 DEN viruses, the
candidate vaccine failed to infect Ae. aegypti after oral feeding. A placebo con-
trolled clinical trial was conducted with an identical design to the Phase I study of
rDEN1D30 described in the preceding paragraph [116]. Overall the results of this
study painted a consistent picture. Asymptomatic rash was seen in 45%, transient
neutropenia in 35%, and mildly elevated ALT in 15% of the subjects. There were no
cases of dengue fever, and the incidence of adverse events other than rash and the
laboratory abnormalities did not exceed that in placebo treated subjects. Viremia
was low (55% viremic; mean peak 0.6 log10 PFU/mL; mean duration 3.2 days).
Neutralizing antibody responses were seen in 100% of subjects with moderate
GMTs (147 and 237, on days 28 and 42, respectively).
The DEN3 chimeric vaccine [rDEN3(prME)/4D30] turned out to be more
problematic. Preclinical evaluation in rhesus monkeys indicated that the virus
was probably overattenuated (no viremia, low Day 28 GMT of 58), and indeed
this proved to be the case in a clinical trial. Two dose levels of the vaccine (3 and 5
log10 PFU) were administered SC; only 5 (25%) of 20 subjects at the 5 log10 PFU
dose and 6 (30%) of 20 subjects given 3 log10 PFU developed neutralizing anti-
bodies in this study. Consequently, further development of rDEN3/4D30 was
stopped. However, based on the original strategy of modifying the DEN 3
30 NCR, two additional candidates had been developed. In the first case, the
D30,31 mutant, two deletions at nucleotides 173-143 and 258-228 in the DEN 3
Sleman/78 virus were made. A second construct replaced the entire DEN 3 30 -NCR
with that of DEN4D30, designated rDEN3-30 DEN4D30 [26]. Both the rDEN3D30/
31 and rDEN3-30 DEN4D30 mutant viruses were similarly attenuated as the suc-
cessful rDEN4D30 vaccine for SCID-HuH-7 mice. In rhesus monkeys the new
candidates were highly attenuated compared to wild type DEN3 and produced no
viremia. The rDEN3D30/31 candidate elicited a moderately strong antibody
response (Day 28 GMT 304) whereas immunogenicity of rDEN3-30 DEN4D30
was weaker (GMT 77). rDEN3D30/31 was also strongly attenuated for mosquitoes.
394 T.P. Monath
Both rDEN3D30/31 and rDEN3-30 DEN4D30 mutant viruses are being evaluated in
clinical studies.
Several different tetravalent formulations were evaluated in rhesus monkeys and
compared with a mixture of wild-type DEN viruses [124]. The vaccine formula-
tions included: (1) a mixture of DEN1 4 each with a D30 mutation in the 30 NCR
(recall that the DEN2 and 3 components when tested alone were not sufficiently
attenuated); (2) a mixture of DEN1D30 and DEN4D30 mutants and two chimeras,
rDEN2/4D30 and rDEN3/4D30 using the mutant DEN4 backbone; and (3) a
mixture of DEN1D30, DEN2D30, DEN4D30 and a tenfold higher concentration
(6 log10 PFU) of the chimera rDEN3/4D30 (recall that this construct had been
shown to be overattenuated). None of these tetravalent formulations represent the
current mix of vaccine candidates that is being pursued clinically.3 In monkeys, all
three formulations tested were highly attenuated compared to a tetravalent mix of
wild-type DEN viruses. Tetravalent immunization was feasible, although a broad
response with neutralizing antibody levels above 1:100 to all serotypes could not be
achieved with a single dose of any of the three formulations. The least immuno-
genic of the formulations was number two above (having chimeric DEN2 and 3
viruses), which resulted in poor antibody responses to DEN2 and 3. However, when
a booster dose of formulation 2 or 3 was given at 4 months, broad high titer
responses and full protection against challenge was achieved. No booster effect
was observed when the second vaccination was given at 1 month, possibly due to
interference by cross-protective heterologous antibodies.
These studies illustrated once again the problem of interference between com-
ponents of a tetravalent vaccine. The lower neutralizing antibody response to the
chimeric DEN2 and 3 components in the tetravalent formulation were not predicted
by studies of these candidates when tested as monovalent vaccines [91, 123]. When
tested for replication in the SCID-HuH7 model, and compared with monovalent
components, no interference between viruses in the tetravalent formulation was
observed. This observation was consistent with the lack of interference between
DEN/YF chimeric components for replication in mouse brain [101]. If interference
is the result of innate immune responses in lymphoid tissues following peripheral
inoculation, the very different SCID-HuH7 and mouse brain models may not reveal
such effects.
At the present time, two candidates for inclusion in a tetravalent vaccine include
those shown below (Whitehead S pers. comm. 2009). All components will be at a
dose of 3.0 log10 PFU. The two formulations differ in the DEN4 component
(rDEN4D30 in Tetravalent 1 and rDEN4D30-200,201, which was more attenuated
in a trial of the monovalent component [113] in Tetravalent 2).
3
However, the tetravalent formulations (described below) that are planned for clinical develop
ment are currently being tested in rhesus monkeys.
Tetravalent 1
Dengue type 1 rDEN1D30 30 NCR deletion in wild type DEN 1 Western Pacific
Dengue type 2 rDEN2/DEN4D30 Chimeric dengue 4 having D30 30 NCR deletion,
with prM-E replaced by corresponding genes of wild-type DEN 2 New Guinea C
Dengue type 3 rDEN3-30 DEN4D30 wild-type DEN 3 Sleman/78 with DEN4D30
30 NCR
Dengue type 4 rDEN4D30 30 NCR deletion in wild type DEN 4 814669
Tetravalent 2
Dengue type 1 rDEN1D30

Dengue type 2 rDEN2/4D30
Dengue type 3 rDEN3-30 DEN4D30
Dengue type 4 rDEN4D30-200,201 additional mutations at NS5 200 and 2001
The safety and immunogenicity profiles in clinical studies with monovalent com-
ponents for DEN1,2 and 4 at a dose of 3.0 log10 PFU are compared in Fig. 10. The
two tetravalent formulations have been tested in rhesus monkeys in formulations
Fig. 10 Summary of clinical data with monovalent NIH dengue vaccines that will be formulated
and tested in trials of tetravalent vaccines. Vaccines administered by the SC route at a dose of
3.0 log10 PFU. The DEN3 vaccine candidates are in ongoing clinical trials
396 T.P. Monath
containing 5 log10 PFU of each component and both showed seroconversion to all
four viruses in 75 100% of the monkeys after a single dose and no significant
differences between formulations (Whitehead S pers. comm. 2009). With respect to
the choice of a DEN3 candidate for the first tetravalent trials, the outcome of
clinical studies comparing rDEN3-30 DEN4D30 and rDEN3D30/31 are underway,
and the latter vaccine will be substituted in the tetravalent formulations if immuno-
genicity and safety are superior to rDEN3-30 DEN4D30.
Interference phenomena in the monkey model were most apparent after the
initial dose, and a booster dose of tetravalent vaccine formulations induced a
broad response and higher antibody titers to all serotypes [124]. To achieve high
antibody responses to all serotypes, it may be that two or more doses of the
tetravalent vaccine will be required, but this decision must await clinical data.
The conclusion that multiple doses would be required had been reached during
development of the empirically derived live, attenuated vaccines at Mahidol
University [125]. Preferably booster doses would be given at sufficiently long
intervals (6 or 12 months) to avoid the transient cross-protection phenomenon
[106] that would prevent “filling-in” of missing serotype immunity. A study in
monkeys given the NIH tetravalent vaccine at 0 and 6 months showed, in contrast to
the 0 1 month schedule, a boost in titer to all four serotypes, with respectable
GMTs > 100 for all four serotypes, with the highest rise in dengue 3 (which had the
lowest level of antibody after primary immunization).
The NIH group also conducted studies with monovalent vaccine candidates in
humans to investigate the ability of a second dose to boost homotypic immunity.
Fifty subjects received 3 log10 PFU of the rDEN1D30 vaccine at an interval of 4 6
months. The first dose was followed by the expected viremia (in 67%), asymptom-
atic rash (27%), neutropenia (43%), seroconversion (92% on Day 42), GMT (98),
titer range (19 844). The subjects were protected from side effects after the second
dose with no cases of rash, no viremia and only 9% neutropenia. Interestingly, none
of the subjects had a boost in antibodies after the second dose, indicative of sterile
immunity. This was true even for subjects who developed very low titers after
primary immunization, and contrasts with the results of the monkey experiment
employing tetravalent vaccine described above. Possibly the presence of hetero-
typic antibodies, or a larger pool of B cells recognizing cross-reactive determinants
explains the difference.
Some subjects that had participated in trials of monovalent vaccines were given
a booster dose of a different (heterologous) monovalent vaccine. Enhanced clinical
signs or disease were not observed after the heterologous boost, indicating that live
attenuated DENV vaccines should be safe for use in populations with pre-existing
DENV antibody. As might be expected from the studies of sequential YF and
monovalent, bivalent or tetravalent YF/DEN chimeras [98, 105], heterologous
prime-boost using the NIH vaccines resulted in a broad multivalent neutralizing
antibody response and enhanced GMTs. In sum, these studies suggest that the
second or third inoculations of tetravalent vaccine, at the appropriate interval
(longer appears better), may result in a broad tetravalent neutralizing antibody
response, a conclusion similar to that reached for the DEN/YF chimeras.
A meticulous effort has been invested in the construction and testing in non-
human primates and humans of a series of monovalent vaccines with acceptable
safety, tolerability and immunogenicity. The first clinical studies of the NIH
tetravalent vaccine, using the best combinations of monovalent vaccines derived
from this large body of work, are planned in 2010 in the US and also in Brazil at the
Butantan Institute. The results are awaited with great interest.
3.2.5 Chimeric Dengue Vaccines Employing Attenuated Dengue

Type 2 Vector
The chimeric DEN2 vectored vaccines were developed originally at CDC, Ft

Collins and were licensed to Inviragen Inc. Inviragen has collaborated with Shantha
Biotech (India) for manufacture and control of its vaccine. Inviragen recently
merged with SingVax, a Singapore-based vaccine company. Inviragen plans to
execute an initial proof of concept clinical study of a tetravalent formulation in the
US in 2010. The Inviragen tetravalent vaccine is named DENVax™.
A live, attenuated DEN2 vaccine, DEN2 PDK53 was developed at Mahidol
University, Thailand by empirical passage of the wild-type 16681 strain 53 times in
primary dog kidney cells. The PDK53 vaccine candidate has small plaque pheno-
type, is temperature sensitive (ts), attenuated for infant mice inoculated IC, and has
crippled replication in C6/36 cells. DEN2 PDK53 has been evaluated in various
Phase I and II clinical trials as a monovalent vaccine [97, 125, 126] or in multivalent
combinations [127]. DEN2 PDK53 had a good safety and tolerability profile and
elicited moderately high PRNT50 titers of 215 230 on day 60 in adult human
subjects. PDK53 was therefore a suitable candidate as the backbone for construc-
tion of chimeric viruses having prM-E genes of heterotypic dengue viruses. DEN2
PDK53, like YF 17D and rDEN4D30, had the advantage that clinical data existed
indicating safety and immunogenicity of the vector strain. Full-length cDNA clones
were constructed from the DEN2 PDK53 virus and the parental DEN2 strain 16681
from which it was derived [128]. Sequence analysis showed that the PDK53
vaccine contains two variants designated PDK53-V and PDK53-E. PDK53-V
contains all 9 NS gene mutations that occurred during derivation of the vaccine,
while PDK53-E has these mutations except for a parental 16681 residue at NS3
250. PDK53-V is being pursued as the vaccine candidate for DEN2 and vector for
intertypic chimeras.
The crippled replication of PDK53 virus in C6/36 cells and attenuation for mice
are determined primarily by mutations in the 50 -NCR (50 -NCR57 C!T) and at
NS153 G!D [129]. The ts phenotype of PDK-53 virus is caused by to the NS153
G!D and NS3250 E!V mutations. All three mutations contribute to the small-
plaque phenotype of PDK53. Restoration of at least two of the three loci to the wild-
type sequence was required to revert to the wild type characteristics of DEN2 16681
in vitro [129]. Genetic stability of the three attenuating mutations in PDK53 was
398 T.P. Monath
investigated by making multiple serial passages [130]. Instability of the 50 NCR

mutation was found but the other attenuating mutations were stable.
Since attenuation of the PDK53 virus depended on mutations in the NS gene
backbone and 50 -NCR, a reasonable hypothesis was that the PDK53 backbone
would confer an attenuated phenotype on chimeras constructed with wild-type
DEN 1,3 and 4 structural gene sequences. To confirm this, chimeric viruses were
constructed that contained the structural genes of an empirically derived attenuated
DEN1 PDK13 vaccine or its parental wild-type DEN1 16007 strain in the NS gene
background of either DEN2 PDK53 or the DEN2 16681 parental DEN2 virus [92].
DEN1 PDK13 had been assessed in clinical trials and was the most attenuated of all
four live vaccine candidates, requiring 10,000 PFU to immunize 50% of subjects
(compared to about 5 PFU for DEN2 PDK53 [131]. There are eight amino
acid differences, five in the E protein, between the DEN1 PDK13 and its parental
DEN1 16007 strain. The chimeric virus containing both donor and vector wild-
type sequences [DEN1 (16007)/DEN2(16681)] grew less efficiently than either
parental virus in C6/36 cells, demonstrating the attenuating effect of chimerization
between heterologous dengue virus genes. The DEN1 (16007)/DEN2(PDK53)
and DEN1 (PDK13)/DEN2(PDK53) chimeras retained the DEN2 PDK53 attenua-
tion markers of small plaque morphology, ts in LLC-MK2 cells, and inefficient
replication in C6/36 cells, as compared to wild type DEN1 16007. These findings
validated the hypothesis that the PDK53 backbone would confer a fully attenuated
phenotype when wild-type prM-E donors were used for construction of chimeras.
The question was whether the chimeric constructs were overattenuated with respect
to immunogenicity.
The immunogenicity of the DEN1 16007, DEN1 PDK13 and their derived
chimeras was analyzed in 3-week-old outbred (ICR) mice [92]. Mice immunized
with two doses ( 4 log10 PFU) of DEN1(16007)/DEN2(PDK53) or with parental
16007 virus developed high neutralization titers of 2,560 10,240. However, use of
the attenuated DEN1 C-prM-E genes from PDK13 in the DEN2 PDK53 backbone
evoked substantially lower PRNT60 titers of 80 160 under the same conditions. The
five amino acid changes identified in E separating the PDK13 from parental 16007
virus contributed to the reduced replication of the DEN1 (PDK13)/DEN2(PDK53)
chimera not only in vitro (in LLC-MK2 and C6/36 cells) but also in mice. The highly
immunogenic DEN1 (16007)/DEN2(PDK53) chimera containing the C-prM-E genes
of wild type DEN1 16007 was a promising vaccine candidate. The virus did not cause
viremia, but stimulated DEN1 neutralizing antibodies and protected cynomolgus
monkeys against viremia after challenge with wild-type DEN1 [132].
Multiple DEN virus chimeras were then constructed containing only the prME
genes of wild-type DEN1 (16007), DEN3 (16562) or DEN 4 (1036) and the
backbone of wild type DEN 2 (16681) and the attenuated dengue 2 PDK53-V
and PDK53-E variants [133]. The chimeric viruses with prME elicited higher
antibody titers in mice than constructs with C-prME. The DEN3/DEN2 (PDK53)
chimera was mutagenized at residue E345 H!L to enhance growth in cell culture.
At first, the DEN4/DEN2 (PDK53) chimeras could be recovered only from C6/36
mosquito but not mammalian cells. Adaptation in Vero cells was required for
adequate growth, and this was accompanied by mutations (at C100, E364, and
E447) that were incorporated in vaccine constructs to ensure high yields in Vero
cells used for manufacturing vaccine lots.
In summary, chimerization per se was insufficient to produce suitable vaccine
candidates, as the constructs with the wild-type 16681 backbone did not have
markers of attenuation (ts, reduced replication in vitro, and lack of neurovirulence
for infant mice). In contrast, the intertypic chimeras with the DEN2 PDK53
backbone demonstrated attenuated phenotypes for all dengue serotypes. The
DEN1/DEN2 (PDK53) chimera elicited higher neutralizing antibodies than the
DEN 3 or 4 chimeras, and a two dose immunization schedule was required to
generate high antibody titers in AG129 (interferon a/b and g receptor deficient)
mice. A tetravalent formulation was tested in AG129 mice, with antibody responses
similar to those following monovalent vaccination. These responses were robust
after a single dose against DEN 1, 2 and 3 and after two doses to all four serotypes.
There were no differences between the V and E variants.
To prepare vaccine for clinical development, Inviragen has plaque-purified
infectious clone-derived DEN2 PDK53 and DEN1, 3 and 4 chimeras in the
DEN2 PDK53 backbone and prepared Master Virus Seeds at P8. Preclinical
evaluation of tetravalent formulations at different doses (e.g., 5,5,5,5 and 3,3,3,3
log10 PFU) and at ratios favoring the less active components (e.g., 3,3,5,5 log10
PFU) of the different components has been performed in AG129 mice and nonhu-
man primates. In addition, a comparison of ID and SC inoculation was performed.
A two dose schedule (Day 0, 42) was used in these experiments. The conclusions
from these studies follow: (1) DEN2 PDK53 is associated with highest viremias and
antibody responses, while the chimeric viruses are more attenuated; (2) among the
four serotypes, the immune response to DEN4 was most severely inhibited on
priming, but improved with the second (day 42) dose; (3) higher viremia was
observed at lower doses (e.g., 3,3,3,3 vs. 5,5,5,5 log10 PFU); (4) partial protection
against challenge was seen after one dose and full protection after two doses of
tetravalent vaccine; (4) the ID route was more effective than SC without inducing
higher viremia.
Inviragen’s Phase 1 trial in healthy young subjects will investigate SC and ID
routes of inoculation using a tetravalent formulation designed to limit interference
by the DEN component (5,4,5,5 log10 PFU).
3.2.6 Single Vector Constructs That Induce Immunity to Multiple

Dengue Serotypes
To avoid interference phenomena associated with simultaneous inoculation of four

live viruses, it would be ideal to have protective epitopes of all DEN viruses in a
single vector. Gene shuffling directed evolution techniques were employed to
derive chimeric prM-E sequences containing segments of all for dengue serotypes
[108]. Plasmids containing shuffled sequences express prME subviral particles
in vitro and presumably in vivo. Mice immunized IM four times with tetravalent
400 T.P. Monath
chimeric DNA produced antibodies to all four DEN serotypes. Monkeys also
developed low titers of neutralizing antibodies to all serotypes and exhibited partial
protection to DEN1 (but not DEN2) challenge [107]. Ideally, the shuffled, tetrava-
lent chimeric DNA could be used to construct a live vaccine by insertion in an
appropriate vector such as YF 17Dm rDEN4D30, or DEN 2PDK53, which would
be more likely than DNA immunization to elicit strong immune responses. How-
ever, the shuffled sequences may present some significant barriers to viral envelope
assembly and replication. To date successful construction of replication competent
viruses has not been reported.
Khanam et al. designed a recombinant defective adenovirus type 5 virus with
in-frame E protein domains III of both DEN2 and 4 [134] or all four serotypes
[135]. The vector expressed all proteins and elicited modest levels of neutralizing
antibodies in mice. This approach will be constrained by the usual issues surround-
ing anti vector immunity.
3.2.7 Vaccines Against West Nile
Chimeric West Nile/Yellow Fever 17D Vaccine
The strategy for development of vaccine against WN virus was similar to that
described for JE and DEN. Development efforts at Acambis were initiated within
months of the recognition of WN in North America in 1999 and a candidate vaccine
entered preclinical studies in 2000 [136]. Initially, the prME genes of wild-type
(383 99, isolated from a flamingo at the Bronx Zoo, 1999) WN virus were inserted
into the YF 17D infectious clone. The construction was achieved using the standard
two-plasmid system [137, 138]. The WN/YF chimera containing the wild-type
prME WN sequence was less neurovirulent for 3 4 week-old mice than YF 17D,
once again illustrating the attenuating effect of chimerization and showing the
dominant influence of the YF 17D vector on phenotype of chimeras derived
therefrom. Mice inoculated with 5.5 log10 PFU exhibited mortality ratios of 20%
whereas mice given <1 log10 PFU of YF 17D showed 100% mortality. However, in
rhesus monkeys inoculated IC neuropathologic scores were similar to those caused
by YF 17D [17].
The WN/YF chimeric virus was immunogenic for mice [17] and hamsters [139]
and immunized animals were protected against challenge with virulent WN virus
(Fig. 11). Baboons inoculated SC with 5.2 log10 PFU developed viremias lasting
1 4 days ranging between 1 and 2.5 log10 PFU/mL, whereas YF 17D in this species
caused no detectable viremia (Acambis, unpublished); the baboons all mounted
strong WN neutralizing antibody responses. The results raised questions about the
safety of the candidate, as it appeared to be marginally attenuated compared to YF
17D with respect to neurotropism for rhesus monkeys [17] and viscerotropism
(viremia) in baboons. At this stage of the vaccine development program, it was
decided to evaluate the chimera with a wild-type WN prME gene in horses, which
Neurovirulence Viremia PRNT50 (to WN virus unless specified) Protection
Mouse1 Monkey2 Rhesus Baboon Mouse Hamster Rhesus Baboon Mouse Hamster Rhesus
wt WN 5´ 3´ 100% - 3.1; 6.53 - - 970 - - - - -
YF 17D 5´ 3´ 100% 0.52-0.60 2.4; 3.5 <1.0; 0 - - 6404 - - - 0%;50%5
ChimeriVaxTM 5´ 3´ 83% 0.49 - 2.1; 3.3 197 299 - 114 100% 100% -
(veterinary) E107 L →F
5´ 3´ 0% - 2.2; 5.0 - - - 640 - - - 100%;100%

E316 A → K
5´ 3´ 25% - - - - - - - - - -
E440 K → R
5´ 3´ 83% - - - - - - - - - -
E316K
E440R
5´ 3´ 40% - 1.8; 3.5 - - - 135 - - - 100%;100%
E107F
E316K
ChimeriVaxTM E440R
WN02 5´ 3´ 0% 0.13 1.4; 4.5 - 37 - 381 80 100% - 100%;100%
1
3-4 week-old mouse, survival ratio, ~ 4 log10 PFU IC
2 Rhesus or cynomolgus macaque IC ~ 4 log PFU IC, mean combined lesion score at 30 days
10
3 Mean peak (log PFU/ml); duration (mean days)
10
4
Titer to yellow fever
5% animals protected against viremia; against death
Fig. 11 Site directed mutagenesis of a WN/YF chimeric infectious clone to develop a human WN vaccine candidate. The nonmutagenized virus was used as a
veterinary vaccine for horses. Attenuation and immunogenicity are indicated. From [17, 139] and unpublished data
402 T.P. Monath
were impacted severely in the recurring summertime outbreaks in the US, but to
further attenuate the vaccine candidate for use in humans.
The feasibility of immunizing horses was demonstrated in 2001 (Bowen R,
Arroyo J, Monath TP, unpublished results). Horses given two doses of the
WN/YF chimera (wild-type prM-E) failed to become viremic, but developed
neutralizing antibodies and were protected against viremia and illness/death on
challenge with WN virus introduced directly into the central nervous system by
intrathecal inoculation. The development of this vaccine for veterinary use is
described further below. Further attenuation of the vaccine (as proposed for
humans) was not indicated since host restriction in the horse of the highly pri-
mate-adapted YF 17D vector limited replication, as evidenced by absence of
vaccine viremia following SC inoculation and relatively low antibody responses.
The WN/YF chimera (wild-type prM-E) failed to infect Culex (Cx.) tritaenior-
hynchus or Cx. pipiens (p.) quinquefasciatus by the oral route; in contrast, wild-type
WN virus infected these mosquitoes and reached high titers of 5.8 6.7 log10
PFU/mosquito [140]. Aedes (the natural vector of wild-type YF virus) are some-
what more susceptible to infection with the chimeric or attenuated YF 17D
strains. Fourteen days after blood feeding, a low proportion of Ae. aegypti and
Ae. albopictus was infected with YF/WN or YF 17D virus; however, the virus titer
was very low (1.6 1.8 log10 PFU/mosquito) and virus did not disseminate to head
tissue. In contrast Aedes spp. ingesting a blood meal containing wild-type WN virus
became infected, virus replicated to high titer and disseminated to head tissue.
3.3 Development of a Suitably Attenuated WN/YF Vaccine

for Humans
To further enhance the safety profile of the WN/YF chimera, mutations in the
E gene were inserted at sites predicted to attenuate the virus based on knowledge of
the molecular structure of the closely related JE virus [17, 137, 138]. The objective
was to introduce multiple independently attenuating mutations, so that reversion at
one or two sites would retain the attenuation phenotype. The selection of nucleo-
tides for mutagenesis was derived from studies of ChimeriVax™-JE [17]. The
ChimeriVax™-JE prME genes were donated by an attenuated SA14-14-2 vaccine
containing mutations in the E gene at 6 amino acid residues (E107, E138, E176,
E279, E315, and E439) that play a role in attenuation. The WN and JE wild-type
gene sequences are conserved at these residues, suggesting that mutations intro-
duced at these sites in WN virus would have the same attenuating effects as they did
in JE SA14-14-2.
Mutagenesis of the WN residues E107, E280 (corresponding to E279 in JE
virus), and E316 (corresponding to E315 in JE virus) caused further attenuation
of neurovirulence. Mutation of the E protein at E440 (corresponding to E439 in JE
virus) from K!R, a conservative residue change, also reduced neurovirulence
for mice. Surprisingly, while a mutation at E138 (E!K) was associated with
attenuation of JE virus [15, 16], a corresponding mutation did not reduce the
neurovirulence of the WN/YF virus. A construct with three mutations F, V, and R
at positions E107, E316, and E440 respectively, designated ChimeriVax™-WN02,
was selected as the candidate for human studies (Fig. 11). The mutated virus was
significantly less neurovirulent than the original chimera with wild-type E sequence
for infant and weanling mice inoculated IC [17]. On histopathological evaluation
of weanling mouse brains following IC inoculation, the mutant vaccine had sub-
stantially lower neuronal degeneration and perivascular inflammation in the hippo-
campus (principal target area for mice) than the chimera with wild-type E gene and
YF 17D (Xiao S, unpublished data, 2005). ChimeriVax™-WN02 was also attenu-
ated for cynomolgus macaques following IC inoculation, causing significantly
lower neuropathological scores than YF 17D and was thus distinguished from the
original wild-type prME chimera [17].
During the manufacturing steps of the vaccine, it was noted that the Production
Virus Seed (at P4 after electroporation of chimeric RNA) contained a population of
approximately 10% of plaques with a small plaque phenotype. This virus was
favored for growth in Vero cells, so that the next passage (the P5 vaccine lot)
contained an equal number of large and small plaques. The small plaque population
had a mutation (L!P) at M66 located in the C terminus of the M protein [17]. The
two plaque variants were isolated by plaque purification and further characterized
in mice and hamsters. Neither virus was pathogenic for hamsters (including ham-
sters immunosuppressed with cyclophosphamide) but the large plaque M66L vari-
ant caused a higher viremia in hamsters than the nonmutated small plaque virus
(M66P). Both viruses were fully attenuated and not statistically different with
respect to neurovirulence in the sensitive 8 day-old mouse test.
Key nonclinical experiments then focused on the monkey model. The vaccine
candidate was evaluated in GLP studies for safety by inoculation of cynomolgus
macaques by the SC route and by IC inoculation (using the WHO standard test for
YF17D neurovirulence). Interestingly, viremia levels, while still low, were higher
in monkeys inoculated SC with the ChimeriVax™-WN02 virus than with YF 17D,
and a similar phenomenon was observed in hamsters (unpublished data, Acambis),
a model for WN infection [141]. It will be recalled that viremia of the nonmutated
WN/YF virus had been higher than YF 17D in baboons. Thus, while the mutated
chimeric virus had significantly lower neurotropism, the ability to replicate in
peripheral tissues and to cause viremia appeared to be enhanced, although without
any pathological consequences as determined in a GLP toxicology study in
monkeys. Since wild-type WN has been reported on one occasion to cause a
hepatitis syndrome in humans, and YF17D can also in rare cases of vaccine-
associated viscerotropic disease, it was of interest to compare the biodistribution
of ChimeriVax™-WN and YF 17D in monkeys [39]. Both viruses replicated in skin
at the site of inoculation, draining lymph nodes, and central lymph nodes, but were
rarely found in visceral organs (and never in liver in the case of ChimeriVax™-
WN02). ChimeriVax™-WN02 was cleared more rapidly from all sites than YF
17D. Thus, with the limitations of small sample size and uncertain relevance of the
404 T.P. Monath
Table 14 Immunization and challenge of rhesus macaques with YF 17D or YF/WN chimeric
viruses having one, two or three E gene mutations
Vaccine N Vaccine viremia PRNT50 Challenge Wild type WN NY99
(GMT)
to WN
% Mean Mean Day Day Viremia Illness Death PRNT50
viremic peak duration 30 63 (15 days)
None 2 100% 100% 100%
YF VAX® 4 100% 2.4b 3.5 NT <40 100% 50% 50% >640
YF/WN E107 4 100% 2.2 5.0 640 453 0% 0% 0% 2305
YF/WN E316/ 4 100% 1.8 3.5 135 453 0% 0% 0% 1076
E440
ChimeriVax™ 4 100% 1.4 4.5 381 190 0% 0% 0% 1280
WN02a
The triple mutant virus (E107, 316, 440) is the human vaccine candidate (ChimeriVax™ WN02).
Challenge (day 63) was performed by IC inoculation of 5.4 log10 PFU of wild type WN virus
(strain NY99 35262 11, flamingo, New York, 1999), a severe test of immunity. Data from [17]
a
Mutations at all three sites E107, 316, 440
b
log10 PFU/mL
Table 15 Viremia in humans following SC inoculation of 3.0 or 5.0 log10 PFU of ChimeriVax™
WN02
Parameter Placebo CV WN02 CV WN02 YF VAX®
(n 30) 5 log10 PFU 3 log10 PFU (n 5)
(n 30) (n 15)
Cmax (PFU/mL) Mean SD 0.0 0.0 97.3 159.2 187.3 164.8 90.0 81.9
AUC (D1 to 14) Mean SD 0.0 0.0 173.0 251.8 311.7 259.4 168.0 156.4
PFU.D/mL
Duration (days) Mean SD 0.0 0.0 5.1 2.9 4.7 1.9 3.6 3.5
Number of days Mean SD 0.0 0.0 4.0 2.2 4.4 1.8 3.2 3.0
viremic
Number (percentage) 0 27 (90%) 15 (100%) 4 (80%)
of subjects viremic
models to humans, the vaccine appeared to have biological properties of an

acceptable vaccine.
To determine efficacy, a study was performed in rhesus macaques inoculated
with YF 17D (control) or three different chimeric viruses containing one mutation
(E107), two mutations (E316 and E440), or all three mutations (i.e., ChimeriVax™-
WN) [17]. All animals developed neutralizing antibodies and were completely
protected from clinical signs and death after IC challenge 2 months following
vaccination with wild-type WN virus (Table 14).
A double-blind Phase 1 clinical trial was conducted in healthy young adults in
the US [39]. A total of 75 subjects were randomized to four treatment groups and
received a single SC inoculation [placebo (N ¼ 15), YF-VAX® (N ¼ 5), Chimer-
iVax™-WN02 high dose (5.0 log10 PFU) (N ¼ 30) and low dose (3.0 log10 PFU)
(N ¼ 15)]. The chimeric vaccine was well tolerated and there were no differences
a
100000 ChimeriVax-WN02 5.0 log
ChimeriVax-WN02 3.0 log
10000
PRNT50
1000
100
10
0 10 20 30 40 90 180 365
Day
b 1100
IFN-g producing cells per million PBMC
1000
500
400
300
200
100
Day 0 Day 14 Day 28
Fig. 12 (a). Neutralizing antibody levels in individual subjects, and GMT; clinical trial of
ChimeriVax™ WN02. (b). T cell responses. From [39] with permission
in the incidence of adverse events across treatment groups. The observation of

higher viremias in hamsters and cynomolgus monkeys associated with the chimeric
vaccine was not recapitulated in humans (Table 15). High titers of neutralizing
antibodies developed without relationship to vaccine dose, peaked at approximately
3 weeks and persisted for >1 year (Fig. 12a). Strong T cell responses were also
documented (Fig. 12b).
The mutation at M66 (L ! P) that was responsible for the small-plaque pheno-
typic change arose spontaneously during early passages after electroporation of
RNA to prepare virus seeds. Since the large plaque variant was more viremogenic
in animals than the small plaque virus, it was decided to attempt to remove it from
the vaccine seed stock by plaque purification. New ChimeriVax™-WN02 SP (small
plaque) master and production seeds were produced and vaccine lots manufactured
at P13. This process produced a vaccine that was predominantly small plaque, and
406 T.P. Monath
the large plaque M66L virus was not detectable by consensus sequencing. How-
ever, it was evident that genetic instability at this locus was a feature of the vaccine
virus and likely an adaptation to growth in Vero cells under serum free conditions.
This finding was in concert with experience with ChimeriVax™-JE virus. During
serial passages of that virus during manufacture of a new seed stock in serum-free
Vero cells, the virus also developed a single mutation at a similar site in the carboxy
terminus of the membrane protein M (an R to C change at residue M60). The M60
mutation was shown to significantly increase the rate of virus replication in serum-
free Vero cells, but like M66 in the WN vaccine the M60 mutation did not increase
neurovirulence of the ChimeriVax™-JE for suckling mice.
A sensitive MAPREC assay [142] was developed to monitor the presence of
large plaque virus in batches of ChimeriVax™-WN02, which was <2% in the P13
vaccine lots. Despite a significant effort, it was not possible to entirely eliminate the
large plaque variant; the virus was unstable at this locus, but the ratio of large and
small plaque virus could be controlled during manufacture. It was expected that
reduction of large plaque virus from 50 to <2% in the vaccine formulation would
improve safety. Nonclinical tests on the ChimeriVax™-WN02 SP vaccine, includ-
ing a GLP monkey neurovirulence test, confirmed that it was attenuated and
efficacious. These studies in monkeys indicated that the small-plaque vaccine
produced a similar level of neutralizing antibody response as that of mixed plaque
vaccine which had been evaluated in monkeys and in the Phase 1 trial [39], but
produced lower viremia levels than the mixed plaque vaccine.
A Phase II clinical trial was performed with the ChimeriVax™-WN02 SP
vaccine. The results of this study have not yet been reported.
3.4 Development of a Veterinary Chimeric WN/YF Vaccine
West Nile is a severe disease with a 28 38% case fatality rate in horses, and large
numbers of animals were affected across North America following introduction
of the virus in 1999. In 2002, Acambis initiated a collaboration with, and then
licensed the ChimeriVax™-WN vaccine to Intervet for veterinary indications. This
collaboration led to commercialization of the vaccine for prevention of WN disease
in horses in 2007 under the brand-name of Prevenile®. This collaboration between
human and veterinary health companies is an outstanding example of the principles
of the One Health Initiative [143].
As described above, Prevenile® was constructed by replacing the prME genes of
YF 17D with the corresponding genes of the wild-type 383-NY WN without the
addition of attenuating mutations. The natural host restriction for replication in
horses of YF 17D and chimeric viruses derived therefrom was highly attenuating, as
shown by initial exploratory studies in horses showing absence of viremia and
lower neutralizing antibody levels than seen in primate hosts. Safety of the vaccine
was exhaustively studied, with emphasis on transmission to nontarget animals.
Horses were given an overdose of virus, euthanized and examined for virus in
tissues and pathological effects at 14 days postvaccination [144]. In addition,

overdosed horses were evaluated for virus shedding and transmission to sentinel
animals. Over 900 horses (including foals <4 months old) were vaccinated under
field conditions and followed for adverse events. These studies showed that the
vaccine was safe and well tolerated, was neither shed from mucosal surfaces nor
infectious for contacts, and did not persist in tissues of the host. Immunogenicity of
the vaccine was evaluated in multiple studies. All horses inoculated with vaccine
(5 log10 PFU) developed neutralizing antibodies at titers of 4 64 (GMT ~10) within
7 14 days, and antibodies persisted for at least 12 months [145]. Given the low
antibody levels following a single inoculation, it was important to employ a severe
test of immunity to determine protection against virulent WN virus infection.
Vaccinated and control horses were therefore challenged by intrathecal injection
of a high dose (5 log10 PFU) of WN NY99 4132 virus (isolated from an infected
crow in 1999) [145, 146]. Other studies used natural challenge by infected mosqui-
toes. Vaccinated horses were protected from viremia, fever, clinical signs, but
developed high titers of neutralizing antibodies after challenge. Rarely (in <10%)
mild clinical signs and brain histopathological lesions were observed in vaccinated-
challenged horses, whereas in unvaccinated controls, WN NY challenge was 80%
fatal and caused viremia in 90 100% of animals. Protection was observed when
challenge was performed early after vaccination (10 days) or late (12 months).
In a comparative study of horses vaccinated with two doses of Prevenile® or
two other commercial WN vaccines (canarypox-vectored, Merial) and formalin-
inactivated vaccine (Ft. Dodge), Prevenile® appeared superior to the inactivated
vaccine in protecting animals against clinical signs following intrathecal WN virus
challenge [146].
West Nile virus is highly pathogenic for crows and a number of other wild avian
species, as well as valuable exotic zoo birds, for which artificial vaccination is
indicated. The ChimeriVax™ vaccine (wild type prME) was tested but found not to
be effective in an avian species (fish crows) [147]. The vaccine given at high dose
to these birds elicited neutralizing antibodies in only 12% of birds. No evidence of
protection against viremia or death was observed after SC challenge with virulent
WN virus. Severe host restriction for replication, determined by YF 17D NS genes,
precludes us of this vaccine for birds.
A chimeric DEN4/WN vaccine candidate was also restricted for growth in birds
(see below).
3.4.1 Dengue Type 4 D30 Vectored Vaccine Against West Nile Virus
Chimeric viruses were constructed by inserting the prME genes of WN NY99 into
the DEN4D30 vector [148 150]. Chimeric viruses constructed with wild-type
DEN4 as well as the mutant DEN4 backbones were attenuated for mice, caused
reduced viremia in monkeys, and had restricted ability to infect Cx. tarsalis and Ae.
aegypi mosquitoes (though curiously grew in Ae. albopictus) [151]. Both constructs
induced neutralizing antibodies and protected monkeys against viremia following
408 T.P. Monath
parenteral challenge with wild-type WN virus [151]. Several mutations appeared

during the passages to make clinical grade vaccine using the DEN4D30 backbone,
one in the E gene (E603 G!R) and three in the DEN4 backbone (one in NS2B, 2 in
NS4B). The vaccine candidate was not neuroinvasive in weanling SCID mice and
had markedly reduced neurovirulence and ability to replicate in brain tissue of
5-day-old mice inoculated IC [19]. The chimeric vaccine did not cause viremia in
rhesus monkeys, but elicited high neutralizing antibody titers. Prior immunity to
DEN did not modulate the anti-WN antibody response.
The chimeric vaccine was not infectious for and did not elicit antibodies in
domestic geese (an economically important species that develops severe and fatal
infection with WN virus) [150].
3.4.2 Dengue Type 2 PDK53 Vectored Vaccine Against West Nile Virus
This vaccine is under development by Inviragen Inc. The prME sequence of the
NY99-35262 strain was inserted into the DEN2 infectious clone. Three backbones
were constructed (wild-type 16681, and PDK53-E and V). In addition, constructs
were made with engineered mutations at M58 (M!L) and E 191 (E!A) to
enhance growth in Vero cells [19]. The chimeras with PDK53 backbones were ts
and restricted for growth in C6/36 cells. Chimeras with 16681 or PDK53 backbones
were less neurovirulent for infant mice than WN99 and wild-type DEN2 16681.
When inoculated IP into outbred mice, the chimeras elicited respectable neutraliz-
ing antibody titers after a single dose (GMT 40 108 for the PDK53 vectored
vaccines) in 75 88% of the mice and protected 88 100% against lethal WN virus
challenge. Two sequential immunizations substantially boosted titers (GMT
580 4,695) in all mice and fully protected against challenge.
3.4.3 Chimeric Vaccines Against Tick-Borne Encephalitis
Medically important members of the TBE antigenic complex include Russian

spring summer encephalitis virus (RSSE, the Far Eastern subtype), and Central
European Encephalitis (CEE) virus, louping ill (LI), Kyasanur Forest disease
(KFD), Omsk hemorrhagic fever (OHF), and Powassan (POW) viruses. These
viruses have E gene homologies of 78% or greater and share protective epitopes
[152], suggesting that a Jennerian approach to vaccine development might be
employed, using a naturally attenuated member of the antigenic complex to con-
struct a cross-protective vaccine against other TBE Complex viruses. The incidence
of TBE in Europe has increased in countries that are not practicing routine vacci-
nation against the disease. Two highly effective, purified, formalin-inactivated
vaccines prepared from the CEE virus grown in chick embryo cells and adsorbed
to alum are approved for use in Europe [153]. The vaccines are believed to cross-
protect against RSSE virus. Since they require two doses for primary immunization
and boosters at regular intervals, there has been interest in developing improved
vaccine strategies, described below. However, children are the principal target for
vaccination in industrialized countries endemic for TBE, and any new vaccine must
have extraordinary assurances of safety to displace the existing, highly effective
inactivated vaccines.
3.4.4 Chimeric TBE/DEN4 Vaccine
The first chimeric vaccine investigated for immunization against TBE employed an
unmodified, wild type DEN4 backbone into which either the C-prM-E or prM-E
genes of a virulent CEE virus (Sofjin strain) were inserted [33]. As shown for the
DEN2/DEN4 chimeras, the chimera with C-prM-E from TBE was less competent
for replication in cell culture than the prM-E construct. The TBE (prM-E)/DEN4
chimera was significantly less virulent than parental TBE virus, which had an IP
LD50 of 14 PFU, whereas the chimera was not lethal by IP injection. However,
when inoculated IC, the chimera caused lethal encephalitis in suckling and adult
mice showing that the prME genes of TBE conferred an neurovirulent phenotype.
The introduction of mutations in prM or E or in the DEN4 NS1 gene of the chimera
reduced neurovirulence for mice and also reduced replication in cell culture [154].
The chimera with or without attenuating mutations induced immunity and protected
mice against lethal challenge with TBE [154, 155]. Given the safety concerns
around use of pathogenic viruses in constructing a live vaccine, attention refocused
on alternative strategies.
3.4.5 Chimeric LGT(TP21)/LGT and LGT(E5)DEN4 viruses
To improve upon the safety phenotype of DEN/TBE chimera, the prM-E genes
from strains of the more attenuated Langat (LGT) virus (a member of the TBE
antigenic complex originally isolated from ticks in Malaysia) were inserted into the
wild-type DEN4 backbone. The prototype LGT TP21 strain had actually been
evaluated on its own in Russia for Jennerian vaccination against TBE [155], but
had been associated with serious adverse events (vaccine-associated encephalitis)
[156]. A derivative, the E5 strain, had been attenuated by serial passage in eggs to a
point where it was less neurovirulent for mice than TBE virus and was considered a
candidate vaccine strain [157]. LGT E protein is approximately 88% homologous
with RSSE virus and neutralizing antibodies cross protect but require antibody
titers fourfold higher than antibody against homologous strains of TBE virus [154].
Chimeric viruses were constructed inserting the prME genes of the LGT TP21 or
E5 viruses into DEN4 814669 [158]. The resulting chimeras were significantly less
neurovirulent than the parental TP21 and E5 viruses by IC inoculation in suckling
mice and were not neuroinvasive. Adult mice immunized IP with as little as 1
log10 PFU developed neutralizing antibodies and were protected against lethal IP
challenge with 1,000 LD50 of homologous TP21.
410 T.P. Monath
The vaccine candidates were subsequently adapted for growth in Vero cells
and investigated for their ability to protect against heterologous challenge with
virulent CEE (Sofjin) as well as the RSSE (Absettarov) strain [159]. The chimeric
viruses carrying LGT prME induced neutralizing antibodies to LGT, but were
substantially less effective in eliciting heterologous protective immunity to Sofjin
or Absettarov viruses. After a single vaccination, partial protection was achieved,
while complete protection required two sequential vaccinations. Moreover, the
chimera with prME derived from the more attenuated E5 strain was less effective
than the LGT(TP21)/DEN4 chimera. The latter conferred complete protection
after two inoculations, whereas only 67% of mice inoculated with two doses
of LGT(E5)/DEN4 survived TBE challenge. These studies illustrated the predom-
inant influence of the structural gene sequence, and the difficulty achieving a
correct balance of attenuation and immunogenicity. They also raised questions
about the use of a heterologous E gene in providing cross-protection against
virulent TBE strains.
The results were nonetheless considered sufficiently promising to proceed with
advanced preclinical evaluation. The chimeric LGT(TP21)/DEN4 vaccine
prepared in Vero cells was tested in rhesus macaques [160]. The vaccine was
highly attenuated in this host following SC inoculation. Only 1 of 12 monkeys in
groups of four immunized SC with 3, 5 or 7 log10 PFU developed detectable
viremia, whereas viremia was detected in all monkeys inoculated with 5 or 7
log10 PFU of parental LGT TP21 or 5 log10 PFU of DEN4 virus. Monkeys in the
groups immunized with LGT(TP21)/DEN4 developed high PRNT60 titers of
372 2,344 against LGT TP21 or 320 640 against heterologous TBE virus.
When challenged 6 weeks after vaccination with 5 log10 PFU of LGT TP21, all
were protected against viremia.
A comprehensive study in cell cultures, mice, and rhesus monkeys compared the
safety and protective activity of TBE/DEN4, LGT(TP21)/DEN4, and a construct in
which wild-type TBE (Sofjin) virus prME was inserted into the DEN4 backbone
having the attenuating 30 NCR D30 deletion [152]. The genomes of the TBE/DEN4
and TBE/DEN4D30 viruses differed at four amino acid residues in prM, NS3 and
NS4B. The LGT(TP21)/DEN4 differed from the other chimeras and wild-type LGT
virus in being restricted for growth in murine and human neuroblastoma cells. The
virus was also significantly less neurovirulent for suckling mice (IC LD50 2.4 3.2
log10 PFU) than either TBE/DEN4 or TBE/DEN4D30 chimeras (IC LD50 <
1 log10 PFU), and none of the vaccine constructs were neuroinvasive. The safety
and immunogenicity of a single dose of the chimeric viruses were compared
with commercial inactivated TBE vaccine given as three sequential vaccinations
on days 0, 7 and 21 (Table 16). The take-away messages were: (1) as determined by
vaccine viremia and antibody response, the LGT(TP21)/DEN4 and TBE/
DEN4D30vaccines were more highly attenuated than TBE/DEN4; (2) homologous
antibody responses were ~5-fold higher than heterologous responses; (3) the TBE/
DEN4D30 vaccine elicited very low antibody responses to heterologous LGT virus
and to homologous TBE virus, was thus over-attenuated, and did not confer
Table 16 Viremia and immune response of rhesus monkeys following immunization with chimeric virus or inactivated TBEV vaccine and after subsequent
challenge with wild –type LGT strain TP21 (From [152], with permission)
Immunizing No of Response to immunizationa Response to challenged
virus monkeys
No of viremic Mean no of Mean peak Geometric mean serum NT No of viremic Mean no of Mean peak Geom mean NT
monkeys viremic days virus titer antibody titer (reciprocal monkeys after viremic days virus titer antibody titerc on
per monkey (log10 PFU/ dilution)c against LGT per monkey (log10 day 21 post
ml)b challenge PFU/ml) challenge
aLGT aTBEV/DEN4 aLGT aTBEV/
DEN4
Day 0 Day 42 Day 0 Day 42
LGT/DEN4 4 3 10 09 <10 177 <10 14 0 0 <0 7 2801 834
TBEV/DEN4 4 4 35 31 <10 227 <10 1125 0 0 <0 7 1372 6457
TBEV/DEN4 4 3 08 07 <10 17 <10 59 1 0 25 07 767 2951
D30
“Encepur” 4 0 0 <0 7 <10 454 <10 281 0 0 <0 7 1125 1804
Control 4 0 0 <0 7 <10 <10 <10 <10 4 20 19 357 97
a
Groups of rhesus monkeys were inoculated SC with 105 PFU of indicated virus or L-15 medium (control) in a 1 mL dose on day 0. Another group of monkeys was
inoculated SC with a formalin-inactivated TBEV vaccine “Encepur” in three doses (3 0.5 mL) on day 0, 7, and 21. Serum used to measure viremia was collected
daily for 10 days
b
Virus titer serum was determined by plaque-forming assay on LLC-MK2 cells. The lower limit of detection was 0.7 log10 PFU/mL. For purpose of calculating the
mean peak titer, a titer of 0.6 log10 PFU/mL was assumed for monkeys serum with undetectable viremia. Viremia was not detected in any monkey after day 4
c
Plaque reduction (60%) neutralizing antibody titers were determined against wild-type LGT TP21 strain or chimeric TBEV/DEN4 virus as indicated. Serum for
neutralization assay was collected on days 0
d
On day 43, all monkeys were inoculated SC with 105 PFU of wild-type LGT TP21. Serum used to measure LGT viremia was collected daily for 10 days. LGT
virus titer in serum was determined by plaque-forming assay on LLC-MK2 cells, and viremia was not detected in any monkey after day 4 post-inoculation
412 T.P. Monath
complete protection against LGT TP21 challenge; (4) administration of three doses
of inactivated vaccine induced broadly cross-reactive, high antibody titers and was
fully protective against LGT TP21 challenge.
Tick-borne flaviviruses do not infect mosquitoes; thus it was of interest to
determine whether chimeras of tick and mosquito-borne viruses would be compe-
tent for replication. The chimeric LGT (TP21)/DEN4, as well as parental LGT
TP21 were incapable of infecting Toxorrhynchites mosquitoes after intrathoracic
infection, while DEN4 virus replicated efficiently [158], showing that the chimera
would not be transmissible by mosquitoes and that the prME genes from a tick-
borne flavivirus determined specificity for arthropod infection.
Based on safety (reduced neurovirulence for suckling mice, lack of neuroinva-
siveness in mice, and low viremia in monkeys) and immunogenicity for monkeys,
the chimeric LGT (TP21)/DEN4 vaccine was selected for testing in a clinical
trial [161]. The principal objectives of the study were to confirm the safety of
this construct and to determine whether the vaccine would elicit antibodies to
the LGT insert, and more importantly to TBE virus. Twenty healthy young
adults received 3 log10 PFU of the chimeric vaccine by SC injection and eight
received a placebo. The vaccine was safe and well tolerated, and, as predicted
by monkey studies, highly attenuated, viremia being observed in only one sub-
ject (5%). In concert with this level of attenuation, immunogenicity was relatively
poor. Only 80% of the vaccines seroconverted to LGT and 35% seroconverted
to TBE. Neutralizing antibody GMT to LGT and TBE were low 63 and 9,
respectively.
Two subsequent studies cast a level of doubt on the safety of this vaccine
construct, despite the attenuation observed in mice (inoculated IC) and in
humans. Pripusova et al. [162] reported that while the LGT (TP21)/DEN4 vaccine
had 40,000-fold reduced neurovirulence in mice compared to LGT TP21 or TBE
(Absettarov) viruses, African green monkeys inoculated IC demonstrated virus
replication, histopathological lesions and clinical signs of encephalitis. In a
separate analysis in rhesus macaques inoculated IC, the TBE/DEN4D30 (which
had shown overattenuation with respect to antibody response in the same
species), was compared to YF 17D [58]. Interestingly, the TBE/DEN4D30 con-
struct caused higher neuropathological lesion scores than YF 17D. These studies
suggest that chimeras with structural genes derived from tick-borne viruses retain
residual neurovirulence for primates. While the low viremias caused by these
vaccines would likely prevent neuroinvasion in humans, from a regulatory per-
spective the residual neurovirulence is problematic. Moreover, these studies call
into question the ability to correlate neurovirulence of virus vaccines in mice and
monkeys [34].
Recent attempts have been made to develop mutagenized LGT E5 virus
with reduced neurovirulence properties that could ultimately be used to construct
more attenuated chimeras [163]. However, given the overattenuated phenotype of
the chimera having unaltered TP21 prM-E it is unlikely that the answer to a
successful construct lies in this direction.
3.4.6 Chimeric Vaccines Against Other Flaviviruses of Medical Importance
Chimeric viruses were constructed using prM-E genes donated from virulent
(MSI-7) or naturally attenuated (CorAn9124) strains of St. Louis encephalitis
(SLE) virus in the YF17D vector backbone [164]. The phenotype of these constructs
was predictable based on previous work with related JE and WN chimeras. The
chimera with the MSI-7 insert had reduced neuroinvasiveness for weanling mice
compared to wild-type MSI-7 and was ~1,000 less neurovirulent when inoculated
IC. The chimera constructed with the naturally attenuated CorAn9124 genes was
not neuroinvasive and was 100,000 less neurovirulent compared to wild-type
CorAn9124 parent. No evaluation has been performed of the utility of these
constructs as vaccines.
As noted previously, cross-protection has been demonstrated between closely
related viruses in the JE antigenic complex (including WN, SLE and MVE). It is
likely that a live, chimeric vaccine against JE could be employed in an emergency
to protect against SLE, WN or MVE, or conversely a vaccine against WN
could be employed against JE or SLE. These applications could extend to
both veterinary and human disease indications. Now that chimeric vaccines are
becoming commercially available, studies of cross-protection are increasingly
relevant.
4 Alphavirus Vaccines
Like the flaviviruses, alphaviruses (family Togaviridae) have single-strand, posi-

tive sense genomes and the viral RNA is infectious following transfection of cells in
culture. The viral RNA of about 12 kb can be reverse transcribed and cDNA cloned
for engineering in bacterial plasmids. It is relatively simple to insert coding regions
for foreign genes, and several groups have explored the use of Sindbis (SIN),
Semliki forest, or attenuated Venezuelan equine encephalitis (VEE) virus as vec-
tors. Site directed mutagenesis has also been used to derive attenuated alphavirus
strains of virus that could be used as vectors for foreign genes [165]. In this section,
we consider the use of alphavirus vectors constructed with donor genes from
another alphavirus to make live, replicating chimeric vaccines against alphaviruses
of medical importance, such as VEE, EEE and WEE, respectively, RRV, and
CHIK. The approach is thus analogous to that described for flavivirus vaccine
development. Interestingly, WEE and all but one of the other New World members
of the WEE antigenic complex originally evolved through a recombination event in
nature between a SIN-like virus and EEE [166]. The E1 and E2 genes of WEE are
derived from SIN, whereas the C and NS genes are derived from EEE. This natural
experiment demonstrated in advance that viable chimeric constructs would be
achievable in the laboratory.
414 T.P. Monath
Live, attenuated vaccines have been developed against VEE [167] and CHIK
[168] using empirical passage. TC-83 has been used as an investigational vaccine
in over 8,000 humans [169], but is highly reactogenic and fails to immunize about
18% of subjects. The VEE vaccine (TC-83) has also been approved for use in
Equidae. A candidate VEE vaccine (V3526) was developed by introducing
mutations in the PE2 furin cleavage site of the virus, and this candidate
has been tested in rodent models and horses [170]. An inactivated VEE vaccine
(C-84) is not protective in animals against respiratory challenge. It is used only
to immunize TC-83 vaccinees who failed to seroconvert but requires multiple
doses and frequent boosters to achieve adequate titers. The live CHIK vaccine
(181/25) appears to be highly immunogenic, but transient arthralgia was observed
in ~9% of subjects [171], and there are concerns about genetic stability of the
virus. A formalin inactivated CHIK vaccine has also been evaluated [172].
Investigational, formalin inactivated vaccine against WEE and EEE are in limited
use in laboratory workers. Improved vaccines against all these alphaviruses
are required.
The genome organization of alphaviruses differs from flaviviruses in that the
four NS proteins encoding transcriptases and replicases are translated from the 50
two-thirds of the full-length 42S messenger RNA, whereas the structural genes
encoding capsid (C) and envelope proteins (usually two E1 and E2, but some-
times three) are translated from a 26S subgenomic messenger RNA transcribed
from the 30 one-third of the genome. Viable, replication-competent chimeric viruses
are constructed using the 50 and 30 noncoding regions and polyA tail, subgenomic
promoter and NS genes of the vector and the 50 noncoding region of the 26S
subgenomic RNA and structural protein genes (capsid, E1 and E2) from the
virus against which immunization is desired. Some variations on this scheme have
been used and will be described below. As for flaviviruses, the absence of structural
genes of the vector may preclude interference due to antivector immunity; however,
this has not been assessed empirically, for example by sequential immunization
with different chimeras sharing the same backbone vector. It is known that
prior immunization with EEE or WEE interferes with subsequent vaccination with
live VEE vaccine [173, 174], so similar effects could apply to the use of chimeric
viruses.
The chimerization process itself significantly attenuates the resulting virus, and
use of attenuated gene donor and vector strains or introduction of mutations can
further optimize biological phenotype. Limited experience to date suggests that
chimeric alphavirus vaccines are attenuated even when two virulent viruses are
used in their construction. A chimeric virus containing the structural genes of EEE
virus and the nonstructural genes of WEE virus was attenuated compared to the two
parental strains in mice [175]. Thus, the principles of use are similar to those
employed for flaviviruses. The live, attenuated alphavirus vaccines would be
expected to induce strong innate and adaptive immune responses, immunize with
a single dose, produce long-lived immunity, would be unlikely to revert to viru-
lence, and wound be in expensive to manufacture.
4.1 Chimeric Vaccine Candidates Using Sindbis Virus

as a Vector
Sindbis (SIN) and several closely related subtypes are mosquito-borne alphaviruses
distributed widely in Europe, Africa and Australia. These viruses are human
pathogens, causing endemic and epidemic illness characterized by a self-limited
fever rash arthritis syndrome (variously named Okelbo disease, Pogosta disease,
and Karelia fever in Scandanavia and Russia) [176]. Given the pathogenic potential
of SIN virus, and the lack of an animal model for the natural disease syndrome in
humans, use as a vector requires demonstrated reduction in other virulence markers
in animals and, ultimately proof of safety and tolerability in nonhuman primates
and humans. SIN is naturally attenuated for many laboratory animals, so that it is
difficult to judge whether a chimeric vaccine is more attenuated than the vector.
However, wild-type SIN virus is neurovirulent for infant mice and attenuation of
chimeric viruses can be assessed in this model. In addition, a model of lethal SIN
infection in interferon-a/b and interferon-g receptor deficient mice [177] provides a
possible approach to the investigation of attenuation of chimeric vaccines. These
mice develop a hemorrhagic diathesis, which is atypical for human SIN infections,
although rare cases have been reported [178]. Wild-type SIN virus is not known to
cause disease in domestic animals and thus it is a vector of interest for veterinary
vaccines against VEE, EEE and WEE.
A number of studies using SIN as a vector for vaccines against VEE, EEE, WEE
and CHIK viruses have been reported (Table 17). Viable chimeras of RR and SIN
were also constructed [179] but have not been evaluated for immunogenicity. The
largest body of work on SIN vectors has been conducted by Scott Weaver and his
collaborators at the University of Texas Medical Branch, Galveston. A VEE/SIN
chimera with structural genes of the live, attenuated TC-83 strain of VEE inserted
in the SIN (Toto1 101) vector was shown to be attenuated, immunogenic and
protective in mouse models [180]. The parental TC-83 vaccine was neurovirulent
and neuroinvasive for infant (6-day-old) mice, whereas the VEE(TC-83)/SIN
chimera was not. The chimera elicited high titers of neutralizing antibodies and
protected mice against lethal SC challenge with VEE subtype IC as well as the more
distantly related VEE subtype ID. The work was extended to investigate various
chimeric vaccines in which structural genes were derived from TC-83, the virulent
parental (TrD) strain, or VEE ID virus [181]. In addition, a construct was prepared
with 3 point mutations in the SIN backbone that are present in SAAR86, a wild-type
SIN strain that is unusual in being virulent for mice by the peripheral route of
injection [185]. None of the chimeras killed adult mice after IC inoculation, and
they were attenuated for neurovirulence and neuroinvasiveness in the 6-day-old
mouse model compared to TC-83 (Table 17). A single SC inoculation elicited
neutralizing antibodies in mice and there was little or no rise in antibodies after a
booster dose given at 8 weeks, indicating sterile immunity (Table 18). The VEE
(ID)/SIN and VEE(TrD)/SIN vector with three SAAR86 mutations were more
immunogenic than the other constructs. However all mice were protected against
Table 17 Biological features, immunogenicity and protective activity of live, chimeric alphavirus vaccine candidates
Target Designation Structure Replication Virulence/attenuation Neutralizing antibody Protection vs Reference
in vitro Neurovirulence Neuroinvasiveness, Other Seroconversion PRNT mean challenge
(mice), mice (SC or IP) rate or range of
mortality (%) (mortality (%) titers 4 wks
Ross RR/SIN RR structural genes, Replicates to high 0/22 (0%)a 0/28 (0%) [179]
River chimera SIN backbone titer Tenfold
lower growth
in Vero, CEF
but higher
growth in C6/
36 than
parental SIN
Growth in
C6/36 similar
to RRV
SIN parental strain derived 0/9 (0%) 0/9 (0%)
from infectious
clone
RR parental strain derived 23/23 (100%) 28/28 (100%)
from infectious
clone
VEE VEE(TC83)/ VEE structural genes Replicates to high 0%c 0% 60–960d 100% protected vs [180]
SINb (from attenuated titer Tenfold challenge SC
TC-83 vaccine), lower growth with 6
SIN (Toto1 101) in Vero and log10 LD50 of
backbone BHK than either VEE ID
TC-83, but to (ZPC738)
similar or (N ¼ 5) or
higher titer VEE IC (SH3)
than SIN (N ¼ 5)
VEE TC-83 Vaccine strain 100% 20% 480 100% protected vs
challenge SC
with 6
log10 LD50 of
either VEE ID
(ZPC738)
(N ¼ 5) or
VEE IC (SH3)
(N ¼ 5)
VEE VEE(TC83)/ VEE structural genes 0%e 0% ~5 log10 PFU/gf see Table 15 see Table 15 [181]
SINb (from attenuated
TC-83 vaccine),
SIN (Toto1 101)
backbone
VEE(TrD)/ VEE structural genes 20% 0% ~7 log10 PFU/g see Table 15 see Table 15
SIN (from virulent
Trinidad donkey
subtype IAB), SIN
backbone
VEE(ZPC)/ VEE structural genes 40% 0% ~7 7 log10 PFU/g see Table 15 see Table 15
SIN (from ZPC738
subtype ID), SIN
backbone
VEE(TrD) VEE structural genes 30% 0% ~6 5 log10 PFU/g see Table 15 see Table 15
/SIN (from virulent
(SAAR, Trinidad donkey
mutated) subtype IAB), SIN
backbone contains
three attenuating
mutations in 50
NCR, nsP1, nsP3/
nsP4 present in a
virulent strain of
SIN (SAAR86)
VEE TC-83 vaccine strain 100% 100% ~9 5 log10 PFU/g see Table 15 see Table 15
EEE EEE (NA)/ EEE structural genes All 3 viruses 100%g No viremia in ~8 3 log10 PFU/gh 70–100%i 125–660 80–100% [182]
SIN (North American grew to 6–7 Prolonged 8 week-old [20–50 protected
subtype), SIN log10 PFU/ AST mice vs across three
Ar339 mL inoculated SC EEE dose groupsi;
nonstructural Replication (SA)] all sham
genes of the EEE immunized
(SA)/SIN controls died
EEE (SA)/ EEE structural genes chimera and 90% Prolonged No viremia in ~7 log10 PFU/g 50–100% 28–308 100% protected;
SIN (Sorth American parental SIN AST 8 week-old [20–36 all sham
subtype), SIN in Vero and mice vs immunized
Ar339 C7/10 inoculated SC EEE controls died
nonstructural mosquito (NA)]
genes cells was
(continued)
Table 17 (continued)
SIN Ar339 similar; EEE 100% ~7 log10 PFU/g
(NA)/SIN
grew to ~10-
fold higher
titer
EEE North American 100% ~9 log10 PFU/g
subtype (FL93-
939)
EEE South American 100% ~8 log10 PFU/g
subtype
(BeAr436087)
WEE WEE(CO92) WEE (CO92-1356) All 3 chimeric 80%k 30%k 6 2 log10 PFU/gl 50–100%m 60–190m 50–100% [183]
/SIN structural genes, viruses grew protection; all
SIN Ar339 to 7 sham animals
nonstructral genesj log10 PFU/ diedm
WEE(McM) WEE (McMillan) mL and had 100% 600–604 100% protection
/SIN/SIN structural genes similar
except for amino- growth
terminal domain curves in
of C gene of SIN Vero and C7/
Ar339, SIN 10 mosquito
nonstructral genesj cells
WEE(McM) WEE (McMillan) 100% 100% 8 1 log10 PFU/g 80–100% 416–420 100% protection
/EEE/ structural genes
SIN except for amino-
terminal domain
of C gene of EEE
(436087), SIN
nonstructral genesj
WEE CO92-1356 Culex 100% 100% 8 5 log10 PFU/g
tarsalis Colorado
1992
WEE McMillan Human, 100% 100% 9 4 log10 PFU/g
Canada, 1941
SIN Ar339 100% ~7 log10 PFU/g
CHIK CHIK/SIN CHIK (LR) structural All viruses grew 0%k 0% Low viremia, no 100%o 43o 100% protectionp [184]
genes, SIN to high titer replication in
(Ar339) CHIK/VEE knee joint or
nonstructral genes and CHIK/ brain 2–10 days
CHIK/VEE CHIK (LR) structural EEE grew to 0% 0% after SC 100% 136o 100% protectionp
p
genes , VEE (TC- titers ~4-fold inoculation of 224–260
83 vaccine strain) higher than 3–4 day-old
nonstructral genes CHIK/SIN in micen No
CHIV/EEE CHIK (LR) structural Vero cells 0% 0% viremia or 100% 116o 100% protectionp
genes , EEE (SA illness in 3 112–200p
subtype week-old C57/
BeAr436087) Bl6 or Swiss
nonstructral genes mice inoculated
SC with
5 3–5 8
log10 PFU
CHIK LR, human, La 45% 0% Higher viremia,
Réunion, 2006 replication in
knee and brain
of suckling
mice (see
above)
SIN Ar339 100%
VEE TC-83 100%
CHIK (181/25 attenuated 0% Higher viremia, 100% 144p
vaccine) replication in
knee and brain
of suckling
mice (see
above)
a
CD1 mice 7 days of age inoculated with 3 log10 PFU IC or (neuroinvasiveness study) SC in footpad
b
Author’s designation is SIN-83
c
Mice (unspecified strain and number), 6 days old, inoculated IC or (neuroinvasiveness) SC with 6.3 log10 PFU
d
Mice inoculated SC at 6 days old (publication may be in error and corectly mean 6-weeks old) were tested for antibody at day 21 prior to challenge
e
Mice (NIH Swiss), 10/group, 6 days old, inoculated IC or (neuroinvasiveness) SC with 6.7 log10 PFU
f
Brain virus titer, peak
g
NIH Swiss mice 6 days of age inoculated IC with 5 log10 PFU and assessed for illness and death
(continued)
Table 17 (continued)
h
Brain tissue virus titers 1–2 days after IC inoculation (6 day old mice)
i
NIH Swiss mice 8 weeks of age inoculated SC with 3.7–3.8, 4.7–4.8, or 5.7–5.8 log10 PFU, bled for antibody at 4 weeks and challenged IP with 6 log10 PFU of EEE(NA)
challenged
j
k
Mice (NIH Swiss) 6 or 4 days of age inoculated, respectively IC (neurovirulence) or SC (neuroinvasiveness) with ~5 log10 PFU
l
Brain virus titer 1–2 days after IC inoculation of 6 day old mice
m
Neutralizing antibody mean PRNT80 titers 6 week-old mice 4 weeks after a single SC immunization with 4.8 or 5.8 log10 PFU; mice challenged IN at 4 weeks with
5.3 log10 PFU WEE TBT235
n
3–4 day old Swiss mice inoculated SC with 5 log10 PFU
o
3 week old Swiss (N ¼ 10) vaccinated SC with 5.3–5.8 log10 PFU,bled 3 weeks after immunization for antibody
p
3 week old C57/Bl6 (N ¼ 5) vaccinated SC with 3.9, 4.9 or 5.9 log10 PFU,bled 3 weeks after immunization for antibody and challenged IN with 6.5 log10 PFU CHIK (Ross)
Table 18 Viremia, immune responses, and protective activity of chimeric vaccines using SIN virus as a vector for structural genes of VEE virus. Data from
Paessler et al. [181]
Virusa Age Dose/route (N) Viremia PRNT80 mean, vs. TC-83 virus Mice given single inoculation of vaccine,
(peak) challenged at 8 wks with VEE subtype ID
(ZPC738) 5.3–6 log10 PFU
Single Booster (N ¼ 6) Illness rate (Mortality) by challenge route
immunization
(N ¼ 6)
4 weeks 8 weeks 4 weeks 8 weeks SC (N ¼ 5) IN (N ¼ 5) IC (N ¼ 5)
p.i. p.i. p.i. p.i.
VEE(TC83)/SIN 6 weeks 5.7 log10 PFU SC 0 55 73 100 160 0% (0%) 20% (0%) 40% (20%)
VEE(TrD)/SIN (N ¼ 12 for 0 37 57 50 73 0% (0%) 0% (0%) 0% (0%)
VEE(ZPC)/SIN immunization; 0 187 253 253 487 0% (0%) 0% (0%) 0% (0%)
VEE(TrD)/SIN(SAAR, N ¼ 15 for 0 126 167 160 152 0% (0%) 0% (0%) 0% (0%)
mutated) challenge)
Mock 100% (100%) 100% (100%) 100% (100%)
a
See Table 15
422 T.P. Monath
challenge with virulent VEE ID virus by various routes, including the respiratory
route. Hamsters were also vaccinated with the chimeric viruses or with TC-83. The
animals given TC-83 developed a self-limited illness and had viremia ~1,000-fold
higher than those caused by the chimeras. All animals survived lethal SC challenge
with VEE ID. Overall, these results indicate that the chimeric constructs had an
improved safety profile compared to TC-83.
Chimeric EEE/SIN viruses were evaluated by Wang et al. [182]. The structural
genes were derived from North American and South American subtypes of EEE
virus, the latter being a naturally attenuated virus and antigenically distinct from
North American virus. In the 6-day-old mouse model, the chimeras were more
attenuated than parental EEE virus strains based on survival times (though mortal-
ity ratios were high) (Table 17). The chimeras did not cause viremia in adult mice,
but elicited high neutralizing antibody titers to the homologous virus and protected
against challenge. Although these viruses are interesting, considerable work will be
required to assess safety, and it is likely these viruses are insufficiently attenuated.
The authors included some benchmarks in their evaluation, including TC-83 and
the V3526 [170] VEE vaccine candidate. The EEE/SIN chimeras were intermediate
in neurovirulence compared to these vaccines. The chimeras were substantially
restricted for disseminated infection after oral feeding in one mosquito vector of
EEE virus (Ae. taeniorhynchus) compared to parental EEE and SIN viruses, but
disseminated infection occurred in another species (Ae. sollicitans) [186]. It was
concluded that chimeric alphaviruses may have selective competence for mosquito
vectors. This would be a potential issue if viremia levels in target hosts for
vaccination are sufficient for mosquito infection, since recombinational events
could result in the arthropod vector.
SIN was also investigated as a vector for WEE structural genes [183]. A first
generation construct was made using the SIN (prototype Ar339 strain) backbone
and structural genes from a recent mosquito isolate of WEE (CO92-1356). Two
second generation chimeric viruses were also constructed in which the amino-
terminal portion of the C gene which contains the RNA-binding domain was
replaced with the corresponding sequence of SIN or EEE virus (Table 17). The
chimeras were less attenuated than similar constructs with other structural genes
(e.g., from CHIK or VEE TC-83), showing that the structural gene inserts
determined the virulence phenotype. In suckling mice, the WEE(CO92-1356)/
SIN chimera was somewhat more attenuated than the other constructs, but still
killed 80% and 30% of the mice after IC or SC inoculation, respectively. It
replicated to lower titer in mouse brain, with titers similar to parental SIN. The
lower neuroinvasiveness in suckling mice compared to parental WEE (CO92-
1356) showed that chimerization per se was attenuating. Unfortunately the mor-
tality ratio for the SIN Ar339 vector in infant mice inoculated SC was not
presented, so that it is difficult to determine whether addition of WEE structural
genes in the chimera enhanced neuroinvasiveness compared to SIN virus. The
less attenuated chimeras with the amino-terminal C proteins from SIN or EEE
elicited higher neutralizing antibody responses and afforded higher grade
protection against a severe IN challenge with WEE virus than the more attenuated
WEE(CO-1356)/SIN chimera.
Finally, a chimeric SIN virus with structural genes from CHIK has also been
evaluated in mice [184]. Unlike the other alphavirus chimeras, the CHIK/SIN virus
was nonpathogenic in infant mice. This high level of attenuation is likely due to the
fact that the wild-type CHIK structural gene donor is relatively attenuated (45%
mortality ratio in suckling mice inoculated IC). Chimeric viruses were also con-
structed using VEE TC-83 and the South American subtype of EEE as vectors. The
chimeras containing CHIK structural genes were also not neurovirulent, whereas
parental gene donor strains were highly lethal, showing that the CHIK genes
modulated virulence in these backbone viruses as well. Attenuation of the chimeras
was compared to CHIK in suckling mice after SC inoculation. Wild-type CHIK
replicates in skeletal muscle and joints as well as brain. All chimeras were attenu-
ated compared to parental CHIK for replication in these tissues. The constructs
induced modest neutralizing antibody levels in Swiss and C57/Bl6 mice; however,
the CHIK/SIN construct was least immunogenic. Vaccinated mice resisted chal-
lenge with CHIK viruses.
The results of these investigations lead to the following conclusions: (1)
Chimeric viruses using SIN as a vector can be readily constructed and are
replication efficient in cells that could be used for manufacturing vaccines; (2)
While SIN is one of the less pathogenic alphaviruses for humans, and no fatal
human cases are recorded, its use as a vector is challenging due to lack of
biomarkers for attenuation for humans; (3) chimeric viruses in which structural
genes of heterologous alphaviruses are inserted have variable attenuation profiles
in mouse models; (4) in general, such chimeras are more attenuated than the
donor structural gene parent, but may be more virulent than the SIN backbone
parent; (5) the chimeras are highly immunogenic and protect against severe
challenge infection.
The observation that both the structural and nonstructural genes play a role in
determining virulence is supported by several investigations of VEE chimeras, in
which genes of low-virulence enzootic strains (less frequently associated with
equid or human disease) were combined with genes from epizootic, virulent
viruses [187, 188]. This observation comes as no surprise, based on the more
extensive experience with flavivirus chimeras described in this chapter, and it
simply emphasizes the requirement in vaccine development to carefully charac-
terize the biological phenotype of each construct not only in mice, but in other
models. The use of a benchmark vaccine virus as the backbone vector with
known biological characteristics in humans (or domestic animal species if they
are the target for vaccination), such as TC-83 [169] or possibly VEE V3526 [189]
or CHIK 181/25 [171], will facilitate development. This is the principle that was
used in the case of YF 17D, as it was useful to show that even when donor genes
from a virulent virus like JE (Nakayama) or WN (NY99) were inserted in the YF
17D backbone, virulence was attenuated or at least similar to that of the 17D
vaccine strain.
424 T.P. Monath
5 Use of Chimeric Viruses in Diagnostic Tests
The plaque reduction neutralization test is the most specific serological assay and
the presence of neutralizing antibodies (or a rise/fall in titer between paired sera)
provides the most precise means of differentiating past infections with close
antigenic relatives and sympatric viruses (like SLE and WN); moreover neutraliz-
ing antibodies are the best immune correlate of protective immunity in vaccine
studies. Unfortunately, use of the test is limited by a number of factors, including
poor plaquing efficiency of some viruses (e.g., dengue), select agent status (JE), and
the need for high-level biocontainment of pathogenic viruses, such as JE, SLE,
and WN. In contrast, the corresponding ChimeriVax™ viruses plaque very well,
and, being designated as BSL-2 agents, do not require high level laboratory
practices and containment. For this reason, they have been deployed by the Centers
for Disease Control and a number of State Health Laboratories for use in serological
tests. Several reports document their use for surveillance of human and equid cases
and infection in wildlife caused by St. Louis and Japanese encephalitis and West
Nile viruses [164, 190, 191].
Similar use of chimeric alphaviruses as safer diagnostic reagents was reported by
Ni and coworkers [192]. They compared attenuated VEE/SIN chimeras with
parental VEE viruses in a variety of serological tests, including PRNT, hemagglu-
tination-inhibition and complement fixation using subtype specific sera and found
that it functioned as well as the parental viruses. The study included the chimeras
incorporating structural genes from VEE IAB (TrD or TC-83), and VEE ID
(ZPC738) [177], as well as new chimeras constructed with enzootic VEE subtypes
IE and IF.
6 Recombination Events and Mutagenesis: Cause

for Concern?
Some authors have raised concerns about the potential for recombination of chime-
ric flavivirus vaccines in hosts or arthropod vectors undergoing dual or sequential
infections, for reversion of attenuation as a result of mutation, or for serious adverse
events in individuals with genetic or acquired susceptibility to infection [193].
Some of the critics are conflicted by virtue of their own work, which is aimed at
nonreplicating vaccine development [194]. Similar concerns could be raised for
chimeric alphaviruses, but these vaccines have not reached a point of development
that attracts adversarial commentary. The positive side of such criticism is that it
has stimulated a healthy debate and scientific exploration to determine the likeli-
hood and impact of untoward events. It is, of course, impossible to prove a negative,
so the probability and consequences of recombination, reversion, and spread
(arthropod infection) must be assessed and then put into context considering not
only risks but also the benefits of vaccination. The debate has been framed in
published responses to the theoretical concerns [194, 195], and will not be reiterated
here in full. The conclusion that may be drawn is that the risks of virus mutation (or
recombination) or host-dependent susceptibility to adverse events associated with
the chimeric Flavivirus vaccines are certainly no greater than (and are likely far less
than) those associated with use of nonrecombinant live vaccines which have long
been in use [191, 192].
Several studies were designed to investigate the phenotypic consequences of a
hypothetical “worst-case” recombination event resulting in the substitution of
genes from a highly virulent virus strain with genes from an attenuated chimeric
vaccine based on the YF 17D vector. The results are summarized in Fig. 13. To
investigate the outcome of a recombination event with a wild-type Australian
flavivirus and ChimeriVax™-JE (which was being tested clinically in Australia),
a virulent Kunjin (KUN) prME cassette was inserted into the YF17D backbone and
the SA14-14-2 prME genes of ChimeriVax™-JE were inserted into the KUN
backbone. The resulting chimeric viruses were somewhat less attenuated than the
original ChimeriVax™-JE virus but were similar to or more attenuated than parental
YF 17D. Both chimeras were attenuated compared to wild-type KUN virus.
A similar examination of potential worst-case recombination was conducted by
McGee and colleagues [30], who made chimeras incorporating virulent wild-type
Fig. 13 Construction of “worst case” chimeras representing hypothetical recombination events

with virulent flaviviruses or strains. (a). Chimeras representing intertypic recombinants between
ChimeriVax™ JE and Kunjin virus (from Pugachev et al. [32]). (b). Chimeras representing
recombinantional events between ChimeriVax™ DEN and YF Asibi (from McGee et al. [30, 31])
426 T.P. Monath
YF Asibi prME genes on the YF17D backbone, or conversely inserted YF17D prME
into YF Asibi (Fig. 13). In addition, wild-type DEN4 prME was inserted into the YF
Asibi backbone. The constructs were injected SC into cynomolgus macaques. The
YF17D/YF (Asibi) and YF (Asibi)/YF17D chimeras, as well as the DEN4/YF
(Asibi) chimera were highly attenuated compared to YF Asibi . The only finding
of note was minimal to mild liver inflammation and transient proinflammatory
cytokine elevation in monkeys given the YF17D/YF(Asibi) virus. Overall, these
studies showed that even in the unlikely event of an intertypic recombination event
involving an exchange of a full structural or NS gene set with a highly virulent virus,
the attenuating effects of the structural or nonstructural genes contributed by the
vaccine, together with the chimerization effect, would result in an attenuated
phenotype of the resulting virus.
An important feature of YF17D virus is its inability to cause disseminated
infection of Aedes mosquitoes after oral ingestion of a virus-containing blood
meal [196], whereas wild-type YF viruses are efficiently transmitted by these
mosquitoes. A number of chimeric vaccines with heterologous prME genes in the
YF 17D backbone were shown to be highly restricted for mosquito transmissibility
[77, 103, 140, 197], due, in part to multiple mutations in the YF 17D backbone that
occurred during passages during the development of 17D. Indeed, substitution of
YF Asibi prME genes in the YF17D backbone does not restore high grade infecti-
vity for mosquitoes [31, 198]. These findings, coupled with the very low viremia
levels in the vaccinated host that would preclude infection, as well as the resistance
of cells to superinfection with flaviviruses, makes recombination highly unlikely
and the outcome of such an event innocuous.
Less is known about the potential for recombination of chimeric alphavirus
vaccines, and little work has been done to assess the capacity for mosquito
transmission [180] or the molecular determinants underlying susceptibility of
mosquitoes to infection. Whereas intertypic recombination of flaviviruses in nature
resulting in a viable virus has not been described, recombination as an evolutionary
theme among alphaviruses is well documented [166]. The live, attenuated VEE TC-
83 vaccine virus was isolated from mosquitoes following a vaccination campaign in
horses [199], indicating the potential for transmission and recombination of a
vaccine that is not severely restricted for viremia and/or mosquito infection.
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Index
A Antigen 85B, 136, 137, 139, 140, 142,

A/Anhui/2/2005, 209 143, 147 150, 152
A/Ann Arbor/6/60, 208 30 kDa Antigen 85B major secretory
A/bar headed goose/Qinghai/2/05, 209 protein, 148
Absence of proofreading enzymes, 70 Antigen expression, 260
ACAM2000, 61 Antigens delivery, live vectors, 22 23
Acute hepatitis, 73 Antimicrobial agents, 72
Adenovirus 4, 7, 60 Antivector immunity, 349, 355
Advanced age, 75 A/Puerto Rico/8/34
Advantages A/PR/8/34 delNS1, 216
of BCG as a vector, 135 A/PR/8/34 NS1 99, 216
of live viral vaccines, 63, 66 67 aro, 117, 118
Adventitious viruses, 63, 72 74 aroD, 122
Adverse events, 59, 63, 69 72, 74 75 A/swine/Texas/4199 2/98
A/eq/KY/5/02 TX/98/NS1 126, 218
KY/02/NS1 73, 219 A/Sydney/5/97, 211
KY/02/NS1 99, 219 A/Texas/36/91
KY/02/NS1 126, 219 TX/91/NS1 126, 223
Aerobactin operon (iuc iut), 118 Attenuated investigational vaccines, 62
Age, 75 Attenuating mutation, 59, 63, 64
A/Hong Kong/491/1997, 209 Attenuation, 27, 28, 32 34, 39 41, 46, 47,
AIDS, 144, 145 62 65, 70, 71, 95 96, 315, 325
AIM2 (inflammasome), 66 Attenuation of virulence, 63
A/Indonesia/5/05, 213 A/turkey/Oregon/71 delNS1, 220
A/Leningrad/134/57, 210 Atypical environmental mycobacteria, 134
Allergic adverse events, 72 Avian leucosis or retrovirus sequences, 73
Allergy, 147, 150, 153 Avian leukosis virus, 73
Alphaherpesviruses A/Vietnam/1194/04, 213
EHV 1, 235 A/Vietnam/1203/04, 213
HSV 1, 236
HSV 2, 236 B
PRV, 235, 237 Bacillus Calmette Guérin (BCG), 17,
VZV, 235, 236 131 170
Alphavirus, 343 356 with altered intracellular pathway, 151
Alphavirus vaccines, 427 435 history of, 132 133
Animal derived proteins, 72 Bacterial contamination, 72
Animal models, 92 94 B/Ann Arbor/1/66, 208
Anterograde spread, 239 B cell activating factor (BAFF), 67

DOI 10.1007/978 3 0346 0277 8, # Springer Basel AG 2011
440 Index
B cell deficiency, 69, 71 Cold passaged temperature sensitive RSV

B cell responses, 67 (cptsRSV), 254
BCG. See Bacillus Calmette Guérin Commensals, 19
Biosafety, 99 100 Commercially licensed vaccines, 312
Bladder cancer, 132, 147, 149 150 Complement protein C1qB, 66
Booster, 317 Congenital rubella syndrome, 68
immunisation, 313, 318, 319 Control of such contamination, 72
Bovine serum, 72, 74 Control of viral RNA replication, 190
Bovine spongiform encephalopathy, 73 cellular chaperone “Jiv,” 190
Bovine tuberculosis, 143 144 CSFV attenuation, 197
Bovine viral diarrhea virus (BVDV), 74 effect on viral biotype, 191
Bronchiolitis, 250 NS2 3 processing, 190
B/USSR/60/69, 210 NS2 protease, 190
B/Yamagata/1/73 suppression of innate immune response,
Yam/88/del NS1, 217 191 195
Yam/88/NS1 80, 217 Coronavirus, 85 101
Yam/88/NS1 110, 217 Cottontail rabbit papillomavirus (CRPV),
157, 167
C Cowpox, 60, 61
5076 1C, 122 Cross protection, 196, 198
Cache Valley virus, 74 against HSV 1, 301
Caliciviruses, 73 CVD 1203, 120
Campenot cultures, 240 CVD 1204, 121
Candidate vaccines, 112, 115 123 CVD 1207, 121
Capsid deleted, 313, 316, 318, 319, 321, CVD 1208, 121
323 325 CXCL10, 66
Capsid deleted genomes, 313, 316 320, 325 Cynomolgus macaques, 394, 395
Capsid deleted nucleic acids, 319 Cytokine gene insertion, 259
Capsid deleted RNA, 316, 319 322, 324 Cytotoxic T cell responses, 59, 67
Capsid deletion mutants, 313, 317, 318
Capsid gene, 313, 316 318, 322 325 D
Carrell, 61 Defective helper, 348, 350, 351, 353, 354
Cathepsin S, 151 152 Defects in innate immunity, 75
Cell culture methods, 61, 73 delNS1, 215 217, 220
Chemokine receptor 5 (CCR5), 75 Dendritic cell, 214
Chicken pox, 43 Dendritic cells, 66
Chikungunya, 62 Dengue, 62
Chikungunya virus (CHIKV), 435 Dengue hemorrhagic fever (DHF), 386
Chimeric flavivirus vaccines Dengue vaccines
in diagnostic tests, 436 chimeric flavivirus vaccines,
principles for use and properties, 386 387
363 364 yellow fever (YF) vaccines
veterinary WN/YF vaccine, 418 425 construction approaches, 390
yellow fever 17D vaccine (see Yellow fever cynomolgus macaques, 394, 395
17D vaccine) growth curves of, 399
Chimeric vaccines, 315 monovalent, phase 1 clinical trial of,
ChimeriVax™ DEN, 387 400 388
Cholera, 273 287 mortality ratios, 393
Circoviruses, 73 mutations in, 392
Classical, 59 75 preclinical studies of, 391
Classical live viral vaccines, 59 75 viremia, 396
Codon deoptimization, 100 viremia and neutralizing antibody
Cold adapted live intranasal influenza, 69 responses, 394
Index 441
Dent, 120 F
Development of classical vaccines, 63 64 fep, 120
Diarrhea, 36, 37, 39, 42 fes, 120
Dicer, 212 Fetal bovine serum (FBS), 74
DicsA, 119, 120 Flagellin, 283 287
Different BCG strains, 134 Flavivirus
Differentiate infected from vaccinated chimeric vaccines
animals (DIVA), 223 in diagnostic tests, 436
Disease, 86 principles for use and properties,
DIVA. See Differentiate infected from 363 364
vaccinated animals (DIVA) molecular construction and rationale
dl5 29 virus, 299 300, 303 design
DNA based vaccine, 315, 322 324 attenuation and immunogenicity,
Drosha, 212 366
d106 vectors, 305 epitopes, 367
17D YFV. See Yellow fever 17D virus target product profile for, 368
recombination events and mutagenesis,
E 436 438
Eastern equine encephalitis (EEE), 434 virus like particles (VLPs), 319, 320
EcSf2a 2, 122 West Nile, 412 414
Efficacy WN/YF vaccine
of BCG, 132 135, 139, 143, 147 149, attenuation development, 414 418
154 165, 167, 169 site directed mutagenesis of, 413
of yellow, 66 veterinary chimeric development,
Electroporation, 348, 350, 351, 354 418 425
Embryonated egg, 61, 72 yellow fever 17D vaccine
Empirical passages in, 64 dengue, 386 387
Encephalomyocarditis virus (EMCV), dengue type 2 vector, 409 411
158, 167 DEN/YF vaccines, 387 400
Enders, J.F., 61, 63 JE/YF vaccine, 370 386
Enhanced antigen presentation, 151 152 multiple dengue serotypes,
Enteroviruses, 68, 71 single vector constructs, 411 412
Envelope (E) protein, 362, 364, 370, 371 NIAID, 400 409
Eradication of polio, 68 fnr, 120
Erns, 187, 192 195 Fomites, 69
dimerization, 194 Formalin inactivated RSV (FI RSV), 253
membrane anchor, 187, 193 Fowlpox virus Woodruff, 61
prevention of interferon response, Fusion proteins, 139, 141 142, 149,
195, 196 150, 157, 168
RNase, 187, 193
role for persistence, 195 G
secretion, 187, 193 Gag, Nef, Env, Pol, and RT, 153, 154
serum level, 193 Gates Grand Challenge grants, 72
Eukaryotic translation factor 2 alpha Gene activation signatures, 66, 67
kinase 4 (EIF2AKA) and solute Gene deletions, 89 98
carrier family 2, 66 NS1, 261
ETS, 66 NS2, 257
Evasion of innate immune response, 191 SH, 256
Erns, 192 195 Gene signatures, 66, 67
Npro, 192 Genetic basis of attenuation, 64 65
type 1 inferferon, 191, 192 Genetic instability, 63, 70 71, 74
Expanded Program of Immunization, 68, 72 Genomes, 312, 313, 315 321, 324, 325
Extracellular protein hypothesis, 137 Glycerol phenol preservatives, 72
442 Index
GM CSF. See Granulocyte macrophage IFNa. See Interferon a

colony stimulating factor IFN antagonist, 214 216
Goodpasture, 61 IFNg. See Interferon g
Granulocyte macrophage colony stimulating IL 2. See Interleukin 2
factor (GM CSF), 147 148, 150, IL 4. See Interleukin 4
151, 165 IL 15. See Interleukin 15
guaAB, 118, 121 IL 18. See Interleukin 18
Guinea pig model of pulmonary tuberculosis, IL 1a, 66
134, 137 Immune deficiency, 71, 75
Immune evasion, 302 303
H Immune response, 96
Hamster kidney cell substrate, 70 Immune stimulatory molecules,
Helminths, 134 135 coexpression, 303
Hemagglutinin Immunity
cleavage sites, 334 335 antibodies, 251
proteolytic cleavage, 334 elderly, 251
Hemorrhagic fever with renal syndrome, 62 IgG, 276, 277
Hepatitis C virus (HCV), 157, 167 infants, 250
Herpes simplex virus (HSV), replication secretory IgA, 276
defective mutant strains Immunocompromised individuals, 136, 145
amplicons, 305 306 Immunoprophylaxis, 253
cross protection, 301 Inapparent, apparent infection ratio, 59
durability, 300 Incidence, 59, 62, 68, 70, 71, 73 75
history of, 298 299 Inconsistent, 133 135, 143
HSV 2 strains, genetic diversity of, Infection, 250
303 304 Infectious cDNA clones, 64, 313 317, 325
immune evasion, 302 303 Infectious clones, 315, 318
immune stimulatory molecules, Infect mosquitoes, 69
coexpression of, 303 Influenza, 61 63, 67, 69
immunization, route of, 301 Influenza A vaccine, 69
pre existing immunity, 301 Influenza A viruses
replication impaired, 299 300 highly pathogenic avian, 334
safety, 300 low pathogenic avian, 334
single cycle mutant viral vaccine, 302 M2 protein, 336
as vaccine vector, 304 305 NS1 protein, 337
Herpes zoster, 43 Influenza vaccine
High mutation rates, 70 in adults, 211, 212
HIV, 62, 132, 144 146, 153 156, 164, 166 168 in children, 210 212
HIV/AIDS, 74, 75 cold adapted, 208 212
HIV positive persons, 144 146 in elderly, 207, 208, 212
H5N1, 209, 213, 219 221 in immunocompromised host, 212
Horsepox, 61 inactivated, 208, 209, 211, 212, 223,
“Hot spots” for mutation, 70 225, 226
HSV 1 gEnull vaccine, 243 Influenza virus
HSV 1 glycoprotein E, 238 matrix protein (M1, M2), 213
HSV 1 IgG Fc receptor, 242 nucleoprotein (NP), 208, 209, 212, 213,
Human papillomavirus, 62 220, 221
Hybrid live vector, 121 122 polymerase basic 1 protein (PB1),
Hydrolyzed porcine gelatin, 72 208, 221
polymerase basic 2 protein (PB2),
I 208, 220, 221
ICDDR,B, 282 Innate immune system, 67
icsA, 114, 117 121 Interferon a (IFNa), 66, 149 150
Index 443
Interferon g (IFNg), 139, 140, 142, antigens, delivery of, 22 23

147 152, 154 161, 163 168 history, 16 17
Interleukin 2 (IL 2), 147 150, 155, mice, 21 22
157 159, 161, 162, 165 modern approaches, 17 18
Interleukin 4 (IL 4), 135, 147 149, 159 rational attenuation, 18 20
Interleukin 15 (IL 15), 149 transmission electron micrograph,
Interleukin 18 (IL 18), 148 151 salmonella, 16
Internal ribosomal entry site (IRES), typhoid, 20 21
187, 197 Local inflammatory state, 122
Intranasal vaccine, 67 Louis Pasteur, 61
ipa, 114, 117, 121 LPS. See Lipopolysaccharide
J M
Japanese encephalitis virus (JEV) vaccines, Maassab, H.F., 63
62, 64, 65, 68 70, 312, 321 Maitland, 61
Japanese encephalitis (JE)/yellow fever (YF) Malaria, 159, 167
vaccine Malnutrition, 134
amino acid differences in, 371 MARTX, 286
clinical studies, 380 381 MAVS, 216
intranasal (IN) and IC routes, 377 Measles, 59 64, 67 69, 72, 73, 75
neurovirulence of, 373, 376, 377 Measles and yellow fever 17D, 73
non human primates of, viremia and Measles vaccine, 61, 67 69, 75
antibody response, 378 Microbial contamination, 71 72
phase 3 trial of, 385 micro RNA (miRNA), 212 213
seroconversion rate M2KO virus, 214
and geometric mean antibody titer Monkey safety test, 70
(GMT), 384 Motility, 275, 280, 282, 285 286
and mean neutralizing antibody mTOR, 66
levels, 383 Mucosal, 66 68
Jenner, E., 60 62 Mucosal immunity, 66, 67
Jennerian vaccination, 61 Multifocal leukoencephalopathy, 74
JEV vaccines. See Japanese encephalitis Multigenic nature of virulence, 64
virus vaccines Mumps, 59, 61, 62, 64, 68 70, 73
Junı́n Argentine hemorrhagic fever, 60 Mutant hybrid, 116
Mutation, 59, 63, 64, 70, 71, 74
K Mycobacterium bovis, 132, 133, 139, 141,
Key metabolic pathways, 117 143 144, 148
Koprowski, 63 Mycobacterium tuberculosis 30 kDa major
Kunjin (KUN) virus, 437 secretory protein, 136, 147
Kyasanur forest disease (India), 60, 62
N
L Nebraska Calf Diarrhea Virus, 61
Langat (LGT) virus, 421 424 Neuroinvasion, 70
Leishmaniasis, 160, 167, 168 Neurovirulence, 62 64, 70, 71
Leprosy, 139, 143 144 Neutralising antibody, 318, 323
Life long immunity, 66 NIH, 402 403, 407
Lipopolysaccharide (LPS), 115, 118 121 Nobel Prize, 63
Listeriolysin, 151, 152 Nonstructural (NS) proteins, 97 98, 362,
Live, 367, 409, 426
Live attenuated, 111 123 Npro, 192
Live attenuated vaccine, 116, 121 deletion, 192, 194
Live vaccines, 59 64, 66 75 prevention of interferon response, 194, 195
Live vaccines, role role for persistence, 194 195
444 Index
NS1 truncated vaccines epitope exchange, 198

in birds, 219 223 Erns dimerization, 194
in guinea pigs, 224 226 mutation of IRES or 30 UTR, 197
in horses, 219 Npro deletion, 192
in macaques, 209, 223 225 Pestivirus persistence, 188 196
in mice, 209, 210, 212 217, 221 acquired immunotolerance, 188
in pigs, 218 219, 224 226 BDV, 196
CSFV, 196
O evasion of interferon response, 191,
O antigen, 115, 117, 119, 121, 122 192, 194 196
Obstacles to development of live vaccines, 62 importance of viral biotype, 190, 195
20 50 Oligoadenylate synthetase1 (OAS1), 75 prerequisites, 186, 188 196
Oral polio, 63, 67 71 transplacental infection, 189, 196
Oral poliovirus vaccine, 70, 71 Pestivirus vaccines
Oral route, 115 chimeric viruses, 198
Orthomyxoviridae, 333 commercially available, 186
Other viral diseases, 154 158, 167 double mutants, 195, 196
Overattenuated, 70 marker vaccines, 199, 200
replicons, 199
P PGAI 42 1 15, 121
Packaging cell line (PCL), 348, 354 Phase 1 human study, 139
PAMP. See Pathogen associated molecular Phase I human trial of rBCG30, 140
patterns Phenotypic/genotypic stability, 261
Pandemic, 28 30, 36 12p40, IL 6 and interferon a, 66
Parainfluenza, 62 Pioneer Vaccine Candidates, 116 117
Parasitic diseases, 159 161, 167 168 PKR, 217
Passage histories, 65 Polio, 61 62, 68
Pasteur, 61 63 Polio vaccine, 63, 67 69, 71
Pathogen associated molecular patterns Polio vaccine excrete virus in feces, 69
(PAMP), 283 Poliovirus, 61 62, 68
Pathogenesis, 29 30, 36 38, 44, 51 Poliovirus vaccination, 68
Pathogenicity, 334 336, 338 Poliovirus vaccines, 64, 74
Pathogenicity island, 114, 117 Polymerase fidelity, 262
Pattern recognition receptors (PRR), 283 Polymerase gene, 70
PDK53 virus, 409 411 Polyomaviruses, 73
Perfringolysin O, 152 Pooled normal human serum, 73
Pestivirus Postvaccinal encephalitis, 70, 71
biotypes, 186 Precautions and contraindications, 75
chimeric viruses, 198 Pregnancy, 75
classification, 187 Prevenile®, 418
diseases, 186 p70 ribosomal S6 protein kinases, 66
fetal infection, 186 Primary dog kidney, 70
genome, 187 prM/E sub viral particles, 317
nonstructural proteins, 187, 188 Productenhanced reverse transcriptase
polyprotein, 187 (PERT), 73
receptor, 187 Proinflammatory cytokines, 66
recombinant viruses, 188, 198, 199 Proteases
replicons, 200 elastase, 336 339
structural proteins, 187 factor X, 334, 338
types of available vaccines, 186 furin, 334, 335
Pestivirus attenuation, 191, 194, 196 198 HAT, 334
change of glycosylation, 198 plasmin, 334
chimeric viruses, 198 proprotein convertases, 334, 335
Index 445
TMPRSS2, 334 Recombination, 99 100

tryptase Clara, 334 Recombination events and mutagenesis,
Protect, 315, 317, 319, 321 chimeric flavivirus vaccines,
Protection, 313, 315 317, 319, 321, 323 436 438
Protective immunity, 67, 68, 137 139, Reduced virulence of the 17D vaccine
145, 152, 158, 160, 168, 169, strain, 64
315, 322, 325 Regulatory pathway for live vaccines, 63
Protein, 134 150, 152, 154, 156 162, 164, Reinfection, 250
166 169 Replicase gene, 97
PRR. See Pattern recognition receptors Replication competent virus (RCV),
PsynII promoter, 213 349 354
Replication deficient, 317
Q Replicon, 346 348, 350 355
Quasi species, 70 RepliVAX D2, 321, 322
RepliVAX JE, 321
R RepliVAX WN, 321
Rabies, 62, 63 Respiratory disease, 28
RANTES, 75 Respiratory syncytial virus (RSV), 62, 63,
rBCG, 136 153, 155, 157, 160, 162 169 158, 167
rBCG30, 136 140, 143 145, 147, 148, 152 Respiratory syndrome, 85 101
rBCG (mbtB)30, 145 147 Responses, 59, 61, 66, 67, 75
rBCG (panCD) 30, 145 Retrograde spread, 239
rBCG/Antigen 85A, 139 140, 143, 152 Reverse genetics, 209, 214, 216, 218 220,
rBCG/Antigen 85B, 143 254
rBCG/Antigen 85B ESAT 6, 142 Reversion, 99 100, 261
rBCG/Antigen 85B Mpt64190 198 Mtb8.4, Revertant strains, 71
142 RF7, 66
rBCG/Antigen 85C, 140 141 Rhesus macaques, 416
rBCG/Cathepsin S, 151 152 Rift Valley fever, 62
rBCG expressing HIV and SIV antigens, RIG I, 66, 216
153, 164 RIG I like RNA helicase, 66
rBCG/72f, 142 Risk factors, 69, 71, 74, 75
rBCG/GM CSF, 147 148 RNA, insertion, 335
rBCG/IFNa, 149 150 RNA viruses, 70, 71
rBCG/IFN and rBCG30/IFN, Robbins, 61
rBCG/IFNg, 149 Rotavirus, 61, 62
rBCG/IFNg and rBCG30/IFNg, 148 Rotavirus vaccines, 61, 62
rBCG/IL 2, 148, 150 Rubella, 59, 63, 64, 68
rBCG/IL 15, 149 Rubella vaccines, 64
rBCG/IL 18, 148 151
rBCG/19 kDa protein, 142 S
rBCG/mIL 18, 150, 151 SA14 14 2, 64, 65, 68, 69
rBCG/MPB51, 143 Sabin, 63
Reassort, 69 SA14 14 2 Japanese encephalitis, 64, 68
Reassortment, 28 30 Salmonella, transmission electron
Recombinant BCG expressing foreign micrograph, 16
antigens, 152 153 Salmonella typhi, 20 21
Recombinant BCG expressing Salmonella typhimurium, 23
immunomodulatory cytokines, SARS CoV, 85 101
147 151 SC599, 120
Recombinant BCG overexpressing native SC602, 118 122
proteins and escaping the Scarification, 68
phagosome, 152 Schistosomiasis, 160, 167
446 Index
SCID mouse, 145 Strain EcSf2a 2, 122

Secreted G, 260 Strain 2457T, 120, 121
Secretory IgA, 115 Streptomycin dependent (SmD) mutants, 16
Secretory IgA antibodies, 67 Structure, 251
Semliki Forest virus (SFV), 344, 346, stxAB, 120
350 352, 355 SV40 in human cancers, 74
sen, 117, 119, 121 Systemic immunity, 67
Sen, 115
Sequenced, 64 T
Serious adverse events, 63, 70 T cell immunity, 31, 50
Serious infections, 72 T cell response, 59, 66, 67
Seroconversion rates, 68 Temperature sensitive virus population, 63
Serotypes, 36 39, 41 TH1, 134, 135, 148 150
Serum free cell culture methods, 73 TH2, 134, 142, 148, 150
Serum IgG, 115 Th1 and Th2 helper T cell response, 66
set gene, 114, 119, 121 Theiler, 61 63
Set expression, 121 Thermolabile, 72
Shed, 68 70 Thermostability, 63, 71 73
Shedding, requirements, 72
Shedding of live viral vaccines, 68 Th2 oriented helper T cell, 67
ShET1, 115, 121 Thymectomy, 75
ShET2, 120, 121 Tick borne encephalitis (TBE), 62, 420 421
Shigella, 111 123 TLR5. See Toll like receptor 5
Shigella boydii, 112 TNFRSF17, 67
Shigella dysenteriae, 112 Toll like receptor 5 (TLR5), 283 287
Shigella ETEC vaccine, 114 Toll like receptors (TLR), 66
Shigella flexneri, 112, 113, 116, 121 Toxoplasmosis, 161, 167
Shigella flexneri 2a strain T32 Istrati, 116, 117 bacterial diseases, 162, 168 169
Shigella sonnei, 112, 119 bacterial toxins, 169
Shingles, 43 lyme borreliosis, 169
Shingles vaccine, 236 Trans complemented, 322
Side effects, 98 99 Transcription factors, 66
Simian virus 40 (SV40), 74 Transcription regulation, 98
Sindbis (SIN) virus Trans encapsidated replicon, 320
biological features, immunogenicity and Transmission electron micrograph,
protective activity of, 428 432 salmonella, 16
viremia, immune responses, and Transmission, influenza virus, 224 226
protective activity of, 433 Trans packaged VLPs, 320
Sindbis virus (SINV), 344, 346, 350, 351, TRIM 25, 216
353, 355 Type VI secretion, 274, 286
Single round infectious particles (SRIPs), Typhoid, 20 21
313, 316, 318, 322 326
Site directed mutagenesis, of WN/YF U
vaccine, 413 Uncontrolled passage, 61, 70
SIV, 62 Upregulation of class I and II MHC, 148
SLC2A6, 67 Urabe strain of mumps, 70
Smallpox, 59 63, 67, 68, 72, 73
Smallpox vaccine, 60, 62, 67, 68, 72, 73 V
SRIP producing DNA vaccine, 322, 323, Vaccina virus Ankara (MVA), 213
325, 326 Vaccine associated paralytic poliomyelitis
Stability of, 71, 72 (VAPP), 69, 71, 74
Stabilizers, 73 Vaccines
STAT1, 66 inactivated, 336
Index 447
killed whole cell vaccine, 277 Viral vaccines approved for use, 60
live attenuated vaccine Viremia, 68 70, 396, 416, 433
Bengal 15, 281 283, 285 virF, 117
CVD101, 279 Virion morphogenesis, 252
CVD110, 279 Virulence genes, 94 95, 117
CVD 103 HgR, 279 Virulence plasmid, 114, 115, 117
Peru 14, 280 Virus, 88 89
Peru 15, 280 283, 285, 286 Virus attenuation, 315, 317
reactogenicity, 273 287 Virus like particles (VLPs), 313, 316,
Texas Star, 278 318 322, 325
Vaccines against TBEV, 312 Virus replicon particles (VRP), 347 354
Vaccine strategy, 112
Vaccinia, 61, 68, 72, 74, 75 W
VAPP. See Vaccine associated paralytic Wag533, 144
poliomyelitis Weller, 61
Varicella, 27 52, 59, 63, 64, 69, 72, 75 Western equine encephalitis
Varicella vaccine (Oka), 64 WestNile, 62
Vector, 343 356 West Nile virus (WNV), 412 414
Vectored RSV vaccines and YF vaccine
adenovirus, 257 attenuation development, 414 418
parainfluenza viruses, 258 site directed mutagenesis of, 413
paramyxoviruses, 258 veterinary chimeric development,
vaccinia virus, 257 418 425
Vectored vaccine, 87 88 World Health Organization, 72
Venezuelan equine encephalitis, 62 WRSF2G11, 119
Venezuelan equine encephalitis (VEE) virus, WRSS1, 119, 120
425, 426, 433 WRSs2, 119, 120
Venezuelan equine encephalitis virus (VEEV), WRSs3, 119, 120
344, 346, 347, 350 353, 355
Veterinary chimeric WN/YF vaccine Y
dengue type 4 D30 vectored vaccine, YEL AVD. See Yellow fever vaccine
419 420 associated viscerotropic adverse
dengue type 2 PDK53 vectored vaccine, events
420 Yellow fever, 59, 61 66, 68 75
flaviviruses, of medical importance, 425 Yellow fever 17D vaccine, 59, 65, 66, 70,
LGT(TP21)/LGT and LGT(E5)DEN4 71, 73 75, 367 370
viruses, 421 424 dengue, 386 387
TBE/DEN4 vaccine, 421 dengue type 2 vector, 409 411
tick borne encephalitis, 420 421 DEN/YF vaccines, 387 400
Vibrio cholerae JE/YF vaccine, 370 386
classical, 274, 277, 279 multiple dengue serotypes, 411 412
ecology, lytic phage, 276 NIAID, 400 409
El Tor, 274, 277 281, 286 Yellow fever 17D virus, 69 71, 73 75, 312,
non O1, non O139, 274 315
O1, 274, 276 281, 286 Yellow fever, influenza, 61
O139, 274, 276, 277, 282 Yellow fever vaccine associated neurotropic
virulence disease, 71
cholera toxin (CT), 275, 276, 278 280 Yellow fever vaccine associated viscerotropic
CTX Phage, 280 adverse events (YEL AVD), 70, 75
toxin co regulated pilus (TCP), 275 YF VAX®, 388, 396
ToxR, 275
ToxT, 275 Z
Viral transcription, 252 Zoster, 60

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Birkhäuser Advances in Infectious Diseases

Stefan H. E. Kaufmann, Max-Planck-Institut für Infektionsbiologie,

Manfred H. Wolff, University Witten/Herdecke, Faculty of Biosciences,

For further volumes:

Dr. Rino Rappuoli

ISBN 978 3 0346 0276 1 e ISBN 978 3 0346 0277 8

# Springer Basel AG 2011

Cover design: deblik, Berlin

Printed on acid free paper

Cambridge, MA, USA Philip R. Dormitzer

Part I Today’s Live Attenuated Vaccines

Live Vaccines and Their Role in Modern Vaccinology . . . . . . . . . . . . . . . . . . . . . 3

Live Attenuated Vaccines: Influenza, Rotavirus and Varicella

Classical Live Viral Vaccines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 47

Part II Genetically Attenuated Micro-Organisms as Vaccines

Recombinant Live Vaccines to Protect Against the Severe Acute

Live-Attenuated Shigella Vaccines. Is Encouraging Good Enough? . . . . . . 99

New Generation BCG Vaccines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 119

Part III Manipulating Host-Pathogen Interactions to Make Vaccines

Live Attenuated Influenza Virus Vaccines: NS1 Truncation

An Attenuated HSV-1 Live Virus Vaccine Candidate That

Live Attenuated Vaccines for Respiratory Syncytial Virus . . . . . . . . . . . . . . . 237

Live Attenuated Cholera Vaccines: Flagella and Reactogenicity . . . . . . . . 261

Part IV New Types of Replicating Vaccines

Replication-Defective Herpes Simplex Virus Mutant Strains

Nucleic Acid-Based Infectious and Pseudo-Infectious Flavivirus

Application of Cleavage Activation Mutants of Influenza Virus

Alphavirus Particle-Based Vaccine Vectors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 331

Recombinant, Chimeric, Live, Attenuated Vaccines

Ann M. Arvin Departments of Medicine, Pediatrics and Microbiology and Immu-

Clayton W. Beard Novartis Vaccines and Diagnostics, 350 Mass Ave,

D. Ewen Cameron Department of Microbiology and Molecular Genetics, Harvard

Marta L. DeDiego Centro Nacional de Biotecnologia (CNB), CSIC, Campus

Luis Enjuanes Centro Nacional de Biotecnologia (CNB), CSIC, Campus Univer-

Harvey M. Friedman Infectious Disease Division, Department of Medicine,

Yves Germani Unitié Pathogénie Microbienne Moléculaire INSERM U786/

Harry B. Greenberg Joseph D. Grant Medicine and Microbiology & Immunology,

Lindsay J. Hall Alimentary Pharmabiotic Centre, Biosciences Institute, Universi-

Marcus A. Horwitz Division of Infectious Diseases, Department of Medicine,

Jose M. Jimenez-Guardeño Centro Nacional de Biotecnologia (CNB), CSIC,

Alexander A. Khromykh Centre for Infectious Disease Research, School of

Hans-Dieter Klenk Institute of Virology, Philipps-University, Marburg,

David M. Knipe Department of Microbiology and Molecular Genetics, Harvard

John J. Mekalanos Department of Microbiology and Molecular Genetics,

Jose L. Nieto-Torres Centro Nacional de Biotecnologia (CNB), CSIC, Campus

Peter Palese Department of Microbiology, Mount Sinai School of Medicine, New

Natalie Pica Department of Microbiology, Mount Sinai School of Medicine, New

Philippe J. Sansonetti Unitié Pathogénie Microbienne Moléculaire INSERM

Juergen Stech Friedrich-Loeffler-Institut, Greifswald Insel Riems, Germany

John Steel Department of Microbiology, Mount Sinai School of Medicine, New

Michael N. Teng Divisions of Translational Medicine and Allergy & Immunology,

Michael V. Tullius Division of Infectious Diseases, Department of Medicine,

Elizabeth E. Zumbrun Center for Aerobiological Sciences, US Army Medical

Gordon Dougan, David Goulding, and Lindsay J. Hall

G. Dougan (*) and D. Goulding,

P.R. Dormitzer et al. (eds.), Replicating Vaccines, 3

2 Live Vaccines, a Brief History

Originally, the microorganisms on which live vaccines were developed originated

3 A Brief Summary of the Modern Perspective

previously unimaginable. Routine whole genome sequencing could be relatively