The Thai Phase III HIV Type 1 Vaccine Trial (RV144)
Regimen Induces Antibodies That Target Conserved
Regions Within the V2 Loop of gp120
Nicos Karasavvas,1Erik Billings,2Mangala Rao,3Constance Williams,4Susan Zolla-Pazner,4,5
Robert T. Bailer,6Richard A. Koup,6Sirinan Madnote,1Duangnapa Arworn,1Xiaoying Shen,7
Georgia D. Tomaras,7Jeffrey R. Currier,2Mike Jiang,8Craig Magaret,8Charla Andrews,2
Raphael Gottardo,8Peter Gilbert,8Timothy J. Cardozo,4Supachai Rerks-Ngarm,9
Sorachai Nitayaphan,10Punnee Pitisuttithum,1 1Jaranit Kaewkungwal,12Robert Paris,3, 13
Kelli Greene,14Hongmei Gao,14Sanjay Gurunathan,15Jim Tartaglia,15Faruk Sinangil,16
Bette T. Korber,17David C. Montefiori,14John R. Mascola,14Merlin L. Robb,2
Barton F. Haynes,7Viseth Ngauy,1Nelson L. Michael,3Jerome H. Kim,3
and Mark S. de Souza,1for the MOPH TAVEG Collaboration
The Thai Phase III clinical trial (RV144) showed modest efficacy in preventing HIV-1 acquisition. Plasma col-
lected from HIV-1-uninfected trial participants completing all injections with ALVAC-HIV (vCP1521) prime and
AIDSVAX B/E boost were tested for antibody responses against HIV-1 gp120 envelope (Env). Peptide micro-
array analysis from six HIV-1 subtypes and group M consensus showed that vaccination induced antibody
responses to the second variable (V2) loop of gp120 of multiple subtypes. We further evaluated V2 responses by
ELISA and surface plasmon resonance using cyclic (Cyc) and linear V2 loop peptides. Thirty-one of 32 vaccine
recipients tested (97%) had antibody responses against Cyc V2 at 2 weeks postimmunization with a reciprocal
geometric mean titer (GMT) of 1100 (range: 200–3200). The frequency of detecting plasma V2 antibodies declined
to 19% at 28 weeks post-last injection (GMT: 110, range: 100–200). Antibody responses targeted the mid-region of
the V2 loop that contains conserved epitopes and has the amino acid sequence KQKVHALFYKLDIVPI (HXB2
Numbering sequence 169–184). Valine at position 172 was critical for antibody binding. The frequency of V3
responses at 2 weeks postimmunization was modest (18/32, 56%) with a GMT of 185 (range: 100–800). In
contrast, naturally infected HIV-1 individuals had a lower frequency of antibody responses to V2 (10/20, 50%;
1Department of Retrovirology, U.S. Army Medical Component, Armed Forces Research Institute of Medical Sciences (USAMC-AFRIMS),
2U.S. Military HIV Research Program (MHRP), Henry M. Jackson Foundation for the Advancement of Military Medicine, Rockville,
3USMHRP, Walter Reed Army Institute of Research, Silver Spring, Maryland.
4Departments of Pathology and Pharmacology, NYU School of Medicine, New York, New York.
5Veterans Affairs Harbor Healthcare System, New York, New York.
6Immunology Laboratory, Vaccine Research Center, National Institute of Allergy and Infectious Diseases, National Institutes of Health,
7Duke Human Vaccine Institute, Duke University Medical Center, Durham, North Carolina.
8Vaccine and Infectious Disease Division, Fred Hutchinson Cancer Research Center, Seattle, Washington.
9Department of Disease Control, Ministry of Public Health, Nonthaburi, Thailand.
10Royal Thai Army Component, Armed Forces Research Institute of Medical Sciences (AFRIMS), Bangkok Thailand.
11Vaccine Trial Center and Department of Clinical Tropical Medicine, Faculty of Tropical Medicine, Mahidol University, Bangkok,
12Center of Excellence for Biomedical and Public Health Informatics (BIOPHICS), Faculty of Tropical Medicine, Mahidol University,
13Walter Reed National Military Medical Center, Bethesda, Maryland.
14Department of Surgery, Duke University Medical Center, Durham, North Carolina.
15Sanofi Pasteur, Swiftwater, Pennsylvania.
16Global Solutions for Infectious Diseases, South San Francisco, California.
17Theoretical Division, Los Alamos National Laboratory, Los Alamos, New Mexico.
AIDS RESEARCH AND HUMAN RETROVIRUSES
Volume 28, Number 11, 2012
ª Mary Ann Liebert, Inc.
p=0.003) and a higher frequency of responses to V3 (19/20, 95%), with GMTs of 400 (range: 100–3200) and 3570
(range: 200–12,800), respectively. RV144 vaccination induced antibodies that targeted a region of the V2 loop
that contains conserved epitopes. Early HIV-1 transmission events involve V2 loop interactions, raising the
possibility that anti-V2 antibodies in RV144 may have contributed to viral inhibition.
VAXB/E (bivalent rgp120) was associated with a 31.2% re-
duction in HIV-1 acquisition at 42 months in the modified
intent to treat population and provided the first opportunity
to study the immune correlates of HIV-1 vaccine-induced
protection in humans.1–3Evaluation of the vaccine regimen in
Phase I/II studies showed that this vaccine induced both
cellular and humoral immune responses.4,5Immunogenicity
studies have demonstrated the induction of neutralizing an-
tibodies against tier 1 strains of HIV-1, antibody-dependent
cell-mediated cytotoxicity (ADCC), antigen-specific lympho-
proliferative responses, and CD8+cytotoxic T lymphocyte
(CTL) responses.4–9Vaccination with recombinant Env pro-
(Vax004 trial) generated strong humoral immune responses
but did not decrease HIV-1 acquisition in Thai injecting drug
users or North American men who have sex with men, re-
The role of antibodies in preventing HIV-1 acquisition has
been suggested by passive13–16and active immunization
studies in nonhuman primates17,18as well as by preliminary
confirmed as a general principle of protection in humans.
Recentlyit wasfound thatantibodiesagainstsubtypeBgp120
V1V2 loops appeared to correlate with decreased risk of in-
fection in RV14420; however, broadly neutralizing antibodies
were not seen in that study,21and neither neutralizing anti-
bodies nor antibody-directed cell-mediated cytotoxicity ap-
peared to correlate with infection risk.20Nonetheless, several
regions of the HIV-1 gp120 glycoprotein contain epitopes that
induce antibodies that may play a role in protection. Epitopes
found in the second variable (V2) loop have been associated
with HIV-1 Env functions that are important in infectivity,
CD4 receptor and CCR5/CXCR4 coreceptor usage, escape
from antibody neutralization, the formation of Env trimers,
and the protection of the coreceptor binding site.22–28Se-
quence variation within the V1/V2 loops alters neutralization
resistance and viral escape is associated with V2 loop muta-
tions.29–33Compared to viruses circulating in chronically in-
to have shorter V1/V2 regions and fewer N-linked glycosyl-
The V2 loop contains a putative a4b7integrin binding motif
(LDI/V) at amino acid (aa) residues 179–181 (HBX2 num-
bering system) that was shown to interact with the gut mu-
cosal homing receptor.23,24Under physiological conditions,
the a4b7receptor facilitates the migration of lymphocytes to
the gut and some have postulated that HIV-1 exploits this
interaction to infect lymphocytes localized at mucosal tis-
sues.23,24,37,38However, further studies are needed to confirm
the role of a4b7in viral transmission with unliganded gp120
accination with the heterologous prime-boost
vaccine regimen ALVAC-HIV (vCP1521) and AIDS-
Broadly neutralizing antibodies that interact with quater-
nary neutralizing epitopes (QNE) of trimeric native Env
spikes have been shown to interact with conserved aa em-
bedded within the variable (V) loops of gp120 including the
V2 loop.40–47Furthermore, antibodies found in individuals
that exhibit potent cross-clade neutralizing activity have been
shown to target conserved regions of the V1, V2, and V3
loops, providing evidence that V2 contributes to the forma-
tion of QNE that can induce potent cross-clade neutralizing
antibodies.25,43The structure of scaffolded V1/V2 complexed
with the broadly neutralizing monoclonal antibody PG9 has
been resolved.47This potent antibody binds to glycans and aa
sequences within the V1/V2 regions. Antibodies against the
V2 loop target several conserved residues that are sensitive to
glycosylation patterns and changes within the loop alter the
efficiency by which antibodies neutralize HIV-1.26,30,41,48
mononuclear cells (PBMCs) from HIV-1-uninfected RV144
vaccine recipients using the interferon-gamma ELISpot assay
demonstrated preferential CD4+T cell responses to the V2 loop
region compared to other regions of gp120.49Antibody epitope
similar preferential targeting of regions within gp120.
In this study we report that the RV144 vaccine regimen
generated antibodies to the V2 region of gp120. Using cyclic
as well as linear peptides, we demonstrate that the vaccine-
role for these antibodies in protection from HIV acquisition
was recently suggested by a genetic sieve analysis of subjects
who enrolled in RV144 and became HIV-1 infected.50
Materials and Methods
Study subjects for phase III Thai clinical trial (RV144)
The design and findings of the RV144 Phase III clinical trial
have been published previously.1In this study we used three
sets of RV144 plasma samples (sets A, C, and Z) that were
randomly generated by the Statistical Center for HIV/AIDS
Research Prevention (SCHARP), Seattle, WA, and the
EMMES Corporation, Rockville, MD from volunteers who
received all immunizations according to schedule and were
HIV seronegative at the end of the study (Table 1). It was not
possible to use the same sample set for all studies due to the
limited volumes of sample available. However, all plasma
separations were performed at the same centralized labora-
tory. Each plasma set consisted of uninfected subjects in the
per-protocol population with ratios of 4:1 vaccine:placebo for
both male and female subjects. Simple random sampling was
used within each of the four strata. For peptide microarray
and biotinylated linear peptide analyses, plasma samples
from 80 vaccine and 20 placebo recipients, designated plasma
set A and set C, were used, respectively (Table 1). For cyclic
(Cyc) peptide analysis, plasma samples from 32 vaccine and 8
placebo recipients, designated plasma set Z, were used (Table
1). RV144 plasma samples from three different time points
RV144 VACCINE REGIMEN INDUCES ANTI-V2 ANTIBODIES1445
were tested: baseline or prevaccination (visit 1), 2 weeks (visit
8), and 28 weeks (visit 9) post-final inoculation. The RV144
trial was conducted under a protocol approved by IRBs from
the Thai Ministry of Public Health, Mahidol University, the
Scientific Subcommittee of the National AIDS Committee,
Thailand, the Royal Thai Army, Thailand, and the U.S. Army
Medical Research and Materiel Command, United States
(http:/ /clinicaltrials.gov number NTC00223080).
HIV-1 naturally infected individuals
1 circulating recombinant form (CRF) 01_AE (WRAIR proto-
Recombinant proteins and peptides
CHO-expressed recombinant gp120 proteins derived from
MN and A244 were kindly provided by Dr. Marc Gurwith,
Global Solutions for Infectious Disease, San Francisco, CA.
HIV-1 CRF01_AE 92TH023 gp120 protein was expressed in
293T cells and purified using Galanthus nivalis lectin columns.
Peptide microarrays andCyc peptides were synthesized by
JPT PeptideTechnologies. Peptides werecyclized by disulfide
bond formation (Fig. 1A) and the purity was determined to be
greater than 90% by high-pressure liquid chromatography
and mass spectrometry. The aa sequences of Cyc V2 and V3
peptides were based on vCP1521 Env glycoprotein of HIV-1
CRF01 AE (92TH023 strain) GenBank accession number
as those with scrambled mid-region (Scr MR) or scrambled
flanking regions (Scr Fl) were synthesized with or without
biotin at the amino terminus of the peptide. HIV-1 strains
92TH023 and A244 have identical V2 loop mid-regions
(Fig. 1B). Cyc V3 peptide was not biotinylated. Cyclic non-
biotinylated peptides were used in all ELISAs and biotiny-
lated peptides were used for all Biacore binding studies. The
sequences of Cyc peptides are shown in Table 2. The aa se-
quences of the scrambled regions of the mid-region of Cyc V2
Scr MR and the flanking regions of Cyc V2 Scr Fl are shown in
bold (Table 2). The integrin binding motif, LDI, is underlined.
Peptides representing the more sequence-conserved seg-
ment in the first two-thirds of the V2 loop were selected from
the set of all recorded V2 sequences in the Los Alamos Na-
tional Laboratory (LANL) database in order to maximize
predicted functional diversity. The sequences of the linear V2
peptides are shown in Table 2, and the integrin binding motif
in each of the linear peptides is underlined. Polar and charged
amino acids mediatemost chemical functions ofproteins such
as solvent interactions (surface accessibility), binding, post-
translational modification, and catalysis. Peptide 1 was se-
lected as the V2 loop mid-region from a strain with the most
charged and polar aa among circulating strains from subtype
A, strainQB585.2102M.Ev1v5.C. Peptide3wasselected asthe
V2 loop mid-region sequencefound in circulating strainswith
the most polar and charged aa and also exhibiting the most
common V2 length of 39 aa from subtype A, strain 01TZA341.
Peptide 2 represents the sequence most commonly found in
circulating subtype B strains recorded in the LANL database
as of May 2011, strain 878v3_2475. Peptide 4 represents the
Table 1. Definition of Plasma Sets from RV144 Subjects
and the Assays for Which They Were Used
Biotinylated linear peptide
Whole gp120 ELISA cyclic
peptide analysis: ELISA,
surface plasmon resonance
Plasma sets used for the analysis of the V2 loop and gp120
responses by microarray, ELISA, and Biacore.
sequence of the cyclic (Cyc) V2 loop of the HIV-1 CRF01_AE 92TH023 strain. The flanks and mid-region are labeled. (B)
Alignment of V2 loop amino acid sequences. Sequences that vary from 92TH023 are boxed and the first (157) and last (196)
amino acids of the V2 are shown on top of the alignment. Numbering is based on the HXB2 strain. Cyclic V2 peptides were
synthesized based on clone 92TH023. Peptides for microarray analysis were based on consensus (Con) sequences and
peptides 1–6 represent linear N-linked biotinylated peptides.
Graphic representation of the cyclic V2 loop and alignment of V2 loop amino acid sequences. (A) Amino acid
1446 KARASAVVAS ET AL.
consensus sequences for V2 in the region covered by peptides
1–3. Peptide 6 contains the N-terminal 14 aa of peptide 1.
Peptides were biotinylated at the N-terminus during synthe-
sis and were purchased from Genemed Biotechnologies, Inc.,
South San Francisco, CA.
Carboxymethylcellulose 5 (CM5) chips, streptavidin (SA)
chips, and the amine coupling kit were purchased from GE
Healthcare, Piscataway, NJ. Lysozyme, Costar Spin-X centri-
phosphate-buffered saline (D-PBS), Tween 20, and Thimerosal
were purchased from Sigma-Aldrich, St. Louis, MO. Affinity
purified sheep antihuman IgG (gamma chain specific) anti-
bodies were purchased from The Binding Site, Birmingham,
UK. Skim milk was from Applichem, St. Louis, MO. ABTS
peroxidase substrate solution was from KPL, Gaithersburg,
MD. Horseradish peroxidase-conjugated goat antihuman IgG
antibodies were from Bethyl Laboratories, Montgomery, TX.
Strepta Well plates were from Roche, Mannheim, Germany.
Alkaline phosphatase-conjugated goat antihuman IgG was
purchased from SouthernBiotech, Birmingham, AL. Anti-IgG
Cy5 antibody was from Jackson ImmunoResearch Labora-
tories, West Grove, PA. Superblock T20 PBS blocking buffer,
were purchased from Thermo Fisher Scientific, Rockford, IL.
Each array consisted of 1423 tiled Env peptides arranged in
three identical subarrays, allowing for triplicate evaluation of
the entire set of peptides. Peptide sequences covered the entire
gp160 subtype consensus sequences from six HIV-1 group M
M gp160, Con-S,51for a total of 1423 peptides (15-mers over-
vaccine strains, as we wished to focus on cross-reactive re-
sponses rather than type-specific responses to the vaccine im-
munogens. The peptides were generated using an alignment of
the seven consensus sequences with LANLs PeptGen tool
(www.hiv.lanl.gov), so that peptides remain in register
throughout the Env despite insertions and deletions, and
identical peptides found in more than one subtype were re-
presented only once. Microarray development was performed
Switzerland). Allarrays wereblocked withSuperblock T20 PBS
blocking buffer for0.5hat30?C,followedby a2-hincubationat
30?C with heat-inactivated plasma diluted 1:100 in Superblock
T20. Arrays were then incubated for 45min at 30?C with a
secondary antibody, 1.5mg/ml anti-IgG Cy5 antibody diluted
1:1000 with Superblock T20. Washes between all steps were
with PBS containing 0.1% Tween. Arrays were scanned at a
wavelength of 635nm using an Axon Genepix 4300 Scanner
(Molecular Devices, Sunnyvale, CA) at a PMT setting of 600,
50% laser power. Images were analyzed using Genepix Pro 7
software (Molecular Devices). Spot intensities were corrected
for prevaccine results.
Peptide microarray spot intensities were extracted from the
raw images using the GenePix software, and peptide inten-
sities were calculated as follows: (1) Spot intensities were
defined as foreground median intensity minus background
median intensity. (2) Resulting intensities were log2trans-
formed and centered by subtracting the average (log2) inten-
sities of the empty (control) spots on the slide. (3) For each
unique peptide, a single intensity was defined as the median
of the triplicate intensities on the slide for that peptide. To
Table 2. Cyclic and Linear Peptides Used in the Study
Cyclic peptides based
on 92TH023 strain
acid numberingAmino acid sequence
Cyc V2 (42 aa)
Cyc V2 (25aa)
Cyc V2 (16aa)
Cyc V2 Scr FI
Cyc V2 Scr MR
Cyc V3 (35aa)
acid numbering Amino acid sequence
Subtype A and B
The amino acid sequence of cyclic V2 and V3 loop peptides is based on HIV-1 CRF01_AE strain 92TH023. Scrambled regions are shown in
bold and the integrin binding motif LDI/V is underlined. Linear biotinylated peptide sequences are based on the sequences of HIV-1 strains
deposited at the LANL databse, except for peptide 4, which is a consensus V2 loop sequence. The specific strains pertaining to peptides 1–3
are listed in Materials and Methods. Peptide 6 is the 14 amino acid N-terminal fragment of peptide 1.
RV144 VACCINE REGIMEN INDUCES ANTI-V2 ANTIBODIES 1447
remove effects unrelated to true peptide-binding activity,
such as nonspecific binding of primary antibody in the sam-
ple, array normalization was performed in an attempt to re-
move such effects. Nonspecific binding may be related to
physicochemical properties of the peptide, we used the z-
scales tocorrect fornonspecific binding biases.52To arrive at a
summary z-scale score for each peptide, the z-scale values of
the individual aa comprising that peptide were summed.
Here, the normalization was done in two stages: (1) a re-
gression model for the probe intensities was derived from
their overall z-scores and (2) each probe was normalized by
subtracting its predicted intensity(representing thebias)from
the observed intensity. A linear model with t4distributed
errors was used, which provides more robustness against
probe outliers as described previously.53This normalization
was performed on each array separately. Once the data were
normalized, the probe intensities were smoothed and a score
was calculated for each peptide that was used to detect
binding regions. Based on the assumption that an antibody
binds a region of approximately 9 aa or more, normalized
peptide intensities were smoothed using a sliding window
mean statistics of size 9 aa, i.e., peptides with HXB2 positions
within 9 aa of one another were grouped together. This
A) or across all subtypes to borrow strength across multiple
peptides. For baseline correction, samples were corrected by
subtracting, for each sample, the peptide intensities measured
prevaccination for that sample.
Once scores have been calculated and corrected for base-
line, the score cutoff to call positive peptides can be set arbi-
trarily, determined based on simple validation, or based on a
false detection rate (FDR) cutoff. Here the placebo group from
the RV144 data was used to estimate the FDR and set at an
appropriate threshold. Peptides with log2-fold change greater
than 1.1 (with respect to baseline) were called positive, which
led to an FDR of approximately 5%.
ELISA for cyclic peptides and recombinant gp120
at 4?C overnight. Wells were washed three times with wash
buffer (PBS, 0.1% Tween 20, and 0.01% Thimerosal, pH 7.4)
using Microplate Washer ELX405, Bio Tek, Winooski, VT, and
blocked with blocking buffer (D-PBS, 5% skim milk) for 2h at
buffer and then serial 2-fold dilutions were performed and ad-
ded to wells for 2h at room temperature. Wells were washed
five times with wash buffer and HRP-conjugated goat antihu-
man IgG (1:25,000) was added to wells for 1h at room temper-
ature. Plates were washed five times with wash buffer, 100ll/
well of substrate was added, and color was allowed to develop
at room temperature for 1h in the dark. Plates were read at
A405nm using an ELISA reader Spectramax 340 PC, Molecular
being defined as the reciprocal of the highest dilution that yiel-
ded an absorbance value above 2.5 times the background value
(wells that did not contain recombinant protein or peptides).
Biotinylated linear peptide ELISA
biotinylated linear V2 peptides for 1.5h at 37?C and then
washed six times with PBS containing 0.05% Tween-20, pH
diluted 1:100 in RPMI media containing 15% fetal bovine se-
rum. The plates were washed six times and alkaline phos-
phatase-conjugated goat antihuman IgG (1:2,000) was added
for 1.5h at 37?C. After washing, 10% diethanolamine sub-
strate was added for 30min to develop color, and the plates
50ll/well and each sample was run in duplicate.
Data analysis for ELISA assays
ELISA antibody titers were calculated as the reciprocal
plasma dilution using serial 2-fold dilutions of plasma from
1:100 to 1:12,800and are expressed asthe reciprocal end-point
titer. Geometric mean titers (GMT) were calculated from the
reciprocal end-point titers. Data analyses and graphs were
generated using Graphpad Prism version 5. Statistical com-
parisons were made using nonparametric tests and a two-
sided p-value of <0.05 was considered significant.
Surface plasmon resonance
To confirm the findings of the ELISA studies, surface plas-
mon resonance (SPR) measurements were conducted with a
Biacore T100 using CM5 or SA chips prepared as described
below. Lysozyme was immobilized onto a CM5 chip using the
amine coupling kit. Unbound free amines were quenched with
ethanolamine. All solutions used during the immobilization
steps had a flow rate of 10ll/min and all experiments were
performed at 25?C. The immobilization wizard packaged
within the T100 control software was used to immobilize
500nM lysozyme in 20mM sodium phosphate, pH 7.4 (5min
contact time) in flow cell 1. Cyc V2 biotinylated peptides were
prepared at 0.1–1lM in Tris-buffered saline, pH 7.4, and al-
of the SA chip, which resulted in immobilization of approxi-
mately 895, 1,401, and 2,073 response units (RU), respectively,
MR (1900 RU) and Cyc V2 Scr Fl peptides (1,850 RU) were
immobilized to the respective flow cells.
Plasma samples were heat inactivated (56?C, 45min),
centrifuged, and the supernatant filtered prior to use. The
plasma was diluted 1:50 in Tris-buffered saline, pH 7.4. The
diluted plasma samples were passed over the chip surface at
30ll/min for 3min followed by a 5-min dissociation period.
At the end of the 5-min period, a 50nM solution of affinity-
purified gamma chain-specific sheep antihuman IgG anti-
body was passed over the peptide-coated Ig-bound surface
for 2min at a flow rate of 10ll/min. After a 70-s dissociation
period, the biotinylated peptides were regenerated using a
30-s pulse of 50mM HCl, a 30-s pulse of 100mM EDTA in
20mM Tris, pH 7.4, another 30-s pulse of 50mM HCl, fol-
lowed by a 1min injection of Tris-buffered saline, pH 7.4.
Nonspecific binding was subtracted and data analysis was
performed using the BIA evaluation 4.1 software. The re-
ported RUs for the IgG-specific values are the difference be-
tween the average value of a 5-s window taken 60s after the
end of the anti-IgG injection and the average value of a 5-s
window taken 10s before the beginning of the anti-IgG in-
plasma samples. A representative experiment of three inde-
pendent experiments is shown.
1448KARASAVVAS ET AL.
RV144 antibody responses to recombinant
To investigate the antibody responses induced by the
RV144 vaccine regimen, we performed ELISAs using the
vaccine matched recombinant gp120 proteins from the three
HIV-1 vaccine strains, MN, 92TH023, and A244. A244 and
MN comprised the protein boost and 92TH023 was in the
canarypox vector. Set Z plasma samples from visit 1 (pre-
vaccination), visit 8 (two weeks), and visit 9 (28 weeks) post-
last inoculation were tested against gp120 proteins (Fig. 2).
The visit1 responses were negative except for three vaccinees’
samples where the responses were above the cutoff value
and were considered nonspecific. Of the 32 vaccinees, 31
(97%) had robust antibody responses against all three re-
combinant proteins at visit 8 with reactivity against MN and
A244 being the highest followed by 92TH023 (Fig. 2A). One of
the eight placebo subjects had antibody responses to both
92TH023 and A244 proteins at all three visits but no re-
sponses to MN (Fig. 2B). Although the frequency of positive
responses at visit 9 did not change 6 months after adminis-
tration of the last vaccination, the magnitude of antibody re-
sponses against all three gp120 proteins declined with
antibody levels to 92TH023 being the lowest followed by
A244 and MN (Fig. 2A).
Detection of V2-specific antibody responses in RV144
by peptide microarray analysis
Binding antibodies in RV144 set A plasma samples were
assessed by peptide microarray analysis using linear over-
lapping peptides covering the entire V2 loop of all major ge-
netic subtypes and CRFs of gp120 (Fig. 3). Of the seven sets of
HIV-1 gp120 peptides tested, six displayed antibody re-
sponses against V2. Notably, there was little or no reactivity
with the V2 peptides of subtype B despite the fact that the
vaccinees had received gp120 protein boosts derived from
both subtypes B and CRF01_AE (Fig. 3A). Based on this
analysis, the RV144 vaccine induced antibody responses that
specifically targeted 15-mer peptides covering the V2 loop
with 25% (20/80) of the vaccinated subjects having antibodies
to this region (Fig. 3B).
Antibody responses in RV144 target the mid-region
of the V2 loop
Based on the antibody responses to linear peptides that
span the V2 loop in peptide microarray analysis, we con-
cluded that antibodies interacted with the mid-region of the
V2 loop having the following aa sequence—KQKVHAL-
The mid-region of the V2 loop contains highly conserved aa
including the a4b7integrin binding motif LDI/V23and ALFY,
but also a less conserved region (KQKVH). To investigate
further the induction of V2 loop-specific antibodies in RV144,
we synthesized Cyc V2 peptides based on the canarypox Env
glycoprotein 92TH023 (see Materials and Methods, Table 2,
Absorbance 405 nm
Absorbance 405 nm
proteins in RV144 vaccinees and placebo recipients. (A) Anti-
body responses to recombinant Env proteins gp120 MN, A244,
and 92TH023 at visits 1, 8, and 9 determined by ELISA using
plasma samples from set Z at a dilution of 1:100. Absorbance at
405nm is shown on the y-axis. (B) Antibody responses to the
same recombinant proteins and visits in placebo recipients.
Dotted line indicates the cutoff for the assay in the absence of
the capturing protein. Each sample was run in triplicate and
data represent the average of two independent experiments.
Bars represent the standard error of the mean.
IgG antibody responses to recombinant HIV-1 Env
analysis was done using plasma from 80 HIV-1-uninfected vaccine recipients and 20 placebo recipients (set A). (A) Reactivity
of plasma with peptides from seven HIV-1 Group M subtypes (M consensus, A, B, C, D, CRF01, and CRF02). (B) Overall
reactivity of RV144 plasma samples to the V2 loop. Peptide positions are shown on the x-axis and percent responses are
shown on the y-axis. The peptide positions are aligned with the amino acid sequence of HXB2 protein and the sequence of
92TH023 V2 loop is shown for comparison.
Evaluation of antibody responses to overlapping peptides of V2 by peptide microarray analysis. Microarray peptide
RV144 VACCINE REGIMEN INDUCES ANTI-V2 ANTIBODIES1449
1450 KARASAVVAS ET AL.
and Fig. 1). 92TH023 and A244 V2 loops vary by two aa at
positions 188 and 189 (HXB2 numbering) but the mid-region
is identical. The Cyc V2 peptide contained 42 aa extending
from aa 158 to 199 (corresponding to HIV-1 HXB2 aa 157–
196). To analyze the antibody responses targeting the flanks
and mid-region of the V2 loop, two additional Cyc peptides
were used (Table 2).
Plasma samples from visits 1 and 8 were tested against the
Cyc V2 peptides by ELISA (Fig. 4). No antibody responses to
CycV2 were detected in prevaccinated (visit 1) plasma. Of the
32 vaccinated subjects tested at visit 8, 31 had positive IgG
antibody responses against Cyc V2 (97%) at visit 8 (Fig. 4A).
Antibody responses to the Cyc V2 Scr Fl peptide were almost
identical to those against the Cyc V2 peptide. Unlike Cyc V2
and Cyc V2 Scr Fl peptides, the IgG antibody responses
against Cyc V2 Scr MR were significantly reduced with only
16/32 (50%) plasma samples being above background values
(p<0.001) indicating that the majority of antibody responses
against V2 targeted the mid-region of the loop (Fig. 4A).
Identical responses were seen with Biacore for all three pep-
tides (data not shown).
To better characterize the antibody-binding epitope within
the Cyc V2 peptide, two shorter Cyc V2 peptides, Cyc V2
(25aa) and Cyc V2 (16aa), were synthesized and tested by
Biacore analysis (Table 2 and Fig. 4C). There was a single
positive responder among the vaccinees at visit 1 to the Cyc
V2 (25aa) peptide (Fig. 4C). Both low-frequency and weak
binding were observed with the Cyc V2 (25aa) peptide (5/32,
16%; p<0.001) but no binding was observed with Cyc V2
(16aa) (Fig. 4C). No antibody responses were detected in
plasma samples from visits 1 and 8 of placebo recipients to
any of the Cyc V2 peptides (Fig. 4C).
Durability of RV144 antibody responses to the V2 loop
To determine the durability of antibody responses against
the V2 loop, the antibody responses at visits 8 and 9 were
compared using the Cyc V2 peptide. The frequency of anti-
1,100 and a range of 200–3,200 (Fig. 5A). The frequency and
magnitude of antibody responses decreased significantly at
visit 9 (6/32, 19%) compared to visit 8 (p<0.001) with a GMT
of 110 and a range of 100–200 (p<0.001). Biacore analysis
using the same peptide and plasma samples showed similar
results with antibody responses to V2 diminishing by visit 9
(Fig. 5B). Reactivity was not detected in placebo recipients or
vaccinees prior to immunization by either ELISA or Biacore
assays (Fig. 5A and B).
Cyc V2 Scr MR (42aa)
Cyc V2 Scr Fl (42aa)
Cyc V2 (42aa)
Absorbance 405 nm
Cyc V2 Scr MR (42aa)
Cyc V2 Scr Fl (42aa)
Cyc V2 (42aa)
Absorbance 405 nm
ELISA and Biacore. (A) Antibody responses at visits 1 and 8
to cyclic peptides Cyc V2, Cyc V2 Scr Fl, and Cyc V2 Scr MR
by ELISA. (B) Antibody responses to Cyc V2, Cyc V2 Scr Fl,
and Cyc V2 Scr MR by ELISA using 20 infected and four
uninfected subjects. (C) Antibody responses to shorter cyclic
V2 peptides by Biacore. In Biacore, plasma samples were
used at a 1:50 dilution and the values are reported as re-
sponse units. A representative experiment of three inde-
pendent experiments is shown. ELISA and Biacore analyses
were performed using the identical plasma samples (set Z).
In ELISAs, plasma samples were used at a dilution of 1:100
and responses were considered positive if they exceeded 2.5
times the background (absence of capturing peptide) and are
indicated by the dotted line. Each sample was tested in
triplicate and the results represent the average of at least two
independent experiments. Absorbance at 405nm is shown on
Antibody responses to cyclic V2 loop peptides by
RV144 VACCINE REGIMEN INDUCES ANTI-V2 ANTIBODIES1451
Antibody responses to the V2 loop in vaccinated
subjects detected with linear peptides
Antibody responses to V2 were also examined using five
linear biotinylated V2 loop-derived peptides that are heter-
ologous in sequence to the RV144 immunogens (Table 2 and
Fig. 6). In this ELISA, RV144 plasma samples from set C were
used (80 vaccine and 20 placebo recipients). Antibody re-
sponses against all peptides were detected at visit 8 with
peptide 6 having the highest frequency (93%) of responses
followed by peptides 1 (84%), 3 (68%), 4 (39%), and 2 (38%),
respectively (Fig. 6A). Poor responses against linear peptides
4, 6, and consensus B V2 peptides (microarray analysis) in-
dicated that the presence of valine at position 172 is important
for antibody binding. The responses to linear peptides de-
clined tobackground levelsatvisit9 andweresimilartothose
observed with the V2 cyclic peptide using ELISA and Biacore
(Fig. 5). Two samples from placebo recipients reacted with the
linear peptides 1 and 6 and one sample reacted with peptide 2
(Fig. 6B). The sample reacting with peptides 1 and 6 (shorter
version of peptide 1) was not the same as with peptide 2.
These responses were considered nonspecific.
Antibody responses to V2 in HIV-1-infected
V2-specific IgG antibody responses generated by the
RV144 vaccine regimen were compared to those induced by
HIV-1 infection. Antibodies to Cyc V2, Cyc V2 Scr MR, and
Cyc V2 Scr Fl were measured using plasma samples from
20 HIV-1 CRF01_AE-infected individuals and four HIV-1-
uninfected Thai subjects. Ten of the 20 infected subjects (50%)
had antibodies to Cyc V2 (GMT: 400; range: 100–3,200) (Fig.
4B), which were significantly lower than the frequency and
magnitude for the RV144 vaccine group (p<0.001). Similar
antibody-binding responses in frequency and magnitude
time. (A) Antibody responses to Cyc V2 peptide at visits 1, 8,
and 9 by ELISA. Responses were considered positive if they
exceeded 2.5 times the background (absence of capturing
peptide) and are indicated by the dotted line. (B) Antibody
responses to Cyc V2 peptide at visits 1, 8, and 9 by Biacore.
Plasma samples were used at a 1:50 dilution and the values
are reported as response units. A representative experiment
of three independent experiments is shown. ELISA and
Biacore analyses were done using plasma sample set Z.
Antibody responses to cyclic V2 loop decline over
Peptide 2 Peptide 1 Peptide 6 Peptide 4
Absorbance 405 nm
Peptide 1 LRDKKQRVYSLFYKLDVVQIN
Peptide 2 IRDKVQKEYALFYKLDVVPID
Peptide 3 LRDKKQQVYSLFYRLDIEKIN
Peptide 4 IRDKKQKEYALFYKLDVVPID
Peptide 6 LRDKKQRVYSLFYK
Absorbance 405 nm
cline over time. (A) Antibody responses to linear biotinylated
peptides 1, 2, 3, 4, and 6 at visits 1, 8, and 9 by ELISA using
plasma sample set C. Absorbance at 405nm is shown on the
y-axis. (B) ELISA using the same peptides and plasma of 20
placebos, set C. Plasma was used at a dilution of 1:100 and
peptide aa sequences are shown in the right upper corner of
(B). Samples were run in duplicate and are the average of
Antibody responses to linear V2 loop peptides de-
1452 KARASAVVAS ET AL.
the antibody responses in HIV-1 infection also target the mid-
region of V2 loop and are similar to those generated by the
RV144 vaccine regimen (Figs. 4A and 5). The magnitude of
antibody responses to the Cyc V2 Scr MR peptide was low,
in HIV-1-uninfected controls (Fig. 4B).
Biacore analysis using plasma samples from 18 of the 20
HIV-1-infected subjects tested by ELISA gave similar results
(data not shown).
Antibody responses to the V3 loop in HIV-1-uninfected
vaccinated subjects and HIV-1-infected subjects
Antibody responses to V2 were also compared to those
eight placebo recipients at visits 1 and 8 were tested against
Cyc V3 (Table 2 and Fig. 7). The frequency (18/32, 56%) and
magnitude (GMT: 200; range: 100–800) of antibody responses
against V3 were lower in vaccinated subjects (Fig. 7). In
comparison, the Cyc V3 antibody responses in HIV-infected
individuals were both higher in frequency (19/20, 95%) and
magnitude (GMT: 3,600; range: 200–12,800) (Fig. 7). Antibody
reactivity against the Cyc V3 peptide was not detected in
uninfected control subjects or vaccine recipients at visit 1.
Unlike HIV-1-uninfected RV144-vaccinated subjects, HIV-1-
infected individuals had a higher frequency of antibody re-
sponses against 92TH023 V3 (95%) compared to V2 (50%;
Comparison of V2 and V3 responses in HIV-1-infected
and vaccinated subjects
The levels of Cyc V2 and V3 antibody responses were
compared in the same HIV-1-infected individuals. Seven out
of 20 (35%) subjects tested had Cyc V3 loop antibody re-
sponses with a GMT of 4,800 (range: 1,600–12,800) but un-
detectable levels of antibodies against the Cyc V2 loop (Fig.
8A). In 3/20 (15%) subjects, the V2 response was high (GMT:
3,200) but still below the V3 antibody responses (GMT: 5,100;
range: 3,200–6,400). All HIV-1-infected subjects had higher
responses to Cyc V3 than to Cyc V2 (Fig. 8A). Of the 20 in-
fected subjects, one (5%) did not have an antibody response to
either V2 or V3 loop peptides.
Of the RV144 vaccinated subjects, 31/32 (97%) had an an-
tibody response to the Cyc V2 peptide, but only 18/32 (56%)
had antibody responses to the Cyc V3 peptide (Fig. 8B). None
of the 32 vaccinated subjects had a V3 loop antibody response
individuals did not have antibody responses to either the V2
or V3 loops.
The estimated efficacy in the RV144 vaccine trial was
31.2% by 42 months in the modified intent to treat popula-
tion,1but data through months 12 and 18 showed estimated
efficacy of 60% and 44%, respectively.54Initial studies
Visit 1 Visit 8UninfectedInfected
nated uninfected subjects, HIV-1-infected and HIV-uninfected.
Antibody responses to Cyc V3 peptide (strain 92TH023) using
plasma samples from RV144 visits 1 and 8 (set Z) and from 20
HIV-1-infected and four HIV-1-uninfected subjects. Plasma
samples were used at a starting dilution of 1:100. Cutoff val-
ues are indicated by the dotted line. Panels represent the av-
erage of at least two independent experiments.
Antibody responses to cyclic V3 peptide in vacci-
PL# 0454 PL# 0845PL# 1595 PL# 3125PL# 3184 PL# 3444 PL# 4249 PL# 4718PL# 4978PL# 4995PL# 5008 PL# 5160PL# 5360PL# 5901 PL# 6170PL# 8105PL# 8172PL# 9259 PL# 9936
Absorbance 405 nm
Z01Z02 Z03 Z04Z05Z06 Z07 Z08Z09Z10 Z11Z12Z13 Z14 Z15Z16Z17Z18 Z19Z20Z21 Z22Z23 Z24 Z25Z26 Z27Z28 Z29Z30 Z31 Z32
Absorbance 405 nm
loops in RV144 vaccinees and HIV-1 naturally infected sub-
jects. (A) Comparison of antibody responses to gp120 V2 and
V3 loops from HIV-1-infected individuals. (B) Antibody re-
sponses to gp120 V2 and V3 loops in RV 144 vaccinated
individuals. Plasma dilutions were at 1:100 and absorbance
at 405nm is shown on the y-axis. Cutoff values are indicated
by the dotted line.
Comparison of antibody responses to V2 and V3
RV144 VACCINE REGIMEN INDUCES ANTI-V2 ANTIBODIES1453
demonstrated that binding antibody to vaccine gp120 anti-
vaccination but waned substantially over the next 6 months.
Using peptide microarray analysis, ELISA, and Biacore
studies we demonstrated that the immunogens used in the
RV144 trial induced antibodies to the V2 loop of gp120 from
diverse HIV-1 subtypes. These responses were high in fre-
quency and magnitude but also declined rapidly to nearly
undetectable levels at 28 weeks post-last vaccination. Haynes
et al.20have shown that IgG against a conformational V1/V2
epitope from 2 weeks postvaccination was inversely corre-
lated with infection rate in the RV144 trial. While diminution
of theV2 antibodymay havecorresponded to waningvaccine
efficacy in RV 144, a mechanistic link between the two ob-
servations is purely speculative at this point. Further research
pursue this hypothesis.
Using scrambled cyclic and linear peptides of the V2 loop
we determined that antibodies induced by RV144 target the
mid-region of the loop, which includes highly conserved aa
including the integrin-binding motif LDI/V but also less
conserved regions. It is likely that RV144 antibodies have
multiple binding epitopes within the mid-region and further
studies are needed to characterize their binding specificities.55
Two monoclonal antibodies isolated from RV144, CH58, and
CH59 have different but partly overlapping binding.56It is
likelythatnot allantibodyresponses targeting theV2loopare
protective and certain epitopes could be more protective than
others in preventing HIV-1 acquisition. Furthermore, volun-
teers who develop antibodies to Cyc V2 may or may not de-
velop antibodies to gp70, a scaffold glycosylated protein
containing the V1/V2 loops used in the case control study.20
frequency or magnitude of binding in vaccinated and infected
subjects indicating that the majority of the antibodies target
epitopes within the mid-region. To further define the binding
epitopes of these antibodies, Cyc V2 (16aa) and Cyc V2 (25aa)
peptides were used. Of these two peptides, weak responses
were observed only to Cyc V2 (25aa). Peptide Cyc V2 (25aa)
N terminus of the peptide, suggesting that these four aa could
be important for epitope recognition by V2 antibodies. Al-
ternatively, conformational changes of shorter cyclized pep-
tides may not bind to antibodies. It is possible that certain
conformational antibodies induced by the recombinant pro-
teins used in the vaccine regimen may not bind to peptides.
Furthermore, antibodies targeting glycosylated epitopes
within the V1/V2 loops were not investigated in this study.
Recently, a proposed structure of scaffolded V1/V2 loops
complexed with the broadly neutralizing monoclonal anti-
body PG9 has been reported.47V1/V2 loops form an inter-
twined four-stranded b sheet domain composed of four
with N-linked glycans on the V1/V2 loop but also interacts
with aa within strand C, which is part of the V2 loop region.
Strand C is part of the mid-region of the V2 loop and interacts
strongly with antibodies induced by the RV144 vaccine regi-
men. In addition to its interaction with PG9, strand C con-
tributes to the formation of a nonglycosylated hydrophobic
core and participates with the other three strands to form a
single topological entity.47The consequence of interaction of
this topological entity with antibodies is currently unknown.
Comparing antibody responses and sequence alignments
of peptides used in the study, we conclude that valine at po-
sition 172 (HXB2 numbering) could be important in epitope
formation and reactivity. Valine at this position is character-
istic of CRF01_AE V2 loops and differs from the glutamate
found most commonly at this position in subtype B strains.
Linear and cyclic peptides lacking valine at position 172 were
poorly reactive or nonreactive with the samples tested.
The generation of antibody responses against the mid-
region of the V2 loop was also confirmed by linear biotiny-
lated peptides. Of the five peptides tested, peptide 6 had the
highest response followed by peptides 1 and 3. Peptide 6 is a
shorter version of peptide 1 and lacks the LDI motif. The high
antibody response to this peptide suggests that the core epi-
tope recognized by antibodies to linear peptides does not in-
clude LDI; however, this does not imply that either the LDI is
is not sterically blocked by V2 antibodies to the core epitope.
In general, linear peptides had weaker responses than the
92TH023 Cyc V2 peptide possibly due to the lack of high
homology with the aa sequences of the V2 loop used in the
vaccine and to conformational differences as cyclic peptides
may assume a more complex conformational folding.57–61
Using microarray analysis, RV144 vaccinee plasmas were
reactive with six of the seven sets of overlapping V2 peptides
from consensus gp160 proteins from group M and subtypes
A, C, D, CRF01, and CRF02; only those from subtype B were
negative. The frequency of antibody responses to the V2 loop
by microarray peptide analysis was much lower than that
observed for cyclic V2 peptides using ELISAs and Biacore.
The difference in binding could be attributed to aa sequence
variability but also to conformation, adsorption, or peptide
In contrast to the V2-specific IgG antibody responses in-
duced by the RV144 vaccine regimen, the Cyc V3 loop re-
sponses in RV144 were of lower magnitude (GMT of 200) and
frequency (56%), thus indicating that RV144 did not induce
vigorous antibody responses to the clade E V3 loop region.
However, responses to the cyclic V3 loop in HIV-1-infected
individuals were higher in magnitude (GMT of 3,600) and
frequency (95%). Infected subjects had robust antibody re-
sponses to the Cyc V3, but 35% of those having strong anti-
body responses to V3 lacked antibodies to the Cyc V2. There
was an 18-fold higher response to the Cyc V3 peptide in HIV-
1-infected individuals compared to the RV144 vaccinees.
However, V2 loop-specific antibodies in infected individuals
were much less frequent (50%) and of lower magnitude than
those induced by the RV144 vaccine regimen. Whether this
observation of high V2 and low V3 responses in vaccinees
compared to HIV-1-infected subjects is related to the vaccine
antigen is unknown. Vaccine-induced antibody responses
have many similarities to antibodies found in infected indi-
viduals but generally are not neutralizing in cell assays.62
Heterosexual transmission is usually a single infectious
event and productive infection reflects the expansion of
the founder virus.36,63,64The V2 loop has a number of features
that might involve initial events in infection. It has a
highly conserved length among early or transmitted/founder
isolates and length and glycosylation of the V2 loop are
commonly associated with escape from neutralizing anti-
bodies.30,35,65Furthermore, V2 contains a putative a4b7
integrin-binding motif of uncertain significance.39Although
1454 KARASAVVAS ET AL.
V2 has been implicated in early virus transmission the precise
mechanisms of attachment and cell-to-cell dissemination
are not well defined.23,24,36,65–67Rolland et al. have reported
that sieve mutations in HIV-1 breakthrough infections in
RV144 contain mutations at positions 169 and 181 in V2.50T
cell epitope mapping studies using PBMCs from HIV-1-
uninfected RV144 vaccine recipients and the interferon-gam-
maELISpotassayalsoshowed preferentialtargeting oftheV2
loop and that the responding cells were CD4+.49Taken to-
gether with the potential role of antibody to V2 as a correlate
of protection,17,20these findings suggest that the RV144 vac-
cine regimen may have induced protection by targeting the
The generation of humoral immune responses by ALVAC-
HIV and AIDSVAX B/E to the mid-region of the V2 loop
raises hypotheses to test in regard to correlates of protection
against HIV-1 infection. The V2 loop (like the V3 loop)
contains many conserved features, and several anti-V2
monoclonal antibodies show broad cross-reactivity41,48,68
target conserved elements in the V2 loop and that this region
of Env might be pivotal for the induction of protective anti-
bodies bysuccessful vaccines.Since replacement of variousaa
in the V2 loop can alter the structure and function of the Env
rendering HIV-1 less infectious or more sensitive to neutrali-
zation,33,48,69the choice of Env and/or V2 sequences used in
rational vaccine design should be carefully considered.
The authors thank SCHARP for assisting with the micro-
array analysis, James Swetnam for providing the biotinylated
subtype B linear peptides, Kelly Soderbergfor coordination of
constructive reading of the manuscript, Robert Parks, Ryan
Meyerhoff, and Krissey Lloyd for ELISA technical assistance,
and Ellen Turk for performing the peptide microarray anal-
ysis. These studies were supported in part by an Interagency
Agreement Y1-AI-2642-12 between U.S. Army Medical Re-
search and Materiel Command (USAMRMC) and the Na-
tional Institutes of Allergy and Infectious Diseases. In
addition, this work was supported by a cooperative agree-
ment (W81XWH-07-2-0067) between the Henry M. Jackson
Foundation for the Advancement of Military Medicine and
the U.S. Department of Defense. This research was funded, in
part, by the U.S. National Institute of Allergy and Infectious
Melinda Gates Foundation’s Collaboration for AIDS Vaccine
Discovery (OPP38744) and the NIH Vaccine Research Center.
S.Z.P. and T.J.C. receive funding from NIH Grants HL59725
(S.Z.P.) and AI084119 (T.J.C.) and the Department of Veterans
Affairs. Supported in part bygrants from the Bill and Melinda
Gates Foundation Collaboration for AIDS Vaccine Discovery
to Dr. Haynes.
MOPH-TAVEG Collaboration: MOPH: Dr. Supachai
Rerks-Ngarm, Dr. Prayura Kunasol, Dr. Nakorn Premsri, Dr.
Chawetsan Namwat, Prof. Prasert Thongcharoen Mahidol,
Prof. Punnee Pitisuttithum, Dr. Jaranit Kaewkungwal. RTA:
MG Sorachai Nitayaphan, MG (ret) Chirapa Eamsila. MHRP:
Dr. Nelson L. Michael, Dr. Jerome H. Kim, Dr. Robert C
O’Connell, Dr. Merlin L. Robb, Dr. Robert Paris, Charla An-
drews, Dr. Jeffrey Currier.
AFRIMS: Dr. Viseth Ngauy, Dr. Mark S. de Souza, Dr.
Nicos Karasavvas, Ms. Rapee Trichavaroj, Ms. Susan T. Ma-
son, Ms. Bessara Nuntapinit, Ms. Nampueng Churikanont,
Dr. Michael Benenson, Ms. Patricia Morgan.
Abstract presentations: AIDS Vaccine 2011: Global Colla-
boration and Coordination to Advance HIV Vaccine Re-
search, September 12–15, 2011, Bangkok, Thailand.
Disclaimer: The views expressed in this article are those of
the authors and do not reflect the official policy of the De-
partment of the Army, Department of Defense, the Depart-
ment of Veterans Affairs, or the U.S. Government.
Author Disclosure Statement
No competing financial interests exist.
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