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 Vaccine 39 (2021) 1310–1318

Contents lists available at ScienceDirect

Vaccine
journal homepage: www.elsevier.com/locate/vaccine

Proof-of-concept of a low-dose unmodified mRNA-based rabies vaccine

formulated with lipid nanoparticles in human volunteers: A phase 1 trial
Cassandra Aldrich a, Isabel Leroux–Roels b, Katell Bidet Huang c, Mihai Alexandru Bica c,1, Edde Loeliger c,
Oliver Schoenborn-Kellenberger c, Lisa Walz c, Geert Leroux-Roels b, Frank von Sonnenburg a,2,
Lidia Oostvogels c,⇑
a
Division of Infectious Diseases and Tropical Medicine, University Hospital, LMU Munich, Munich, Germany
b
Center for Vaccinology, Ghent University and Ghent University Hospital, Ghent, Belgium
c
CureVac AG, Tübingen, Germany

a r t i c l e i n f o a b s t r a c t

Article history: Introduction: In a first-in-human study immune responses to rabies virus glycoprotein (RABV-G)-mRNA
Received 23 October 2020 vaccine were dependent on the route of administration, necessitating specialized devices. Following suc-
Received in revised form 22 December 2020 cessful preclinical studies with mRNA encapsulated in lipid nanoparticles (LNP), we tested an mRNA-LNP
Accepted 24 December 2020
formulation (CV7202).
Available online 22 January 2021
Methods: In this phase 1, multi-center, controlled study in Belgium and Germany we enrolled 55 healthy
18–40-year-olds to receive intramuscular injections of 5 lg (n = 10), 1 lg (n = 16), or 2 lg (n = 16)
Keywords:
CV7202 on Day 1; subsets (n = 8) of 1 lg and 2 lg groups received second doses on Day 29. Controls
Vaccine
mRNA
(n = 10) received rabies vaccine, Rabipur, on Days 1, 8 and 29. Safety and reactogenicity were assessed
Lipid nanoparticles up to 28 days post-vaccination using diary cards; immunogenicity was measured as RABV-G-specific
Rabies neutralizing titers (VNT) by RFFIT and IgG by ELISA.
Results: As initially tested doses of 5 lg CV7202 elicited unacceptably high reactogenicity we subse-
quently tested 1 and 2 lg doses which were better tolerated. No vaccine-related serious adverse events
or withdrawals occurred. Low, dose-dependent VNT responses were detectable from Day 15 and by Day
29%, 31% and 22% of 1, 2 and 5 lg groups, respectively, had VNTs  0
5 IU/mL, considered an adequate
response by the WHO. After two 1 or 2 lg doses all recipients had titers  0.5 IU/mL by Day 43. Day
57 GMTs were not significantly lower than those with Rabipur, which elicited adequate responses in
all vaccinees after two doses. CV7202-elicited VNT were significantly correlated with RABV-G-specific
IgG antibodies (r2 = 0.8319, p < 0.0001).
Conclusions: Two 1 lg or 2 lg doses of CV7202 were well tolerated and elicited rabies neutralizing anti-
body responses that met WHO criteria in all recipients, but 5 lg had unacceptable reactogenicity for a
prophylactic vaccine.
ClinicalTrials.gov Identifier: NCT03713086.
Ó 2020 Published by Elsevier Ltd.

1. Introduction pathogens through human vectors with international travel as evi-

denced by the 2020 Covid-19 pandemic [2], or animal vectors with
Recent infectious disease outbreaks caused by Zika, Ebola, climate change illustrated by the increasingly wide range of den-
chikungunya and SARS-CoV-2 viruses have highlighted the poten- gue, chikungunya and Zika virus endemicity [3–5]. The develop-
tial global threat to human health posed by emerging human infec- ment of new platforms to allow rapid production of novel
tious diseases [1]. There are increasing risks of spread of novel vaccines, preferably avoiding the use of live pathogenic viruses
and chemical inactivation steps which may modify the natural epi-
⇑ Corresponding author at: Vice President Area Head Infectious Diseases, Clinical topes, is a major priority of vaccine research [1].
Development, CureVac AG, Schumannstrasse 27, 60325 Frankfurt, Germany. One promising technology with the potential to overcome
E-mail address: [email protected] (L. Oostvogels). many of these limitations is the use of messenger ribonucleic acid
1
Current address: Department of Molecular and Developmental Medicine, Univer- (mRNA) coding for the required antigen [6], which offers several
sity of Siena, Italy. advantages for vaccine manufacture. Production of mRNA using
2
In memory of Professor Frank von Sonnenburg.

https://doi.org/10.1016/j.vaccine.2020.12.070
0264-410X/Ó 2020 Published by Elsevier Ltd.
C. Aldrich, I. Leroux–Roels, Katell Bidet Huang et al. Vaccine 39 (2021) 1310–1318

well-defined manufacturing techniques allows the same facilities injection of either a first or second dose of CV7202 administered
to be used to prepare different mRNA molecules for vaccines to healthy adults in a range of increasing dosages starting at
against various other pathogens using the same manufacturing 5 mg. Main secondary objectives are ongoing evaluation of safety
platform. As this may lead to lower production costs this may be up to two years after vaccination, and comparison of the immune
of particular importance for vaccines destined for low-income response to CV7202 with the licensed rabies vaccine, RabipurÒ,
countries, including rabies vaccines. administered in its recommended three dose schedule. For the lat-
CureVac has developed a proprietary mRNA platform, RNActiveÒ, ter we used proportions of each study group achieving the WHO-
for use in the development of safe and effective prophylactic vaccines required level of response, a rabies-specific serum virus neutraliz-
for humans [7]. Following preclinical demonstration of the feasibility ing titer  0.5 IU/ml. An exploratory objective was the characteri-
of this approach the first clinical investigation assessing the potential zation of the humoral immune responses in terms of the
of RNActiveÒ for a variety of vaccine targets used the rabies virus gly- immunoglobulin IgG isotype against RABV-G.
coprotein (RABV-G) as a model antigen. Use of RABV-G presents sev-
eral advantages in the early stages of development: the antigen has 2.2. Study design and participants
been clearly defined and characterized, and there is a WHO-
defined level of immune response that is considered adequate for Eligible participants were 18–40 year-old adults of either gen-
the assessment of new vaccines. Furthermore, the virtually 100% der who were healthy at enrolment according to medical history
fatality outcome of rabies disease means that volunteers in phase 1 and examination, with a BMI  18.0 and  32.0 kg/m2. Female vol-
trials, providing they have no history of rabies vaccination, will rep- unteers were required to have a negative pregnancy test (serum
resent an immunologically naïve population. hCG) at screening and negative urine hCG tests before vaccination
We demonstrated the proof-of-concept of mRNA for human on Days 1 and 29, and to agree to use approved contraception
vaccines in the first-ever human clinical trial of RABV-G mRNA throughout the study. Male volunteers were required to use barrier
using an initial formulation (CV7201) with the cationic protein contraception (condom) until three months after their last
protamine [8]. CV7201 was generally well tolerated but the induc- vaccination.
tion of adequate immune responses was dependent upon the mode The main exclusion criteria included participation in any other
of administration of the vaccine, notably requiring intradermal or clinical trial, receipt of other vaccines either 14 days (inactivated
intramuscular administration with specialized devices. Further vaccines) or 28 days (live vaccines) before Day 1, any history of
preclinical research in animal models has found that formulation rabies vaccination, or planned travel to countries for which rabies
of the mRNA in lipid nanoparticles (LNP) protects the mRNA and vaccination is recommended or where there is a high risk of rabies
enhances the immune response [9]. Preclinical studies found that exposure. Other exclusions included any immunosuppressive ther-
CV7202, a novel mRNA-LNP formulation which includes the same apy within six months of study start, medication except for inhaled
mRNA antigen as CV7201 encapsulated in LNP, elicits immune or nasal steroids or topically applied steroids, any history of an
responses in non-human primates comparable to those induced immunodeficient condition or potentially immune-mediated dis-
by licensed vaccines (CureVac, data on file). We now report on ease and any known allergy to any vaccine component.
the first use of this new formulation in adult human volunteers The study was initially designed to be a dosage escalation with
in an ongoing phase 1 study to assess the safety and immunogenic 5 lg as the lowest starting dosage; the first ten participants were
potential of this new vaccine model, which is now being applied to enrolled in a staggered manner to receive the lowest anticipated
other novel pathogenic viruses, notably SARS-CoV-2. dosage of CV7202, 5 lg, and this was to be followed by
sequentially-enrolled groups of 16 volunteers each to receive
2. Methods higher dosages with the first eight participants enrolled into each
group then receiving a second dose 28 days after the first. A control
This is a non-randomized, open-label, controlled, dose-escalation, group enrolled without stagger received three doses of a licensed
multi-center phase 1 study done at the University Hospital LMU rabies vaccine according to the manufacturer’s recommendations.
Munich, Germany and the University of Ghent, Belgium from October The planned staggered enrolment consisted of the first partici-
2018. The objective was to determine the safety, reactogenicity and pant in the 5 lg group receiving their first vaccination, followed
immunogenicity of different dosages of CV7202 when administered by the second and third participants two working days later. The
as intramuscular injections in one- or two-dose regimens. The study fourth and fifth participants were then enrolled and vaccinated
protocol was approved by the respective Ethical Committees of the one week after the first. Two days after these vaccinations the iSRC
two institutions and registered on ClinicalTrials.gov and DSMB reviewed the safety data from the first five participants
(NCT03713086). Trial procedures were done in accordance with before agreeing to the enrolment of further participants in this
International Conference on Harmonization (ICH) and Good Clinical group. This process was to be repeated for the next groups, with
Practice guidelines, and applicable regulatory requirements. An inter- the exception that safety data for sentinel safety groups compris-
nal safety review committee (iSRC) consisting of internal medical ing the first four participants was considered by the iSRC before
experts and the investigators, and an independent data safety mon- enrolment of the remaining participants in each group. As
itoring board (DSMB) consisting of external vaccine experts reviewed described in results, following observation of excess reactogenicity
safety data on a regular basis and made recommendations regarding to 5 lg CV7202 the protocol was modified to assess lower (1 lg
the sequential enrolment of participants into dose escalation or de- and 2 lg) rather than higher dosages.
escalation groups according to the DSMB charter and the protocol.
The study is ongoing at the time of this report for long-term safety 2.2.1. Vaccine
and immunogenicity follow-up, but database lock for the presented CV7202 is composed of mRNA encoding the RABV-G from the
data was May 2020, at least four weeks after the second dose of Pasteur strain (GenBank accession number: AAA47218.1) with four
experimental vaccine, CV7202. lipid components—cholesterol, 1,2-distearoyl-sn-glycero-3-
phosphocholine (DSPC), PEGylated lipid and a cationic lipid—pro-
2.1. Outcomes vided as a sterile solution in a 2 mL glass vial. The stock solution
of CV7202 was mixed with 0
9% sodium chloride by a study phar-
The primary objective was assessment of safety and reacto- macist to produce a 10 lg/mL solution, and volumes injected were
genicity up to 28 days after administration by intramuscular 0
5 mL, 0
2 mL and 0
1 mL for the 5 mg, 2 mg and 1 mg doses of
1311
C. Aldrich, I. Leroux–Roels, Katell Bidet Huang et al. Vaccine 39 (2021) 1310–1318

mRNA, respectively. The control vaccine was RabipurÒ (GSK Vacci- post-Day 1 safety data are available. Immunogenicity analyses
nes GmbH, Marburg, Germany), a licensed, inactivated rabies vac- were done on the Full Analysis Set (FAS) comprising participants
cine containing  2
5 IU per ml reconstituted dose, administered who provided a valid baseline sample and at least one additional
according to the manufacturer’s recommended schedule of 1, 8 blood sample for VNT analysis. Seroconversion was defined as
and 29 days. All vaccines were administered by intramuscular observation of the adequate response titer (0
5 IU/mL) any time
injection with a standard syringe/needle in the deltoid of the after vaccination in a subject confirmed seronegative at baseline.
non-dominant arm. Geometric mean titers (GMT) of rabies-specific VNTs were calcu-
lated for each group at each study time-point with 95% confidence
2.2.2. Safety assessments intervals (95% CI) as well as the percentages of each group achiev-
Following vaccination on Day 1 participants were monitored for ing a VNT  0
5 IU/mL. GMTs and seropositivity rates of RABV-G
4 h and were only discharged when vital parameters were within specific IgG were calculated assigning values of half the LLoQ
the normal ranges and similar to pre-vaccination levels. Injection (780 EU/mL) for samples below that value. Spearman correlations
site reactogenicity was assessed 1 h after vaccination on Day 1, between VNT and IgG responses to first and second doses of
and on Day 2 for 1 mg and 2 mg doses, or Day 3 for the 5 mg dose. CV7202 were calculated. All analyses were done using SAS (version
Participants were provided with diary cards in which they 9.4) and GraphPad Prism software.
recorded solicited local reactions (pain, redness, swelling and itch-
ing) and systemic adverse events (AE; fever, chills/shivers, nausea/
vomiting, diarrhea, headache, fatigue, myalgia and arthralgia) for 3. Results
seven days post vaccination, and any unsolicited AEs for 28 days
after each vaccination. In the two dose subsets and the Rabipur A total of 69 volunteers were screened of whom 55 were
group this monitoring schedule was applied for each vaccination. enrolled, screen failures mainly being due to inability to meet all
All solicited and unsolicited AEs were graded according to severity, protocol requirements (Fig. 1). Two enrolled participants did not
using mild, moderate or severe as categories using the FDA toxicity receive any vaccination, one due to non-compliance with the pro-
grading scale [10]. Serious adverse events (SAE) or adverse events tocol and one who withdrew for personal reasons. The original
of special interest (AESI; AEs with a suspected immune mediated staggered enrolment plan was followed for the 5 lg dose group,
disease etiology) were recorded throughout the study duration and ten volunteers were enrolled and received one 5 lg vaccina-
and reported immediately to the sponsor or investigator. tion. However, concerns over the reactogenicity profile in this
group led to a temporary hiatus in the study while the reactogenic-
2.3. Virus-neutralizing antibodies ity and immunogenicity data from this group were assessed. Fol-
lowing an extensive root cause analysis the iSRC and DSMB
Serum samples prepared at baseline (Day 1) and subsequently recommended continuing with 1 lg and then 2 lg dosages, and
on Days 8, 15, 29, 36, 43, and 57 (pre-vaccination for applicable the control Rabipur group. Results for all groups are reported
time-points), were stored at 80 °C for assessment of immune below. Group demographics of the 53 volunteers enrolled and
responses. Rabies virus-specific serum neutralizing titers (VNT) assigned to the four groups were similar (Table 1). One participant
were measured using the WHO-recommended rapid fluorescent from the Rabipur control group was lost to follow up after Day 1,
focus inhibition test (RFFIT) [13] at the accredited Kansas State and one participant in the 5 lg CV7202 group withdrew consent
University Rabies Laboratory. Titers are expressed as International at Day 15 for personal reasons. The remaining 51 participants com-
Units per mL (IU/mL), with a titer  0
5 IU/mL considered an ade- pleted study procedures through to the Day 57 visit.
quate response to vaccination [11].

2.4. ELISA for RABV-G-specific immunoglobulins 3.1. Safety

Levels of RABV-G-specific IgG were measured by enzyme-linked There were no immediate reactions or AEs during the four-hour
immunosorbent assay (ELISA) at the same timepoints. Briefly, post-vaccination surveillance period in any participant, and there
plates were coated overnight with recombinant RABV-G protein have been no vaccine-related SAEs or AESIs throughout the study
expressed in HEK293 cells diluted to 1 mg/ml in phosphate- to date. One Rabipur participant was hospitalized with appendici-
buffered saline (PBS). Wells were blocked in phosphate-buffered tis, which was not considered to be related to the vaccine. No par-
saline (PBS) + 0.05% Tween-20 (PBST) + 5% skimmed milk for ticipant withdrew from the study due to an AE.
1.5 h before incubation of duplicate serial dilutions of serum sam- As noted, following the administration of one dose of vaccine in
ples in PBST + 2% skimmed milk for 2 h at room temperature. After the 5 lg group there was a high rate of early onset reactogenicity
four washes with PBST, horseradish peroxidase (HRP)-labelled which affected 9 (90%) of the 10 participants (Table 2). All but one
detection antibodies specific for human IgG (clone EFE 565, of the participants reported pain at the injection site, which was
ThermoFischer Scientific) were added for 1 h. Plates were washed graded as severe in one case. There was only one report of any
five times with PBST before colorimetric detection using tetram- other local reaction—a case of mild redness in one participant—
ethylbenzidine substrate (Biolegend). Reactions were stopped with with no reports of injection site swelling or itching. There was a
2 N H2SO4 and absorbance read at 450 nm with 620 nm as refer- high rate of solicited systemic adverse events in this group, with
ence wavelength. Antibody titers were calculated based on isotype 41 events reported by 9 participants, 32 (78%) of which were
control calibration curves, with titers expressed as arbitrary ELISA described as mild or moderate, but 9 (22%) of which were
units per mL (U/mL). described as ever in 6 participants. The most frequent systemic
AEs were headache, fatigue, myalgia and chills, the majority with
2.4.1. Statistics onset within 24 h of vaccination on Days 1 or 2. Severe cases of
In this exploratory phase 1 trial we only used descriptive statis- fatigue, chills and myalgia which occurred became moderate or
tics, with no confirmatory statistical inference planned or per- mild within 24 h of onset, except for one case of severe fatigue that
formed. The primary outcome was analysed on the Safety Set, lasted for 29 h, and a second case of severe fatigue lasting three
defined as those participants who received at least one dose of days with onset on Day 5 that was related to an acute
the CV7202 or the active control RabipurÒ and for whom any gastroenteritis-like illness with nausea and vomiting.
1312
C. Aldrich, I. Leroux–Roels, Katell Bidet Huang et al. Vaccine 39 (2021) 1310–1318

69 Screened
14 screen failures
• 1 withdrew consent
• 13 did not meet
eligibility criteria

55 Enrolled

2 not vaccinated
• 1 non-compliance
Clinical hiatus • 1 withdrew consent

6 enrolled 5 enrolled

10 enrolled 11 enrolled 16 enrolled 16 enrolled

Day 1 Day 1 Day 1 Day 1

10 received 11 received 16 received 16 received
one dose one dose one dose one dose
5 μg CV7202 Rabipur 1 μg CV7202 2 μg CV7202

1 lost to follow up

Day 8
10 received
second dose
Rabipur
Day 15
1 consent
withdrawal
Day 29 Day 29 Day 29
10 received 8 received 8 received
third dose second dose second dose
Rabipur 1 μg CV7202 2 μg CV7202

Day 57 Day 57 Day 57 Day 57

9 completed 10 completed 16 completed 16 completed

Fig. 1. Trial profile.

Table 1
Demographics of the enrolled study population.

CV7202 Dose groups Rabipur group

Parameter 5 lg 2 lg 1 lg
N= 10 16 16 11
Age, years
Mean (±SD) 26
1 (±4
0) 28
3 (±5
8) 27
1 (±5
6) 25
5 (±4.2)
Range [20, 33] [21, 38] [19, 38] [21, 36]
Male
n (%) 5 (50) 4 (25) 7 (44) 4 (36)
Ethnicity
White, n (%) 10 (100) 16 (100) 15 (94) 11 (100)
Height, (cm)
Mean (±SD) 174 (±4
4) 172 (±
8
4) 171 (±8
4) 174 (±12
4)
Weight, (kg)
Mean (±SD) 72
4 (±9
2) 72
1 (±14
4) 70
0 (±8
9) 71
4 (±15
1)
BMI, (kg/m2)
Mean (±SD) 23
8 (±2.6) 24
4 (±3
6) 24
0 (±2
5) 23
2 (±2.2)
Range [20, 28] [19, 32] [18, 28] [20, 27]

Unsolicited adverse events were reported by 9 (90%) of the 10 syncopal episode, hot flushes, thirst, lack of thirst, perceived dehy-
participants; 7 (70%) of these were considered to be related to vac- dration, weakness, and lethargy—some in the same participant.
cination, including 5 (50%) graded as severe. The most frequent of The majority of unsolicited AEs resolved within 24 h of onset and
these were three cases of lack of appetite (all severe), three cases of the only medication used for treatment of such AEs was ibuprofen,
night sweats (two severe), two cases of dizziness (one severe), and used by 4 of 10 (40%) participants.
two cases of tachycardia (one severe). There were single cases of Following the study hiatus, 1 and 2 lg dosages of CV7202 were
severe AEs—neck stiffness, excessive sweating, hip stiffness, pre- administered and were associated with a much lower reactogenic-

1313
C. Aldrich, I. Leroux–Roels, Katell Bidet Huang et al. Vaccine 39 (2021) 1310–1318

Table 2
Solicited local reactions and systemic AEs in the 5 lg CV7202 group.

5 lg CV7202 (N = 10)
Reaction or adverse event, n (%) Any Mild Moderate Severe
Any local reaction 9 (90) 3 (30) 5 (50) 1 (10)
Pain 9 (90) 3 (30) 5 (50) 1 (10)
Redness 1 (10) 1 (10) 0 0
Swelling 0 0 0 0
Itching 0 0 0 0
Any solicited systemic adverse event 9 (90) 7 (70) 9 (90) 6 (60)
Fever 5 (50) 3 (30) 1 (10) 1 (10)
Headache 7 (70) 0 7 (70) 0
Fatigue 7 (70) 1 (10) 3 (30) 3 (30)
Chills 7 (70) 2 (20) 2 (20) 3 (30)
Myalgia 5 (50) 1 (10) 2 (20) 2 (20)
Arthralgia 4 (40) 3 (30) 1 (10) 0
Nausea/vomiting 3 (30) 2 (20) 1 (10) 0
Diarrhea 2 (10) 1 (10) 1 (10) 0
Any unsolicited systemic adverse event 9 (90) 6 (60) 6 (60) 6 (60)

ity profile (Table 3). In the 1 lg CV7202 group there were no severe ients, the total incidence in the study population was 5 of 53 (9%).
AEs reported, although 4 (25%) of the 16 participants in this group When these five participants were interviewed for more detail two
reported moderate AEs. Three unsolicited AEs were reported by reported that episodic jaundice and/or bilirubin elevations had
two participants—one had a single episode of loose stool and been present ‘since youth’.
another had two episodes of lower back pain—that were consid- In three of these five subjects, we observed isolated, elevated
ered to be possibly related to the vaccination. Amongst the 16 par- total bilirubin elevations prior to the first vaccination (either at
ticipants of the 2 lg CV7202 group there was one (6%) report of screening or at baseline immediately prior to vaccine administra-
severe injection site pain and three (19%) participants reported tion. Three of the five had one or more bilirubin elevations that
severe solicited systemic AEs; chills (two), headache (two) and were marked as clinically significant during the conduct of the
myalgia (one). All but one of these reports occurred after the first study, two of which were present at baseline. The third described
dose, with onset on Days 1 or 2, all had improved to mild or mod- previous clinically significant elevations which were added to their
erate by Day 3 within 48 h and all but one had resolved by Day 4. medical history.
One participant reported two severe unsolicited AEs, palpitations Split bilirubin analysis was requested in the three cases of clin-
and tachycardia after their first 2 lg dose on the day of vaccination. ically significant bilirubin elevation to substantiate the suspected
All these AEs resolved without sequelae. MGS: one participant had a split bilirubin typical for MGS and in
Each of the three doses of Rabipur were associated with injec- combination with a history of raised elevations the diagnosis of
tion site pain in about half of the participants (Table 3), none of MGS was marked in the medical history without genetic confirma-
which were severe, with no other local reactions. There were also tion. In a second suspected MGS participant split bilirubin was
cases of mild to moderate headache, fatigue and myalgia associ- requested at a visit when total bilirubin was normal and there
ated with Rabipur. were no further elevations observed. Because of a childhood his-
The major finding from the laboratory safety assessments was tory of intermittent icterus on further questioning, suspected
the observation of transient lymphopenia at Day 2, the day after MGS was marked in their medical history. Split bilirubin analysis
vaccination in 9 of 16 and 14 of 16 in the 1 and 2 lg CV7202 will be done at any future study visit where total bilirubin is ele-
groups, respectively, and 6 of 8 and 8 of 8 in those groups at Day vated and the subject will be referred to their GP for further test-
30, one day after the second dose. This was not observed in the ing. In the third participant with clinically significant elevation of
5 lg group as the first blood sampling in this group was conducted bilirubin results of the split bilirubin analysis was not typical for
on Day 3, rather than Day 2, when lymphopenia was no longer pre- MGS, but the participant had a recent symptomatic acute CMV ill-
sent. These transient changes were not considered to represent ness. Considering the pattern of bilirubin elevations, overall clinical
toxicity but rather redistribution of lymphocytes due to the vac- picture and prevalence in Munich, per exclusionem MGS is still sus-
cine’s mode of action as has been observed for other vaccines pected in this participant who has been referred to their GP for fur-
[12,13]. ther testing. The results are as yet unknown.
An anomaly which was not considered to be vaccine-related Split bilirubin levels will be analyzed for both participants with
was the observation of asymptomatic, isolated elevated bilirubin non-clinically significant bilirubin elevations at their next sched-
levels in several young male participants recruited at the Munich uled site visit. Until then, both subjects remain suspected for
site which were not temporally associated with vaccination. These MGS and might be referred for further testing.
were present in prevaccination (baseline) samples and post-
vaccination samples in no specific pattern and with no particular
association with any study vaccine. The site observed that this is 3.2. Immunogenicity – Neutralizing antibodies
‘‘very common’’ in their experience with an incidence substantially
(2–4x) higher than reported in most western settings and is asso- VNT responses were detected in all four study groups as illus-
ciated with Meulengracht-Gilbert Syndrome (MGS), a benign trated in Fig. 2. Following a 5 lg dose of CV7202, VNT
genetic disorder which is present in approximately 10% of young levels  0
5 IU/ml were observed from Day 29 in two of nine
men in Bavaria and affects bilirubin processing leading to elevated (22%) participants and these responses were maintained up to
levels of unconjugated bilirubin in the blood [14–16]. In our study, Day 57, the last timepoint assessed (Fig. 3).
the incidence was 5 of 45 (11%) CV-7202 vaccinated participants, The 1 lg and 2 lg CV7202 groups also displayed small detect-
and as there were no suspected MGS cases amongst Rabipur recip- able responses following the first dose. These were more pro-
nounced in the 2 lg group in which 5 of 16 (31%) had VNT
1314
C. Aldrich, I. Leroux–Roels, Katell Bidet Huang et al. Vaccine 39 (2021) 1310–1318

Table 3
Numbers (%) of participants reporting solicited local reactions and systemic adverse events (AE) in the 7 days after receiving CV7202 or Rabipur.

5 lg 1 lg 2 lg Rabipur
CV7202 CV7202 CV7202
Reaction or AE, n (%) Dose 1 Dose 1 Dose 2 Dose 1 Dose 2 Dose 1 Dose 2 Dose 3
N= 10 16 8 16 8 11 10 10
Any solicited local reaction 9 (90) 13 (81) 5 (63) 15 (94) 6 (75) 5 (45) 6 (60) 5 (50)
Pain 9 (90) 13 (81) 5 (63) 15 (94) 6 (75) 5 (45) 6 (60) 5 (50)
Redness 1 (10) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0)
Swelling 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0)
Itching 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0)
Any solicited systemic AE 9 (90) 11 (69) 5 (63) 13 (81) 7 (88) 8 (73) 5 (50) 4 (40)
Fever 5 (50) 0 (0) 1 (13) 3 (19) 2 (25) 0 (0) 0 (0) 0 (0)
Headache 7 (70) 8 (50) 4 (50) 11 (69) 5 (63) 4 (36) 2 (20) 3 (30)
Fatigue 8 (80) 6 (38) 4 (50) 9 (56) 5 (63) 5 (45) 5 (50) 1 (10)
Chills 7 (70) 0 (0) 2 (25) 6 (38) 2 (25) 0 (0) 0 (0) 1 (10)
Myalgia 5 (50) 6 (38) 4 (50) 7 (44) 5 (63) 2 (18) 0 (0) 0 (0)
Arthralgia 4 (40) 3 (19) 3 (38) 3 (19) 1 (13) 0 (0) 0 (0) 0 (0)
Nausea/vomiting 3 (30) 1 (6) 0 (0) 1 (6) 1 (13) 1 (9) 0 (0) 0 (0)
Diarrhea 2 (20) 1 (6) 0 (0) 1 (6) 2 (25) 0 (0) 0 (0) 0 (0)
Any unsolicited systemic AE 9 (90)* 9 (56) 4 (50) 14 (88) 5 (63) 4 (36) 4 (40)
Related to vaccination 7 (70) * 2 (13) 1 (13) 9 (56) 2 (25) 1 (9) 0 (0)
Any medically attended AE 1 (10) * 4 (25) 1 (13) 2 (13) 1 (13) 0 (0) 0 (0)
*
Events reported over the whole 56-day study period.

Fig. 2. Geometric mean virus neutralizing titers (with 95% CI) in the four study groups after immunization (indicated by arrows) with CV7202 or Rabipur. Dashed line
indicates level considered adequate by the WHO (0.5 IU/mL).

levels  0
5 IU/ by Day 29, whereas no participants in the 1 lg Day 36 in both groups and were further increased at Days 43 and
group displayed an adequate response (0
5 IU/mL) by Day 29 after 57. Peak GMTs were achieved at Day 43 with 1 lg (4
8 IU/mL
one dose (Fig. 3). Responses were markedly increased following the [95% CI:1
77–13
0]) and 2 lg (4
2 IU/mL [1
02–17
2]) of CV7202.
second dose on Day 29 such that 5 of the 8 (63%) participants who All participants in the Rabipur group had titers  0
5 IU/mL by
received a second dose of 1 lg and 7 of 8 (83%) participants who Day 15, 7 days after the second vaccination and this 100% rate
received a second dose of 2 lg had titers  0
5 IU/mL at Day 36. was maintained up to Day 57.
All participants (100%) in both the 1 lg and 2 lg groups reached Rabipur achieved a peak GMT of 13
5 IU/mL [5
95–30
6]) IU/mL
this level at Day 43 (Fig. 3). GMTs were higher than 0
5 IU/mL at at Day 15, 7 days after the second dose. The GMT did not further

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C. Aldrich, I. Leroux–Roels, Katell Bidet Huang et al. Vaccine 39 (2021) 1310–1318

Fig. 3. Responder rates (percentages of each group with a VNT  0.5 IU/mL) in the four study groups after immunization with CV7202 or Rabipur. Rates represent the
numbers of participants achieving the protective VNT of 0.5 IU/mL. The 1 and 2 lg CV7202 groups consisted of 16 participants each for Days 8, 15 and 29, and 8 participants
each for Days 36, 43 and 57. The 5 lg CV7202 group consisted of 10 participants for Days 8 and 15, 9 participants for Days 29, 36, 43 and 57. The Rabipur group had 10
participants at each timepoint.

increase following a third dose of Rabipur but was maintained at els [9], we assessed the safety, reactogenicity, and immunogenicity
9
1 IU/mL through to Day 57. Day 43 GMTs after two doses of of CV7202, a novel mRNA-LNP formulation, in adults in compar-
CV7202 were not statistically significantly lower than those ison with a licensed rabies vaccine. Following observations of high
achieved with three doses of Rabipur (p = 0.2831 for 1 mg, reactogenicity when using the 5 lg dose of CV7202 we found 1 and
p = 0.3507 for 2 mg; Mann-Whitney test). 2 lg dosages were better tolerated, with no safety concerns and
two doses elicited immune responses in terms of neutralizing
activity and IgG antibodies that were comparable with three doses
3.3. Immunogenicity – ELISA RABV-G-specific immunoglobulin
of licensed rabies vaccine. Preliminary investigation of the 5 lg
antibodies
response suggest that high innate immune responses driven by
type 1 interferon and cytokines and strong induction of toll-like
As illustrated in Fig. 4 anti-RABV-G IgG antibodies displayed the
receptor signaling pathways observed in most participants, might
same pattern of responses as VNT. There were detectable increases
have contributed to unfavorable reactogenicity and immunogenic-
after one dose with 6 of 16 (38%), 11 of 16 (69%) and 8 of 9 (89%)
ity profiles. This will be investigated in further studies.
participants in the 1, 2 and 5 lg dosage groups developing low
In this small trial CV7202 appeared to be safe, with no vaccine-
levels of RABV-G-specific IgG, respectively. GMTs were 853 U/mL
related SAEs or withdrawals due to AEs. Although over half the
(95% CI: 455–1599), 1581 U/mL (899–2780), and 2409 U/mL
recipients of the highest dose of CV7202 reported severe solicited
(1113–5215) at Day 29 after the first dose in the 1, 2, and 5 lg
systemic or unsolicited AEs, the reactogenicity profiles of the lower
CV7202 groups, respectively, and these levels did not further
doses of CV7202 (1 and 2 lg) were more acceptable. The 2 lg dose
increase in one dose groups. Much larger increases were observed
elicited a limited number of severe AEs in the first 24 h post-
after second vaccinations, peaking at 34,186 U/mL (13253–88185)
vaccination. Local reactions to these dosages consisted almost
and 20,707 U/mL (5592–76678) at Day 43 in the 1 and 2 lg groups,
exclusively of transient mild to moderate injection site pain. Sys-
respectively. There were highly significant positive Spearman cor-
temic AEs mainly consisted of transient mild or moderate head-
relations between VNT and IgG titers (Fig. 5), particularly after two
ache, fatigue and chills, and any cases that were described as
doses of CV7202 (r2 = 0
8319, p < 0
0001).
initially severe rapidly moderated and resolved, most within 48–
An IgG response was not detected one week after the first Rabi-
72 h and all within the 7-day reporting period. There were no
pur vaccination; GMTs were 461 and 464 at Days 1 and 8, respec-
major changes in reactogenicity after the second dose when com-
tively, but rapidly increased to 12,460 U/mL (95% CI: 6575–23611)
pared with the first, although the numbers of participants are
at Day 15, 7 days after the second dose. A further incremental
small. This contrasts with recent reports of mRNA-LNP vaccines
increase to 33,373 U/mL (21236–52447) was observed after the
against the SARS-CoV-2 virus responsible for the COVID-19 pan-
third dose and this level was sustained to Day 57. As for the
demic which had similar rates of reactogenicity and in which reac-
VNT, RABV-G IgG GMTs at Day 43 after two doses of CV7202 were
togenicity noticeably increased after a second dose [17,18].
not statistically significantly lower than those achieved with three
Importantly, we showed that all recipients, despite the low
doses of Rabipur (p = 0
9654 for 1 mg, p = 0
2031 for 2 mg by Mann-
amount of mRNA included in the vaccine, had functional antibody
Whitney test).
responses after two 1 or 2 lg doses of CV7202, with GMTs of both
rabies-specific neutralizing and RABV-G-specific IgG antibodies
4. Discussion that were not significantly lower than those observed after three
doses of licensed rabies vaccine. The neutralizing response profile
Following demonstration that encapsulation of RABV-G mRNA of CV7202 displayed a strong correlation with production of
in lipid nanoparticles improved immune responses in animal mod- RABV-G-specific IgG antibodies following two doses. There were

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C. Aldrich, I. Leroux–Roels, Katell Bidet Huang et al. Vaccine 39 (2021) 1310–1318

Fig. 4. GMTs (with 95% CI) of RABV-G-specific Ig responses assessed by ELISA. IgG concentrations after immunization with one (red arrow) or two (open arrow) doses of
CV7202 or three doses of Rabipur (blue arrows). Dotted lines indicate LLOQ. (For interpretation of the references to colour in this figure legend, the reader is referred to the
web version of this article.)

Fig. 5. Correlation of titers of RABV-G-specific neutralizing activity (VNT) and IgG antibodies after one or two doses of CV7202.

also transient increases in IgM antibodies but not IgA after second responses evident 7 days after the second vaccination, and their
doses of Rabipur and CV7202 (data not shown), but direct compar- direct correlation with the neutralizing response suggest the first
ison of the kinetics of these responses is complicated by the differ- dose of lower dosages of CV7202 had primed B cells to respond
ent vaccination schedules—1 and 8 days for licensed vaccine and 1 to the second vaccination with an anamnestic response. Although
and 29 days for CV7202—and no second 5 lg dose. The large IgG responses with two doses of 1 or 2 lg CV7202 were not signifi-

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C. Aldrich, I. Leroux–Roels, Katell Bidet Huang et al. Vaccine 39 (2021) 1310–1318

cantly lower than those induced by three doses of Rabipur, it may Acknowledgements
be interesting to compare CV7202 and Rabipur responses when
used in the same schedule of three doses at Days 1, 8 and 29. The authors are grateful to all the participants in Munich and
This interim report presents the immune responses up to four Ghent who volunteered for this study and to Dr Mirjam Schunk
weeks after the second dose, but participants will be monitored (Munich), Dr Cathy Maes (Ghent), and Dr Aurelio Garofano (Cure-
for two years to assess long-term safety and persistence of the Vac), and the respective study teams, trial coordinators and nurses
immune response. Further investigations of antibody responses for their expert assistance. We thank Keith Veitch (keithveitch
after a booster vaccination, possibly with lower doses, will be nec- communications, Amsterdam, the Netherlands) for assistance in
essary to determine whether long-term immune memory has drafting the manuscript. This paper is dedicated to the memory
developed, together with a qualitative comparison of avidity, IgG of Professor Frank von Sonnenburg who passed away on 21 August
subclasses and B cell responses for CV7202 and the licensed 2020.
vaccine.
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