Antibody-Dependent Cellular Cytotoxicity-Mediating Antibodies from an HIV-1 Vaccine Efficacy Trial Target Multiple Epitopes and Preferentially Use the VH1 Gene Family

Article (PDF Available)inJournal of Virology 86(21):11521-32 · August 2012with85 Reads
DOI: 10.1128/JVI.01023-12 · Source: PubMed
The ALVAC-HIV/AIDSVAX-B/E RV144 vaccine trial showed an estimated efficacy of 31%. RV144 secondary immune correlate analysis demonstrated that the combination of low plasma anti-HIV-1 Env IgA antibodies and high levels of antibody-dependent cellular cytotoxicity (ADCC) inversely correlate with infection risk. One hypothesis is that the observed protection in RV144 is partially due to ADCC-mediating antibodies. We found that the majority (73 to 90%) of a representative group of vaccinees displayed plasma ADCC activity, usually (96.2%) blocked by competition with the C1 region-specific A32 Fab fragment. Using memory B-cell cultures and antigen-specific B-cell sorting, we isolated 23 ADCC-mediating nonclonally related antibodies from 6 vaccine recipients. These antibodies targeted A32-blockable conformational epitopes (n = 19), a non-A32-blockable conformational epitope (n = 1), and the gp120 Env variable loops (n = 3). Fourteen antibodies mediated cross-clade target cell killing. ADCC-mediating antibodies displayed modest levels of V-heavy (VH) chain somatic mutation (0.5 to 1.5%) and also displayed a disproportionate usage of VH1 family genes (74%), a phenomenon recently described for CD4-binding site broadly neutralizing antibodies (bNAbs). Maximal ADCC activity of VH1 antibodies correlated with mutation frequency. The polyclonality and low mutation frequency of these VH1 antibodies reveal fundamental differences in the regulation and maturation of these ADCC-mediating responses compared to VH1 bNAbs.
Antibody-Dependent Cellular Cytotoxicity-Mediating Antibodies from
an HIV-1 Vaccine Efficacy Trial Target Multiple Epitopes and
Preferentially Use the VH1 Gene Family
Mattia Bonsignori,
Justin Pollara,
M. Anthony Moody,
Michael D. Alpert,
Xi Chen,
Kwan-Ki Hwang,
Peter B. Gilbert,
Ying Huang,
Thaddeus C. Gurley,
Daniel M. Kozink,
Dawn J. Marshall,
John F. Whitesides,
Chun-Yen Tsao,
Jaranit Kaewkungwal,
Sorachai Nitayaphan,
Punnee Pitisuttithum,
Supachai Rerks-Ngarm,
Jerome H. Kim,
Nelson L. Michael,
Georgia D. Tomaras,
David C. Montefiori,
George K. Lewis,
Anthony DeVico,
David T. Evans,
Guido Ferrari,
Hua-Xin Liao,
Barton F. Haynes
Duke University Medical Center, Durham, North Carolina, USA
; Harvard Medical School, Boston, Massachusetts, USA
; Statistical Center for HIV/AIDS Research and
Prevention, Fred Hutchinson Cancer Research Center, Seattle, Washington, USA
; Tropical Hygiene, Mahidol University, Bangkok, Thailand
; Armed Forces Research
Institute of Medical Sciences (AFRIMS), Bangkok, Thailand
; Clinical Tropical Medicine, Mahidol University, Bangkok, Thailand
; Department of Disease Control, Ministry of
Public Health, Nonthaburi, Thailand
; U.S. Military HIV Research Program, Rockville, Maryland, USA
; and Institute of Human Virology, University of Maryland School of
Medicine, Baltimore, Maryland
The ALVAC-HIV/AIDSVAX-B/E RV144 vaccine trial showed an estimated efficacy of 31%. RV144 secondary immune correlate
analysis demonstrated that the combination of low plasma anti-HIV-1 Env IgA antibodies and high levels of antibody-depen-
dent cellular cytotoxicity (ADCC) inversely correlate with infection risk. One hypothesis is that the observed protection in
RV144 is partially due to ADCC-mediating antibodies. We found that the majority (73 to 90%) of a representative group of vac-
cinees displayed plasma ADCC activity, usually (96.2%) blocked by competition with the C1 region-specific A32 Fab fragment.
Using memory B-cell cultures and antigen-specific B-cell sorting, we isolated 23 ADCC-mediating nonclonally related antibodies
from 6 vaccine recipients. These antibodies targeted A32-blockable conformational epitopes (n 19), a non-A32-blockable con-
formational epitope (n 1), and the gp120 Env variable loops (n 3). Fourteen antibodies mediated cross-clade target cell kill-
ing. ADCC-mediating antibodies displayed modest levels of V-heavy (VH) chain somatic mutation (0.5 to 1.5%) and also dis-
played a disproportionate usage of VH1 family genes (74%), a phenomenon recently described for CD4-binding site broadly
neutralizing antibodies (bNAbs). Maximal ADCC activity of VH1 antibodies correlated with mutation frequency. The poly-
clonality and low mutation frequency of these VH1 antibodies reveal fundamental differences in the regulation and maturation
of these ADCC-mediating responses compared to VH1 bNAbs.
he RV144 ALVAC-HIV (vCP1521) prime/AIDSVAX B/E
boost clinical trial provided the first evidence of vaccine-in-
duced protection from acquisition of human immunodeficiency
virus type 1 (HIV-1) infection (39). Analysis of immune correlates
of risk of infection demonstrated that antibodies (Ab) targeting
the Env gp120 V1/V2 region inversely correlated with infection
risk, while IgA Env-binding antibodies to Env directly correlated
with infection risk (17). In addition, in secondary immune corre-
late analyses, low plasma IgA Env antibody levels in association
with high levels of antibody-dependent cellular cytotoxicity
(ADCC) were inversely correlated with infection risk (17). Thus,
one hypothesis is that the observed protection in RV144 is due, in
a subset of vaccinees, to ADCC-mediating antibodies.
The importance of ADCC responses has been reported in
chronically HIV-1-infected individuals (3, 13, 22) and in HIV-1
vaccine studies in nonhuman primates (14, 15, 18, 45). Baum et al.
reported an inverse correlation between titers of HIV-1 gp120-
specific ADCC antibodies and the rate of disease progression in
humans (3). Moreover, HIV-1-infected elite controllers who had
undetectable viremia showed higher ADCC antibody titers than
infected individuals with viremia (22). In nonhuman primates,
administration of vaccine candidates elicited ADCC antibody ti-
ters that correlated with control of virus replication after mucosal
challenge with a pathogenic simian immunodeficiency virus (SIV)
(2, 15). More recently, different groups have reported that titers of
nonneutralizing ADCC antibodies are associated with control of
viremia against primary SIV infection (14, 18, 45). While antibod-
ies against multiple epitopes can mediate ADCC, it has been re-
cently reported that the A32 monoclonal antibody (MAb), recog-
nizing a conformational epitope in the C1 region of HIV-1 Env
gp120 (53), could mediate potent ADCC activity and could block
a significant proportion of ADCC-mediating Ab activity detect-
able in HIV-1-infected individuals (13).
We have recently observed that ADCC-mediating Ab re-
sponses are detectable as early as 48 days after acute HIV-1 infec-
tion (37). This early appearance of ADCC-mediating Abs after
acute HIV-1 infection contrasts with HIV-1 broadly neutralizing
antibodies (bNAbs) that appear approximately 2 to 4 years after
HIV-1 infection (16, 27, 42).
In this study, we have defined a series of modestly somatically
mutated ADCC-mediating antibodies induced by the ALVAC-
HIV/AIDSVAX B/E vaccine (34, 39), most of which are directed
against conformational A32-blockable epitopes of the gp120 en-
Received 24 April 2012 Accepted 24 July 2012
Published ahead of print 15 August 2012
Address correspondence to Mattia Bonsignori,
M.B., J.P., M.A.M., G.F., H.-X.L., and B.F.H. contributed equally to this work.
Copyright © 2012, American Society for Microbiology. All Rights Reserved.
November 2012 Volume 86 Number 21 Journal of Virology p. 11521–11532 11521
velope glycoprotein. This group of antibodies displayed preferen-
tial use of the variable heavy 1 (VH1) gene segment, a phenome-
non similar to that recently described for highly mutated CD4
binding-site (CD4bs)-specific bNAbs (41, 52).
Plasma and cellular samples from vaccine recipients. All trial partici-
pants gave written informed consent as described for both studies (34,
39). Samples were collected and tested according to protocols approved by
the institutional review boards at each site involved in these studies.
Plasma samples were obtained from volunteers enrolled in the phase I/II
clinical trial (34) and in the community-based, randomized, multicenter,
double-blind, placebo-controlled phase III efficacy trial (39); both trials
tested the prime-boost combination of vaccines containing ALVAC-HIV
(vCP1521) (Sanofi Pasteur) and AIDSVAX B/E (Global Solutions for In-
fectious Diseases). Plasma samples collected at enrollment (week 0) and 2
weeks after the last immunization (week 26) were selected by simple ran-
dom sampling with a vaccine/placebo ratio of 40:10 for both men and
Peripheral blood mononuclear cells (PBMCs) from six vaccine recip-
ients enrolled in the phase II (n 3) and phase III (n 3) trials whose
plasma showed ADCC activity were used for isolation of memory B cells
and monoclonal antibodies (MAbs). Subjects T141485, T141449, and
T143859 participated in the phase II trial; subjects 609107, 210884, and
347759 were enrolled in the phase III trial. All six subjects had negative
serology for HIV-1 infection at the time of collection.
Competition binding assay. To determine the presence of A32 bind-
ing Ab in the plasma of the vaccine recipients, we modified the previously
described full-length single-chain (FLSC) assay (9). Briefly, biotinylated
A32 was used at a limiting dilution of 0.173 g/ml to compete for the
binding of plasma Ab to a single-chain complex (FLSC) captured (Aby
D7324) on a plate. Plasma samples from 80 vaccine recipients and 20
placebo recipients were initially screened at a 1:50 final dilution. For
plasma samples that blocked binding of biotinylated A32 MAb, the ability
to mediate 50% of A32 blocking at 1:50 dilution was used as the crite-
rion for inclusion in this study. Seventy-nine plasma samples met this
criterion (data not shown) and were tested in a serial dilution to calculate
the 50% inhibitory dose (ID
) titer.
ADCC-luciferase (ADCC-92TH023) assay. Plasma was evaluated for
ADCC activity against cells infected by HIV-1 92TH023 in an assay that
employs a natural killer (NK) cell line as effectors. The NK cell line was
derived from KHYG-1 cells (Japan Health Sciences Foundation) (54).
These cells were transduced with a retroviral vector to stably express the
V158 variant of human CD16a (FCGR3A). The target cells were
cells (AIDS Research and Reference Reagent Program,
Division of AIDS, NIAID, NIH; contributed by Alexandra Trkola) (47),
which were modified to express Firefly luciferase upon infection. Target
cells were infected with HIV-1 92TH023 by spinoculation (35) 4 days
prior to use in assays. NK effectors and 92TH023-infected targets were
incubated at an effector/target (E/T) ratio of 10:1 in the presence of trip-
licate serial dilutions of plasma samples for 8 h. Wells containing NK cells
and uninfected targets without plasma defined 0% relative light units
(RLU), and wells with NK cells plus infected targets without plasma de-
fined 100% RLU. ADCC activity was measured as the percent loss of
luciferase activity with NK cells plus infected targets in the presence of
Recombinant gp120 HIV-1 proteins. Where indicated, CEM.
target cells were coated with recombinant gp120 HIV-1 protein
from the CM243 isolate representing the subtype A/E HIV-1 envelope
(GenBank accession no. AY214109; Protein Sciences, Meiden, CT). The
optimum amount to coat target cells was determined as previously de-
scribed (36).
Virus and IMCs for ADCC GTL assay. HIV-1 reporter viruses used
were replication-competent infectious molecular clones (IMC) designed
to encode subtype A/E, B, or C env genes in cis within an isogenic back-
bone that also expresses the Renilla luciferase reporter gene and preserves
all viral open reading frames (10). The Env-IMC-LucR viruses used were
subtype A/E NL-LucR.T2A-AE.CM235-ecto (IMC
) (GenBank ac
cession no. AF259954.1; plasmid provided by Jerome Kim, U.S. Military
HIV Research Program), subtype B NL-LucR.T2A-BaL.ecto (IMC
(1), subtype C NL-LucR.T2A-DU422.ecto (IMC
; GenBank acces
sion no. DQ411854), and subtype C NL-LucR.T2A-DU151.ecto
; GenBank accession no. DQ411851). Reporter virus stocks
were generated by transfection of 293T cells with proviral IMC plasmid
DNA and titrated on TZM-bl cells for quality control.
ADCC-GTL assay. ADCC activity was detected according to our pre-
viously described ADCC-GranToxiLux (GTL) procedure (36). We used
the following target cells: CM243 gp120 coated (ADCC-CM243 assay)
and IMC
-, IMC
-, IMC
-, and IMC
-infected CEM.
cells (ADCC-E.CM235, ADCC-B.BaL, ADCC-C.DU422, and
ADCC-C.DU151 assay, respectively) (47). All of the PBMC samples from
the seronegative donors used as effector cells were obtained according to
the appropriate institutional review board protocol. We used 10,000 tar-
get cells per well, and E/T ratios of 30:1 and 10:1 were used for whole
PBMC and purified NK effector cells, respectively. MAb A32 (James Rob-
inson, Tulane University, New Orleans, LA), palivizumab (MedImmune,
LLC, Gaithersburg, MD; used as a negative control), and vaccine-induced
MAbs were tested as six 4-fold serial dilutions starting at a concentration
of 40 g/ml (range, 40 to 0.039 g/ml). For the Fab blocking assay, the
target cells were incubated for 15 min at room temperature in the presence
of 10 g/ml A32, 19B (31), and 17B (46) Fab fragments, which were
produced by Barton Haynes. The excess Fab was removed by washing the
target cell suspensions once before plating with the effector cells as previ-
ously described (13). A minimum of 2.5 10
events representing viable
gp120-coated or infected target cells was acquired for each well. Data
analysis was performed using FlowJo 9.3.2 software. The results are ex-
pressed as percent granzyme B (GzB) activity, defined as the percentage of
cells positive for proteolytically active GzB out of the total viable target cell
population. The final results are expressed after subtracting the back-
ground represented by the percent GzB activity observed in wells contain-
ing effector and target cell populations in the absence of MAb, IgG prep-
aration, or plasma. The results were considered positive if the percent GzB
activity after background subtraction was 8% for the gp120-coated cells
or 5% for the CM235-infected target cells.
Isolation of ADCC-mediating monoclonal antibodies. Monoclonal
antibodies were isolated either from IgG
memory B cells cultured at
nearly clonal dilution for 14 days (4), followed by sequential screenings of
culture supernatants for HIV-1 gp120 Env binding and ADCC activity, or
from memory B cells that bound to the HIV-1 group M consensus
Env sorted by flow cytometry (16).
Subject 210884 was tested using IgG
memory B-cell cultures isolated
and cultured at nearly clonal dilutions as previously described (4). Briefly,
57,600 IgG
memory B cells were isolated from frozen PBMCs by select
ing CD2
, CD14
, CD16
, CD235a
, IgD
, and IgG
cells through two
rounds of separation with magnetic beads (Miltenyi Biotec, Auburn, CA)
and resuspended in complete medium containing 2.5 g/ml oCpG
ODN2006 (tlrl-2006; InvivoGen, San Diego, CA), 5 M CHK2 kinase
inhibitor (Calbiochem/EMD Chemicals, Gibbstown, NJ), and Epstein-
Barr virus (EBV; 200 l supernatant of B95-8 cells/10
memory B cells).
After overnight incubation in bulk, cells were distributed into 96-well
round-bottom tissue culture plates at a cell density of 8 cells/well in the
presence of ODN2006, CHK2 kinase inhibitor, and irradiated (7,500 cGy)
CD40 ligand-expressing L cells (5,000 cells/well). Cells were refed at day 7
and harvested at day 14.
Subjects T141485, T141449, T143859, and 609107 were tested using
antigen-specific memory B-cell sorting as previously described (16), with
the following modifications. The group M consensus gp140
Env la
beled with Pacific Blue and Alexa fluor 647 (Invitrogen, Carlsbad, CA) was
used for sorting. Memory B cells were gated as Aqua vital dye
, CD3
, CD16
, CD235a
, CD19
, and surface IgD
; memory B cells
Bonsignori et al.
11522 Journal of Virology
stained with gp140
in both colors were sorted as single cells as de
scribed previously (16). A total of 137,345 memory B cells were screened
using this method: 32,766 from subject T141485, 54,621 from subject
T141449, 20,629 from subject T143859, and 29,329 from subject 609107.
For subject 347759, memory B cells were screened using both meth-
ods: 57,600 cells were cultured at nearly clonal dilution and 69,400 mem-
ory B cells were sorted. Sorted cells were previously enriched for IgG
memory B cells as described above, incubated overnight in complete me-
dium containing 2.5 g/ml oCpG ODN2006, 5 M CHK2 kinase inhib-
itor, and EBV (200 l supernatant of B95-8 cells/10
memory B cells), and
then stimulated for 7 days at a cell density of 1,000 cells/well in the pres-
ence of ODN2006, CHK2 kinase inhibitor, and irradiated CD40 ligand-
expressing L cells (5,000 cells/well).
Isolation of V(D)J immunoglobulin regions. Single-cell PCR was
performed as previously described (25, 50). Briefly, reverse transcription
(RT) was performed using Superscript III reverse transcriptase (Invitro-
gen, Carlsbad, CA) and human constant region primers for IgG, IgA
, IgM, IgD, Ig, and Ig; separate reactions amplified individual V
, and V
families from the cDNA template using two rounds of PCR.
Products were analyzed with agarose gels (1.2%) and purified with PCR
purification kits (Qiagen, Valencia, CA). Products were sequenced in for-
ward and reverse directions using a BigDye sequencing kit using an ABI
3700 device (Applied Biosystems, Foster City, CA). Sequence base calling
was performed using Phred (11, 12); forward and reverse strands were
assembled using an assembly algorithm based on the quality scores at each
position (32). The estimated PCR artifact rate was 0.28 or approximately
one PCR artifact per five genes amplified. Ig isotype was determined by
local alignment with genes of known isotype (44); V, D, and J region
genes, CDR3 loop lengths, and mutation rates were identified using SoDA
(48), and data were annotated so that matching subject data and sort
information were linked to the cDNA sequence and analysis results.
Expression of recombinant antibodies. Isolated Ig V(D)J gene pairs
were assembled by PCR into linear full-length Ig heavy- and light-chain
gene expression cassettes (25) and optimized as previously described for
binding to the Fc receptors (43). Human embryonic kidney cell line
293T (ATCC, Manassas, VA) was grown to near confluence in 6-well
tissue culture plates (Becton Dickson, Franklin Lakes, NJ) and transfected
with 2 g per well of purified PCR-produced IgH and IgL linear Ig gene
expression cassettes using Effectene (Qiagen). The supernatants were har-
vested from the transfected 293T cells after 3 days of incubation at 37°C in
5% CO
, and the monoclonal antibodies were purified as previously de
scribed (25).
Direct binding ELISAs. Three hundred eighty-four-well plates
(Corning Life Sciences, Lowell, MA) were coated overnight at 4°C with 15
l of purified HIV-1 monomeric gp120 envelope glycoprotein (E.A244
gp120, B.MN gp120, and A.92TH023 gp120) antigen at 2 g/ml and
blocked with assay diluent (phosphate-buffered saline [PBS] containing
4% [wt/vol] whey protein–15% normal goat serum– 0.5% Tween 20
0.05% sodium azide) for1hatroom temperature.
Ten l/well of purified MAbs was incubated for2hatroom temper-
ature in serial 3-fold dilutions starting at 100 g/ml for the determination
of 50% effective concentrations (EC
) and then washed with PBS– 0.1%
Tween 20. Thirty l/well of alkaline phosphatase-conjugated goat anti-
human IgG in assay diluent was added for 1 h, washed, and detected with
30 l/well of p-nitrophenyl phosphate substrate diluted in 50 mM
plus Na
(1:1, vol/vol), pH 9.6, 10 mM MgCl
. Plates were
developed for 45 min in the dark at room temperature and read at an
optical density of 405 nm (OD
) with a VersaMax microplate reader
(Molecular Devices, Sunnyvale, CA).
Epitope mapping studies were performed using 15-mer linear pep-
tides spanning the gp120 envelope glycoprotein of the MN and 92TH023
HIV-1 strains obtained from the AIDS Reagent Repository as coating
antigens, horseradish peroxidase goat anti-human IgG as secondary anti-
body, and 3,3=,5,5=-tetramethylbenzidine (TMB) substrate for detection.
Statistical analyses. The analysis of the ADCC-mediating Ab re-
sponses in the plasma of the vaccine recipients was conducted as follows.
For each time point of a subject, partial area under the activity versus the
log10 (dilution) curve (AUC) was estimated nonparametrically for each
assay. For ADCC-CM243 assay using gp120-coated target cells, the AUC
was calculated based on percent GzB activity across dilution levels of 50,
250, 1,250, 6,250, 31,250, and 156,250; for ADCC-92TH023 assay using
infected cells, the AUC was calculated based on percent loss of luciferase
activity across dilution levels of 32, 100, 316, and 1,000. Two-sample t test
allowing for unequal variance was used to test the mean difference in AUC
between the vaccine and placebo groups at week 26. A paired t test was
used to test the mean difference in AUC between week 26 and week 0
among vaccinees. For each of the vaccine and placebo groups and for each
time point, the positive response rate was estimated by the observed frac-
tion of subjects that have a positive response (defined as peak percent GzB
greater than 8% for the ADCC-CM243 assay and peak percent loss of
luciferase activity greater than 9% for the ADCC-92TH023 assay). A 95%
confidence interval (CI; computed by the Agresti-Coull method) was pro-
vided around each response rate. An exact P value from McNemar’s test
was used to evaluate whether the response rate differs for the week 26 time
point versus the week 0 time point among vaccinees. Fisher’s exact test was
used to provide a P value to test whether the response rate differed be-
tween the vaccine and placebo groups at week 26.
The other statistical analyses conducted in this study were performed
using Prism software v5.0c (GraphPad Software, Inc.), and the appropri-
ate methods are listed throughout the manuscript.
Vaccine-induced ADCC responses. We studied 50 simple ran-
domly sampled plasma specimens drawn from subjects enrolled
in the RV144 vaccine trial at enrollment (week 0) and 2 weeks after
the last immunization (week 26), including 10 placebo recipients
(5 male and 5 female) and 40 vaccine recipients (20 male and 20
female; four injections of recombinant canarypox vector vaccine
ALVAC-HIV [vCP1521] and two booster injections of recombi-
nant gp120 subunit [AIDSVAX B/E]) (34, 39). The frequency of
ADCC responders (Table 1) and the AUC for ADCC activity (Fig.
1A to D) of both vaccine and placebo recipients were measured
using two ADCC assays, CEM.NKR
target cells either coated
with HIV-1 AE.CM243 gp120 (ADCC-CM243) (36) or infected
with the AE.92TH023 HIV-1 strain (ADCC-92TH023) (17).
The ADCC response rate measured with the ADCC-CM243
assay increased from 0% at week 0 to 90% at week 26 among the
vaccine recipients (Table 1). Similarly, the ADCC-92TH023 assay
detected activity in 72.5% (29/40) of vaccine recipients at week 26
(Table 1). For both assays, the frequency of positive responses
among the vaccine recipients was significantly higher comparing
baseline (week 0) to postimmunization (week 26) (P 0.0001 for
both assays).
TABLE 1 Frequency of ADCC responders among vaccine and placebo
recipients before and after vaccination
Recipient group
(n) and time
Assay result (no. of responders [%, 95% CI])
Vaccine (40)
Wk 0 0 (0, 0–31) 4 (10, 2.8–23.7)
Wk 26 36 (90, 76–97) 29 (72.5, 56.1–85.4)
Placebo (10)
Wk 0 1 (10, 0–44.5) 0 (0, 0–31)
Wk 26 1 (10, 0–44.5) 1 (10, 0.3–44.5)
Characterization of HIV-1 Vaccine-Induced ADCC Abs
November 2012 Volume 86 Number 21 11523
We next evaluated the AUC of a dilution of antibody in the
assay (see the statistical analysis in Materials and Methods). In
both the ADCC-CM243 and ADCC-92TH023 assays, AUC values
of vaccinated subjects at week 26 were significantly higher than
both of those in the vaccine recipients at week 0 and in the placebo
group at week 26 (P 0.0001 and P 0.001, respectively) (Fig. 1A
to D). Thus, the ALVAC-HIV/AIDSVAX B/E vaccine induced an-
ti-HIV-1 gp120 ADCC activity in 70 to 90% of vaccine recipi-
ents depending on the assay utilized. This frequency of responders
among vaccinees is similar to that reported in earlier phase II
studies as well as in RV144 (17, 21). It is important to note that the
92TH023-infected target cell ADCC assay was used in the RV144
immune correlate primary analysis, and in the secondary analysis,
high activity in this assay associated with low plasma anti-Env IgA
responses inversely correlated with infection risk (17).
Plasma ADCC activity is blocked in part by MAb A32. Since
MAb A32 can block plasma ADCC responses during chronic in-
fection (13), we sought to determine whether A32-like antibodies
were produced by RV144 vaccine recipients. We first evaluated the
ability of plasma samples collected at week 26 postvaccination
from simple random samples drawn from both RV144 vaccine (n
79 out of 80; one sample was not studied because of less than 50%
inhibition at screening) and placebo (n 20) recipients for
their ability to block the binding of biotinylated A32 MAb to
B.BaL Env. Plasma Ab blocked A32 MAb binding in 76/79
(96.2%) of the vaccine recipients with an average 50% inhibi-
tory dose (ID
) titer of 119 (95% CI, 95 to 130) (Fig. 2A
These data demonstrated the presence of A32-like antibodies in
the plasma of vaccine recipients.
We then evaluated the effect of pretreatment of CM243 gp120-
coated target cells with A32 Fab on plasma-mediated ADCC (13).
Thirty vaccine recipients whose plasma samples were previously
identified to mediate ADCC were selected to represent each tertile
(low, medium, and high response) of the range of ADCC activities
observed. These plasma samples were tested to determine the di-
lution that provided maximum ADCC activity (data not shown).
When tested at the optimal dilution, these plasma samples in-
duced granzyme B (GzB) activity against AE.CM243 gp120-
coated target cells ranging from 8.0 to 34.6% (mean standard
deviation [SD], 20.4 6.6) (Fig. 2B). When the cells were pre-
treated with 10 g/ml of A32 Fab, ADCC activity was reduced or
completely abrogated for each plasma sample (GzB activity,
3.2%; P 0.001 versus untreated samples) (Fig. 2B). Similar
treatment with a control Fab made from palivizumab (19) did not
affect plasma ADCC activity (range, 9.0 to 35.8%; mean SD,
21.1% 6.7%) (Fig. 2B). However, preincubation with 10 and 50
g/ml of A32 Fab did not block plasma ADCC activity at the peak
of responses (1:50 dilution) in ADCC assays using target cells in-
fected with either the E.92TH023 or the E.CM235 HIV-1 strains
(data not shown). This lack of inhibition may be due to unfavor-
able kinetics for Fab epitope recognition on infected cells in the
FIG 1 Vaccine-induced ADCC responses. To measure plasma ADCC activity
induced by the ALVAC-HIV(vCP1521)/AIDSVAX B/E vaccine, plasma sam-
ples from 40 vaccine recipients and 10 placebo recipients were collected before
immunization (week 0) and 2 weeks after the last boost (week 26). ADCC
activity was measured using the ADCC-CM243 assay (A and B) and ADCC-
92TH023 assay (C and D) as described in Materials and Methods. Results are
reported as areas under the curve (AUC). Each dot represents one sample. The
lines connect samples obtained from the same donor.
FIG 2 Recognition of the A32 epitope in plasma of ALVAC-HIV(vCP1521)/AIDSVAX B/E vaccine recipients. (A) Plasma samples collected at week 26 from 20
placebo recipients and 79 vaccine recipients were evaluated for the presence of Abs with A32-like binding specificities by competition ELISA. We defined plasma
samples that inhibited 50% of A32 MAb binding as positive (red dots). While none of the placebo recipients displayed A32-like responses, the plasma of 76/79
vaccine recipients (96.2%) competed for A32 MAb binding to its cognate epitope. The whisker boxes show the average plasma ID
titer and the 95% confidence
intervals for each test group. (B) Inhibition of plasma ADCC activity by epitope blocking with the MAb A32 Fab fragment was evaluated in the ADCC-CM243
assay (see Materials and Methods). Plasma samples were collected at week 26 from 30 vaccine recipients and were tested at dilutions corresponding to peak
activity. Data are reported as maximum percent GzB activity detected using CM243-gp120-coated targets without pretreatment (no Fab pretreatment; left) or
treated with 10 g/ml MAb A32 Fab (center) or palivizumab Fab (negative control; right). Lines and error bars represent the mean percent GzB activity SD.
The P values were obtained using repeated-measure analysis of variance. pos, positive; neg, negative; n.s., not significant.
Bonsignori et al.
11524 Journal of Virology
presence of polyclonal antibodies in plasma. To better define the
nature of the antibodies responsible for the observed ADCC ac-
tivity, we isolated ADCC-mediating MAbs from ALVAC-HIV/
AIDSVAX B/E vaccine recipients.
Isolation of ADCC-mediating antibodies from ALVAC-HIV/
AIDSVAX B/E vaccinees. We isolated a total of 23 MAbs that
mediated ADCC from memory B cells of six vaccine recipients
enrolled in the RV135 phase II (n 3) (21, 34) or RV144 phase III
(n 3) (39) ALVAC-HIV/AIDSVAX B/E clinical trials. Nine
MAbs (CH49, CH51, CH52, CH53, CH54, CH55, CH57, CH58,
and CH59) were obtained from cultured IgG
memory B cells
that bound to one or more of the E.A244, B.MN, and E.92TH023
gp120 envelope glycoproteins, while the remaining 14 were ob-
tained from group M consensus gp140
Env-specific flow-cy
tometric single-memory-B-cell sorting (4, 16). Two of the 23
ADCC-mediating MAbs were against the gp120 Env V2 region
and are the subject of a separate report (H.-X. Liao, M. Bon-
signori, B. F. Haynes, unpublished data).
ADCC activity of the remaining 21 MAbs, purified and ex-
pressed in a codon-optimized IgG1 backbone, was measured us-
ing both E.CM243 gp120-coated (ADCC-CM243) and E.CM235-
infected (ADCC-CM235) target cells in the flow-based assay
described in Materials and Methods. The maximum percent GzB
activity of the 21 MAbs ranged from 38.9% (CH54) to 6.0%
(CH92) (Fig. 3A). Remarkably, 11/21 MAbs displayed a maxi-
mum percent GzB activity greater than that of A32 MAb (16%) in
duplicate assays: CH54 (38.9%), CH55 (31.4%), CH57 (31.3%),
CH23 (31.2%), CH49 (26.7%), CH51 (25.9%), CH53 (24.4%),
CH52 (23.9%), CH40 (22.6%), and CH20 (21.0%). The endpoint
titers of each of the 21 MAbs (Fig. 3B) ranged from 20 ng/ml to
30.3 g/ml (means SD, 4.1 8.8 g/ml).
None of the ADCC-mediating MAbs were heavily somatically
mutated: the mean nucleotide mutation frequencies of the heavy
and light chains were 2.4% (range, 0.5 to 5.1%) and 1.8% (range,
0.4 to 4.3%), respectively (Table 2). These data demonstrate that
the ALVAC-HIV/AIDSVAX B/E vaccine induced polyclonal anti-
body responses capable of mediating moderate to high levels of
ADCC activity without requiring high levels of ADCC antibody
affinity maturation.
Epitope mapping of vaccine-induced ADCC-mediating anti-
bodies. To define the specificity of ADCC-mediating MAbs, we
asked if they recognized linear epitopes by testing their ability to
bind to overlapping linear peptides spanning the gp120 envelope
glycoprotein of the B.MN or E.92TH023 HIV-1 strain. Each MAb
bound to one or more of the vaccine gp120 envelope glycopro-
teins, which included the B.MN and E.92TH023 strains (Table 3).
We found that 19/20 MAbs (CH53 was not tested) did not react
with any of the B.MN or E.92TH023 peptides, while one (CH23)
reacted with the clade E V3 loop (NTRTSINIGRGQVFY). As pre-
viously described, we used the A32 Fab blocking strategy in the
ADCC-CM235 assay to determine whether the ADCC activity of
the 20 MAbs not specific for the V3 loop was mediated by target-
ing conformational epitopes expressed on infected cells that could
be blocked by the A32 MAb (Fig. 4). As a control, we also tested the
ability of these 20 MAbs to block the ADCC activity mediated by
17B and 19B Fab fragments, which target the CD4-induced
(CD4i) and V3 epitopes, respectively (Fig. 4). In contrast to
plasma ADCC activity, which could not be blocked by A32 when
tested against CM235-infected target cells, A32 Fab blocking in-
hibited between 73 and 100% (means SD, 92% 9%) of the
ADCC activity mediated by 19/20 (95%) non-V3 MAbs (Fig. 4).
CH20 was not inhibited by any of the A32, 17B, or 19B Fab frag-
ments (Fig. 4). None of the MAbs displayed substantial loss of
ADCC activity (defined as 20% inhibition) when E.CM235-in-
fected target cells were preincubated with Fab fragments of MAb
17B or 19B (Fig. 4).
To confirm the results observed with the ADCC assay, we
tested the ability of the ADCC-mediating MAbs to block A32
binding to the AE.A244 gp120 envelope glycoprotein and found
that 16 MAbs blocked 20.7 to 94% of A32 binding to gp120 Env
(Fig. 5). As expected, MAb CH20 did not block MAb A32 binding
to gp120 Env, consistent with the inability of A32 Fab to block
CH20-mediated ADCC activity. Of note, CH29 and CH57 did not
reciprocally block A32 binding to the envelope, even though A32
Fab blocked their ADCC activity (Fig. 4) and MAb A32 blocked
their binding to Env (Table 3).
FIG 3 ADCC activity of vaccine-induced MAbs. ADCC activity mediated by
monoclonal antibodies isolated from memory B cells of ALVAC-HIV-
(vCP1521)/AIDSVAX B/E vaccine recipients. Twenty-three MAbs were iso-
lated from six vaccine recipients. Each bar is color-coded by subject: T141485
(light blue), T141449 (red), T143859 (brown), 609107 (green), 210884 (or-
ange), and 347759 (dark blue). MAb A32 (positive control) and palivizumab
(negative control) are shown in black and white, respectively. (A) The plots
show the maximum percent GzB with the threshold of positivity (5%) indi-
cated by the black line. (B) The endpoint titer expressed in g/ml for each
MAb. The threshold of positivity was determined by averaging the results
obtained by testing more than 70 MAbs with different binding capacities to
gp120 and infected cells. Data shown refer to the results obtained with the
ADCC-CM235 assay with the exception of MAb CH23, for which results of the
ADCC-CM243 assay are shown. ADCC activity of all MAbs was confirmed in
the ADCC-CM235 assay with a 6-h incubation (not shown; P 0.001 by
Spearman correlation analysis).
Characterization of HIV-1 Vaccine-Induced ADCC Abs
November 2012 Volume 86 Number 21 11525
We found that 6/19 (32%) of the A32-blockable MAbs partially
blocked the binding of soluble CD4 (sCD4) and/or MAb b12 to
gp120 envelope glycoproteins (Table 3). This activity ranged from
22% (CH77) to 46% (CH40) blocking of sCD4 binding to
AE.A244 gp120 Env and from 25% (CH40) to 40% (CH55) block-
ing of b12 binding to B.JRFL gp120 Env; in some cases blocking
was higher than that seen for A32 (Table 3). These data suggest that
these ADCC-mediating MAbs interfere with binding of CD4bs-di-
rected MAbs either by inducing conformational changes on the
gp120 envelope glycoprotein or by partially blocking access to the
CD4bs. The combination of blocking and binding data indicate that
the ALVAC-HIV/AIDSVAX B/E vaccine induced a group of anti-
bodies that mediate ADCC by targeting distinct but overlapping
Env epitopes that are mostly A32 blockable.
Moreover, it should be noted that the original isotypes of CH29
and CH38 were IgA
and IgA
, respectively (Table 2). When CH29
and CH38 were expressed as IgG
MAbs, they mediated ADCC
activity (GzB activities of 6.4% [CH29] and 12.4% [CH38]) that
was directed against the gp120 C1 region, as demonstrated by
blocking with the A32 Fab (Fig. 4).
Cross-clade ADCC activity of RV144-induced antibodies.
We next studied the ability of the 21 MAbs to mediate ADCC
against viruses from different HIV-1 subtypes. MAb A32 medi-
ated ADCC against all four tested isolates with an endpoint titer of
0.039 g/ml against all strains (Fig. 6). Each of the 21 MAbs de-
rived from vaccinees were able to mediate ADCC against target
cells infected with the subtype A/E strain virus AE.CM235, while
14/21 MAbs (67%) mediated ADCC against those infected with
B.Bal. When tested against subtype C virus isolates, 4/21 (19%)
mediated ADCC against C.DU151-infected target cells while a
single recovered MAb (CH54) mediated ADCC against C.DU422-
infected target cells (Fig. 6). The patterns of cross-clade ADCC
activity, combined with the patterns observed in binding and
blocking experiments, demonstrate that the RV144 immunogen
elicited a diverse set of antibodies directed at epitopes overlapping,
but not identical to, that of MAb A32.
VH1 gene family members are overrepresented among
ADCC-mediating monoclonal antibodies recovered from vac-
cine recipients. Association of anti-HIV-1 ADCC activity with the
use of a specific VH family gene has not been previously reported.
It was therefore quite surprising to find that 17/23 (74%) of
ADCC-mediating MAbs isolated from the vaccine recipients uti-
lized the VH1 family gene (Fig. 7); this group includes the two
anti-V2 MAbs that are described in a separate report (23), which
did not use VH1. In contrast, only 19/111 (17.1%) heavy chains
isolated from memory B-cell cultures that did not mediate ADCC
used VH1 family gene segments. The frequency of VH1 family
gene usage was significantly lower than that for the 23 ADCC-
mediating antibodies (P 0.0001 by Fisher’s exact test), demon-
strating that the high frequency of VH1 gene usage among ADCC-
mediating MAbs was not reflective of a disproportionate use of
VH1 among recovered antibodies from vaccinees.
TABLE 2 Characteristics of the V(D)J rearrangements of vaccine-induced ADCC-mediating monoclonal antibodies
Characteristics of rearrangement by chain
Heavy Light
Isotype V D J CDR3
(%) Type V J CDR3
T141485 CH20 G1 1-69*02 6-6*01 4*02 15 2.6 2-23*02 3*02 10 0.4
T141449 CH77 G3 1-2*02 2-OF15*02 6*02 15 2.3 4-1*01 4*01 8 0.8
CH89 G3 1-2*02 3-22*01 4*02 12 2.1 1-39-*01 4*01 9 1.4
CH92 G1 1-2*02 2-15*01 4*02 19 1.7 1D-12*01 5*01 9 2.6
CH80 G1 1-2*02 1-IR1*01C 4*02 12 1.6 1-27*01 4*01 10 1.1
CH29 A2 1-2*02 2-15*01 4*02 12 0.8 1-39*01 1*01 9 0.6
CH78 G1 1-2*02 322*01 4*02 19 0.7 3-11*01 1*01 9 1.1
CH94 G1 1-46*02 5-12*01 6*02 23 2.2 1-39*01 2*01 9 1.7
CH90 G1 1-46*01 3-10*01 4*02 14 1.5 1-13*02 1*01 9 4.3
CH91 G1 4-31*03 4-17*01 3*02 15 2.0 2-11*01 3*02 11 1.4
T143859 CH23 G1 3-66*01 3-OR15*3 1*01 11 4.5 6-57*01 3*02 10 2.2
609107 CH81 G1 1-8*01 3-10*01 4*02 19 0.5 1-39*01 2*01,02 9 1.4
CH40 G1 1-46*02 6-6*01 5*02 15 3.6 3-20*01 4*01 5 0.9
210884 CH49 G1 1-2*02 1-26*01 4*02 16 5.1 2-11*01 3*02 10 3.1
CH53 G1 1-2*02 2-2*01,02 4*02 16 2.3 2-11*01 2*01 10 2.4
CH52 G1 1-2*02 6-13*01 4*02 13 1.4 3-20*01 2*01 10 1.8
CH55 G1 1-46*01 1-1*01 5*02 15 4.3 3-15*01 5*01 10 1.5
CH54 G1 1-58*02 1-26*01 5*02 14 2.1 1-39*01 2*01 9 1.4
CH51 G1 4-34*12 3-10*01 4*02 14 0.5 3-20*01 1*01 8 0.6
347759 CH57 G1 1-2*02 1-1*01 6*02 12 3.4 1-39*01 1*01 9 4.0
CH38 A1 3-23*01 3-10*01,02 1*01 12 4.7 2-14*03 3*02 10 3.6
PTID, participant identity.
CDR3, complementarity determining region 3. Length is expressed as amino acids according to the Kabat numbering system (20).
Nucleotide mutation frequency in V gene as determined by SoDA (48).
Bonsignori et al.
11526 Journal of Virology
The frequency of VH1 gene usage among vaccine-induced
HIV-specific ADCC-mediating antibodies also was higher than
those of other published data sets: in HIV-1-negative subjects,
Brezinschek and colleagues reported the frequency of VH1 genes
to be approximately 13% (9/71 reported in reference 6; P 0.0001
by Fisher’s exact test comparing the ADCC-mediating antibod-
ies), while in chronically HIV-1-infected subjects the frequency of
VH1 usage in anti-HIV-1 antibodies was reported to be 39% (76/
193 reported in reference 5; P 0.003 by Fisher’s exact test com-
paring the ADCC-mediating antibodies). We have recently re-
ported frequencies of HIV-1 reactive antibodies using VH1 gene
segments of 16.4% (11/67) in HIV-1 acutely infected subjects,
which is similar to VH1 usage reported in the National Center for
Biotechnology Information database (15.2%; 5,238/34,384) (24),
and 38.2% (13/34) in vaccine recipients enrolled in an unrelated
HIV-1 vaccine trial (29). In both cases, the frequency of VH1 gene
segment usage in ALVAC-HIV/AIDSVAX B/E-induced ADCC-
mediating antibodies was significantly higher (P 0.0001 and P
0.014, respectively, by Fisher’s exact test). In the present study, none
of the recovered ADCC antibodies were clonally related, and VH1
antibodies were recovered from 5/6 vaccinees studied. Thus, the high
frequency of usage of VH1 heavy-chain genes among antibodies that
mediate ADCC suggests that B cells using those genes have been pref-
erentially selected by the vaccine trial Env proteins.
It is possible that this phenomenon relates to properties of
gp120 more generally. Analysis of a different HIV-1 vaccine trial
resulted in the recovery of 13/34 (38%) MAbs that used VH1
genes, including 2 MAbs with ADCC activity and 1 with neutral-
izing activity (29). In contrast, only 12/252 (5%) influenza virus-
specific antibodies recovered after influenza immunization (30)
used VH1 genes.
ADCC activity of antibodies using VH1 genes correlated
with the degree of somatic mutation. A number of recent studies
have suggested that highly somatically mutated anti-CD4bs
bNAbs preferentially use the VH1 gene, in particular the VH
1-2*02 and 1-46 segments, and common amino acid sequence
TABLE 3 HIV-1 Env binding of vaccine-induced ADCC-mediating MAbs and blocking of sCD4 and b12 binding to Env
and MAb
of MAbs to HIV-1 Env
% Blocking by MAb
A244 gp120 92TH023 gp120 MN gp120
sCD4 binding to
A244 gp120
sCD4 binding to
JRFL gp120
b12 binding to
JRFL gp120
CH20 ⫺⫺⫺
CH77 ⫹⫹ ⫹⫹⫹ ⫹⫹⫹ 22 ⫺⫺
CH89 ⫺⫺⫺
CH92 ⫺⫺⫺
CH80 ⫹⫺ 23 26
CH29 ⫹⫹⫹
CH78 ⫹⫹ ⫹⫹⫹ ⫹⫹ 27 36 29
CH94 ⫹⫹⫹ ⫹⫹⫹ ⫹⫹⫹
CH90 ⫹⫹⫹
CH91 ⫹⫹ ⫹⫹⫹ ⫹⫹⫹
CH23 ⫹⫹⫹ ⫹⫹⫹ ⫹⫹⫹ 36 ⫺⫺
CH81 ⫹⫹⫹
CH40 ⫹⫹⫹ ⫹⫹ ⫹⫹⫹ 46 20 25
CH49 ⫹⫹⫹
CH53 ⫹⫹⫹ ⫹⫹⫹ ⫹⫹⫹
CH52 ⫹⫹⫹ ⫹⫹⫹ ⫹⫹⫹ 32 30
CH55 ⫹⫺ 31 40
CH54 ⫹⫹⫹
CH51 ⫹⫹ ⫹⫹⫹
CH57 ⫹⫹⫹ ⫹⫹⫹
CH38 ⫹⫹ ⫹⫹⫹ ⫹⫹⫹
A32 29 23
Palivizumab ⫺⫺⫺
VRC-CH31 97 67 70
PTID, participant identity.
⫹⫹⫹, 50% inhibitory concentration (IC
)of10 nM; ⫹⫹,IC
between 10 and 100 nM; ,IC
between 0.1 and 1 M; , negative/no binding/no blocking.
Characterization of HIV-1 Vaccine-Induced ADCC Abs
November 2012 Volume 86 Number 21 11527
motifs (HAAD motifs) have been described for both the heavy and
light chains of such anti-CD4bs bNAbs (41, 51). It was striking
that among the ADCC-mediating VH1 antibodies that we recov-
ered, 10/17 (59%) used the VH1-2*02 gene segment (Fig. 7). None
of the MAbs recovered from this group of participants had broad
neutralizing activity, and of the MAbs reported here, only the
V3-specific MAb CH23 (VH3-66) displayed tier 1 strain-specific
neutralizing activity (28). We sought to determine if this group of
antibodies shared the previously described HAAD motifs with the
potent CD4bs bNAbs (41). Alignments of the amino acid se-
quences of the 17 vaccine-induced ADCC-mediating antibodies
that used VH1 with the heavy- and light-chain HAAD consensus
motifs showed a high degree of similarity (range, 46 to 57 match-
ing amino acids [aa] of 68 aa for the heavy chain, 68 to 84%, and 37
to 46 matching aa of 53 aa for the light chain, 70 to 87%) (Fig. 8A,
red circles), which was comparable to the levels of similarity of the
CD4bs bNAbs (Fig, 8A, black crosses). We also analyzed a group
of three non-HIV-1-reactive VH1-2 anti-influenza virus antibod-
ies that mediate broad influenza virus neutralization (49), which
showed a similar degree of heavy-chain homology (52 to 55 matching
aa, 76 to 81%) but less homology for the light chain (31 to 32 match-
ing aa, 58 to 60%) (Fig. 8A, blue diamonds). Thus, the similarity of
the RV144 vaccine-induced antibodies to the HAAD motif may not
reflect functional selection but rather similarities in Env selection of B
cells with similar heavy- and light-chain pairings.
Since the broadly neutralizing CD4bs antibodies are also
highly mutated, we sought to determine if the degree of somatic
mutation in the RV144-induced antibodies correlated with func-
tion. We found that the ability to block sCD4 binding did not
correlate with the degree of somatic mutation (Fig. 8B). In con-
trast, the overall strength of ADCC activity, as measured by max-
imal percent GzB activity against CM235-infected CD4
T cells,
did correlate with heavy-chain somatic mutation (Spearman cor-
relation, ␳⫽0.56; P 0.02) (Fig. 8B).
The induction of neutralizing antibody (NAb) and cytotoxic T-
lymphocyte (CTL) responses are key goals for HIV-1 vaccine de-
FIG 4 Monoclonal antibody competition of A32, 17B, and 19B Fab ADCC activity. The 20 ADCC-mediating MAbs that did not bind to linear epitopes were
tested for their ability to inhibit ADCC mediated by Fab A32 (left), 17B (middle), and 19B (right) in the ADCC-E.CM235 assay. The y axis shows the average
inhibition of ADCC activity in duplicate assays, and each bar is color-coded by subject as described for Fig. 3.
FIG 5 Monoclonal antibody competition of A32 MAb binding to HIV-1 AE.A244 gp120 envelope glycoprotein. The ADCC-mediating MAbs (with the
exception of CH55 and CH80) were tested for their ability to compete for MAb A32 binding to AE.A244 gp120 envelope glycoprotein. The y axis shows the
percentage of blocking of binding activity, and each bar is color-coded by subject as described for Fig. 3. The data shown are representative of duplicate
independent experiments.
Bonsignori et al.
11528 Journal of Virology
velopment. Recently, the phase III efficacy trial of the prime-boost
combination of vaccines containing ALVAC-HIV and AIDSVAX
B/E has offered the first evidence of vaccine-induced partial pro-
tection in humans (39). The vaccine appeared to induce NAb
responses with a narrow specificity profile and minimal CD8
CTL responses (39), suggesting that nonneutralizing Ab and cel-
lular responses other than those of CD8
CTL have played a role
in conferring protection.
A number of studies have suggested that ADCC play an impor-
tant role in the control of SIV and HIV-1 infection. Several studies
have shown that the magnitude of ADCC Ab responses correlates
inversely with virus set point in acute SIV infection in both unvac-
cinated macaques (45) and in vaccinated animals after challenge
(2, 7, 14, 15). In humans, ADCC-mediating Abs have been shown
to protect against HIV-1 infection in mother-to-infant transmis-
sion (26, 33) and to correlate with both control of virus replication
(22) and lack of progression to overt disease (3). In contrast,
weakly neutralizing and nonneutralizing antibodies were shown
to not protect against vaginal simian-human immunodeficiency
virus (SHIV) challenge in macaques (8).
ADCC is one of the mechanisms that might have conferred
protection from infection in RV144 (17). For this reason, we
sought to isolate MAbs that can mediate ADCC from ALVAC-
HIV/AIDSVAX B/E vaccine recipients and determine their speci-
ficity, clonality, and maturation. In this study, we have demon-
strated that the ALVAC-HIV/AIDSVAX B/E vaccine elicited
antibodies that mediate ADCC in the majority of the vaccinated
subjects, which is in line with previous observations (17, 21), and
that gp120 C1 region-specific A32-like antibodies significantly
contributed to the overall ADCC responses. By isolating 23
ADCC-mediating MAbs from multiple vaccine recipients, we also
demonstrated the presence of ADCC-mediating MAbs of addi-
tional specificities. In addition, we determined that the ADCC-
mediating MAbs underwent limited affinity maturation and pref-
erentially used VH1 gene segments.
Antibody responses that mediate ADCC were directed toward
A32-blockable conformational epitopes (n 19), a non-A32-
blockable conformational epitope (n 1), the gp120 Env V2 re-
gion (n 2) (23), and a linear epitope in the gp120 V3 region (n
1). The conformational epitope recognized by the A32 MAb is a
dominant target of HIV-1-positive plasma ADCC antibodies (13),
and A32-like MAbs are among the anti-HIV-1 CD4i Ab responses
that are detected following HIV-1 transmission (38, 40). The iden-
tification of A32-like MAbs in vaccine recipients suggests that the
gp120 epitope recognized by the A32 MAb is an immunodomi-
nant region not just in response to natural infection but also upon
vaccination. Our data suggest that this A32-binding region reacts
with antibodies that have a diverse binding profile, suggesting that
the RV144 vaccine targeted multiple related but distinct confor-
mational epitopes on gp120. These epitopes have been shown to
be upregulated on the RV144 immunogen and to be efficiently
presented by novel Env designs (S. M. Alam, unpublished data),
thus it will be possible to test this vaccine strategy in future vaccine
trials targeted to different HIV-1 subtypes.
In contrast to ADCC-mediating antibodies, HIV-1 bNAb re-
sponses have been reported to appear an average of 2 to 4 years after
HIV-1 transmission (16, 27, 42), suggesting that different levels of Ab
FIG 6 Cross-clade activity of RV144-induced, ADCC-mediating MAbs. Twenty-one MAbs isolated from six vaccine recipients were tested against CEM.
target cells infected with E.CM235 (black bar), B.BaL (red bar), C.DU422 (blue bar), and C.DU151 (green bar) using the GTL assay. The plot shows
the average endpoint titer from duplicate values expressed in g/ml for each MAb and calculated as previously described for Fig. 3.
FIG 7 VH gene segment usage of the ADCC-mediating monoclonal antibod-
ies. The pie chart shows the distribution of VH gene segments and allele usage
of the 23 ADCC-mediating MAbs. Each antibody is color-coded by subject of
origin using the same color scheme as that described for Fig. 3. The yellow fill
indicates all MAbs that used VH1.
Characterization of HIV-1 Vaccine-Induced ADCC Abs
November 2012 Volume 86 Number 21 11529
maturation are required to mediate ADCC and neutralizing activi-
ties. Indeed, the mutation frequencies observed in the MAbs isolated
from the ALVAC-HIV/AIDSVAX B/E vaccine recipients in our study
were low (0.5 to 5.1%) and well below the 6% changes in variable-
domain amino acid sequences commonly seen as greater affinity for
the cognate antigen is acquired (30, 50). We did, however, find that
higher degrees of VH somatic mutation correlated with greater max-
imal percent GzB activity (Fig. 8B), consistent with vaccine-driven
affinity maturation. Whether repeated boosting of vaccine recipients
would result in on-going maturation of these antibodies to further
increase ADCC activity, CD4 blocking, or addition of neutralizing
activity remains to be determined.
Finally, while ADCC-mediating MAbs were isolated that used di-
verse VH genes, we observed a clear preferential usage of the VH1
heavy-chain gene (74%) similar to that of potent bNAbs directed
against the CD4bs (41, 52). Therefore, while these findings prove that
the ADCC-mediating response in these subjects was not restricted to
a specific VH gene family and are consistent with there being no
obvious strong regulatory mechanisms that would inherently limit
the generation of antibodies with ADCC activity, the preferential use
of the VH1 gene raises the possibility that the Env proteins used in
RV144 or Env gp120 proteins in general preferentially induce the use
of the VH1 gene family. Whether a vaccine regimen can be developed
that will harness the observed Ig VH1 gene-using B cells to also induce
CD4bs antibodies with a high degree of mutation is currently un-
known. It is interesting that we were able to recover ADCC antibodies
with a degree of CD4 blocking activity that had low levels of mutation,
suggesting that B cells expressing those antibodies can be harnessed to
produce the desired potent CD4-blocking antibody response under
the right conditions.
In summary, the ALVAC-HIV/AIDSVAX B/E vaccine induced
potent ADCC responses mediated by modestly mutated and pre-
dominantly A32-blockable MAbs that have overlapping but distinct
binding profiles. This response is qualitatively similar to anti-HIV-1
responses observed during chronic HIV-1 infections and may have
been partly responsible for the modest degree of protection observed.
ADCC-mediating MAbs predominantly utilized the VH1 Ig heavy-
chain family, which has been previously reported for CD4bs-directed
broadly neutralizing antibodies. This observation raises the possibil-
ity that continued boosting with this vaccine formulation leads to
further somatic mutations of VH1 gp120-specific antibodies and,
perhaps, to an enhanced ability to augment any protective effect they
might have had to limit HIV-1 acquisition.
We thank Robert J. Parks, Krissey E. Lloyd, Bradley Lockwood, Mark
Drinker, Lawrence Armand, Joshua Eudailey, Neil Meguid, Brandy Ward,
and Faraha Brewer for excellent technical assistance. We also thank the
following members of the Thai AIDS Vaccine Evaluation Group: (i) from
AFRIMS, N. Sirisopana, S. Sukwit, S. Tabprasit, A. Kleebmontha, V. Ka-
monsin, P. Panjapornsuk, S. Akapirat, W. Kaneechit, C. Chuenchitra, P.
Chanbancherd, W. Lokpicaht, R. Paris, B. Merrell, J.-L. Excler, S. Wong-
kamhaeng, A. Triampon, P. Buapunth, S. Chinaworapong, R. Tricha-
varoj, S. Chantakulkij, N. Khaochalod, S. Mason, P. Srisaengchai, S.
Chanthong, Y. Poangngern, and A. Brown; (ii) from the Vaccine Trials
Centre, Mahidol University, W. Supamaranond, S. Naksrisuk, W. Pe-
onim, N. Thantamnu, and R. Muanoum; (iii) from Siriraj Hospital, Pra-
sert Thongcharoen; and (iv) from the Research Institute of Health Sci-
ences, Chiang Mai University, Vinai Suriyanon.
The views expressed in this article are those of the authors and do not
reflect the official policy of the Department of the Army, Department of
Defense, or the U.S. Government.
This work was supported by the National Institutes of Health (NIH),
National Institutes of Allergies and Infectious Diseases (NIAID), the Di-
vision of AIDS with the Center for HIV/AIDS Vaccine Immunology
(CHAVI) (grant U19 AI067854); by a Collaboration for AIDS Vaccine
Discovery (CAVD) grant to B.F.H. from the Bill and Melinda Gates Foun-
dation; in part by Interagency Agreement Y1-AI-2642-12 between U.S.
Army Medical Research and Materiel Command (USAMRMC) and the
NIAID. In addition, this work was supported by a cooperative agreement
FIG 8 Characteristics of antibodies that used VH1 gene segments. (A) Amino acid sequences of ADCC-mediating antibodies that used VH1 gene segments were
aligned to the heavy- and light-chain consensus HAAD motifs previously identified for CD4bs bNAbs, which were described to preferentially use the VH1 gene,
in particular the VH 1-2*02 and 1-46 segments (41). The consensus HAAD motifs of the heavy and light chains are 68 and 53 amino acids long, respectively. Data
are plotted as the number of identical amino acids for heavy chain (x axis) and light chain (y axis). Black X, CD4bs bNAbs (41); red circles, VH1 ADCC mediating
antibodies (range, 46- to 57/68-aa identity for heavy chain, 68 to 84%, and 37- to 46/53-aa identity for light chain, 70 to 87%); blue diamonds, influenza virus
broadly neutralizing antibodies (49) (52- to 55/68-aa identity for heavy chain, 76 to 81%, and 31- to 32/53-aa identity for light chain, 58 to 60%). (B) Maximal
percent GzB activity is correlated with HC mutation frequency (Spearman correlation, ␳⫽0.56; P 0.02). Antibodies that blocked sCD4 binding to gp120 are
shown as red diamonds and were found throughout the range of mutation frequencies; those without blocking activity are shown as black circles.
Bonsignori et al.
11530 Journal of Virology
(W81XWH-07-2-0067) between the Henry M. Jackson Foundation for
the Advancement of Military Medicine, Inc., and the U.S. Department of
Defense. Additional support was provided by the CAVD from the Bill and
Melinda Gates Foundation (grants 38619 to G.F. and D.C.M. and 38650
to G.F.). J.P. was supported by the NIH, NIAID grant AI07392.
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Bonsignori et al.
11532 Journal of Virology
    • "Analysis of the correlates of protection in the RV144 vaccine trial suggested that decreased HIV-1 acquisition was linked to increased ADCC activity in protected vaccinees (Haynes et al., 2012). Accordingly, potent ADCC-mediating antibodies (Abs) targeting anti-cluster A epitopes were isolated from some RV144 vaccinees (Bonsignori et al., 2012) and were shown to preferentially recognize the HIV-1 envelope glycoproteins (Env) in their CD4-bound conformation (Veillette et al., 2014b ). The CD4- bound Env conformation is also recognized by non-neutralizing CD4-in- duced (CD4i) ADCC-mediating antibodies present in sera ( Richard et al., 2015; Richard et al., 2016; Williams et al., 2015), breast milk (Richard et al., 2015) and cervicovaginal lavages (Batraville et al., 2014; Richard et al., 2015 ) of HIV-1-infected individuals . "
    [Show abstract] [Hide abstract] ABSTRACT: HIV-1 developed sophisticated strategies to conceal vulnerable epitopes of its envelope glycoproteins (Env) recognized by antibody-dependent cellular cytotoxicity (ADCC)-mediating antibodies. CD4-mimetics (CD4mc) were shown to sensitize HIV-1-infected cells to ADCC induced by HIV + sera. Here we show that this response requires a sequential opening of Env at the surface of HIV-1-infected cells. Co-receptor binding site antibodies, also present in HIV + sera, are required to expose ADCC-mediating epitopes recognized by anti-cluster A antibodies upon CD4mc addition. The understanding of the conformational changes required to expose anti-cluster A epitopes might be important in the design of new strategies aimed at fighting HIV-1.
    Full-text · Article · Sep 2016
    • "Subsequent analyses showed that non-neutralizing antibodies to variable loops 1 and 2 (V1V2) regions of HIV-1 Env were associated with a reduced risk of HIV-1 acquisition [12 ,15] , and a genetic sieve analysis demonstrated that the ALVAC-HIV/AIDSVAX B/E regimen had improved efficacy against viruses that matched the immunogen sequence in the V2 location [15]. Moreover, high levels of antibody-dependent cellular cytotoxicity (ADCC) were also shown to correlate with a reduced risk of HIV-1 acquisition in RV144 [36] and V2-specific monoclonal antibodies isolated from RV144 vaccines were shown to mediate ADCC against HIV-1-infected CD4+ T cells [37]. HIV-1 specific IgG3 responses were also noted to correlate with protection in RV144 and distinguish this trial from the VAX003 trial [13]. "
    [Show abstract] [Hide abstract] ABSTRACT: With 2 million people newly infected with HIV-1 in 2014, an effective HIV-1 vaccine remains a major public health priority. HIV-1 vaccine efficacy trials in humans, complemented by active and passive immunization studies in non-human primates, have identified several key vaccine-induced immunological responses that may correlate with protection against HIV-1 infection. Potential correlates of protection in these studies include V2-specific, polyfunctional, and broadly neutralizing antibody responses, as well as effector memory T cell responses. Here we review how these correlates of protection are guiding current approaches to HIV-1 vaccine development. These approaches include improvements on the ALVAC-HIV/AIDSVAX B/E vaccine regimen used in the RV144 clinical trial in Thailand, adenovirus serotype 26 vectors with gp140 boosting, intravenous infusions of bNAbs, and replicating viral vectors.
    Full-text · Article · Aug 2016
    • "The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. in alum [7]. The modest reduction of acquisition of HIV infection is thought to correlate with non-neutralizing antibodies, most likely associated with antibody-dependent cellular cytotoxicity (ADCC) [8][9][10]. The HIVIS/TaMoVac consortium has optimized the delivery of multisubtype HIV-DNA and HIV-MVA vaccine candidates in a series of trials, and have shown them to be safe and potent T-cell and B-cell immunogens [11][12][13][14][15]. "
    [Show abstract] [Hide abstract] ABSTRACT: A vaccine against HIV is widely considered the most effective and sustainable way of reducing new infections. We evaluated the safety and impact of boosting with subtype C CN54rgp140 envelope protein adjuvanted in glucopyranosyl lipid adjuvant (GLA-AF) in Tanzanian volunteers previously given three immunizations with HIV-DNA followed by two immunizations with recombinant modified vaccinia virus Ankara (HIV-MVA).
    Full-text · Article · May 2016
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