2. antibody response (35). Recent studies have further indicated that
BCNAb development in vivo is associated with antibody affinity
maturation and coevolution of virus, resulting in a considerable
degree of somatic hypermutations (19, 20, 23, 26, 35, 42–50). Such
information is crucial for the design and development of suitable
Env-based immunogens capable of eliciting broad and potent
cross-neutralizing antibodies through vaccination.
While a number of studies on the molecular specificities of
broadly neutralizing antibodies obtained from African clade C-in-
fected individuals have been reported (9, 37, 51–62), knowledge
on immune evasion in Indian clade C-infected elite neutralizers is
very limited (63).
In the present study, we examined plasma samples obtained
from two hundred asymptomatic and antiretroviral therapy
(ART) naive Indian HIV-infected donors and identified plasma
with cross-neutralizing antibodies. The molecular specificities of
plasma antibodies obtained from an HIV-1 clade C-infected elite
neutralizer was characterized in detail that displayed exceptional
neutralization breadth across clades of different geographical or-
igins. Interestingly, we found that neutralization breadth was as-
sociated with the presence of unique epitopes on the trimeric
gp120.
MATERIALS AND METHODS
Ethics statement. The blood samples were collected under the IAVI Pro-
tocol G study from slow-progressing ART naive HIV-1-positive donors
from Nellore District of the state of Andhra Pradesh, southern India, by
trained clinicians at the YRG Care Hospital following approval and clear-
ance from the Institutional Review Board (IRB) and the Ethics Commit-
tee. The serum and plasma samples collected were shipped to the HIV
Vaccine Translational Research Laboratory, Translational Health Science
and Technology Institute, for further assessment and research on the neu-
tralizing antibody response.
Plasmids, viruses, antibodies, proteins, and cells. Plasmids encoding
HIV-1 envelopes representing distinct clades are shown in Table 1. Mono-
clonal antibodies used in the study and TZM-bl cells were procured from
the NIH AIDS Research and Reagents Reference program and from the
IAVI Neutralizing Antibody Consortium (NAC). 293T cells were pur-
chased from the American Type Culture Collection (ATCC). Plasmid
DNA encoding BG505-SOSIP.664-D7324, its purified cleaved trimeric
protein (64), and pcDNA5-FRT BG505 furin A (65) were kindly provided
by John Moore, Weill Cornell Medical College, New York. Purified gp120
TripleMut core protein (66) was obtained from Richard Wyatt, The
Scripps Research Institute, through the NAC. HIV-2 7312A and its chi-
meric constructs were provided by Lynn Morris, NICD, Johannesburg,
South Africa.
Purification of monomeric and trimeric Env proteins. Codon-opti-
mized gp120 plasmid encoding clade C 4-2.J41 (67, 68) gp120 was cloned
in pcDNA 3.1/V5-His-TOPO vector and transfected into 293T cells using
polyethyleneimine (PEI). Supernatants containing soluble gp120 were fil-
tered through 0.45-m-pore-size filters and subsequently purified using
nickel-nitrilotriacetic acid (Ni-NTA) agarose matrix (Qiagen Inc.) by elution
with phosphate-buffered saline (PBS) containing 300 mM imidazole (pH
8.0). The purified monomeric gp120 protein was extensively dialyzed with
PBS (pH 7.4), concentrated using Amicon ultracentrifugal filters (Millipore
Inc.) with a 30-kDa cutoff, and stored at Ϫ80°C until further use.
The trimeric BG505-SOSIP.664 protein was purified using 293F
cells essentially as described by Sanders et al. (69). Briefly, the 293F
cells were transfected with plasmid DNA encoding both BG505-SO-
SIP.664 gp140 envelope and furin (65). Supernatant containing solu-
ble BG505-SOSIP.664 gp140 was harvested 72 to 96 h posttransfection,
filtered, and passed through a lectin agarose column obtained from
Galanthus nivalis (Sigma Inc.). The nonspecifically bound proteins then
were washed in PBS (pH 7.4) supplemented with 0.5 M NaCl. The bound
proteins then were eluted using 0.5 M methyl ␣-D-mannopyranoside,
extensively dialyzed with 1ϫ PBS, and concentrated. BG505-SOSIP.664
was further purified by Sephadex G-200 size exclusion chromatography
(AKTA; GE). Trimeric protein fractions were collected and pooled, their
quality was assessed by running in blue native polyacrylamide gel electro-
TABLE 1 Neutralization breadth of Protocol G G37080 plasma samples
collected at two different time points tested against panel of 57 Env-
pseudotyped viruses
HIV-1 Clade C Plasma Confers Cross-Clade Neutralization
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3. phoresis (BN-PAGE), and they were favorably assessed for their ability to
bind to only neutralizing and not to nonneutralizing and MPER-directed
monoclonal antibodies as described elsewhere (64) by enzyme-linked im-
munosorbent assay (ELISA).
Depletion of plasma antibodies by monomeric gp120 and trimeric
gp140 Env proteins. Purified soluble monomeric 4-2.J41 gp120 and tri-
meric BG505 SOSIP.664 proteins, in addition to the MPER peptide (C1C;
encoding clade C sequence) (70), were used for the depletion of plasma
antibodies where purified proteins were covalently coupled to tosylacti-
vated MyOne Dynabeads (Life Technologies Inc.) according to the man-
ufacturer’s protocol. Briefly, 30 mg of beads was coupled with 1 mg of
both monomeric and trimeric Env proteins in coupling buffer [0.1 M
NaBO4, 1 M (NH4)2SO4; pH 9.4] overnight at 37°C for 16 to 24 h. Proteins
bound to magnetic beads were separated from unbound proteins using a
DynaMag 15 magnet (Life Technologies, Inc.). Beads bound to Env pro-
teins next were incubated with blocking buffer (PBS [pH 7.4], 0.1% bo-
vine serum albumin [BSA; Sigma], and 0.05% Tween 20) at 37°C to block
the unbound sites. The antigenic integrity of both 4-2.J41 monomeric
gp120 and BG505-SOSIP.664 bound to the beads was assessed for their
ability to bind VRC01 and 4E10 MAbs (for monomeric gp120) and
PGT121, F105, and 4E10 MAbs (for BG505-SOSIP.664) by flow cytom-
etry (FACSCanto; Becton and Dickinson, Inc.).
For depletion studies, G37080 plasma was diluted to 1:50 in Dulbec-
co’s modified Eagle’s medium (DMEM) containing 10% fetal bovine se-
rum (FBS), and 500 l of diluted plasma was incubated with 20 l of beads
at room temperature for 45 min. Unbound plasma antibodies were sepa-
rated from those that were bound to protein-coated beads using a
DynaMag 15 magnet as described above. This step was repeated 4 to 5
times for the depletion of plasma antibodies by monomeric gp120 and 10
to 12 times in the case of BG505-SOSIP.664-coated beads. As a negative
control, G37080 plasma antibodies were depleted with uncoated beads in
parallel. In addition to ELISA, the percent depletion of G37080 plasma
antibodies was assessed by examining the sequential decrease in binding
of protein-coated beads with depleted plasma antibodies by fluorescence-
activated cell sorting (FACS). PGT121 MAb was taken as a positive con-
trol for checking depletion by BG505-SOSIP.664 trimeric Env.
gp120 and gp140 ELISA. For gp120 ELISA, a high-binding polysty-
rene microtiter plate (Nunc, Inc.) was coated with 100 l of monomeric
4-2.J41 gp120 (1 g/ml) in binding buffer containing 0.1 M NaHCO3 (pH
8.6) and incubated overnight at 4°C. The gp120-plate was washed once
with 1ϫ PBS (pH 7.4) and blocked with 5% nonfat milk for 90 min at
37°C. The plate then was washed three times with 1ϫ PBS followed by the
addition of 100 l of MAbs, as well as the depleted and undepleted plasma
antibodies at different dilutions, and incubated for 1 h at room tempera-
ture. The wells of the ELISA plate were washed four times with PBS con-
taining 0.1% Tween 20 (PBST), followed by the addition of 100 l of
1:3,000-diluted horseradish peroxidase (HRP)-conjugated anti-human
IgG (Jackson ImmunoResearch, Inc.), and further incubated for 45 min at
room temperature. Unbound conjugates were removed by washing with
PBST, and color was developed by the addition of 100 l of 3,3=,5,5=-
tetramethylbenzidine (TMB) (Life Technologies, Inc.) substrate. Absor-
bance was measured at 450 nm in a spectrophotometer.
Binding of antibodies to BG505-SOSIP.664-D7324 trimeric protein
was assessed essentially as described by Sanders et al. (69) in a sandwich
ELISA. Briefly, a high-binding microtiter plate (Nunc, Inc.) first was
coated with D7324 antibody at 10 g/ml (Aalto Bio Reagents, Dublin,
Ireland) followed by blocking extra unbound sites with 5% nonfat milk
for 90 min at 37°C. One hundred microliters of BG505.664-D7324 trim-
eric protein (300 ng/ml) then was added and incubated for 45 min at room
temperature. The extent of binding of G37080 plasma antibodies com-
pared to that of known neutralizing monoclonal antibodies was assessed
by the addition of primary and HRP-conjugated secondary anti-human
antibody as described above.
Neutralization assay. Neutralization assays were carried out using
TZM-bl cells as described before (68). Briefly, Env-pseudotyped viruses
were incubated with various dilutions of depleted plasma antibodies and
incubated for an hour at 37°C in a CO2 incubator under humidified con-
ditions, and subsequently 1 ϫ 104
TZM-bl cells were added to the mixture
in the presence of 25 g/ml DEAE-dextran (Sigma, Inc.). The plates were
further incubated for 48 h, and the degree of virus neutralization was
assessed by measuring relative luminescence units (RLU) in a luminom-
eter (Victor X2; PerkinElmer Inc.).
Amplification, cloning, and mutagenesis of autologous HIV-1 env
genes. Autologous complete env genes were obtained from G37080
plasma as described previously, with slight modifications (68). Briefly,
viral RNA was extracted using a high-pure viral RNA kit (Roche Inc.) by
following manufacturer’s protocol, and cDNA was prepared by reverse
transcription-PCR (RT-PCR) using a Superscript III first-strand synthesis
kit (Invitrogen Inc.). rev-gp160 env genes were amplified using a Phusion
high-fidelity DNA polymerase (New England BioLabs Inc.). The gp160
amplicons were purified and ligated into pcDNA 3.1/V5-His-TOPO (In-
vitrogen Inc.) vector. Chimeric Env proteins were prepared by overlap-
ping PCR, and point substitutions were made with a QuikChange II kit
(Agilent Technologies Inc.) by following the manufacturer’s protocol and
as described previously (71).
Preparation of envelope-pseudotyped viruses. Pseudotyped viruses
were prepared by the cotransfection of envelope-expressing plasmid with
an env-deleted HIV-1 backbone plasmid (pSG3⌬Env) into 293T cells in
6-well tissue culture plates using a FuGENE6 transfection kit (Promega
Inc.). Cell supernatants containing pseudotyped viruses were harvested 48
h posttransfection and then stored at Ϫ80°C until further use. The infec-
tivity assays were done in TZM-bl cells (1 ϫ 105
cells/ml) containing
DEAE-dextran (25 g/ml) in 96-well microtiter plates, and the infectivity
titers were determined by measuring the luciferase activity using Britelite
luciferase substrate (PerkinElmer Inc.) with a Victor X2 luminometer
(PerkinElmer Inc.).
RESULTS
Identification of an elite neutralizer with HIV-1 clade C infec-
tion whose plasma showed exceptional neutralization breadth.
The present study, under IAVI Protocol G, was designed (i) to
screen and identify plasma antibodies obtained from Indian do-
nors chronically infected with HIV-1 clade C, with substantial
breadthtowardneutralizingcross-cladeHIV-1primaryvariants,and
(ii) to elucidate the molecular specificities associated with neutraliza-
tionbreadth.Ourhypothesiswasthatgiventhegeneticdistinctnessof
clade C viruses of Indian and non-Indian origin, as well the likely
differences in host genetics between populations and their ancestral
origins associated with the modulation of humoral immune re-
sponses, the specificities of antibodies developed in vivo associated
with neutralization breadth and potency would be different.
Through screening of two hundred plasma samples obtained
from chronically infected ART naive Indian patients against a
panel of 57 pseudoviruses containing Envs of distinct clades and
geographical origins (Fig. 1A), we identified one donor (G37080)
whose plasma showed exceptional neutralization breadth. Donor
G37080 serum neutralized Ͼ90% of the 57 different pseudovi-
ruses tested, with a median 50% inhibitory dose (ID50) value of
533.03 (Table 1 and Fig. 1B).
Follow-up plasma sample from this donor (G37080) subse-
quently was obtained after 8 months to assess whether the neu-
tralization breadth and potencies, along with their molecular
specificities, were retained and/or improved, as we expected that
during the course of disease, the breadth and potency of neutral-
izing antibodies broadens through somatic hypermutations (72)
and/or clonal selection processes. As shown in Fig. 1B and Table 1,
follow-up plasma antibodies of donor G37080 (referred to as the
visit 2 samples) were found to exhibit neutralization breadth com-
Patil et al.
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4. parable to that of visit 1 plasma. Overall, G37080 BCN plasma was
found to potently neutralize pseudoviruses containing Indian
clade C Env with a neutralization score of 2.5 (13). Furthermore,
the neutralization sensitivity of Env-pseudotyped viruses was
found to be correlated with the serum IgG (data not shown), sug-
gesting that the broad neutralization was associated with IgG-
specific responses. Taken together, our data indicate that a strong
humoral immune response to HIV-1 was mounted in donor
G37080 and was maintained over time.
Evidence that G37080 BCN plasma antibodies do not target
epitopes in CD4bs, MPER, and known glycan and nonglycan
residues in variable domains of Env. We first examined whether
the G37080 BCN plasma contains antibodies directed to the
CD4bs on Env. Plasma samples obtained from both visits were
pretreated with 25 g/ml of TripleMut core protein (66), which
was a concentration that we found to inhibit the neutralization of
25711-2.4 pseudovirus by VRC01 MAb by Ͼ95%. Pretreated
plasma subsequently was used to neutralize pseudovirus 25711-
2.4 Env, and as shown in Fig. 2, no perturbation of G37080 neu-
tralizing activity was observed against pseudovirus 25711-2.4. A
similar observation was made when these plasma antibodies were
pretreated with RSC3 core protein (22). In addition, the G37080
BCN plasma antibodies were found to efficiently neutralize
IgG1b12- and VRC01-resistant viruses (data not shown). Our
data indicated that the G37080 BCN plasma antibodies do not
contain CD4bs-directed neutralizing antibodies.
To elucidate whether the BCN plasma antibodies are directed
to MPER in gp41, we used HIV-2/HIV-1 chimeric viruses (73)
that expressed minimal residues of HIV-1 MPER containing
epitopes required for MPER-directed MAbs, such as 2F5, 4E10,
Z13e, and 10E8. As shown in Table 2, the G37080 BCN plasma
from both visits was found to show modest neutralization of
HIV-2 expressing HIV-1 clade C MPER (7312-C1C), with ID50
values of 306.42 and 371.02, respectively. We also found that the
depletion of G37080 plasma with a clade C MPER peptide (C1C)
completely abolished the sensitivity of 7312A-C1C virus to
FIG1 (A) Genetic divergence of amino acid sequences of 57 HIV-1 Env (gp160) pseudoviruses used to assess neutralization breadth and potency of G37080 BCN
plasma. The maximum likelihood bootstrapped consensus phylogenetic tree was constructed using the Jones-Taylor-Thornton (JTT) substitution model with
50 bootstrapped replicates in Mega 5.2. Bootstrapped values are shown at the nodes of each branch. Hollow circles represent envelopes (16055-2.3 and 92TH021)
resistant to neutralization by G37080 BCN plasma. (B) Neutralization breadth of the G37080 BCN plasma obtained at visit 1 and visit 2 were assessed against
pseudotyped viruses expressing HIV-1 Env representing different clades and origins. Neutralization titers (median ID50 values) were obtained by titrating
Env-pseudotyped viruses against G37080 plasma samples. Values at the top of each bar graph indicate the number of viruses belonging to each clade/origin tested.
FIG 2 Assessing dependence of G37080 BCN antibodies to CD4 binding site (CD4bs) region of HIV-1 Env. G37080 BCN plasma samples and VRC01 MAb
(concentrations that neutralized 25711-2.4 by Ͼ80%) preincubated with different concentrations, as indicated, with TripleMut core (A) and RSC3 (B) proteins
were examined for their ability to neutralize 25711-2.4 Env pseudotyped virus in a TZM-bl cell neutralization assay. Note that while VRC01 preabsorbed with
both TripleMut and RSC3 proteins showed inhibited neutralization of 25711-2.4 in a dose-dependent manner, no such effect was observed with G37080 BCN
plasma, indicating the absence of CD4bs-directed neutralizing antibodies.
HIV-1 Clade C Plasma Confers Cross-Clade Neutralization
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5. G37080 plasma (Table 3). Our data suggest that although the
G37080 BCN plasma neutralized 7312-C1C, the presence of
MPER-directed antibodies was not associated with neutralization
breadth.
We next investigated whether the plasma antibodies of the do-
nor G37080 target residues in variable loops, particularly in V1V2
and V3 regions, which have been shown in several studies to be
epitopes targeted by BCN antibodies on HIV-1 Env. We first
tested the extent of neutralization by G37080 BCN plasma anti-
bodies of Env-pseudotyped viruses lacking glycans at positions
160 (N160) and 332 (N332) in the V2 region and V3 base, respec-
tively, and also R166 and K169 in the V2 region, which are major
targets of recently identified broad and potent neutralizing mono-
clonal antibodies. In order to test this, two clade C Envs (25711-
2.4 and CAP239.G3), containing N160A and N332A substitutions,
were tested (Table 2). Our data indicate that the pseudoviruses con-
taining Env expressing the N160 or N332 substitution have sensi-
tivities identical to those of G37080 plasma antibodies. Similar
observations were found with R166A and K169A in the 93IN905
Env backbone. Taken together, our observations indicate that
G37080 BCN plasma antibodies did not utilize these residues in
V2 and V3 regions for neutralization breadth; these have been
identified as important epitopes recognized by broadly neutraliz-
ing antibodies elicited in clade C infection as described before (35,
41, 56).
AssociationofneutralizationbreadthofG37080plasmawith
recognition of conformational epitopes on cleaved trimeric Env
but not with that in monomeric gp120 or MPER. In order to
examine whether broad neutralization conferred by the G37080
plasma antibodies was through the recognition of epitopes on
monomeric gp120 or cleaved near-native Env trimers, we tested
the binding of G37080 serum IgG to monomeric 4-2.J41 gp120
and soluble gp140 (BG505-SOSIP.664) by ELISA. We found that
in addition to the monomeric 4-2.J41 gp120 (Fig. 3A), G37080
serum polyclonal IgG was found to efficiently bind to the BG505
SOSIP.664-D7324 soluble trimeric Env (Fig. 3B), indicating that
the G37080 plasma primarily contains neutralizing antibodies
that target epitopes on cleaved Env trimers.
We next examined whether binding of the G37080 plasma an-
tibodies to epitopes on cleaved BG505-SOSIP.664 trimeric enve-
lope was associated with neutralization breadth. For this, we tested
the ability of G37080 plasma antibodies depleted of both mono-
meric and trimeric Envs, as well as of MPER peptides, to neutralize
a set of Env-pseudotyped viruses, which were found to be sensitive
to this particular plasma sample. Purified 4-2.J41 monomeric
gp120, BG505-SOSIP.664 trimeric gp140, and C1C MPER pep-
tide bound to the magnetic beads were used to deplete G37080
plasma antibodies as described in Materials and Methods. The
depleted BCN G37080 antibodies first were assessed for their
binding to 4-2.J41 gp120 monomers, BG505-SOSIP.664-D7324,
and C1C peptide and compared to undepleted plasma antibodies
by ELISA. As shown in Fig. 3C and D, G37080 plasma depleted
with monomeric gp120 and trimeric gp140, respectively, had sig-
nificantly reduced binding activity against the respective soluble
proteins. Similar observation was made with MPER peptide (data
not shown). The depleted plasma antibodies subsequently were
assessed for neutralization activity using a panel of 12 Env-pseu-
dotyped viruses that were susceptible to untreated G37080 plasma
antibodies as mentioned above. As shown in Table 3, depletion
with 4-2.J41gp120 monomer and C1C peptide did not show any
change in neutralization breadth of G37080 plasma antibodies,
while depletion with BG505-SOSIP.664 showed a significant re-
duction in virus neutralization. Similar observations were made
with the BG505-SOSIP.664-depleted PGT121 and C1C peptide-
depleted 4E10 MAbs, which lost the ability to efficiently neutralize
Env-pseudotyped viruses (16055 and ZM233.6) and HIV-2/
HIV-1 (7312A-C1C) chimeric virus compared to their undepleted
counterparts (data not shown), validating our data. Interestingly,
C1C peptide-depleted G37080 plasma failed to neutralize HIV-2/
TABLE 3 Degree of shift in sensitivity of Env-pseudotyped viruses to
G37080 BCN plasma depleted with monomeric and trimeric Envs and
C1C peptide
Env-pseudotyped
virus
Fold reduction in neutralization (ID50)a
gp140 trimer
(BG505-SOSIP.664)
gp120 monomer
(4-2.J41)
MPER (C1C
peptide)
25710-2.3 Ͼ10.30 1.3 0.83
25711-2.4 Ͼ8.52 1.4 1.44
3-5.J25 Ͼ7.85 0.9 0.84
4-2.J41 12.11 1.1 1.04
IAVI_C22 Ͼ15.92 1.2 1.18
92BR020 Ͼ35.08 1.1 1.34
93IN905 3.41 1.2 0.94
JRCSF Ͼ8.75 0.5 0.93
Q23.17 Ͼ23.28 1.0 0.98
Du156.12 Ͼ15.73 0.8 1.61
HVTR-PG80v1.eJ7 Ͼ10.03 0.9 1.12
HVTR-PG80v1.eJ19 Ͼ15.60 0.5 1.18
HIV-2 7312A-C1C Ͼ10
a
Fold reduction in virus neutralization was obtained by comparing the neutralization
titer (ID50 values) of panel viruses against undepleted and depleted G37080 plasma.
ID50 values are reciprocal dilutions at which the undepleted and depleted plasma
conferred 50% neutralization of the Env-pseudotyped viruses in TZM-bl cells.
TABLE 2 Examination of specificity of G37080 plasma antibodies
obtained at both visits to HIV Env
HIV type Region
Fold decrease in
ID50
a
for plasma
at visit:
1 2
HIV-1 Env mutants
HIV-1 25711-2.4 N160A V2 1.02 Ͻ1
HIV-1 25711-2.4 R166A Ͻ1 Ͻ1
HIV-1 25711-2.4 K169E Ͻ1 Ͻ1
HIV-1 93IN905 R166A Ͻ1 Ͻ1
HIV-1 93IN905 K169A Ͻ1 Ͻ1
HIV-1 25711-2.4 N332A V3 1.52 Ͻ1
HIV-1 CAP239.G3 N332A 1.35 1.32
HIV-2/HIV-1 chimera Region of HIV-1 ID50 ID50
HIV-2 7312A HIV-2 wild type Ͻ20 Ͻ20
HIV-2 7312A-C1C Clade C MPER 306.42 371.02
HIV-2 7312A-C3 2F5 epitope Ͻ20 Ͻ20
HIV-2 7312A-C4 4E10, Z13e1, and
10E8 epitopes
334.34 371.27
HIV-2 7312A-C6 4E10 minimal epitope Ͻ20 223.90
HIV-2 7312A-C7 2F5 minimal epitope Ͻ20 Ͻ20
a
ID50 values refer to the reciprocal dilution that conferred 50% neutralization of
viruses in a TZM-bl assay. Assays were done in duplicate and were repeated more
than three times.
Patil et al.
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6. HIV-1 (7312A-C1C) chimeric virus, indicating that the presence
of residual traces of MPER-directed antibodies (as shown in
Table 2) is not responsible for neutralization breadth. Further-
more, the examination of sensitive (25711-2.4) and resistant
(16055-2.3 and CAP45.G3) chimeric Envs indicated that the BCN
G37080 plasma antibodies predominantly target epitopes in the
V1V2 region (Table 4) in gp120. Our data clearly indicate a cor-
relation between neutralization breadth and binding of the
G37080 BCN plasma antibodies to the conformational epitopes
on cleaved trimeric gp120, likely in the V1V2 region; however, we
do not rule out the possibility that this BCN plasma targets other
discontinuous epitopes in gp120 but not in MPER.
Mutations in V1 region confer resistance to autologous vi-
ruses to the G37080 plasma antibodies. In order to decipher the
specificity of the G37080 plasma antibodies, we examined the de-
gree of susceptibility of pseudoviruses prepared using env genes
amplified from contemporaneous autologous G37080 plasma ob-
tained at the baseline and follow-up visits. As shown in Fig. 4A,
both of the Env proteins obtained from visit 2 plasma (HVTR-
PG80v2.eJ38 and HVTR-PG80v2.eJ41) were found to be resistant
to its contemporaneous plasma antibodies, while Env proteins
obtained from visit 1 plasma (HVTR-PG80v1.eJ7 and HVTR-
PG80v1.eJ19) were found to be modestly sensitive to visit 2 autol-
ogous G37080 plasma antibodies. To facilitate mapping G37080
BCN antibody specificity, we prepared chimeric Envs between
a sensitive (HVTR-PG80v1.eJ7 and HVTR-PG80v1.eJ19) and a
resistant (HVTR-PG80v2.eJ38) autologous Env by first swap-
ping the V1V2 regions, as their amino acid sequences differed
maximally in this region (Fig. 4B). As shown in Table 4, the
insertion of the V1V2 sequences of HVTR-PG80v1.eJ7 and
HVTR-PG80v1.eJ19 into HVTR-PG80v2.eJ38 conferred Env-
pseudotyped viruses expressing HVTR-PG80v2.eJ38 Env with
sensitivity to G37080 visit 2 plasma antibodies enhanced by
Ͼ25- and Ͼ12-fold, respectively. Conversely, the neutraliza-
tion susceptibilities of the Env-pseudotyped viruses expressing
HVTR-PG80v1.eJ7 and HVTR-PG80v1.eJ19, which contained
HVTR-PG80v2.eJ38 V1V2 sequence corresponding to visit 2
G37080 plasma, were found to be reduced by Ͼ45- and Ͼ23-
fold, respectively. We noted that alterations of regions other
than the V1V2 loop in the autologous Env did not confer any
change in neutralization sensitivity (Table 4). To further spec-
ify residues in the V1V2 loop associated with neutralization
sensitivity and resistance of autologous Envs, chimeric Envs
and point mutants were prepared and tested for their degree of
modulation in susceptibility to autologous G37080 plasma ob-
tained from the second visit. As shown in Table 4, we found that
the V1 sequence, but not the V2 sequence, of the sensitive Envs
(HVTR-PG80v1.eJ7 and HVTR-PG80v1.eJ19) increased sensitiv-
ity to G37080 BCN plasma antibodies by Ͼ50 and Ͼ37-fold, re-
spectively, when transferred to the resistant HVTR-PG80v2.eJ38
FIG 3 Binding of G37080 BCN plasma IgG to 4-2.J41 monomeric gp120 (A) and BG505-SOSIP.664-D7324 cleaved trimeric gp140 (B) soluble proteins was
assessed by ELISA. IgG purified from HIV-negative healthy donor and known MAbs were used as controls. The extent of binding of the depleted and undepleted
G37080 BCN plasma with magnetic beads coated with 4-2.J41 monomeric gp120 (C) and BG505-SOSIP.664 cleaved trimeric gp140 to their respective proteins
by ELISA. Note that binding to trimeric protein by ELISA was assessed by using BG505-SOSIP.664 tagged with the D7324 epitope to maintain the native
conformation of trimeric Env as described before (69).
HIV-1 Clade C Plasma Confers Cross-Clade Neutralization
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7. Env. In agreement with this result, V1 of HVTR-PG80v2.eJ38,
when transferred into the sensitive Envs described above, in-
creased neutralization resistance by Ͼ27- and Ͼ28-fold, respec-
tively, to the G37080 visit 2 BCN plasma antibodies. We observed
that the removal of a glycan at the 138 position in V1 (T140D) in
HVTR-PG80v1.eJ19 mediated the enhanced sensitivity of this Env
to G37080 plasma by 2.64-fold (Table 4). Concurrent with this
observation, we found that the alteration of the V1 region of
PG80v1.eJ19 with the T140D substitution in PG80v2.eJ38 Env
exhibited enhanced susceptibility compared to that of the
PG80v2.eJ38 Env chimera containing the PG80v1.eJ19 V1 loop, as
shown in Table 4. Our data indicate that N138 glycan masks the
PG80v1.eJ19 Env from being efficiently neutralized by the autol-
ogous plasma compared to that of its contemporaneous counter-
part, PG80v1.eJ7 Env. Fine scanning of V1 regions of the autolo-
gous Envs further revealed that the N133 glycan motif and P147
residues in the PG80v2.eJ38 Env played a significant role in neu-
tralization resistance to G37080 BCN autologous plasma antibod-
ies (Fig. 4B). Interestingly, all of the V1 chimeras as well as the
point mutants showed sensitivities to PG9 MAb comparable to
those of their wild types (Table 5), indicating that the shifts in
neutralization susceptibilities were not due to changes in Env con-
formation. Moreover, we noted that both the sensitive and the
resistant autologous Envs contain T332 in the V3 base, clearly
indicating that the absence of N332 was not associated with resis-
tance to autologous neutralization. Similar observations were
made with respect to N160, R166, and K169 amino acid residues,
further consolidating that the neutralization conferred by G37080
BCN plasma antibodies was not associated with antibody target-
ing these epitopes in autologous Envs, and is likely the case for all
of the Envs tested against G37080 plasma antibodies.
DISCUSSION
The identification of the molecular specificities of antibodies elic-
ited in natural infection and that mediate neutralization breadth
and potency is key in the design and development of suitable Env-
based immunogens capable of eliciting similar antibody responses
upon vaccination. In the present study, we characterized the mo-
lecular specificity of plasma antibodies obtained from an Indian
elite neutralizer (G37080) infected with HIV-1 clade C that dis-
played exceptional cross-neutralization of different clades of distinct
geographical origins. The G37080 plasma was found to contain the
most broad and potent cross-neutralizing antibodies among the two
hundred plasma samples obtained from Indian patients chronically
infected with HIV-1. Plasma samples collected from the G37080 do-
nor at two time points at 8 months apart showed similar neutraliza-
tion breadth with modest increase in potency in the follow-up visit,
indicating an association with the sustained maturation of antibody-
producing B cells in this individual.
Since polyclonal plasma antibodies are not suitable for epitope
mapping, we examined the specificity of the G37080 BCN plasma
by making use of mutant viruses with specific point substitutions
of known neutralizing epitopes with nonspecific amino acids and
via depletion with monomeric and trimeric Envs in addition to
MPER peptide. The G37080 plasma antibodies did not show de-
pendence on the N160/K169 and N332 epitopes in the V2 apex
and V3 base, respectively. Our data also are consistent with the
target epitopes of the G37080 BCN antibodies being distinct from
those which are recognized by 2G12 (74), PGT121-128 (17), and
PGT130-131 and PGT135 (19) (e.g., residues at the following po-
TABLE 4 Dissection of specificity for autologous neutralization
resistance
Chimera and point mutant
Neutralization potency
(ID50)
Fold
changea
Effectb
PG80v1.eJ7 Env backbone
V1V2 loop
PG80v2.eJ38 (V1V2) in v1.eJ7 45.75 Decrease
PG80v2.eJ38 (V1) in v1.eJ7 27.97 Decrease
PG80v2.eJ38 (V2) in v1.eJ7 1.35 No effect
Point mutations
PG80v1.eJ7 (D133N) 3.25 Decrease
PG80v1.eJ7 (S143G) 0.87 No effect
PG80v1.eJ7 (D133N ϩ S143G) 2.88 Decrease
PG80v1.eJ7 (T147P) 8.21 Decrease
PG80v1.eJ19 Env backbone
V1V2 loop
PG80v2.eJ38(V1V2) in v1.eJ19 23.05 Decrease
PG80v2.eJ38 (V1) in v1.eJ19 28.61 Decrease
PG80v2.eJ38 (V2) in v1.eJ19 1.87 Increase
Point mutations
PG80v1.eJ19 (D133N) 2.51 Decrease
PG80v1.eJ19 (T139A) 0.99 No effect
PG80v1.eJ19 (T139A ϩ T140D) 2.64 Increase
PG80v1.eJ19 (N143G) 1.38 No effect
PG80v1.eJ19 (T139A ϩ T140D ϩ N143G) 2.16 Increase
PG80v1.eJ19 (T145P) 3.24 Decrease
PG80v2.eJ38 Env backbone
V1V2 loop
PG80v1.eJ7 (V1V2) in v2.eJ38 26.56 Increase
PG80v1.eJ7 (V1) in v2.eJ38 49.62 Increase
PG80v1.eJ7 (V2) in v2.eJ38 1.07 No effect
PG80v1.eJ19(V1V2) in v2.eJ38 12.81 Increase
PG80v1.eJ19 (V1) in v2.eJ38 37.60 Increase
PG80v1.eJ19 (V2) in v2.eJ38 0.94 No effect
Other regions in gp120
PG80v1.eJ7 (V3C3) in v2.eJ38 0.84 No effect
PG80v1.eJ7 (V3C3V4C4) in v2.eJ38 0.90 No effect
PG80v1.eJ7 (C4V5C5) in v2.eJ38 1.08 No effect
PG80v1.eJ19 (V3C3) in PG80v2.eJ38 1.09 No effect
PG80v1.eJ19 (V3C3V4C4) in PG80v2.eJ38 1.15 No effect
PG80v1.eJ19 (C4V5C5) in PG80v2.eJ38 0.96 No effect
Point mutations
PG80v1.eJ19 V1 (T139AϩT140D) in
v2.eJ38
53.91 Increase
PG80v2.eJ38 (N133D) 4.72 Increase
PG80v2.eJ38 (G143S) 0.94 No effect
PG80v2.eJ38 (N133DϩG143S) 3.63 Increase
PG80v2.eJ38 (P147T) 4.11 Increase
Heterologous Env chimera
V1V2 loop
16055-2.3 (25711-2.4 V1V2) 18.38 Increase
25711-2.4 (16055-2.3 V1V2) 2.03 Decrease
CAP45 (25711-2.4 V1V2) 16.84 Increase
25711-2.4 (CAP45-V1V2) 10.54 Decrease
a
Fold changes in reciprocal dilution of plasma mediating 50% virus neutralization
(ID50).
b
Fold increase or decrease in neutralization titer (ID50 values).
Patil et al.
3452 jvi.asm.org April 2016 Volume 90 Number 7Journal of Virology
onMarch11,2016byguesthttp://jvi.asm.org/Downloadedfrom
8. sitions: 295, 297, 301, 332, 334, 386, 388, 392, 394, 448, and 450);
thus, BCN G37080 antibodies appear to target a new epitope. Our
data highlighting the N332-independent development of neutral-
izing antibodies in clade C-infected donor G37080 also differ from
recent findings (57, 75–77) associating N332 with the develop-
ment of broad and potent neutralizing antibody, especially in
clade C infection in African donors. Moreover, recent studies in-
dicating the role of K169 as a target of BCN antibodies obtained
from a clade C-infected South African donor (41, 56) and the
observation that vaccine-induced protection in the RV144 vaccine
trial was associated with antibodies targeting epitopes, including
K169 in the V2 apex (27, 78), prompted us to examine whether
broad neutralization of the G37080 plasma antibodies also was
dependent on the K169 epitope. In the present study, the neutral-
ization potency of G37080 was unaffected by N160A/K169A
knockout mutations, and we also observed that both sensitive and
resistant autologous Envs obtained from both visits contain N160
and K169 in the V2 region. Hence, owing to the lack of association
of neutralization breadth of the G37080 BCN antibodies with
N160, K169, and N332 dependencies, our study further high-
lighted that there is a likelihood of differences in the development
pathway of elicitation of broadly neutralizing antibodies in indi-
viduals infected with HIV-1 clade C, particularly those with eth-
nically distinct variants.
Wibmer et al. (41) recently demonstrated an association be-
tween the evolution of a broadly neutralizing antibody response in
a clade C-infected donor with shifts in antibody specificities from
the recognition of epitopes in V2 to the CD4bs. In the present
study, the G37080 neutralizing plasma antibodies obtained from
both visits were found not to be absorbed by the TripleMut (66,
79) and RSC3 (22) core proteins, which effectively absorb anti-
bodies directed to the CD4bs. This result indicates a lack of devel-
opment of CD4bs-directed neutralizing antibodies during the dis-
ease course in the G37080 donor. Additionally, the absence of
MPER-directed antibodies from G37080 plasma was found, al-
though a negligible antibody titer (1:300 reciprocal dilutions) to
the HIV2/HIV1 (C1C) chimera was observed with plasma sam-
FIG 4 (A) Neutralization susceptibility of autologous Envs to contemporaneous G37080 BCN plasma and its follow-up sample from the same donor.
Neutralization titers (median ID50) were obtained by titrating pseudotyped viruses expressing autologous Envs obtained from visit 1 and follow-up G37080
plasma to contemporaneous plasma antibodies. Note that both of the Envs obtained from follow-up G37080 plasma (visit 2) were found to be resistant to
contemporaneous autologous plasma, while Envs obtained from visit 1 G37080 plasma were found to be sensitive to follow-up plasma antibodies. (B) Alignment
of V1V2 amino acid sequences of sensitive and resistant autologous Envs obtained at both visits was done by using seqpublish, available at the HIV Los Alamos
database (www.hiv.lanl.gov). Key residues that mediate autologous neutralization resistance are highlighted.
TABLE 5 Sensitivity of wild type, chimera, and point mutants of
autologous Envs to PG9 MAb
Env chimera and mutant ID50
PG80v1.eJ7 (wild type) 0.12
PG80v1.eJ19 (wild type) 0.97
PG80v1.eJ19 (T139A ϩ T140D) 0.77
PG80v2.eJ38 (wild type) 0.02
PG80v1.eJ7 (V1) in v2.eJ38 0.04
PG80v1.eJ19 (V1) in v2.eJ38 0.05
PG80v1.eJ19 (V1) (T139A ϩ T140D) in v2.eJ38 0.06
PG80v2.eJ38 (N133D) 0.01
PG80v2.eJ38 (P147T) 0.04
HIV-1 Clade C Plasma Confers Cross-Clade Neutralization
April 2016 Volume 90 Number 7 jvi.asm.org 3453Journal of Virology
onMarch11,2016byguesthttp://jvi.asm.org/Downloadedfrom
9. ples from both visits. However, the neutralization breadth of the
G37080 plasma was not found to be associated with the presence
of MPER-directed antibody. Nonetheless, we do not rule out the
possibility that in the further course of infection, this donor may
be able to develop MPER-directed antibodies.
Recentstudieshaveshownthatneutralizingantibodiesthattarget
conformational epitopes bind exclusively to the cleaved near-native
trimeric Envs (15, 64, 80, 81). In the present study, we found that the
absorption of G37080 plasma antibodies to soluble trimeric BG505-
SOSIP.664 Env was associated with the depletion of neutralizing ac-
tivity in G37080 BCN plasma. However, we do not rule out the pos-
sibility of the presence of 39F-, 19b-, and 14e-like nonneutralizing
antibodies that were reported to bind to BG505-SOSIP.664 trimeric
Env (69). Our findings indicate that the G37080 BCN antibodies tar-
get conformational epitopes in gp120. Our observation also high-
lights that native-like trimeric Envs, such as BG505-SOSIP.664, can
be utilized in selecting antigen-specific memory B cells, as reported
earlier(82),fromdonorG37080towardisolationofMAbcorrelating
with broad neutralization displayed by the plasma antibodies.
We made use of env clones obtained from autologous G37080
plasma from both time points to refine the fine specificity of the
G37080 BCN plasma antibodies. By examining chimeric Envs and
mutant viruses, we identified key residues in the V1 loop associ-
ated with neutralization resistance. Interestingly, the Env chimera
and mutant viruses showed susceptibility to PG9 MAb compara-
ble to that of their respective wild-type Envs, indicating that they
did not alter Env conformation. We identified a glycan at the 133
position and a proline residue at the 147 position within the V1
loop of the resistant Env (PG80v2.eJ38) that were found to be
associated with neutralization escape, which indicated that these
are contact sites for the G37080 BCN plasma antibodies. Thus,
from our study we conclude that changes in V1 loop sequence are
associated with the escape of autologous viruses to the BCN
G37080 plasma. Additionally, an examination of the degree of
susceptibilities of pseudoviruses expressing chimeric heterolo-
gous Envs to the G37080 plasma revealed that the BCN plasma
antibodies predominantly target epitopes in the V1V2 region in
gp120. However, we do not rule out the possibility of the contri-
bution of other discontinuous epitopes in gp120 in mediating
neutralization breadth. The isolation and identification of mono-
clonal antibodies from this elite neutralizer donor (G37080) will
help precisely map specific epitopes associated with neutralization
breadth and potency.
In summary, we identified an HIV-1-infected elite neutralizer
whose plasma showed exceptional neutralization breadth, and we
provided evidence that it targets novel conformational epitopes
on trimeric Env, predominantly in the V1V2 region, not reported
previously. Moreover, the neutralization resistance of the autolo-
gous Envs to G37080 plasma is associated with substitutions of
novel residues within the V1 loop that form the key contact points
of the BCN plasma antibody. The identification of novel epitopes
associated with broad neutralization of HIV-1, in particular the
major circulating clade C strains, will significantly contribute to
efforts toward effective immunogen design.
ACKNOWLEDGMENTS
We thank all of the Protocol G study participants registered with YRG
Care, Chennai, all of the research staff members at the Protocol G clinical
center at YRG Care, Chennai, and all of the IAVI Protocol G team mem-
bers. We sincerely thank Christopher Parks, IAVI Design and Develop-
ment Laboratory, for providing valuable input in preparing the manu-
script, and we also thank G. Balakrish Nair and Sudhanshu Vrati (THSTI),
Shreyasi Chatterjee, and all of the HVTR laboratory members for support.
We thank Albert Cupo, John P. Moore, and the members the SOSIP
trimer HIVRAD team, Weill Cornell Medical College, New York, for pro-
viding us with BG505.SOSIP.664 plasmid DNA and purified protein. We
thank David Montefiori, Lynn Morris, Pascal Poignard, and Richard Wy-
att for making available many reagents used in our study. The following
reagent was obtained through the NIH AIDS Reagent Program, Division
of AIDS, NIAID, NIH, from John C. Kappes and Xiaoyun Wu: pSG3 env.
IAVI’s work was made possible by generous support from many do-
nors, including the Bill & Melinda Gates Foundation, the Ministry of
Foreign Affairs of Denmark, Irish Aid, the Ministry of Finance of Japan,
the Ministry of Foreign Affairs of the Netherlands, the Norwegian Agency
for Development Cooperation (NORAD), the United Kingdom Depart-
ment for International Development (DFID), and the United States
Agency for International Development (USAID). The full list of IAVI
donors is available at www.iavi.org. The contents are the responsibility of
the International AIDS Vaccine Initiative and do not necessarily reflect
the views of USAID or the United States Government.
The International AIDS Vaccine Initiative has filed a patent relating to
the autologous HIV-1 clade C envelope clones (J. Bhattacharya, S. Desh-
pande, S. Patil, R. Kumar, and B. K. Chakrabarti, U.S. patent application
62/254,971).
FUNDING INFORMATION
This study was made possible by the generous support of the American
people through the United States Agency for International Development
(USAID) through the IAVI, support from a THSTI-IAVI HIV Vaccine
Design Program grant through the Department of Biotechnology, Gov-
ernment of India, in part by a grant from the Department of Science and
Technology, Government of India (DST/INT/SAFR/Mega-P3/2011 to
J.B.), and in part by a DBT National Bioscience Research Award [BT/
HRD/NBA34/01/2012-13(iv) to J.B.]. The funders had no role in study
design, data collection and interpretation, or the decision to submit the
work for publication.
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