Phase 1 safety and immunogenicity evaluation of ADMVA, a multigenic, modified vaccinia Ankara-HIV-1 B'/C candidate vaccine.

Abstract

We conducted a Phase I dose-escalation trial of ADMVA, a Clade-B'/C-based HIV-1 candidate vaccine expressing env, gag, pol, nef, and tat in a modified vaccinia Ankara viral vector. Sequences were derived from a prevalent circulating HIV-1 recombinant form in Yunnan, China, an area of high HIV incidence. The objective was to evaluate the safety and immunogenicity of ADMVA in human volunteers.
ADMVA or placebo was administered intramuscularly at months 0, 1 and 6 to 50 healthy adult volunteers not at high risk for HIV-1. In each dosage group [1x10(7) (low), 5x10(7) (mid), or 2.5x10(8) pfu (high)] volunteers were randomized in a 3:1 ratio to receive ADMVA or placebo in a double-blinded design. Subjects were followed for local and systemic reactogenicity, adverse events including cardiac adverse events, and clinical laboratory parameters. Study follow up was 18 months. Humoral immunogenicity was evaluated by anti-gp120 binding ELISA, immunoflourescent staining, and HIV-1 neutralization. Cellular immunogenicity was assessed by a validated IFNgamma ELISpot assay and intracellular cytokine staining. Anti-vaccinia binding titers were measured by ELISA. ADMVA was generally well-tolerated, with no vaccine-related serious adverse events or cardiac adverse events. Local or systemic reactogenicity events were reported by 77% and 78% of volunteers, respectively. The majority of events were of mild intensity. The IFNgamma ELISpot response rate to any HIV antigen was 0/12 (0%) in the placebo group, 3/12 (25%) in the low dosage group, 6/12 (50%) in the mid dosage group, and 8/13 (62%) in the high dosage group. Responses were often multigenic and occasionally persisted up to one year post vaccination. Antibodies to gp120 were detected in 0/12 (0%), 8/13 (62%), 6/12 (50%) and 10/13 (77%) in the placebo, low, mid, and high dosage groups, respectively. Antibodies persisted up to 12 months after vaccination, with a trend toward agreement with the ability to neutralize HIV-1 SF162 in vitro. Two volunteers mounted antibodies that were able to neutralize clade-matched viruses.
ADMVA was well-tolerated and elicited durable humoral and cellular immune responses.
Clinicaltrials.gov NCT00252148.

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Phase 1 Safety and Immunogenicity Evaluation of
ADMVA, a Multigenic, Modified Vaccinia Ankara-HIV-1
B’/C Candidate Vaccine
Sandhya Vasan1,2*, Sarah J. Schlesinger1,2, Zhiwei Chen1,2¤a, Arlene Hurley1,2, Angela Lombardo3, Soe
Than3¤b, Phumla Adesanya3¤c, Catherine Bunce4, Mark Boaz3¤d, Rosanne Boyle3, Eddy Sayeed3, Lorna
Clark6, Daniel Dugin1, Mar Boente-Carrera1, Claudia Schmidt3, Qing Fang1, LeiBa1¤e, Yaoxing Huang1,2,
Gerasimos J. Zaharatos1,2¤f, David F. Gardiner1,5¤g, Marina Caskey2, Laura Seamons7, Martin Ho7, Len
Dally7, Carol Smith7, Josephine Cox6, Dilbinder Gill6, Jill Gilmour6, Michael C. Keefer4, Patricia Fast3,
David D. Ho1,2
1Aaron Diamond AIDS Research Center, New York, New York, United States of America, 2The Rockefeller University, New York, New York, United States of America,
3International AIDS Vaccine Initiative, New York, New York, United States of America, 4University of Rochester Medical Center, Rochester, New York, United States of
America, 5Weill Cornell Medical Center, New York, New York, United States of America, 6International AIDS Vaccine Initiative Core Laboratory, Imperial College, London,
United Kingdom, 7EMMES Corporation, Rockville, Maryland, United States of America
Abstract
Background: We conducted a Phase I dose-escalation trial of ADMVA, a Clade-B’/C-based HIV-1 candidate vaccine
expressing env, gag, pol, nef, and tat in a modified vaccinia Ankara viral vector. Sequences were derived from a prevalent
circulating HIV-1 recombinant form in Yunnan, China, an area of high HIV incidence. The objective was to evaluate the
safety and immunogenicity of ADMVA in human volunteers.
Methodology/Principal Findings: ADMVA or placebo was administered intramuscularly at months 0, 1 and 6 to 50 healthy
adult volunteers not at high risk for HIV-1. In each dosage group [16107(low), 56107(mid), or 2.56108pfu (high)] volunteers
were randomized in a 3:1 ratio to receive ADMVA or placebo in a double-blinded design. Subjects were followed for local and
systemic reactogenicity, adverse events including cardiac adverse events, and clinical laboratory parameters. Study follow up
was 18 months. Humoral immunogenicity was evaluated by anti-gp120 binding ELISA, immunoflourescent staining, and HIV-1
neutralization. Cellular immunogenicity was assessed by a validated IFNc ELISpot assay and intracellular cytokine staining. Anti-
vaccinia binding titers were measured by ELISA. ADMVA was generally well-tolerated, with no vaccine-related serious adverse
events or cardiac adverse events. Local or systemic reactogenicity events were reported by 77% and 78% of volunteers,
respectively. The majority of events were of mild intensity. The IFNc ELISpot response rate to any HIV antigen was 0/12 (0%) in
theplacebogroup,3/12(25%)inthelowdosagegroup,6/12(50%)inthemiddosagegroup,and8/13(62%)inthehighdosage
group. Responses were often multigenic and occasionally persisted up to one year post vaccination. Antibodies to gp120 were
detected in 0/12 (0%), 8/13 (62%), 6/12 (50%) and 10/13 (77%) in the placebo, low, mid, and high dosage groups, respectively.
Antibodies persisted up to 12 months after vaccination, with a trend toward agreement with the ability to neutralize HIV-1
SF162 in vitro. Two volunteers mounted antibodies that were able to neutralize clade-matched viruses.
Conclusions/Significance: ADMVA was well-tolerated and elicited durable humoral and cellular immune responses.
Trial Registration: Clinicaltrials.gov NCT00252148
Citation: Vasan S, Schlesinger SJ, Chen Z, Hurley A, Lombardo A, et al. (2010) Phase 1 Safety and Immunogenicity Evaluation of ADMVA, a Multigenic, Modified
Vaccinia Ankara-HIV-1 B’/C Candidate Vaccine. PLoS ONE 5(1): e8816. doi:10.1371/journal.pone.0008816
Editor: Sean Emery, University of New South Wales, Australia
Received June 22, 2009; Accepted November 11, 2009; Published January 25, 2010
Copyright: ? 2010 Vasan et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: Funding for this study was provided by the International AIDS Vaccine Initiative (IAVI) and its donors, including the generous support of the American
people through the United States for International Development (USAID). IAVI played a direct role in study design, data collection and analysis, decision to
publish, and preparation of this manuscript. Support was also provided by the Rockefeller University and University of Rochester Clinical and Translational Science
Awards.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: svasan@adarc.org
¤a Current address: AIDS Institute, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Hong Kong, Special Administrative Region, People’s Republic of
China
¤b Current address: Pfizer Inc., New York, New York, United States of America
¤c Current address: Novartis Pharmaceuticals, East Hanover, New Jersey, United States of America
¤d Current address: Sanofi Pasteur, Swiftwater, Pennsylvania, United States of America
¤e Current address: Schering-Plough, Kenilworth, New Jersey, United States of America
¤f Current address: McGill University, Montreal, Quebec, Canada
¤g Current address: Bristol-Meyers Squibb Company, Princeton, New Jersey, United States of America
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Introduction
With an estimated 33 million people living with HIV/AIDS
globally, and approximately 2.5 million new infections in 2007
alone, the need for an effective vaccine to prevent or attenuate
HIV-1 infection remains paramount [1]. In the People’s Republic
of China, an estimated 700,000 people are living with HIV/AIDS
in an epidemic spread both through sexual transmission and
injection drug use. The prevalence of HIV infection among
injection drug users in Yunnan province, which borders
Myanmar, Laos, and Vietnam in the ‘‘golden triangle’’ region,
has increased dramatically in the last ten years, to over 40% in
several prefectures [2]. In a separate study, the incidence rate of
new HIV infections among intravenous drug users in Guanxgi
province was found to be 3.1% [3].
For these reasons, our laboratory has pursued the development
of a multigenic vaccine regimen based on the predominant B’/C
circulating recombinant form of HIV-1 from Yunnan, China,
CRF07_BC [4]. After codon-optimization and certain safety
mutations, matched sequences from the env, gag, pol, nef, and tat
genes were inserted into both a naked DNA plasmid backbone
(ADVAX) and a modified vaccinia ankara (MVA) viral vector
(ADMVA), as described by Y. Huang et al. and Z. Chen et al.,
respectively [5,6]. These vectors were initially chosen based on
reports of improved cellular immunogenicity when used in a
prime-boost combination in humans with a variety of antigens
[7–9] and on their ability to control viremia after multiple routes
of SHIV challenge in rhesus macaques [10,11].
The Phase I trial described in this report was designed to assess
the safety, tolerability, and humoral and cellular immunogenicty of
ADMVA alone. A parallel Phase I study of the ADVAX vaccine
alone was conducted separately, as reported in the accompanying
manuscript.
Methods
Study Setting
The study was conducted at the Rockefeller University Hospital
in New York City, USA, and at the University of Rochester
Medical Center in Rochester, New York, USA. The protocol for
this trial and supporting CONSORT checklist are available as
supporting information; see Checklist S1 and Protocol S1. This trial
is registered at clinicaltrials.gov, registry number NCT00252148,
http://clinicaltrials.gov/ct2/show/NCT00252148.
Participants
Healthy men and women aged 18–40 years were eligible for
participation if they were not at high risk for HIV-1, as defined by
having none of the following activities in the six months prior to
enrollment: unprotected vaginal or anal sex with a known HIV-1-
infected person or casual partner, injection drug use, acquisition of
a sexually transmitted disease, or sex work for money or drugs.
Participants agreed to safe sexual practices and to use effective
contraception to avoid pregnancy throughout the duration of the
18-month study. Participants had to demonstrate a clear
understanding of the possibility of HIV-1 seroconversion in the
event of a humoral immune response to encoded HIV-1 antigens.
Exclusion criteria included chronic medical conditions, clinically
significant abnormal laboratory parameters, infection with Hep-
atitis B or C, infection with syphilis, or recent receipt of a vaccine
or blood transfusion. Although MVA has not been associated with
myocarditis or pericarditis to date, due to the rare occurrence of
cardiac events after vaccination with live replicating vaccinia to
prevent smallpox infection [12,13], volunteers with abnormal
electrocardiograms, troponin values, or a history of cardiac
abnormalities were also excluded from this study. Individuals
with a prior history of smallpox immunization were limited to no
more than ten percent of all volunteers.
Ethical Compliance
This study was approved by the Institutional Review Boards of
the Rockefeller University Hospital and the University of
Rochester Medical Center. All participants in this study provided
written informed consent after appropriate review, discussion and
counseling by the clinical study team. The trial was monitored by
the International AIDS Vaccine Initiative (IAVI) and conducted in
compliance with International Conference on Harmonisation -
Good Clinical Practice (ICH-GCP).
Interventions
The ADMVA vaccine is a non-replicating viral vaccine
constructed with the MVA backbone expressing sequences from
the env, gag, pol, nef, and tat genes of HIV-1 B’/C, as previously
described [6]. GMP manufacturing, quality control testing and
real-time stability studies of ADMVA clinical lots were undertaken
at Impfstoffwerk Dessau-Tornau GmbH (IDT-Germany).
The study was randomized, dose-escalating, and double-blinded
with respect to active vaccine or placebo. Study site staff and
volunteers remained blinded with respect to the allocation of
placebo or vaccine, but not dosage group. Safety and tolerability of
ADMVA or placebo in each dosage group were evaluated by an
independent Data and Safety Monitoring Board at least 14 days
after the12th volunteer had received the second injection, and
prior to initiation of enrollment of the next dose group. The study
design is summarized in Table 1.
Objectives
The primary objective was to evaluate the safety and tolerability
of three vaccinations with ADMVA at three different dosage levels
in healthy HIV-uninfected adults. The secondary objective was to
evaluate the humoral and cellular immunogenicity of ADMVA
versus placebo at each dose.
Outcomes
Primary endpoints were designed to evaluate the safety of
ADMVA in human volunteers. Local reactogenicity (including
pain, tenderness, erythema, edema, skin damage, induration,
and formation of crust, scab or scar) and systemic reactogenicity
(including fever, chills, headache, nausea, vomiting, malaise,
fatigue, myalgia, arthralgia, rash, chest pain, palpitations,
reduced exercise, shortness of breath and allergic reaction)
were assessed by telephone two to four days following each
vaccination and by history and physical examination one and
two weeks after each vaccination. Subjects were monitored for
adverse events, general health and laboratory parameters at
each study visit. Due to reports of myo- and pericarditis
following vaccination with live replicating vaccinia virus
[12,13], subjects were also monitored for evidence of cardiac
abnormalities.
Secondary endpoints were designed to evaluate the cellular and
humoral immunogenicity of ADMVA. Cellular immunogenicity
was assessed by IFNc ELISpot on frozen peripheral blood
mononuclear cells (PBMCs) at the IAVI Core Laboratory at the
Imperial College, London, as previously described [14], and as
detailed in the accompanying manuscript in this issue.
Cell stimulation.
ELISpot-positive samples were tested for
phenotype, cytokine secretion, and antigen-specific proliferation
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using
accompanying manuscript in this issue.
Humoral immunogenicity.
(NIH AIDS Reagent Program) were assessed by ELISA at pre-
vaccination baseline and two weeks after each vaccination, as
described by Huang et al. [15]. In parallel, anti-gp160, anti-p24,
or anti-gp36 Group M/O antibodies were assessed using the
Genetic SystemsTMHIV-1| HIV-2 PLUS O EIA Kit (Bio-Rad
Laboratories, Hercules, CA), at the New York State Department
of Health. Those samples that were positive were further evaluated
by the Genetic SystemsTMHIV-1 Western Blot Kit (Bio-Rad
Laboratories, Hercules, CA) and for viral load quantification using
the Roche Amplicor HIV-1 Monitor v1.5 RNA-PCR Kit (Roche
Diagnostic Systems, Indianapolis, IN) to differentiate a response to
vaccine from de novo HIV infection. Results were monitored by an
independent physician to maintain blinding of the clinical study
team.
Serum from pre-vaccination and from four weeks after the third
vaccination was assessed for neutralization of a panel of laboratory
strain and primary HIV-1 Clade C and Clade B isolates at
Monogram Biosciences, Inc. (San Francisco, CA) [16]. Develop-
ment of anti-vaccinia binding antibodies was quantified by a
binding antibody ELISA performed by V-Bio, Inc. (St. Louis,
MO).
Antibodies against conformational envelope were detected by
an immunoflourescent staining assay for Vero cells expressing
envelope. Vero cells were transfected with a DNA plasmid
expressing Clade C/B’ envelope. After 48 hours, cells were fixed
and incubated with undiluted serum for 37uC for one hour.
Antibodies bound to envelope were detected by an anti-human
IgG fluorescent dye (Alexa Fluor 594 goat anti-human IgG,
Invitrogen, Carlsbad, CA).
polychromatic flowcytometry asdescribedinthe
Antibodies to Clade C gp120
Sample Size
In each of the three dosage groups volunteers were randomized
in a 3:1 ratio of active vaccine to placebo. The study design
allowed for a total of 48 volunteers to be enrolled; 36 volunteers
receiving active vaccines and 12 volunteers receiving placebo.
However, up to ten percent over enrollment was permitted to
compensate for discontinuation of vaccinations within 30 days of
enrollment, resulting in an extra vaccine recipient in the low and
high dosage groups, making the total sample size 50. The small
sample size was deemed adequate for an exploratory dose-
escalation study of a novel product while investigating safety and
tolerability of the vaccine. Based on a 10% event rate in the
placebo group (n=12), there was at least 80% power to detect a
significantly greater event rate of 51% or more in the active group
(n=36) at level a=0.05 using Fisher’s exact one-sided test.
Randomization and Blinding
The randomization schedule was prepared by the statisticians at
the Data Coordinating Center at the EMMES Corporation. The
randomization list was sent to Fisher Clinical Services, Inc. for
labeling and packaging of study vaccine and placebo in a double-
blind fashion. Study site staff, volunteers, and laboratories
remained blinded with respect to the allocation of placebo or
vaccine, but not dosage group.
Statistical Methods
Data from all participants, including those lost to follow up and
those not completing the vaccination series, were included in the
analyses. The distribution of overall maximum severity per
volunteer of local and systemic reactogenicity events was used to
assess the differences between dosage groups. Fisher’s exact test
was used for 262 tables, and the Cochran-Armitage trend test was
used to investigate trends in event rates with increasing dosage.
The Kappa statistic and McNemar’s test were used for tests of
agreement.
Results
Participant Flow
As shown in Figure 1, 130 volunteers were screened for this
study, of whom 50 volunteers were enrolled. The majority of the
80 screen failures were due to medical abnormalities: 19 due to
chronic medical conditions, 24 due to abnormalities on screening
laboratories or urinalysis, and 11 due to minor abnormalities on
ECG. Eighteen volunteers withdrew consent after completing the
screening process. Of the remaining eight screen failures, seven
were assessed by the study team as being unable to comply with
the protocol, and one was already enrolled in another clinical trial
of an investigational agent. The average interval from date of
screening to enrollment was 18 days, ranging from 6–42 days. All
13 low dosage volunteers completed the three planned vaccina-
tions. In the mid dosage group, 2 volunteers received only two and
one received only one vaccination. In the high dosage group, one
volunteer received only 2 vaccinations. One placebo recipient
withdrew after the first vaccination due to a non-related serious
adverse event. Another placebo recipient missed the second
vaccination, but received the third. None of the discontinuations
was related to study vaccine.
Recruitment
Enrollment started in January 2005 and was completed in
January 2006. Study follow up ended in August 2007. Baseline
demographic and clinical characteristics for all trial participants
are listed in Table 2.
Reactogenicity and Adverse Events
ADMVA was generally well tolerated at all dosages. Two
volunteers, both randomized to receive placebo, experienced
serious adverse events not related to vaccination (pituitary tumor
and brain tumor, both likely undiagnosed pre-existing conditions).
The remainder of adverse events were mild (132/176 events, 75%)
and not related or unlikely related to vaccine (165/176 events,
94%). There was no clinical or laboratory evidence of pericarditis
or myocarditis.
The percentage of volunteers experiencing local and systemic
reactogenicity after each vaccine is presented in Figure 2. The
most frequently reported local reactogenicity events in all dosage
groups were pain and tenderness. The most frequently reported
systemic reactogenicity events in all dosage groups were headache,
fever, myalgia and fatigue, all of which were generally mild. Local
Table 1. Study design.
Group
Vaccine Dose
(pfu)
Volunteers
Receiving
Vaccine:Placebo
Vaccination
Schedule
(Months)
Total
Follow Up
(Months)
Low1.06107
12:40, 1, 618
Middle 5.06107
12:40, 1, 618
High2.56108
12:40, 1, 6 18
Total 36:12
Note: An over enrollment of 10% was allowed to compensate for
discontinuation of vaccinations within 30 days of enrollment.
doi:10.1371/journal.pone.0008816.t001
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Figure 1. Clinical trial participant flow diagram.
doi:10.1371/journal.pone.0008816.g001
Table 2. Subject demographics.
ADMVA LowADMVA Mid ADMVA High PlaceboAll Subjects
Gender
Male668626
Female7656 24
Age
Mean 27.624.8 25.1 25.825.8
Range 19–4019–4021–3218–4018–40
Race/Ethnicity
Caucasian6266 20
Asian10214
African American1622 11
Hispanic or Latino3422 11
Native American or Alaskan Native10001
Native Hawaiian or Other Pacific Islander10001
Other/Unknown00112
doi:10.1371/journal.pone.0008816.t002
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and systemic reactogenicity events generally resolved within 4 days
after vaccination. The proportion of volunteers with moderate/
severe local reactions increased significantly with increasing dosage
(15%, 33% and 62% in the low, mid and high dose groups,
respectively: p=0.015), whereas dose had no significant effect on
moderate/severe systemic reactogenicity (p=0.129).
Cellular Immunogenicity
IFNc ELISpot results are summarized in Table 3. In the low
dosage group, three of twelve vaccinees (25%) formed ELISpot
responses to HIV envelope (mean 79, range 57–138 SFC/million).
One volunteer in the low dose group was excluded from ELISpot
analysis due to QC failure secondary to high background. Six of
twelve vaccinees (50%) in the mid dosage group (mean 69, range
40–394 SFC/million) and eight of thirteen vaccinees (62%) in the
high dosage group mounted IFNc responses to multiple gene
products (mean 89, range 42–275 SFC/million). There were no
positive responses to any peptide pool among the placebo
recipients. The majority of the responses in the low and mid
dosage groups occurred after at least the second vaccination. In
the high dosage group, IFNc ELISpot responses in 5/8 responders
occurred as early as 1–2 weeks after the first vaccination.
Intracellular cytokine responses were undetectable in all ELI-
Spot-positive volunteers.
Humoral Immunogenicity
Binding antibodies.
volunteers (62%) in the low dosage group, six of twelve volunteers
(50%) in the mid dosage group, and ten of thirteen (77%) in the
high dosage group formed binding antibodies against HIV-1
subtype C gp120. None of the placebo recipients formed positive
responses. Total response rates in the low, mid and high dosage
groups in either the IFNc ELISpot assay or the anti-gp120 binding
assay were 10/13 (77%), 7/12 (58%), and 12/13 volunteers (92%),
respectively. Anti-gp120 binding antibodies were elicited in all
three dose groups after the three injections of ADMVA, although
one responder formed antibodies after one vaccination with high
dose ADMVA, and five responders formed anti-gp120 antibodies
after two vaccinations. All responders formed antibodies to
conformationally intact HIV-1 B’/C envelope expressed on
Vero cells, as measured by immunofluorescent staining.
One ADMVA vaccine recipient in the high dosage group tested
positive on a standard clinical HIV ELISA two weeks after the
third vaccination. This Western Blot showed positive bands
against gp120 and p24, but the viral load as measured by HIV-1
RT-PCR was undetectable. All subsequent ELISA results in this
volunteer were negative. At the final study visit, no volunteers
tested HIV positive.
Neutralizing antibodies.
Table 4 also depicts the frequency
of volunteers with neutralizing antibodies to HIV-1 laboratory
strain SF162, and to a subtype C HIV-1 isolate. After three
vaccinations of ADMVA, two volunteers were able to neutralize
the subtype C viruses, and three volunteers (one placebo and two
high dose volunteers) were able to neutralize HIV-1 laboratory
strain NL43. 21/36 ADMVA recipients (58%) were able to
neutralize the laboratory HIV strain SF162 at Week 28, which
trended towards agreement with the formation of anti-gp120
binding antibodies (Kappa=60%, McNemar’s test p=0.7).
As shown in Table 4, eight of thirteen
Figure 2. Local and systemic reactogenicity by dosage group.
Panels A and B depict the percentage of volunteers experiencing local
or systemic reactogenicity, respectively, by severity and dosage group.
Total responses and (percentage of responses) are depicted above each
bar. There is evidence of increased moderate/severe local reactions with
increasing dose (two-tailed Cochran-Armitage trend test: p=0.015). A
similar comparison of systemic reactogenicity was not statistically
significant (p=0.129).
doi:10.1371/journal.pone.0008816.g002
Table 3. IFNc ELISpot results.
ADMVA dosage groups (pfu)166107
566107
2.566108
Positive volunteers 3/12 (25%) 6/12 (50%) 8/13 (62%)
SFC per million – mean 796989
SFC per million – range(57–138)(40–394) (42–275)
Gag responders020
Env responders346
Pol responders033
Nef-Tat responders013
Response Timing – median (week) 27 276
Response Timing – range (weeks) 6–282–78 1–78
Table 3 summarizes the IFNc ELISpot response rate and magnitude in spot
forming cells per million PBMCs (SFC) among volunteers receiving ADMVA by
dose group. There were no positive responses in placebo recipients. The timing
of IFNc ELISpot responses and distribution of antigens eliciting these responses
are listed.
doi:10.1371/journal.pone.0008816.t003
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Reciprocal geometric mean neutralizing titers were all low (,300
except for one titer of 476) against the SF162 in the low, mid and
high dose groups and ,20 in all placebo specimens.
Anti-vaccinia antibodies.
Figure 3 depicts the average anti-
vaccinia antibody titer over time in each dosage group. As
expected, anti-vaccinia titers increased after each subsequent
immunization in a dosage-dependent manner. In the placebo
group, 1/12 (8.3%) was positive at baseline and throughout the
trial. No other placebos were positive after immunization. In the
low dose group, 3/12 (25%) were positive at baseline and 12/12
after immunization (100%). One volunteer in the low dose group
was excluded from analysis due to unavailability of sample. In the
mid dose group, 1/12 (8.3%) were positive at baseline and 11/12
(91.6%) after immunization. In the high dose group, none were
positive at baseline and 13/13 (100%) were positive after
immunization. Interestingly, there was no correlation between
individuals with a prior history of smallpox vaccination and
positive baseline anti-vaccinia titers.
Discussion
This trial was the first evaluation of ADMVA in human
volunteers. ADMVA was well tolerated at the dosage levels tested,
with no evidence of cardiac toxicity. There were no serious
adverse events related to vaccine. Local and systemic reactoge-
nicity following vaccination was usually mild to moderate and
generally resolved within four days. Local reactogenicity increased
in severity with each dosage group. This dosage-dependent
reactogenicity may indicate an immune response to products of
the HIV gene inserts, to the viral vector, or both. While anti-vector
immunity increased after subsequent vaccinations in each dose
group, a titer of 1:450 in the high dose group after two
vaccinations did not prevent generation of humoral or cellular
immune responses after the third vaccination.
In the mid and low dosage groups, binding antibodies to
gp120 were detected only after the third dose of ADMVA, while
in a subset of volunteers in the high dosage group, binding
antibodies were detected after the first and/or second vaccina-
tions, although the majority of vaccine recipients also required
three injections. Antibody titer peaked two weeks after
vaccination and then waned, but persisted for one year post
vaccination. Binding antibodies were likely functional in part,
given the correlation with the ability to neutralize HIV SF162, a
strain that is relatively easy to neutralize. Given the inability to
neutralize clade-matched HIV isolates in the majority of
volunteers, it is unlikely that this humoral response will be
sufficient on its own to neutralize incoming infection, reduce
viral load set point, or impact disease progression post infection
with HIV-1.
ADMVA elicits a cellular immune response, as quantified by
IFNc ELISpot assay. Responses occurred after one, two and three
vaccinations, and were directed against multiple antigens.
Unfortunately, in both humans and macaques, IFNc ELISpot
responses do not correlate with protection from HIV/SIV or
reduction in viral load [17–19]. The magnitude of the ELISpot
response may also not reflect the quality of the cellular immune
response [20,21]. In our hands, the 16-hour detection platform of
the ELISpot is more sensitive for IFNc detection than the 6-hour
detection platform of the flow assay, which may account for the
lack of detectable responses on intracellular cytokine staining. As
Table 4. Binding and neutralizing antibody response rate.
Vaccine Dose Anti-gp120 Ab (%)SF162 Neutralization (%) HIV-1 Clade C Neutralization (%)
Placebo0/12 (0%) 0/10 (0%)0/12 (0%)
1.06107
8/13 (62%) 5/13 (39%)1/12 (8%)
5.06107
6/12 (50%)6/11 (55%) 1/12 (8%)
2.56108
10/13 (77%)10/12 (83%)0/13 (0%)
doi:10.1371/journal.pone.0008816.t004
Figure 3. Graphs depict the anti-vaccinia binding antibody titer after each vaccination (arrows) by dose group, expressed as
geometric mean titer. Error bars represent SEM. Arrows indicate vaccination time points. As predicted, anti-vaccinia antibody titers increased after
each vaccination and with increasing doses of ADMVA.
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there has been no documented case of natural clearance of HIV-1
in humans, much remains to be understood regarding the
immunologic correlates of protection from HIV-1. Therefore,
many of the current vaccine strategies to induce cellular immune
responses are in effect, proceeding ‘‘blinded’’, as we do not yet
know the desired immune response.
Given the possibility of enhanced susceptibility to HIV infection
in adenoviral vaccine recipients with high pre-existing adenoviral
titers [18,19], studies of HIV vaccines in humans should not be
pursued without sufficient consideration for volunteer safety.
Poxviral vectors have fewer issues with pre-existing immunity, as
such immunity is generally limited to persons who have been
previously vaccinated against smallpox. Since routine smallpox
vaccinations have been discontinued for several decades world-
wide, with the exception of certain groups perceived to be ‘‘at
risk’’, such as military personnel and health care workers [22], the
prevalence of pre-existing immunity to an MVA-based vaccine
would arguably be low, relative to adenovirus-based vaccines [23].
This vaccine was designed to be administered in combination
with ADVAX, a matched Clade C-B’ DNA-based multigenic
vaccine (see the accompanying manuscript). Given that the DNA
prime - MVA boost vaccinations have proven superior to MVA
vaccinations alone in animal models and in humans [7–11,24,25],
it is possible that ADMVA may be more immunogenic when
administered in combination with other DNA or viral vectors.
Supporting Information
Checklist S1
Found at: doi:10.1371/journal.pone.0008816.s001 (0.19 MB
DOC)
CONSORT Checklist.
Protocol S1
Found at: doi:10.1371/journal.pone.0008816.s002 (7.99 MB
PDF)
Trial Protocol.
Acknowledgments
The authors wish to thank the Clinical and Translational Science Awards
at the Rockefeller University and the University of Rochester Medical
Center and their associated staff for assistance in conducting this clinical
trial; the New York City Department of Public Health Laboratories, V-Bio,
Inc, and Monogram, Inc. for assistance with immunomonitoring assays;
Christine Hogan, M.D., Edward Charles, M.D., and Lucio Verani for
monitoring HIV-1 status; members of our Data Safety Monitoring Board;
Delivette Castor, Ph.D., for assistance with statistical analyses; and most
importantly, our dedicated clinical trial volunteers.
Author Contributions
Conceived and designed the experiments: SV SJS ZC AL ST PA MB RB
ES YH JG PF DDH. Performed the experiments: SV SJS ZC AH CB LC
DPD MBC QF LB GJZ DFG MC LS DKG MCK. Analyzed the data: SV
SJS ZC AH AL ST PA MB CS QF LB YH MH LD CS JC DKG JG PF
DDH. Contributed reagents/materials/analysis tools: SV ZC MB RB ES
QF LB YH. Wrote the paper: SV AH AL CS JC MCK PF DDH.
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ADMVA HIV MVA Vaccine Trial
PLoS ONE | www.plosone.org7 January 2010 | Volume 5 | Issue 1 | e8816