M A J O R A R T I C L E
Specificity and 6-Month Durability of Immune
Responses Induced by DNA and Recombinant
Modified Vaccinia Ankara Vaccines Expressing
HIV-1 Virus-Like Particles
Paul A. Goepfert,1,aMarnie L. Elizaga,2,aKelly Seaton,4Georgia D. Tomaras,4David C. Montefiori,4Alicia Sato,2
John Hural,2Stephen C. DeRosa,2,3Spyros A. Kalams,5M. Juliana McElrath,2,3Michael C. Keefer,6Lindsey R. Baden,9
Javier R. Lama,15Jorge Sanchez,15Mark J. Mulligan,10Susan P. Buchbinder,12Scott M. Hammer,7Beryl A. Koblin,8
Michael Pensiero,13Chris Butler,13Bernard Moss,14and Harriet L. Robinson,11for the HVTN 205 Study Group,band the
National Institutes of Allergy and Infectious Diseases HIV Vaccines Trials Network
1Department of Medicine, University of Alabama at Birmingham;2Vaccine and Infectious Disease Division, Fred Hutchinson Cancer Research Center, and
3University of Washington, Seattle, Washington;4Laboratory for AIDS Vaccine Research and Development, Department of Surgery, Duke University
Medical Center, Durham, North Carolina;5Vanderbilt University School of Medicine, Nashville, Tennessee;6University of Rochester School of Medicine
and Dentistry, Rochester, and7Columbia Universityand8New York Blood Center, New York, New York;9Brigham and Women’s Hospital, Harvard Medical
School, Boston, Massachusetts;10Division of Infectious Diseases, Emory University, Atlanta, and11GeoVax Inc., Smyrna, Georgia;12Bridge HIV,
San Francisco Department of Public Health, California;13Division of AIDS and14Laboratory of Viral Diseases, National Institute of Allergy and Infectious
Diseases, National Institutes of Health, Bethesda, Maryland; and15Asociacion Civil IMPACTA Salud y Educacion, Lima, Peru
ticles displaying trimeric membrane-bound envelope glycoprotein (Env) were tested in a phase 2a trial in human immu-
nodeficiency virus (HIV)–uninfected adults for safety, immunogenicity, and 6-month durability of immune responses.
months, respectively (the DDMM regimen); 3 doses of MVA/HIV62B at 0, 2, and 6 months (the MMM regimen); or
Results. At peak response, 93.2% of the DDMM group and 98.4% of the MMM group had binding antibodies for
Env. These binding antibodies were more frequent and of higher magnitude for the transmembrane subunit (gp41)
than the receptor-binding subunit (gp120) of Env. For both regimens, response rates were higher for CD4+T cells
(66.4% in the DDMM group and 43.1% in the MMM group) than for CD8+T cells (21.8% in the DDMM group
and 14.9% in the MMM group). Responding CD4+and CD8+T cells were biased toward Gag, and >70% produced
2 or3 ofthe4 cytokines evaluated (ie,interferonγ, interleukin 2, tumornecrosisfactorα, and granzyme B). Six months
after vaccination, the magnitudes of antibodies and T-cell responses had decreased by <3-fold.
Conclusions.DDMM and MMM vaccinations with virus-like particle–expressing immunogens elicited durable
antibody and T-cell responses.
Clade B DNA and recombinant modified vaccinia Ankara (MVA) vaccines producing virus-like par-
Keywords.HIV/AIDS; vaccines; clinical trial; T cells; antibodies; DNA; recombinant MVA.
A human immunodeficiency virus (HIV) vaccine faces
the challenge of eliciting immune responses that can
prevent the acquisition of virus and the establishment
of latency. Vaccine-induced antibodies (Abs) can
block infection by directly neutralizing virus  and
by binding to virus and virus-infected cells to tag
them for destruction by the innate immune response
[2,3]. Elicited cytotoxic (ie, CD8+) T cells can modulate
the severity of infection and slow disease progression by
recognizing and killing infected cells.
Received 4 October 2013; accepted 19 December 2013; electronically published 7
Presented in part: AIDS Vaccine Meeting, Boston, Massachusetts, September
2012. Abstract 0A09.08 LB.
aP. A. G. and M. L. E. contributed equally to this work.
bAdditional members of the study group are listed at the end of the text.
Correspondence: Paul A. Goepfert, MD, 908 20th St South, CCB 328, Birmingham,
AL 35294 (email@example.com).
The Journal of Infectious Diseases 2014;210:99–110
© The Author 2014. Published by Oxford University Press on behalf of the Infectious
Diseases Society of America. All rights reserved. For Permissions, please e-mail:
HIV Vaccine–Induced Durable Abs • JID 2014:210 (1 July) • 99
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The one vaccine to achieve at least partial prevention of infec-
tion (efficacy, 31.2%) was tested in Thailand in the RV144 trial
. This vaccine regimen consisted of 2 recombinant canarypox
primes(ALVAC-HIVvCP1521),followedby2 canarypox plusbi-
valent gp120 protein in alum boosts (AIDSVAX B/E) . The
elicited Abs had limited ability to neutralize tier 1 HIV isolates,
which are easy to neutralize, and no detectable neutralizing activ-
ity for tier 2 viruses, which are more difficult to neutralize and
characteristic of most currently circulating viruses .In the cor-
circulating Env-specific immunoglobulin A (IgA) appeared to de-
crease vaccine efficacy by competing with immunoglobulin G
(IgG) binding and Fcγ-initiated mechanisms of protection, such
as Ab-dependent cellular cytotoxicity [7,10,11].Nonneutralizing
Ab, induced by the RV144 vaccine regimen, captured infectious
RV144 rapidly waned, fallingby ≥10-fold in the first 6 months [6,
13]. Efficacy also fell with time, from an estimated 60% at peak
vaccine response to <30% by 2.5 years .
The DNA and recombinant modified vaccinia Ankara
(MVA62B) vaccines tested in this phase 2a study produce
virus-like particles that display membrane-bound trimeric
forms of Env [15–17]. Simian immunodeficiency virus (SIV)
prototypes of these vaccines elicited 61%–64% reductions in
the per-challenge risk of intrarectal infection and prevented in-
fection in 25% of the animals receiving 12 weekly rectal admin-
istrations of the heterologous SIVE660 . Phase 1 testing of
these vaccines revealed that both the DDMM regimen, involv-
ing 2 doses of DNA vaccine followed by 2 doses of MVA62B
vaccine, and the MMM regimen, involving 3 doses of
MVA62B vaccine, were well tolerated and replicated the overall
patterns of immunogenicity observed for analogous SIV immu-
nogens in macaques [19,20]. The current phase 2atrial expand-
ed testing of the DDMM and MMM regimens and extended
analyses of both T-cell and Ab responses to include the durabil-
ity of elicited responses at 6 months after vaccination.
SUBJECTS, MATERIALS, AND METHODS
The GeoVax vaccine, GOVX-B11 comprises a DNA prime and
B HIV-HXB-2/BH10 sequences and Env from HIV ADA
sequences (gp41/120 cleavage site intact) from a single transcript
by subgenomic splicing . The vaccine is rendered nonin-
fectious by gene deletions and inactivating point mutations [15,
16]. The MVA component, MVA62B, encodes HIV-1 Gag, pro-
tease, reverse transcriptase, and Env from the same sequences
and also produces immature noninfectious virus-like particles
. In MVA62B, the ADA Env gene is truncated for the 115
C-terminal amino acids of the endodomain of gp41 to enhance
stability of the vaccine insert during manufacture .
HIV Vaccine Trials Network (HVTN) protocol 205 was a ran-
domized, double-blind, placebo-controlled trial conducted at
clinical sites in the United States and Peru among participants
who were considered to be at lower risk for HIV infection (clin-
ical trials registration NCT00820846). The institutional review
boards or ethics committees for each site provided initial and
ongoing approvals and review of the research. Adults aged
18–50 years who were deemed healthy on the basis of medical
history, physical examination findings, laboratory test results,
troponin levels, and electrocardiogram (ECG) findings were en-
rolled. In part A of the study, 180 participants were enrolled, of
whom 120 were vaccinated with 3 mg of DNA at months 0 and
2, followed by 108median tissue culture infective doses of
MVA62B at months 4 and 6 (the DDMM regimen); 60 addi-
tional participants were enrolled and received normal saline as
placebo injections. In part B, 29 enrolled participants received
DDMM, and 75 enrolled participants received MVA62B at
months 0, 2, and 6 (the MMM regimen); 15 additional enrolled
subjects received normal saline as placebo injections.
Vaccines were delivered intramuscularly by needle injection at
a final volume of 1 mL into the deltoid region. Safety evaluations
included physical examinations, standard clinical chemistry and
hematological tests, and cardiac troponin analysis. Postvaccina-
tion chest symptoms were evaluated with a 12-lead ECG, and
findings were interpreted bya central ECG laboratory. Local reac-
togenicity (ie, injection site pain, tenderness, redness, erythema,
and induration) and systemic reactogenicity (ie, malaise, head-
ache, fever, chills, myalgias, arthralgias, nausea, vomiting, and fa-
tigue) were assessed for3 days followingeachvaccination or until
resolution. Adverse events were recorded for 12 months after
the first vaccination for each participant and were graded as
mild, moderate, or severe according to standard criteria (avail-
able at: http://rcc.tech-res.com/safetyandpharmacovigilance/).
Social impact assessments were obtained at each study visit fol-
lowing the first vaccination and consisted of 10 targeted ques-
tions about potential discrimination due to study participation.
Immune Response Assays
Validated binding Ab multiplex assays  for IgG and IgA
were performed according to a prespecified assay study plan
and good clinical laboratory practices guidelines. HIV-specific
anti-IgG Abs were detected with mouse anti-human IgG
(Southern Biotech, Birmingham, AL). Anti-HIV IgA responses
in serum were detected with goat anti-human IgA (Jackson Im-
munoresearch, West Grove, PA) in specimens depleted of IgG
by use of protein G high-performance MultiTrap plates (GE
100 • JID 2014:210 (1 July) • Goepfert et al
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Healthcare Life Sciences, Pittsburgh, PA) according to the man-
ufacturer’s instructions, with minor modifications. Antibody
measurements were performed using a Bio-Plex 200 instrument
(Bio-Rad, Hercules CA), and results are expressed as mean fluo-
rescence intensity. The preset criteriafor inclusion of samples in
data analysis were a coefficient of variation of ≤15% for dupli-
cate measurements and the presence of >100 beads counted per
sample. Positive controls included anti-HIV immunoglobulin
and monoclonal IgA Ab carrying the b12 region (kindly provid-
ed by Drs Dennis Burton and Ann Hessell). Negative controls
were blank beads, HIV-1–negative normal human serum
(Sigma Aldrich, St. Louis, MO), and serum samples obtained
before vaccination. The consensus antigens ConS gp140 and
Con6 gp120 were kindly provided by Drs Larry Liao and Barton
Haynes (Duke Human Vaccine Institute, Durham, NC). Re-
combinant MN gp41 (ImmunoDiagnostics, Woburn, MA),
ADA gp120 (MyBioSource, San Diego, CA), and p24 (BD Bio-
sciences, San Jose, CA) proteins were purchased. To evaluate
vaccine-induced seroreactivity, we performed enzyme-linked
immunosorbent assays (Abbot Laboratories, Abbot Park, IL)
and Western blot testing (Bio-Rad) on specimens obtained
after the final vaccination .
Neutralizing Abs were measured as a reduction in Tat-
regulated luciferase reporter gene expression in either TZM-bl
or A3R5 cells, as described elsewhere . The TZM-bl assay
measured neutralization titers against a panel of heterologous
Env-pseudotyped viruses that exhibit either a tier 1A (MN.3,
SF162.LS, Bal.26, W61D-TCLA.71, and MW965.26), a tier 1B
(SS1196.1), or tier 2 (CAAN5342.A2, REJO4541.67, SC422661.8,
and TRO.11) neutralization phenotype in TZM-bl cells. The
A3R5 assay measured neutralization titers against infectious
molecular clones that exhibit either a tier 1A (9020.A13) or
tier 2 (CH77, RHPA, SC22.3C2) neutralization phenotype in
A3R5 cells. Virus stocks were produced by transfection in 293
T cells. All viruses are clade B, except for MW965.26, which is
Peripheral blood mononuclear cells (PBMCs) were processed
from whole-blood specimens and cryopreserved at the HVTN
clinical site laboratories within 8 hours of venipuncture [24,
25]. Blood specimens for PBMC processing were obtained 2
weeks after each vaccination and 3 and 6 months after the last
vaccination. HIV-specific T-cell responses were measured using
intracellular cytokine staining as previously described [26, 27].
Global potential T-cell epitope peptide pools representing HIV
Env (3 pools), Gag (2 pools), or Pol (3 pools) were used at afinal
concentration of 1 µg per mL for each peptide . Cells were
first stained with the Aqua Live/Dead Fixable Dead Cell Stain
(Invitrogen) and then fixed, permeabilized, and stained with
CD3 PE-TR (Beckman-Coulter), CD4 APC-Cy7, CD8 PerCP-
Cy5.5, interferon γ (IFN-γ) phycoerythrin (PE)–Cy7,
interleukin 2 (IL-2)-PE, tumor necrosis factor α (TNF-α) fluo-
rescein isothiocyanate, perforin Alexa 647, granzyme B Alexa
700, and CD57 Alexa 405 (BD Biosciences for all except CD3
PE-Texas red). Positive responses were identified using 1-
sided Fisher exact test and the numbers of CD4+or CD8+T
cells producing IFN-γ and/or IL-2 in response to peptide
Selection of Samples for Longevity Assays
Longevity studies were conducted at 24 weeks after the last vac-
cination on samples from participants who had positive assay
results 2 weeks after their last vaccination.
For safety, the number and percentage of participants experi-
encing each reactogenicity symptom were tabulated by severity
and vaccination regimen by visit and overall. Each participant’s
reactogenicity was counted once under the maximum severity
antibody; see Subjects, Materials, and Methods for a description of
each regimen. A, The percentage of participants with human immunodefi-
ciency virus (HIV)–specific CD4+and CD8+T-cell responses to any protein
(Gag, Env, or Pol) and immunoglobulin G (IgG) and immunoglobulin A (IgA)
responses to consensus gp140. Data are for samples collected at 2 weeks
following the final vaccination for all participants for whom measurements
could be obtained. B, Summary of response rates shown in panel A.
Abbreviation: CI, confidence interval.
Response rates for DDMM- and MMM-elicited T cells and
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for that injection visit. Adverse events were classified by the
maximum severity or the strongest recorded causal relationship
to treatment, using Medical Dictionary for Regulatory Activities–
preferred terms. Immunogenicity response rates and magni-
tudes among positive responders were compared between
groups using Fisher exact or χ2tests and Wilcoxon rank sum
tests. Response magnitudes for studies on longevity were com-
pared for all participants (not just positive responders), using
Wilcoxon signed rank tests. P values were not adjusted for
multiple comparisons. Wilson score intervals were reported
for binomial proportions .
Participant Accrual, Demographic Characteristics, Tolerability,
The median age of the enrolled participants was 25 years, and
59% were male. The majority of participants were white (59%);
able antibody responses to the indicated proteins (A) is given along with response magnitudes (B) and a response summary for the subset of individuals in
the DDMM group, MMM group, or placebo group (C); see Subjects, Materials, and Methods for a description of each regimen. Response magnitudes for
gp41 and p24 represent low estimates because responses of >23000 are above the range of the assay. Median titers are in fluorescence units and are for
positive responses. Box plots show median values and interquartile ranges (IQRs); the whiskers indicate the lowest datum still within 1.5 IQR of the lower
quartile and the highest datum still within 1.5 IQR of the upper quartile. Abbreviations: MFI, mean fluorescence intensity, NA, not applicable.
The proportion of responders and specificity of vaccine-induced binding antibody responses. A–C, The percentage of participants with detect-
102 • JID 2014:210 (1 July) • Goepfert et al
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13% were African American, and 23% were Hispanic. All par-
ticipants received their initial vaccination, and 274 (91%) re-
ceived all doses.
Both the DDMM and MMM vaccine regimens were safe and
well tolerated. Participants had similar local (injection site) re-
actogenicityafter placebo and JS7 DNA administrations, report-
ing no or mild local symptoms . However, MVA62B was
associated with an increased number of participants experienc-
ing mild (up to 68% of participants) or moderate (up to 27% of
participants) local reactogenicity (Supplementary Figure 1A).
Most of the local reactogenicity was pain at the injection site.
A majority of participants reported no or mild systemic symp-
toms, with a few moderate reactions, similar to placebo recipi-
ents (Supplementary Figure 1B).
Therewere no serious or life-threatening adverse events relat-
ed to the vaccinations (Supplementary Table 1). However, 1
participant experienced an allergic reaction ≤15 minutes after
the second MVA62B vaccination that was considered definitely
related to the MVA62B inoculation (Supplementary Table 1).
The symptoms resolved within 2 hours, and no further vaccina-
tions were given. Fewer than 7% of the participants reported a
social problem, and the majority felt that the problem repre-
sented a minimal impact.
HIV-1–Specific Ab Responses
The initial screen for Ab responses was conducted using gp140,
an antigen that represents the gp120 receptor binding subunit
of Env and the extracellular region of the gp41 transmembrane
domain (Figure 1). The DDMM and MMM regimens elicited
IgG Abs to gp140 in 93.2% and 98.4% of vaccine recipients, re-
spectively. Testing of the specificity of the gp140 IgG response
revealed that it was strongly biased toward gp41, to which
92.5% of subjects in the DDMM group and 95.3% in the
MMM group responded (Figure 2A). In contrast, responses to
Con6 gp120 were detected in 47% in the DDMM group and
70% in the MMM group. Response rates and titers to the
matched ADA gp120 did not differ significantly from those to
the consensus gp120. Titers of gp41-specific IgG were much
higher than titers of gp120-specific IgG (>33-fold higher for
the DDMM group and >17-fold higher for the MMM group;
Figure 2B and 2C).
To assess the durability of the responses, Ab titers were
assessed 2 and 24 weeks following vaccination. During this
period, the proportion of responders for gp140 decreased
by 15.6% for the DDMM group but did not decline in
the MMM group (Figure 3). Over the same period, the
regimen. A, The durability of positive antibody responses targeting gp140, by time after receipt of last vaccination. Box plots show median values and
interquartile ranges (IQRs); the whiskers indicate the lowest datum still within 1.5 IQRof the lower quartile and the highest datum still within 1.5 IQR of the
upper quartile. B, Summary of the number of participants and response rates and titers for antibody responses, by time after receipt of last vaccination.
Throughout, the response rates and magnitudes are for the subset of individuals chosen for durability studies. Response titers are fluorescence units. All
participants selected for this durability analysis had positive responses at 2 weeks. Responses for a few became negative at 24 weeks; summary statistics
include both positive and negative responses. Abbreviation: MFI, mean fluorescence intensity.
The durability of binding antibody responses in the DDMM and MMM groups; see Subjects, Materials, and Methods for a description of each
HIV Vaccine–Induced Durable Abs • JID 2014:210 (1 July) • 103
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magnitudes of Ab responses for both groups declined by 2.7-
fold (Figure 3).
Two weeks after the last vaccination, the serum IgA response
rates for gp140 were similar in the DDMM and MMM groups
(12% vs 13%; Figure 1). The IgA response was also biased to-
ward gp41, with IgA being detected for gp120 in only 2 partic-
ipants. In the 11%–12% of participants with both IgG and IgA
responses, the median ratio of IgG to IgA for gp140 was 17 for
the DDMM group and 34 for the MMM group (data not
Both vaccine regimens induced neutralizing Abs, with 64.4%
in the MMM group and 30.4% in the DDMM group having de-
tectable serum neutralizing responses against ≥1 tier 1 isolate
(Figure 4A). Tier 2 isolates were neutralized by sera from 37%
of participants in the MMM group and 15% in the DDMM
group (Figure 4C). Titers for 2 tier 1 isolates and 1 tier 2 isolate
were higher for the MMM recipients (Figure 4B and 4D).
Vaccine-induced seroreactivity was noted in 84% of the indi-
viduals in the DDMM group and 87.7% in the MMM group
(Supplementary Table 2). The majority of participants who de-
veloped vaccine-induced seroreactivity had a positive Western
blot result, with reactivity to Gag and Env, the 2 major diagnos-
tic determinants of HIV infection.
HIV-1 Specific T-Cell Responses
Two weeks following the final vaccination, 66% of participants
in the DDMM group had detectable CD4+T-cell responses,
compared with 43% in the MMM group (P = .005). Vaccine-
induced HIV-1–specific CD8+T cells were detected in 22% in
antibodies. B and D, Magnitude of these responses 2 weeks after the final vaccination (both regimens). Panels A and B represent tier 1 viral isolates tested
using the TZMBCL cell line, and panels C and D represent a tier 1A and 3 tier 2 viruses analyzed with the A3R5 cell line. See Subjects, Materials, and
Methods for a description of each regimen and for further details. Abbreviation: IC50, half maximal inhibitory concentration.
Vaccine-induced neutralizing antibody responses. A and C, The percentage of participants with human immunodeficiency virus–neutralizing
104 • JID 2014:210 (1 July) • Goepfert et al
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the DDMM group and 15% in the MMM group. The magni-
tudes of positive CD4+and CD8+T-cell responses were similar
between groups (Figure 5B, 5D, and 5E).
In the DDMM group, both CD4+and CD8+T-cell re-
sponses targeted Gag more frequently than Env (Figure 5A,
5C, and 5E). For CD4+T cells, 64.1% of the participants
(A) and CD8+(C) T-cell responses directed against any protein, Gag, Env, or Pol. B and D, The magnitude of responses directed against any protein, Gag, Env,
or Pol among CD4+and CD8+T cells, shown as apercentage of the total CD4+or CD8+T cells. Box plots show median values and interquartile ranges (IQRs);
the whiskers indicate the lowest datum still within 1.5 IQR of the lower quartile and the highest datum still within 1.5 IQR of the upper quartile. E, Median
percentage of participants with CD4+and CD8+T-cell responses for Gag, Env, and Pol. The median magnitude of interferon γ (IFN-γ) and/or interleukin 2
(IL-2) production as a percentage of the total CD4+or CD8+T cells is shown in parentheses.
The proportion of responders and magnitude of vaccine-induced T-cell responses. A and C, The percentage of participants demonstrating CD4+
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responded to Gag, whereas 31.2% responded to Env
(P < .0001). For CD8+T cells, 17.3% responded to Gag and
9% to Env (P = .07). In the MMM group, CD4+T-cell
response rates also were higher for Gag than for Env
(41.5% vs 10.8%; P = .0001), whereas for CD8+T-cell re-
sponse rates, the greater response rate for Gag was only a
trend (Figure 5C and 5E). Pol responses were infrequently
seen in both groups.
studies;seeSubjects, Materials,andMethods fora description ofeachregimen. A andB, Magnitude of vaccine-induced T-cell responsesto GagamongCD4+
(A) and CD8+(B) T cells obtained 2 and 24 weeks after the final vaccination from subjects, shown as a percentage of the total CD4+or CD8+T cells. Box plots
show median values and interquartile ranges (IQRs); the whiskers indicate the lowest datum still within 1.5 IQR of the lower quartile and the highest datum
still within 1.5 IQR of the upper quartile. C, Summary of the number of participants evaluated and CD4+and CD8+T-cell response rates and magnitudes.
analysis had positive responses at 2 weeks. A few became negative at 24 weeks; summary statistics include both positive and negative responses.
The durability of vaccine-induced T-cell responses for the subset of individuals in the DDMM and MMM groups who were chosen for durability
106 • JID 2014:210 (1 July) • Goepfert et al
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Studies on the longevity of the T-cell responses to Gag re-
vealed response rates falling by about 25% in the first 6 months
after vaccination in the DDMM group and undergoing greater
falls of 30% (for CD8+T cells) and 45% (for CD4+T cells) in
the MMM group (Figure 6). During this 6-month period, the
magnitudes of the CD4+and CD8+T-cell responsesto Gag con-
tracted by 1.6–2.1-fold (Figure 6).
scored forcoproduction of IFN-γ, IL-2, TNF-α, and granzyme B
(Figure 7). Two weeks following the final vaccination, CD4+
T cells from the DDMM and MMM groups most commonly ex-
pressed 2 or 3 of these cytokines (Figure 7A). For both regimens,
the most frequent pattern for dual cytokine production was
IFN-γ and TNF-α; triple cytokine production most frequently
added IL-2 (Figure 7A). These patterns of cytokine production
were similar 24 weeks after the last vaccination (Figure 7B).
The CD8+T-cell response showed higher polyfunctionality
than the CD4+T-cell response. Two weeks after the final
responding CD4+(A) and CD8+(B) T cells at 2 weeks (A; upper row) and 24 weeks (A; lower row) after the final vaccination. In each row, the left panel
summarizes the percentage of Gag-specific cells with 1, 2, 3, or 4 expressed cytokines, and the right panel denotes the percentage of Gag-specific cells
expressing each cytokine alone or combinations of 2 and 3 cytokines. Only participants with positive responses as determined for the interferon γ (IFN-γ)
and/or interleukin 2 (IL-2) subset are in the analyses (left-most panels). See Subjects, Materials, and Methods for a description of the DDMM and MMM
regimens. Box plots present median values and interquartile ranges. Abbreviations: GzB, granzyme B; TNF-α, tumor necrosis factor α.
The polyfunctionality of vaccine-induced T-cell responses. A, The number of expressed functions and the cytokine production patterns for Gag-
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vaccination, CD8+T cells from subjects in the DDMM and
MMM groups expressed 3 cytokines slightly more often than
they expressed 2 cytokines, with a minority of cells expressing
1 or 4 cytokines. Triple cytokine–producing CD8+T cells in-
duced by DDMM tended to include more IL-2 coproduction,
whereas the MMM-induced cells included more granzyme
B–coproducing cells (Figure 7C). Six months following the
final vaccination, the differences in granzyme B and IL-2 copro-
duction for the DDMM and MMM CD8+T-cell responses were
no longer significant (Figure 7D). This reflected a higher pro-
portion of granzyme B–coproducing cells in the DDMM
group. Throughout the study, cells that were granzyme B posi-
tive were IL-2 negative (Figure 7). Both CD4+and CD8+T cells
responding to Env showed patterns of cytokine coproduction
similar to those of cells responding to Gag (data not shown).
An important finding for this trial was the durability of the Ab
response to gp140, which declined by <3-fold during the first 6
months after vaccination (the latest time point studied). In trials
using gp120 protein in alum as a boost for a poxvirus prime
(RV144) and gp120 protein in AS02A adjuvant for priming
and boosting , declines in the magnitudes of binding
Abs have been 10-fold in the same period [13, 30]. We hypo-
thesize that the durability of the DDMM- and MMM-elicited
Ab response reflects a number of factors that include the
gp41 dominance of the response and the MVA vector stimu-
lating the generation of survival signals in responding B cells
. The parent for the MVA vector, the smallpox vaccine, is
known for its ability to establish long-lived Ab responses .
The durability of an Env-specific Ab response is important for
an HIV vaccine because Env-specific Abs have the greatest po-
tential to prevent infection.
Interestingly, the vaccines induced Env-specific Ab that was
mainly focused on the relatively conserved gp41 subunit of HIV
Env. The focus on gp41 is consistent with the virus-like particle
vaccine inducing a response that mimics the response to HIV
infection, in which emergence of Ab to gp41 precedes emer-
gence of Ab to gp120 [22, 33]. Sequences in gp41 are targets
for Ab-mediated virion capture . They also include con-
served targets for Ab-dependent cellular cytotoxicity that are
exposed during virus fusion (G. Lewis, personal communica-
tion) . In preclinical studies using a single high-dose
simian/human immunodeficiency virus challenge, the avidity
of Abs for the highly conserved immunodominant epitope of
gp41 correlated with reductions in peak viremia .
Env-specificserum IgA responses were poorly induced in this
study. In the RV144 study, titers of Env-specific serum IgA
correlated with decreased vaccine efficacy, likely due to IgA
competing for binding with IgG and reducing the initiation of
Fcγ-mediated mechanisms of protection [7, 11].
Comparison of Ab responses elicited by the DDMM and
MMM regimens confirmed that the MMM regimen elicited
higher titer gp120 responses and higher titer neutralizing Ab re-
sponses, as previously noted in the phase 1 trial, HVTN 065
. The immunogens expressed by the DDMM and MMM
regimens differ in that the Env expressed by the DNA prime
is a C-terminal complete gp160, whereas the gp150 expressed
by the MVA is truncated for the C-terminal endodomain of
gp41. The increased immunogenicity for gp120 for the MMM
regimen could reflect the gp150-truncated Env having a more
open structure than the gp160 expressed by the DNA prime.
Similar truncations have opened up the structure of Env, as
measured by tests for the binding of Abs to the CD4 binding
site and sensitivity to neutralization .
The magnitude of the T-cell responses also showed good du-
rability, declining byonly 1.6–2.1-fold during the first 6 months
after vaccination. These responses were qualitatively different
than those induced by recombinant adenovirus type 5 vectors
(rAd5), as given in the Step Study  and HVTN 505 trials
,neither of which protected vaccinees against HIV-1 infec-
tion. rAd5 induced an immune response biased toward CD8+T
cells over CD4+T cells, whereas the opposite was true for
MVA62B and other pox vectors in the presence or absence
of a DNA prime [39,40].Also, the DDMM and MMM regimens
induced CD8+T cells that predominantly targeted Gag and
not Env, as elicited in the VRC DNA/rAd5 regimen , or
Pol, as elicited in the Step Study .Targeting of Gag epitopes
by CD8+T cells has been demonstrated in multiple studies to
correlate with viral control [42–45].
elicited more highly polyfunctional cells than rAd5 regimens
have elicited [27,41]. The DDMM and MMM vaccinations elic-
ited dual-producing CD4+T cells most frequently, followed by
triple-producing CD4+T cells and single-producing CD4+
T cells, whereas rAd5 immunizations (in the presence orabsence
of a DNA prime) elicited frequencies of these populations that
were similar to each other. The DDMM and MMM regimens
also elicited predominantly triple-producing CD8+T cells,
whereas dual-producing CD8+cells have been most frequently
elicited by rAd5 regimens. The patterns of T-cell polyfunctional-
ity were remarkably stable with time. An instance in which poly-
functionality changed over 6 months was for more granzyme B
coexpression appearing in the DDMM group.
The comparisons of binding and neutralizing Abs between
the 2 regimens favor the MMM strategy, whereas comparisons
of cellular immunity favor the DDMM approach. Because the
Ab responses after the second MVA inoculation in the MMM
regimen were similar to those after the second (and final)
MVA inoculation in the DDMM regimen , a DDMM regi-
men with a third MVA boost (ie, a DDMMM regimen) will be
moved forward into efficacy trials. The goal of the DDMMM
regimen is to optimize both Ab and T-cell responses.
108 • JID 2014:210 (1 July) • Goepfert et al
by guest on July 10, 2015
STUDY GROUP MEMBERS
In addition to authors of this article, members of the HVTN
205 Study Group consist of Yeycy Donastorg, Li Qin, Dale Law-
rence, Massimo Cardinali, Jin Bae, Renée Holt, Huguette Red-
inger, Jan Johannessen, Gail Broder, Jerri Moody-White, Butch
McKay, Gabriela Calazans, Carter Bentley, Lisa Kakinami, Katie
Skibinski, Scharla Estep, Jenny Tseng, Molly Swenson, Tamra
Madenwald, Edgar Turner Overton, Srilatha Edupuganti, Na-
dine Rouphael, Jennifer Whitaker, C Mhorag Hay, Catherine
A Bunce, Pedro Gonzales, Juan Carlos Hurtado, Raphael
Dolin, Ken Mayer, Steven Walsh, and Jennifer Johnson.
Supplementary materials are available at The Journal of Infectious Diseases
online (http://jid.oxfordjournals.org/). Supplementary materials consist of
data provided by the author that are published to benefit the reader. The
posted materials are not copyedited. The contents of all supplementary
dataare thesole responsibilityofthe authors. Questions ormessagesregarding
errors should be addressed to the author.
bers at the HVTN research clinics.
Health (NIH; grants AI69452 [to the University of Alabama–Birmingham],
AI069418 [to Emory University], AI06970 [to the New York Blood Center
and Columbia University Medical Center], AI069511 [to the University of
Rochester], AI069438 [to the Asociacion Civil Impacta Salud y Educacion],
AI069412 [to Brigham and Women’s Hospital], AI069439 [to Vanderbilt
University], AI069481 [to the Fred Hutchinson Cancer Research Center],
AI069496 [to the San Francisco Department of Public Health], AI068614
[to the HVTN Core] AI068635 [to SCHARP], and AI068618 [to the
HVTN Laboratory]) and the NIH Integrated Preclinical/Clinical AIDS Vac-
cine Development Program (grant U19A1074073 to GeoVax for product
and preclinical studies).
Potential conflict of interest.
GeoVax. M. J. M. works for Emory, which is a stakeholder in GeoVax,
but M. J. M. does not have any direct holdings in GeoVax. All other authors
report no potential conflicts.
All authors have submitted the ICMJE Form for Disclosure of Potential
Conflicts of Interest. Conflicts that the editors consider relevant to the
content of the manuscript have been disclosed.
We thank the study participants and the staff mem-
H. L. R. is a stakeholder in
1. Haynes BF, Kelsoe G, Harrison SC, Kepler TB. B-cell-lineage immuno-
gen design in vaccine development with HIV-1 as a case study. Nat Bio-
technol 2012; 30:423–33.
2. Robinson HL. Non-neutralizing antibodies in prevention of HIV infec-
tion. Expert Opin Biol Ther 2013; 13:197–207.
3. Ackerman ME, Dugast AS, Alter G. Emerging concepts on the role of
innate immunity in the prevention and control of HIV infection. Annu
Rev Med 2012; 63:113–30.
4. Rerks-Ngarm S, Pitisuttithum P, Nitayaphan S, et al. Vaccination with
ALVAC and AIDSVAX to prevent HIV-1 infection in Thailand. N Engl
J Med 2009; 361:2209–20.
5. Pitisuttithum P, Gilbert P, Gurwith M, et al. Randomized, double-blind,
placebo-controlled efficacy trial of a bivalent recombinant glycoprotein
120 HIV-1 vaccine among injection drug users in Bangkok, Thailand.
J Infect Dis 2006; 194:1661–71.
6. Montefiori DC, Karnasuta C, Huang Y, et al. Magnitude and breadth of
the neutralizing antibody response in the RV144 and Vax003 HIV-1
vaccine efficacy trials. J Infect Dis 2012; 206:431–41.
7. Haynes BF, Gilbert PB, McElrath MJ, et al. Immune-correlates analysis
of an HIV-1 vaccine efficacy trial. N Engl J Med 2012; 366:1275–86.
8. Zolla-Pazner S, deCamp AC, Cardozo T, et al. Analysis of V2 antibody
responses induced in vaccineesin the ALVAC/AIDSVAXHIV-1vaccine
efficacy trial. PLoS One 2013; 8:e53629.
9. Liao HX, Bonsignori M, Alam SM, et al. Vaccine induction of antibod-
ies against a structurally heterogeneous site of immune pressure within
HIV-1 envelope protein variable regions 1 and 2. Immunity 2013;
10. Bonsignori M, Pollara J, Moody MA, et al. Antibody-dependent cellular
cytotoxicity-mediating antibodies from an HIV-1 vaccine efficacy trial
target multiple epitopes and preferentially use the VH1 gene family.
J Virol 2012; 86:11521–32.
11. Tomaras GD, Ferrari G, Shen X, et al. Vaccine-induced plasma IgA
specific for the C1 region of the HIV-1 envelope blocks binding and
effector function of IgG. Proc Natl Acad Sci U S A 2013; 110:9019–24.
12. Liu P, Yates NL, Shen X, et al. Infectious virion capture by HIV-1
gp120-specific IgG from RV144 vaccinees. J Virol 2013; 87:7828–36.
13. Karasavvas N, Billings E, Rao M, et al. The Thai Phase III HIV Type 1
Vaccine trial (RV144) regimen induces antibodies that target conserved
regions within the V2 loop of gp120. AIDS Res Hum Retroviruses 2012;
14. Robb ML, Rerks-Ngarm S, Nitayaphan S, et al. Risk behaviour and time
as covariates for efficacy of the HIV vaccine regimen ALVAC-HIV
(vCP1521) and AIDSVAX B/E: a post-hoc analysis of the Thai phase
3 efficacy trial RV 144. Lancet Infect Dis 2012; 12:531–7.
15. Smith JM, Amara RR, McClure HM, et al. Multiprotein HIV-1 Clade B
DNA/MVA Vaccine: Construction, Safety and Immunogenicity. AIDS
Res Hum Retroviruses 2004; 20:654–65.
16. Smith JM, Amara RR, Campbell D, et al. DNA/MVA vaccine for HIV
type1: effectsof codon-optimizationand theexpressionof aggregatesor
virus-like particlesontheimmunogenicityof theDNAprime.AIDSRes
Hum Retroviruses 2004; 20:1335–47.
17. Wyatt LS, Earl PL, Liu JY, et al. Multiprotein HIV type 1 clade B DNA
and MVA vaccines: construction, expression, and immunogenicity in
rodents of the MVA component. AIDS Res Hum Retroviruses 2004;
18. Lai L, Kwa S, Kozlowski PA, et al. Prevention of infection by a granu-
locyte-macrophage colony-stimualting factorco-expressing DNA/mod-
ified vaccinia Ankara simian immunodeficiency virus vaccine. J Infect
Dis 2011; 204:164–73.
19. Goepfert PA, Elizaga ML, Sato A, et al. Phase 1 safety and immunoge-
nicity testing of DNA and recombinant modified vaccinia Ankara vac-
cines expressing HIV-1 virus-like particles. J Infect Dis 2011;
20. Lai L, Kwa SF, Kozlowski PA, et al. SIVmac239 MVA vaccine with and
without a DNA prime, similar prevention of infection by a repeated
dose SIVsmE660 challenge despite different immune responses. Vac-
cine 2012; 30:1737–45.
21. Wyatt LS, Belyakov IM, Earl PL, Berzofsky JA, Moss B. Enhanced cell
surface expression, immunogenicity and genetic stability resulting from
a spontaneous truncation of HIV Env expressed by a recombinant
MVA. Virology 2008; 372:260–72.
22. Tomaras GD, Yates NL, Liu P, et al. Initial B-cell responses to transmit-
ted human immunodeficiency virus type 1: virion-binding immuno-
globulin M (IgM) and IgG antibodies followed by plasma anti-gp41
antibodies with ineffective control of initial viremia. J Virol 2008;
23. Cooper CJ, Metch B, Dragavon J, Coombs RW, Baden LR, Force
NHVTNV-IST. Vaccine-induced HIV seropositivity/reactivity in non-
infected HIV vaccine recipients. JAMA 2010; 304:275–83.
24. Bolard G, Prior JO, Modolo L, et al. Performance comparison of two
commercial BGO-based PET/CT scanners using NEMA NU 2–2001.
Med Phys 2007; 34:2708–17.
HIV Vaccine–Induced Durable Abs • JID 2014:210 (1 July) • 109
by guest on July 10, 2015
25. Bull M, Lee D, Stucky J, et al. Defining blood processing parameters for
optimal detection of cryopreserved antigen-specific responses for HIV
vaccine trials. J Immunol Methods 2007; 322:57–69.
26. Horton H, Thomas EP, Stucky JA, et al. Optimization and validation of
an 8-color intracellular cytokine staining (ICS) assay to quantify antigen-
specific T cells induced by vaccination. J Immunol Methods 2007;
27. McElrath MJ, De Rosa SC, Moodie Z, et al. HIV-1 vaccine-induced im-
munity in the test-of-concept Step Study: a case-cohort analysis. Lancet
28. Li F, Malhotra U, Gilbert PB, et al. Peptide selection for human immu-
nodeficiency virus type 1 CTL-based vaccine evaluation. Vaccine 2006;
29. Agresti C, Bernardo A, Del Russo N, et al. Synergistic stimulation of
MHC class I and IRF-1 gene expression by IFN-gamma and TNF-
alpha in oligodendrocytes. Eur J Neurosci 1998; 10:2975–83.
30. Goepfert PA, Tomaras GD, Horton H, et al. Durable HIV-1 antibody
and T-cell responses elicited by an adjuvanted multi-protein recom-
binant vaccine in uninfected human volunteers. Vaccine 2007;
31. Pulendran B, Ahmed R. Translating innate immunity into immunolog-
ical memory: implications for vaccine development. Cell 2006; 124:
32. Slifka MK, Antia R, Whitmire JK, Ahmed R. Humoral immunity due to
long-lived plasma cells. Immunity 1998; 8:363–72.
33. Yates NL, Stacey AR, Nolen TL, et al. HIV-1 gp41 envelope IgA is fre-
quentlyelicited after transmission but has an initial short response half-
life. Mucosal Immunol 2013; 6:692–703.
34. Burton DR, Hessell AJ, Keele BF, et al. Limited or no protection by
weakly or nonneutralizing antibodies against vaginal SHIV challenge
of macaques compared with a strongly neutralizing antibody. Proc
Natl Acad Sci U S A 2011; 108:11181–6.
35. Finnegan CM, Berg W, Lewis GK, DeVico AL. Antigenic properties of
the human immunodeficiency virus transmembrane glycoprotein dur-
ing cell-cell fusion. J Virol 2002; 76:12123–34.
36. Edwards TG, Wyss S, Reeves JD, et al. Truncation of the cytoplasmic
domain induces exposure of conserved regions in the ectodomain of
human immunodeficiency virus type 1 envelope protein. J Virol
37. Buchbinder SP, Mehrotra DV, Duerr A, et al. Efficacy assessment of a
cell-mediated immunity HIV-1 vaccine (the Step Study): a double-
blind, randomised, placebo-controlled, test-of-concept trial. Lancet
38. Cohen J. AIDS research. More woes for struggling HIV vaccine field.
Science 2013; 340:667.
39. Paris RM, Kim JH, Robb ML, Michael NL. Prime-boost immunization
with poxvirus or adenovirus vectors as a strategy to develop a protective
vaccine for HIV-1. Expert Rev Vaccines 2010; 9:1055–69.
40. Keefer MC, Frey SE, Elizaga M, et al. A phase I trial of preventive HIV
vaccination with heterologous poxviral-vectors containing matching
HIV-1 inserts in healthy HIV-uninfected subjects. Vaccine 2011;
41. Churchyard GJ, Morgan C, Adams E, et al. A phase IIA randomized
clinical trial of a multiclade HIV-1 DNA prime followed bya multiclade
rAd5 HIV-1 vaccine boost in healthy adults (HVTN204). PLoS One
42. Edwards BH, Bansal A, Sabbaj S, Bakari J, Mulligan MJ, Goepfert PA.
Magnitude of functional CD8+ T-cell responses to the gag protein of
human immunodeficiency virus type 1 correlates inversely with viral
load in plasma. J Virol 2002; 76:2298–305.
43. Nqoko B, Day CL, Mansoor N, et al. HIV-specific gag responses in early
infancy correlate with clinical outcome and inversely with viral load.
AIDS Res Hum Retroviruses 2011; 27:1311–6.
44. Perez CL, Milush JM, Buggert M, et al. Targeting of conserved gag-
epitopes in early HIV infection is associated with lower plasma viral
load and slower CD4(+) T cell depletion. AIDS Res Hum Retroviruses
45. Kiepiela P, Ngumbela K, Thobakgale C, et al. CD8+ T-cell responses to
different HIV proteins have discordant associations with viral load. Nat
Med 2007; 13:46–53.
110 • JID 2014:210 (1 July) • Goepfert et al
by guest on July 10, 2015