REVIEW

HIV

Immune correlates of vaccine protection

against HIV-1 acquisition
Lawrence Corey,1* Peter B. Gilbert,2 Georgia D. Tomaras,3 Barton F. Haynes,3
Giuseppe Pantaleo,4 Anthony S. Fauci5
The partial efficacy reported in the RV144 HIV vaccine trial in 2009 has driven the HIV vaccine field to define
correlates of risk associated with HIV-1 acquisition and connect these functionally to preventing HIV infection.
Immunological correlates, mainly including CD4+ T cell responses to the HIV envelope and Fc-mediated antibody
effector function, have been connected to reduced acquisition. These immunological correlates place immunological and genetic pressure on the virus. Indeed, antibodies directed at conserved regions of the V1V2 loop and antibodies that mediate antibody-dependent cellular cytotoxicity to HIV envelope in the absence of inhibiting serum
immunoglobulin A antibodies correlated with decreased HIV risk. More recently, researchers have expanded their
search with nonhuman primate studies using vaccine regimens that differ from that used in RV144; these studies
indicate that non-neutralizing antibodies are associated with protection from experimental lentivirus challenge as
well. These immunological correlates have provided the basis for the design of a next generation of vaccine regimens
to improve upon the qualitative and quantitative degree of magnitude of these immune responses on HIV acquisition.

INTRODUCTION
Over 30 years into the HIV-1 pandemic, the need for a globally effective
HIV-1 vaccine is more compelling than ever. Biomedical interventions
to reduce HIV-1 acquisition, such as population-based antiretroviral
therapy, large-scale circumcision programs, pre- and post-exposure
prophylaxis with tenofovir-based regimens for high-risk individuals,
and the use of antiretroviral drugs to prevent mother-to-child transmission of HIV-1 have favorably influenced the trajectory of HIV-1 infections in several populations throughout the world (1). However,
microepidemics of HIV-1 persist throughout all affected countries,
and overall population prevalence of HIV-1 remains relatively stable,
including in the United States and Europe (2, 3), and UNAIDS (Joint
United Nations Programme on HIV/AIDS) estimates that there were
2.1 million new infections in 2013 (4).
Vaccines are historically the primary public health intervention
for prevention of a wide range of infectious diseases and thus would
provide the most cost-effective, durable, and accepted approach to
reduce HIV-1 infection. However, developing a safe and effective
HIV-1 vaccine has proven to be a considerable scientific challenge
(5, 6). A milestone in the field of HIV-1 vaccine development was
achieved in September 2009 with the report of the RV144 trial, which
evaluated a regimen consisting of a replication-defective canarypox
vector (ALVAC) in combination with a recombinant gp120 protein
(AIDSVAX), administered intramuscularly to more than 16,000 heterosexual men and women at risk of HIV-1 infection in Thailand (7).
This regimen demonstrated a statistically significant, albeit modest,
reduction in HIV-1 acquisitions. Although the first-year vaccine efficacy (VE) approached 60%, efficacy waned over time and overall
VE over the 3.5 years of the trial was 31.2%.
1
HIV Vaccine Trials Network, Vaccine and Infectious Disease Division, Fred Hutchinson
Cancer Research Center, Seattle, WA 98109, USA. 2Statistical Center for HIV/AIDS Research
and Prevention, Vaccine and Infectious Disease Division, Fred Hutchinson Cancer
Research Center, Seattle, WA 98109, USA. 3Duke Human Vaccine Institute, Duke University
Medical Center, Durham, NC 27710, USA. 4Lausanne University Hospital and Swiss Vaccine
Research Institute, Lausanne 1011, Switzerland. 5National Institute of Allergy and Infectious
Diseases, National Institutes of Health, Bethesda, MD 20892, USA.
*Corresponding author. E-mail: [email protected]

The results of this trial were, in many quarters, quite unexpected.

Preclinical testing had indicated that ALVAC was less immunogenic
than many other candidate prototypes in human testing, such as recombinant adenovirus vectors (8, 9), and the AIDSVAX clade B/E protein
vaccine, used as a boost to the ALVAC vector, had proven ineffective
when used alone in preventing HIV-1 among intravenous drug users in
Bangkok (810). Moreover, a bivalent clade B gp120 recombinant protein
vaccine was ineffective in reducing acquisition of HIV-1 in men who have
sex with men (MSM) in North America, Australia, and Netherlands (11).
That the combination of these two immunogens would achieve a statistically significant reduction in HIV-1 acquisition among heterosexual
Thai men and women was met with surprise and, by some, skepticism,
especially because the number of infections was relatively small as compared to the size of the large-trial participant population studied (125 infections in 16,395 participants, of which 51 infections occurred in 8197
vaccinees).
Shortly after announcing the results, the organizers of the RV144
trial [the U.S. Military HIV Research Program (USMHRP) and the Thai
government] in collaboration with the National Institute of Allergy
and Infectious Diseases (NIAID), the primary funder of the trial, mobilized a major scientific effort to evaluate whether one could define
immune responses among vaccinated individuals that were associated
with protection, with the goal of developing biological underpinnings
and/or hypotheses that could lead to a better understanding of the
trial findings. Could these studies provide a road map to construct future iterations of HIV-1 vaccine regimens that could improve upon
the RV144 outcome? Defining such responses would markedly enhance the ability to construct vaccine regimens that could be tested
in areas of the world with markedly higher prevalence and higher exposure rates than those in Thailand.
Over the last 4 years, an international collaboration has produced
a substantial number of provocative scientific findings that raise the
hypothesis that antibody-mediated protection played the predominant role in the efficacy observed in the RV144 trial. This article will
outline these findings, describe how follow-up vaccine trials, planned
to be conducted predominantly in southern Africa, are being designed
to build upon these results, and hopefully provide both benchmarks

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and additional insights into how to develop the globally effective
vaccines that are needed to successfully reduce HIV-1 acquisition
worldwide.

DEFINING CORRELATES OF IMMUNITY

The process of defining correlates of immunity associated with vaccination in the RV144 trial was driven by both laboratory scientists
and statisticians and followed an analysis plan that included examining a large variety of laboratory assays so that statistical validity
could be defined from the available samples. The overall strategy used
a case-control format in which specimens at the week 26 visit (2 weeks
after last vaccination) from all 41 vaccine recipients who acquired
HIV-1 infection after week 26 were included (12). For comparison,
week 26 specimens from a random sample of 205 controls (vaccine
recipients who were HIV-1negative at the end of the study follow-up
period at month 42) were also included. In addition, specimens from
40 placebo recipients were included.
Before beginning the case-control study, a pilot study, which was
conducted between November 2009 and July 2011, evaluated 32 assays from 20 immunology laboratories of innate immunity, T cell,
and antibody responses on four criteria: low false-positive rate (comparing vaccine responses to baseline and to placebo responses),
broad variability of responses across vaccinees, low correlation of responses (nonredundant responses), and high reproducibility of
within-vaccinee replicate samples. The pilot study assessed samples
from both vaccine (n = 80 or n = 40) and placebo (n = 20 or n = 10)
recipients who were HIV-1negative at month 42. Seventeen assays
measuring a wide range of both antibody and T cell responses were
chosen for this initial case-control evaluation, from which six immune response variables were prespecified for the primary correlate
analysis, which then provided evidence for their association with VE:

(i) the binding of immunoglobulin G (IgG) antibodies to the variable

loops 1 and 2 (V1V2) region of gp120; (ii) the binding of plasma IgA
antibodies to envelope (Env); (iii) the avidity of IgG antibodies for Env;
(iv) antibody-dependent cellular cytotoxicity (ADCC); (v) neutralizing
antibodies; and (vi) the magnitude of CD4+ T cells specific for HIV-1 Env.
Immune responses (i) and (ii) were significantly associated with VE after
multiplicity correction. In secondary analyses without multiplicity correction, immune responses (iii) to (vi) were also significantly associated with
VE in vaccinees with low plasma IgA responses (12).

NON-NEUTRALIZING ANTIBODIES AND

CORRELATES OF EFFICACY
The observed protective efficacy in RV144 was surprising given the
low titer and narrow scope of neutralizing antibodies elicited by the
RV144 vaccine regimen (8, 12, 13). None of the sera from RV144 recipients, even at peak levels of antibody response, neutralized a panel
of 20 contemporaneous isolates of HIV-1 circulating in Thailand during
the course of the trial. Although neutralizing antibodies were uncommon, essentially all RV144 vaccine recipients developed binding
antibodies to gp120 (12). Titers of these antibodies peaked shortly after
the 6-month boost and, as seen in Fig. 1, rapidly waned over time, a pattern that tracked the degree of VE. To pursue whether particular regions
of gp120 were more closely associated with protection, several linear
and conformational peptides were evaluated in both the pilot and
case-control studies. Postvaccination sera tested against a linear peptide
array derived from the A244 CF01_AE strain of HIV-1 (used in both the
ALVAC vector and gp120 protein) exhibited a high binding pattern to
peptides in the V1V2 and V3 regions of the HIV-1 Env, a pattern of reactivity that differed considerably from HIV-1infected patients (14, 15).
The secondary case-control analysis indicated that IgG antibody responses to a series of relatively conserved amino acids on the crown

Prevalence of response

100

80

IgG gp120

60
CD4 + Env
40

20

IgG3 V1V2

IgG V1V2

0
0

Pox

Pox

Pox + Env
protein

Pox + Env
protein

12
Time (months)

24
IgG gp120
IgG V1V2 scaffold
IgG3 V1V2 scaffold
CD4 + Env

Fig. 1. Kinetics of vaccine-induced antibody response and vaccine protection in RV144. Schematic representation of selected immune responses to vaccination with RV144 regimen ALVAC (pox) followed by gp120 (protein).
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60

0.6

50

0.5

40

0.4

30

0.3

20

0.2

10

0.1

0.0

Prevalence

Estimated VE[s1s] (%)

Probability of HIV infection

of the V1V2 loop (residues 163 to 178) were associated with reduced
HIV-1 acquisition [relative risk (RR) = 0.57 for reactive versus nonreactive vaccinees; VE for reactive vaccinees = 43% over the 3.5-year
study]. Because of insertion of a herpes simplex virus glycoprotein D
sequence used for purification of the gp120, the A244 CF01_AE gp120 Env
in RV144 contained an N-terminal deletion of 11 amino acids in the HIV1 sequence that enhanced exposure of these V1V2 epitopes (16).
Vaccinees with the highest binding antibody titers were more
likely to be protected than those with lower titers, with a reasonably
linear association between the peak concentration to the V1V2 scaffold
and VE (Fig. 2A). Follow-up studies using a wide variety of V1V2 antigens including antigens derived from a variety of clade C isolates indicated similar associations between high binding to V1V2 and reduced

HIV-1 acquisition (17). Overall, about 84% of vaccine recipients developed antibodies to the V1V2 loop (14). Those with the highest magnitude exhibited greater protection from infection; those with the highest
titers to the original gp70-V1V2 protein in the upper third have a VE of
60% (12), compared to no protection for those with negative or lowest
third titers (Fig. 2B), a result that was recapitulated for titer responses to
eight other gp70-V1V2 proteins (average VE = 47% for the upper third
of responders) (17). The concentration of IgG to the V1V2 correlates in
RV144 was a median of 1.63 mg/ml, with those in the lower tertile below
0.98 mg/ml and those in the upper tertile, associated with a decrease in
HIV infection, having a titer of at least 2.98 mg/ml (Fig. 2C). Notably, the
V1V2 antibody response rate and magnitude waned over time in temporal association with the waning efficacy.

0.010

Placebo
Low
Medium
High

0.008
0.006
0.004
0.002
0.000
0

0
1
2
3
s1 = gp70-V1V2 response (log MFI)

12

24

36

Months

C
100

2.98
1.63
1

0.98

0.1
gp70.B (Case A2)-V1V2
IgG BAMA
RV144 uninfected vaccine recipients (n = 205)

100

10
5.30
3.04
1.91
1

CH58 mAb ( g/ml)

10

CH58 mAb ( g/ml)

CH58 mAb ( g/ml)

100

0.1

10

11.38
8.37
5.00

0.1

gp70.AE (92TH023)-V1V2
IgG BAMA
RV144 uninfected vaccine recipients (n = 205)

Fig. 2. Estimated VE in RV144. (A) Estimated VE in RV144 as a function

of the level of IgG binding antibody to gp70-scaffolded V1V2 in a model
assuming VE of 0% in vaccinees with no V1V2 antibodies (black line) and
the distribution of IgG levels among vaccinees (histogram). MFI, mean
fluorescence intensity. (B) Kaplan-Meier curve of the probability of acquiring HIV infection in vaccine recipients with low, medium, and high
V1V2 scaffold IgG antibody responses, measured by binding antibody

tagsAE (A244)-V1V2
IgG BAMA
RV144 uninfected vaccine recipients (n = 205)

multiplex assay (BAMA) at week 26. (C) The median, upper, and lower
bounds of antibody concentrations to three of the V1V2 antigens that
were correlated to VE: the left panel shows the cross-clade clade B gp70
levels; the middle panel shows the V1V2 response to the AE isolate in the
ALVAC vector used in RV144; and the right panel shows the V1V2 responses to the AE gp120 used in the protein boost in RV144. mAb, monoclonal antibody.

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Epitope mapping of the antibodies to V1V2 indicated that much of
the immune response was directed at a linear epitope interval including
a lysine residue at amino acid 169 in the Env V2 region. Antibodies
from RV144 vaccinees that bound to the K169 V2 region did not neutralize nor capture hard to neutralize (tier 2) AE viruses but did bind to
Env on tier 2 AE virusinfected CD4 T cells and mediate ADCC. Although antibodies to the C1 region themselves did not correlate with
decreased transmission risk (15), addition of conformational C1-specific
antibodies from RV144-induced memory B cells to A244 CF01_AE
infected CD4 T cells synergized with RV144 V2 antibodies for
enhanced ADCC activity (18). These data give rise to the hypothesis
that one candidate of the protective antiHIV-1 antibody effector
function induced by the RV144 vaccine was a nonbroadly neutralizing
and most likely Fc receptor (FcR)mediated action that included ADCC
activity (see below) (19). Isolation of antibodies that bind to the V2
region around K169 demonstrated that all K169-directed antibodies
from humans (20) and rhesus macaques (21) after the RV144 vaccine
regimen use a l light chain CDR2 (complementarity determining
region 2) glutamic acidaspartic acid (ED) motif to bind to K169. Moreover, this l light chain motif is conserved throughout primate phylogeny, suggesting an evolutionary advantage to this dominant recognition
mode. The response to Env induced by the RV144 vaccine regimen was
remarkably dominant to this light chain motif. It is of interest that V1V2
responses were not substantially elicited by the DNA/Ad5 regimen used
in the HIV Vaccine Trials Networks (HVTNs) HVTN 505 vaccine
trial, although these sequences were present in two of the three Env vaccine constructs (22). The low level of V1V2 scaffold responses in association with a vaccine regimen in which no protection was seen suggests
the potential importance of responses to the V1V2 scaffold in protection against HIV acquisition. Finally, in the secondary analysis, strong
but transient linear V2 IgG responses, especially against V2 in A244
CF01_AE, were associated with a lower risk of HIV-1 infection in
RV144 vaccinees.

LINKING OF THE IMMUNE RESPONSE WITH IMMUNE

PRESSURE ON THE VIRUS
Genetic sieve analysis of the viral isolates from subjects who became
HIV-1infected in RV144 revealed that isolates from vaccinated
subjects were less likely to have a lysine at K169 of the Env V2 region
than placebo recipients, and VE was significantly higher against
HIV-1 manifesting a lysine at K169 than against HIV-1 with a different
residue at position 169 (23). This lysine is found in more than 85% of
circulating HIV-1 strains in Thailand, yet was present in only 66% of
RV144 HIV-1infected vaccine recipients, suggesting vaccine immune
pressure at this region of the virus. Thus, both the immunological and
virological data point to the V2 region of HIV-1 as a vulnerability point
for the virus and a target site of protective antibodies associated with VE
of the RV144 regimen.
A similar correlation between vaccine-induced antibody responses
to the third variable (V3) loop region of the circulating A244 CF01_AE
isolates in Thailand and genetic pressure on circulating strains of
HIV-1 in vaccinees was also detected (24). The RV144 regimen elicited
V3-specific antibodies at week 26 (peak point of antibody response,
2 weeks after the fourth dose) in 88 to 95% of recipients, depending upon
the antigen used. Similar to the V2 antibody response, the V3 antibody
response was largely cross-reactive among strains, and similar to the V2

antibody response, the V3 antibody levels markedly declined over time

(reduction in prevalence at week 52 of 50 to 70% depending on the
antigen). Past studies indicated that such antibodies may demonstrate
some cross-clade in vitro neutralizing activity (25). Analogs of the viral
isolates of the A244 CF01_AE breakthrough viruses from 43 vaccine
and 66 placebo recipients demonstrated an 85% VE against viruses with
amino acids mismatching the vaccine at V3 site 317 (P = 0.004 for higher
VE than against F317 matched HIV-1), and 52% VE against viruses
matching the vaccine at V3 site 307 (P = 0.004 for higher VE than
against I307 mismatched HIV-1). When point mutations similar to
those found in breakthrough isolates were made in the V3 crown at isoleucine site 307, the titers of antibodies in RV144 vaccinees to the V3
crown were markedly reduced, demonstrating the specificity of the
RV144 regimen to this region of V3 (24). Together, these data indicate
that the RV144 regimen induced both V2- and V3-specific antibodies
that imposed immune pressure on infectious viruses and provide strong
support for antibody-mediated protection. These observations also
provide both a qualitative and a quantitative basis for evaluating future
vaccine regimens that should be considered for advanced clinical
development.

ISOTYPE-SPECIFIC ANTIBODIES AND PROTECTION

Because IgG subclasses have been implicated in both viral- and vaccineinduced protection (26, 27), a detailed evaluation was initiated of IgG
subclassspecific antibodies comparing the RV144 regimen with that
of the ineffective AIDSVAX B/E gp120 regimen used in VAX003 and
VAX004. ALVAC priming with gp120 boosting was associated with both
significantly higher and broader IgG3 responses to the HIV-1 Env glycoprotein and significantly lower IgG2 and IgG4 response rates than gp120
vaccination alone (27, 28). IgG3 antibody response rates to V1V2 were
higher in RV144 than in VAX003 recipients. Vaccinees with high IgG3
responses were shown to have higher ADCC antibodies, and IgG3 antibodies to the V1V2 region were associated with VE (RR = 0.32 for vaccinees positively versus negatively responding to a gp70-V1V2 scaffold
protein with a lysine placed at position 169; P = 0.003), especially to
viruses in which V169K was present (27). IgG3 responses to HIV-1 Env
declined rapidly over time from 79% prevalence at peak immunity
(week 26) to 0% at year 1, correlating with the rapid loss of VE during
this time period. Notably, there was no significant correlation between
individuals with high V1V2 IgG3 responses and those with high IgG2
and IgG4 responses, suggesting that there are unique determinants to
IgG isotypes. These associations between IgG3 antibodies to HIV-1 Env
and protection offer a potential discrimination between ALVAC priming/
gp120 versus gp120 alone. Subclass-specific antibody responses differ
between rhesus macaques and humans; hence, evaluation of this
potential correlate of risk must continue to be done within the context
of human vaccine trials.

IgA INHIBITORY ANTIBODIES

One puzzling observation from the primary correlates of protection
studies associated with RV144 was the strong direct correlation between
high serum IgA antibodies to specific HIV-1 Envs and an increased risk
of infection. This response was one of the strongest statistical associations influencing VE (estimated VE = 0% for the highest third IgA

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responding vaccinees compared to estimated VE = 43% for the lower
two-thirds of IgA responding vaccinees) (12). Subjects with IgA binding
levels to HIV-1 Env >3000 MFI had a VE of 0%. Why certain serum IgA
antibodies were associated with lack of protection was at first perplexing. Epitope mapping indicated that some of the serum IgA antibodies
seen after vaccination bound to the same conformational C1 region of
gp120 that ADCC antibodies were shown to bind (29, 30). Seventy-five
percent to 90% of RV144 vaccinees exhibited ADCC activity after vaccination. Much of this activity was blocked by C1 regionspecific antibodies to the A244 CF01_AE isolates, suggesting that the IgA antibodies
inhibited functional ADCC activity, providing a direct hypothesis for
the inverse correlation between high serum levels of IgA and increased
HIV-1 acquisition. Moreover, natural IgA antibodies to the C1 region
isolated from RV144 vaccinees also blocked the mediation of ADCC
by RV144 IgG antibodies to the same epitope (30). Additional data
supporting the presence of inhibitory antibodies are that individuals
with low levels of IgA antibodies to Env also had high levels of antibodies to V3 peptides. VE approached 70% among the subgroup of
vaccinees who had low to no serum IgA responses to gp120 and high V3
antibodies (15, 24). High serum IgA responses to HIV-1 gp120 were
also seen with the DNA/Ad5 regimen used in the HVTN 505 vaccine
trial, a regimen that showed no VE in MSM in the United States (22).
Development of regimens that do not induce inhibitory antibodies provides a potential strategy for improving VE, especially if such alterations
resulted in enhanced induction of functional antibodies that mediate
ADCC or neutralization. One of the impediments to a more detailed
evaluation of ADCC responses and HIV vaccine protection is the lack
of information on the correlation of ADCC assays between nonhuman
primates (NHPs) and humans. Greater attention to such issues may allow the field to pursue the observed connection between ADCC and VE
more completely.

important determinants of the non-neutralizing antibody effector function induced by RV144 vaccination. However, the mechanistic link between such a polymorphism and antibody-mediated immune response
remains undefined.

PASSIVE PROTECTION STUDIES USING

NON-NEUTRALIZING MONOCLONAL ANTIBODIES
AFTER RV144 VACCINATION
Although studies using neutralizing antibodies to HIV-1 have shown
protection in NHP models, studies defining the efficacy of nonneutralizing antibodies in protection against experimental challenge
in NHP models are incomplete (35). In established SIV infections in
macaques, polyclonal non-neutralizing anti-SIV IgG, administered
by passive infusion, induced ADCC and an associated decline in SIV
viremia (36). Because the passively administered sera had low levels
of neutralizing activity, the protective nature of ADCC onlymediating
antibodies was not apparent. To explore whether nonbroadly neutralizing antibodies can protect against rapid disease progression, Binley
et al. (37) infused polyclonal anti-SIV IgG purified from macaques
infected with mac251 SIV, at a concentration of 170 mg/kg, into two rhesus
macaques who were rapid progressors infected with the mac251 SIV
strain. After the infusion, the anti-Env antibody levels in these animals
were temporarily restored to levels typically found in nonrapid progressors, and these antibodies killed SIV-infected cells, presumably through
an effector function such as ADCC.
Although non-neutralizing ADCC-mediating monoclonal antibodies
reactive with the A244 CF01_AE infected tier 2 CD4+ T cells are available for testing in passive protection trials, these studies have been hindered by the lack of availability of R5 tier 2 SHIV-1s with the K169 and
full V2 epitope of putatively protective V2 antibodies from RV144.
SHIV-1s are under development (38, 39).

FcR FUNCTIONS AND THEIR ROLE IN HIV-1 ACQUISITION

In 2007, Hessel et al. demonstrated that the Fcg receptor binding
function of a monoclonal antibody to HIV-1 was critical for the ability
of this antibody to protect against experimental challenge with simian/
human immunodeficiency virus (SHIV) in a monkey model (31). More
recently, in a paper evaluating the mechanisms by which a live, attenuated simian immunodeficiency virus (SIV) vaccine protects against
experimental SIV challenge, gp41 antibodies were concentrated by
neonatal FcR in cervical and vaginal epithelium, and such local antibodies transported to epithelial surfaces through the neonatal FcR
were an important correlate of protection (32). Moreover, a second
correlate in this model was the preexistence of virus-specific immune
complexes that correlated with the FcgRIIB inhibition in the epithelium lining the cervix, and these complexes blocked CD4+ T cell recruitment and likely inhibited virus expansion (33). In RV144, individuals
with a CT or TT at a single-nucleotide polymorphism in the FCRGR2C
gene had an estimated 91% VE against HIV-1 with lysine at position
169 in the V2 loop, compared to 15% VE for individuals with a CC
(19). This suggests that RV144 responses selectively blocked infection
of HIV-1 isolates with lysine at position 169 only among CT or TT subjects (19). The prevalence of these polymorphisms is seen in only 28%
of Thais and in an estimated 49% of South Africans (34), perhaps predicting higher efficacy for RV144-like vaccines in Africa. These data
support the assertion that both vaccine-induced and host factors are

T CELL RESPONSES
Recent studies have shown that CD4+ T cell immune responses to
HIV-1 Env independently influenced VE in the RV144 trial (12, 40).
Sixty percent of RV144 recipients who had exhibited CD4+ T cell
responses to Env recognized the V2 peptides, and intracellular cytokine staining confirmed that these responses were mediated by polyfunctional effector memory CD4+ T cells, which produced more than
one cytokine in 58% of the samples, predominantly interleukin-2 (IL-2)
and tumor necrosis factora (TNF-a). The predominant interferon-g
(IFN-g) response (25%) was to the Env V2 region at amino acid positions 145 to 208. The main peptide recognized in the vaccine group
contained the integrin a4b7 binding motif, a region of the Env involved
in the initial encounter of HIV-1 and CD4+ T cells (12). Using more
sophisticated single-cell analyses, vaccinees with CD4+ T cells that secreted IL-2, TNF-a, IFN-g, IL-4, and CD154 to HIV-1 Env peptides
had a reduced rate of infection (RR = 0.58; P = 0.006) versus those who
did not make such a polyfunctional response. Moreover, this response
was an independent correlate of infection after accounting for the primary correlates of IgG binding to V2 and IgA binding to Env (41).
These data indicate the potential importance of initiating a strong helper
T cell response with vaccination, and study of the CD4+ T cellB cell
interaction may provide insights into enhanced immunogenicity.

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BUILDING UPON THE CORRELATES FOR THE
CONSTRUCTION OF NEXT-GENERATION VACCINES
With only one vaccine approach demonstrating efficacy against HIV
acquisition, there is at present uncertainty as to whether the abovedescribed correlates will be effective guideposts for a globally effective
HIV vaccine (42). The differences between the immune responses in
RV144 and those seen in HVTN 505 and VAX003, two VE trials with
no observed efficacy against HIV-1 acquisition, provide indirect evidence of the potential importance of the types of non-neutralizing
antibodies important in reducing HIV-1 acquisition. Thus, although
the possibility of a false-positive result always exists in any given study,
the confidence that RV144 demonstrated real efficacy has increased
substantially on the basis of the viral sieve and immune correlate analyses. A series of recent studies in NHP with different vaccine prototypes
such as a replication-deficient Ad26 alone or in combination with
modified vaccinia Ankara (MVA) have shown a correlation between
non-neutralizing antibodies and protection from experimental challenges with both SIV and SHIV (Table 1) (43). Addition of a trimeric

gp120 protein boost increases several antibody effector functions

and is associated with enhanced protection from experimental challenge (4345). These NHP studies support the notion that enhancing
antibody effector functions and CD4+ T cell responses offer the potential for providing HIV-1 VE.
It was recognized that a series of more rapid iterative trials were
needed to address the multitude of scientific questions to be answered
from the immune correlate analyses (46). This was best done by conducting test-of-concept efficacy trials in populations with higher acquisition rates than those in Thailand, such as in sub-Saharan Africa,
where clade C viruses predominate (47). Because the antibody responses
associated with protection in RV144 were predominantly clade-specific,
the use of regimens that would provide clade C coverage should be
used. Moreover, improving the durability of the Env-specific immune
responses was essential (Fig. 1). Studies with HIV Env and other recombinant proteins have suggested that alternative adjuvants such as MF59
and monophosphoryl lipid Alike adjuvants such as AS01B can enhance
the durability of immune responses over those seen with alum-adjuvanted
proteins (48, 49). The degree of somatic hypermutation, breadth of

Table 1. Comparison of immune responses that influence VE in RV144 versus past and ongoing HIV vaccine trials.
Immune response identified as a
correlate in RV144

Responses in non-RV144
efficacy trials

Responses in vaccines in
current development

Total IgG to V1V2 scaffold

Lower in HVTN 505

compared with RV144

Higher titers in DNA/NYVAC/gp120 than RV144.

Correlates of protection in heterologous NHP
challenge studies with Ad26/Ad35 and
Ad26/MVA studies.

Serum IgA to gp120

(higher IgA = lower VE)

Higher IgA (including IgA to A1ConEnv)

in HVTN 505, compared to RV144

Administration of gp120 at onset of priming with

NYVAC and DNA/NYVAC markedly lowers
serum IgA to gp120. DNA/MVA priming
has lower IgA.

IgG3 to V1V2

Lower in HVTN 505 and

VAX003 than RV144

Under evaluation.

ADCC activity

Minimal ADCC in HVTN 505

ADCC correlates with protection in NHP using

Ad26 trimeric gp120. High ADCC in
DNA/MVA regimen.

Higher frequency in RV144 compared

with HVTN 505

Clade C regions under study.

Not measured in HVTN 505 program. Env

IgG avidity with low IgA correlated
with decreased risk of infection in RV144

DNA/MVA-containing regimens have

high avidity. Other products
under study.

Different cytokine profile in

HVTN 505 versus RV144

DNA/NYVAC and Ad26/MVA increase prevalence

and magnitude of Env-specific CD4+ T cells.

Tier 1 neutralizing
antibodies
High avidity to gp120

CD4+ T cells with

polyfunctional response

Table 2. Post-RV144 vaccine combinations.

Improve frequency, magnitude, and
polyfunctionality of CD4+ T cell response
to HIV-1 Env products
ALVAC (ZM96) DNA
NYVAC (ZM96) DNA
Ad26 MVA

Improve clade-specific antibody

responses to gp120

Improve durability of antibody

response through adjuvants

Bivalent clade C recombinant gp120 protein

(strains 1086 and TV-1) construct

Alum, MF59, AS01B

gp140 clade C trimeric protein gp140

mosaic trimeric protein

Alum, AS01B

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immune response, and functional activities of antibodies induced by
protein immunogenicity can be influenced by adjuvants (50). Hence,
comparative studies of recombinant HIV clade C Env proteins (gp120
and trimeric gp140) adjuvanted with AS01B, MF59, and alum adjuvants
(Table 2) are under way. In addition, more frequent boosting of immune responses by dosing the vaccine regimen more frequently is
under evaluation.
Another approach for improving upon RV144 is to use more immunogenic vector platforms to improve B and T cell priming, especially to Env proteins. Second-generation regimens, including a bivalent
ALVAC (env and gag-pol on separate constructs), NYVAC/DNA constructs, and combinations of replication-incompetent adenoviruses
such as Ad26 in combination with MVA, are in clinical development
(5153). One hope from the ALVAC and Ad26/MVA programs is that
these different vaccine approaches will elicit immune responses that
correlate with each other and with protection, and hence provide a
critical surrogate marker for future vaccine development. The finding that non-neutralizing antibody responses such as binding antibodies to HIV Env, ADCC, and other non-neutralizing functional
antibodies are correlates of protection in these NHP experimental
challenge studies provides some optimism for this approach. Validation
of such a marker would allow the field to do bridging immunogenicity studies as a way of initiating a comprehensive global vaccine
strategy.
Although these developments occur in defining non-neutralizing
antibody approaches to HIV-1 protection, there is also a concerted international effort toward developing immunogens that will elicit
broader neutralizing antibodies than currently demonstrated by immunization with any of the current vaccine prototypes (54). The lack
of broadly neutralizing antibodies elicited by the RV144-like regimens
is an acknowledged deficiency in the immune profile of these approaches. Enhancing basic and translational scientific programs in attempting to elicit such broadly neutralizing antibodies is an important
component of the research agenda of the HIV vaccine effort. Recent
developments in developing stable HIV-1 Env trimers provide evidence
for progress in this area of research (55, 56). The continued elucidation
of novel targets of binding antiHIV-1 neutralizing antibodies is also
providing new insights on how to develop immunogens that can elicit
such antibodies. This agenda includes the use of passively administered
broadly neutralizing antibodies on a 1 to 3 monthly basis (antibodymediated prevention) to reduce HIV acquisition (57, 58). A test-ofconcept study to demonstrate how effective such antibodies would
be in reducing HIV acquisition, and what level of neutralizing activity
is required to reduce HIV-1 acquisition, is an important milestone for
the development of an effective neutralizing antibodybased vaccine.
In summary, the past 3 years have seen an unprecedented scientific effort that has provided a wealth of new information on the immune responses that are potentially associated with and responsible
for protection from HIV acquisition. New understanding of regions of
the HIV Env heretofore largely ignored (such as the V2 loop) and nonneutralizing antibody responses, as well as the role that CD4 T cells play
in B cell maturation, are important concepts that have emerged from
these studies. These data have provided hypotheses and new benchmarks for the development of new vaccine strategies (59). Whether
increased VE will be achieved by these efforts remains to be determined;
however, this collaborative effort has produced a momentum and series of immune targets that will hopefully lead to an effective global
vaccine effort.

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Acknowledgments: We thank A. deCamp, S. Grant, Y. Huang, Y. Huang, and H. Janes for the
statistical analysis for Fig. 2; the HVTN Laboratory led by J. McElrath at Fred Hutchinson Cancer
Research Center; D. Montefiori at Duke University for the neutralizing antibody assays; and
N. Michael who directed the USMHRP RV144 study and its correlates program. We thank A. Ferrara
and M. Miner for help in the preparation of this manuscript. Funding: Research reported in
this publication was supported by the NIAID of the NIH under award nos. UM1AI068618, U01AI
06861405, and UM1AI068635; Duke Center for AIDS Research Immunology Core (AI064518);
and the Bill and Melinda Gates Foundation (OPP1040758, OPP1032144, and OPP1068333). The
content is solely the responsibility of the authors and does not necessarily represent the official views of the NIH.
Submitted 12 June 2015
Accepted 14 August 2015
Published 21 October 2015
10.1126/scitranslmed.aac7732
Citation: L. Corey, P. B. Gilbert, G. D. Tomaras, B. F. Haynes, G. Pantaleo, A. S. Fauci, Immune
correlates of vaccine protection against HIV-1 acquisition. Sci. Transl. Med. 7, 310rv7 (2015).

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