1. The study identified determinants in the V2C2 region of HIV-1 clade C envelopes that conferred altered neutralization susceptibilities to the IgG1b12 and PG9 monoclonal antibodies. 2. Examining chimeric envelopes between a b12-sensitive and b12-resistant envelope, the authors found that residues in the V2 and proximal C2 region modulated susceptibility to b12. 3. Substituting the V2C2 region from a b12-sensitive envelope into unrelated b12-resistant envelopes resulted in varied effects on b12 susceptibility, indicating the effect is context dependent.

1. Determinants in V2C2 region of HIV-1 clade C primary envelopes
conferred altered neutralization susceptibilities to IgG1b12 and
PG9 monoclonal antibodies in a context-dependent manner
Shilpa Patil a,1
, Ipsita Choudhary b,1
, Nakul K. Chaudhary a
, Rajesh Ringe b,2
, Manish Bansal a
,
Brihaspati Narayan Shukla a
, Saikat Boliar a
, Bimal K. Chakrabarti a
, Jayanta Bhattacharya a,n
a
HIV Vaccine Translational Research Laboratory, THSTI-IAVI HIV Vaccine Design Program, Translational Health Science and Technology Institute,
450, Udyog Vihar, Phase-III, Gurgaon 122016, Haryana, India
b
National AIDS Research Institute, Pune, Maharashtra, India
a r t i c l e i n f o
Article history:
Received 31 March 2014
Returned to author for revisions
14 April 2014
Accepted 16 June 2014
Keywords:
HIV-1
Clade C
Envelope
Neutralizing antibody
CD4bs
IgG1b12
VRC01
PG9
PG16
a b s t r a c t
In the present study by examining pseudoviruses expressing patient chimeric envelopes (Envs) made
between an IgG1b12 (b12)-sensitive (2-5.J3) and a b12-resistant (4.J22) HIV-1 clade C envelope, we
identified determinants in the V2C2 region that governed susceptibility to b12 monoclonal antibody, but
not to other CD4 binding site antibodies. Interestingly, when the V2C2 sequence of the 2-5.J3 Env was
transferred to other b12-resistant primary clade C Envs, their susceptibility to b12 varied, indicating that
this effect was context dependent. In addition, we identified determinants within the V2 region in the
b12-resistant envelope that significantly modulated the neutralization of Env-pseudotyped viruses
to PG9/PG16 MAbs. The enhanced neutralization susceptibilities of Envs to b12 and PG9 MAbs were
correlated with increased exposure of their corresponding epitopes highlighting vulnerabilities in the
V2C2 region that altered Env conformation necessary for the efficient accessibility of b12 and PG9
antibodies.
& 2014 Elsevier Inc. All rights reserved.
Introduction
The entry of the Human Immunodeficiency Virus Type 1 (HIV-1)
into the target cells depends primarily on the interaction of gp120 of
trimeric envelope (Env) glycoprotein (gp160) with CD4 and subse-
quently with a coreceptor, usually CCR5 or CXCR4, which facilitates
fusion of viral membrane to cell membrane. In recent years, a
number of broad and potent neutralizing human monoclonal
antibodies were isolated from slow progressing individuals
(Huang et al., 2012; Walker et al., 2011; Walker et al., 2009; Wu
et al., 2010) with unique specificities which has provided clues of
several vulnerabilities associated with immune evasion by HIV-1.
The CD4 binding sites (CD4bs) and quaternary epitopes (QNE) on
the HIV-1 Env trimers are important targets for vaccine develop-
ment. b12 is the first identified neutralizing monoclonal antibody
(MAb) (Burton et al., 1994) whose epitopes in HIV-1 Env overlap
the CD4 binding domain of gp120 (Burton et al., 1994; Mo et al.,
1997). The b12-mediated neutralization has been shown to be
influenced by the mutations in V2 and C3 of gp120 (Mo et al.,
1997). Additionally, altered conformation of Env due to subtle
mutation in variable loops changed the sensitivity of neutraliza-
tion of virus to b12 (Duenas-Decamp et al., 2008; Ly and
Stamatatos, 2000; Utachee et al., 2010). By alanine scanning
mutagenesis, Pantophlet et al. (2003) demonstrated several resi-
dues in gp120 that are important for gp120 recognition by
neutralizing and non-neutralizing MAbs to CD4bs. Other
structure-based studies also demonstrated distinct vulnerable
sites in gp120 conferring b12 susceptibility and/or resistance
(Chen et al., 2009; Liu et al., 2008; Wu et al., 2009; Zhou et al.,
2007). In addition, in monkey models, using SHIV-HXBc2 and
SHIV-89.6P viruses, specific amino acid changes in V2 region
(SHIV-89.6P) and V1, V2, V3, and gp41 regions (SHIV-HXBc2) that
were associated with b12 resistance were documented (Etemad-
Moghadam et al., 1999; Si et al., 2001). Recently, we have also
demonstrated a single amino acid substitution at the 681 position
in the gp41 membrane proximal external region (MPER) enhanced
binding and neutralization of HIV-1 clade A, B and CRF02-AG-235
envelopes to CD4-Ig and b12 MAb, indicating gp41 as referred
Contents lists available at ScienceDirect
journal homepage: www.elsevier.com/locate/yviro
Virology
http://dx.doi.org/10.1016/j.virol.2014.06.018
0042-6822/& 2014 Elsevier Inc. All rights reserved.
n
Corresponding author.
E-mail addresses: JBhattacharya@iavi.org,
JBhattacharya@thsti.res.in (J. Bhattacharya).
1
Equal contribution.
2
Current address: Weill Cornell Medical College, New York, USA.
Virology 462-463 (2014) 266–272

2. above can also modulate HIV-1 to b12 susceptibility (Ringe and
Bhattacharya, 2012). Very recently Utachee et al. (2014) demon-
strated that the presence of aspartic acid at the position 185 in V2
loop confers HIV-1A/E subtype envelopes with enhanced suscept-
ibility to b12 monoclonal antibodies and that this effect was found
to be not dependent on glycan residues present in 186 and 197
positions previously described to be regulating envelope suscept-
ibility to b12 (Utachee et al., 2010). On the other hand, the variable
loops particularly V1, V2 and V3 regions have also been shown to
be important for the recognition of recently discovered broad and
potent MAbs, such as PG9, PG16, PGT121 and PGT145 (Doores and
Burton, 2010; Moore et al., 2011; Ringe et al., 2012; Thenin et al.,
2012; Walker et al., 2011; Walker et al., 2009). Interestingly, HIV-1
clade C has been widely shown to be refractory to neutralization
by b12 MAb in different geographic settings (Gray et al., 2006;
Kulkarni et al., 2009; Ringe et al., 2010; Zhang et al., 2010).
In natural infection, the antibodies usually develop within the first
few weeks of infection (Moore et al., 1994; Tomaras et al., 2008).
However, the antibodies which neutralize autologous viruses were
found to be elicited after several weeks of initial infection (Montefiori
et al., 2007) and have been demonstrated to exert immune selection
pressure on autologous viral variants (Bunnik et al., 2008; Frost et al.,
2005; Gray et al., 2007; Li et al., 2006; Richman et al., 2003; Wei et al.,
2003). It has been demonstrated earlier that HIV-1 variants that
become resistant to autologous neutralizing antibodies in the course
of infection by virtue of their escape strategies developed different
susceptibilities to monoclonal antibodies and/or heterologous serum
or plasma antibodies (Mascola, 2009; Mascola and Montefiori, 2010).
We previously reported that env clones obtained at different time
points from an Indian patient (NARI-IVC2) with early infection was
unusually sensitive to b12, an MAb that targets CD4 binding site
(CD4bs) on HIV-1 Env (Ringe et al., 2010). This observation prompted
us to investigate the determinants in Env responsible for enhanced
sensitivity of these Envs to b12. Since, pseudoviruses expressing all the
envs obtained from the NARI-IVC2 patient at different time points
showed comparable sensitivity to b12, we examined the b12-sensitive
Env, 2-5.J3 with that of 4.J22, also a clade C but b12-resistant Env
obtained from a different patient (NARI-IVC4) towards mapping
determinants responsible for altered sensitivity to b12. Constructing
the chimeric Envs taking both sensitive (2-5.J3) and resistant (4.J22)
isolates and subsequently by fine mapping, we identified amino acid
residues in V2C2 regions that conferred reciprocal susceptibilities to
b12 and PG9/16 MAbs.
Results and discussion
V2C2 region modulated susceptibility of primary patient-derived Envs
to b12 MAb
We previously showed that HIV-1 clade C env clones obtained
at different time points from an Indian patient (IVC-2) with recent
infection showed enhanced susceptibility to b12 MAb (Ringe et al.,
2010). In order to decipher the enhanced sensitivity of IVC-2 Envs
to b12, we prepared chimeric Env constructs by exchanging
different Env fragments between a b12-sensitive (2-5.J3) Env and
a b12-resistant (4.J22) Env (Ringe et al., 2010) which was obtained
from an unrelated patient (Fig. 1). Since all the env clones that we
obtained from this particular patient showed enhanced b12
sensitivity, we used the most sensitive env (2-5.J3 with IC50 of
0.29 mg/ml) along with an env (4.J22 with IC50 of 450 mg/ml)
obtained from an unrelated patient (NARI-IVC4) to prepare chi-
meric Env constructs. Pseudotyped viruses expressing chimeric
Envs were tested for their sensitivities to b12 MAb, which revealed
that residues within V2 and proximal half of the C2 regions of the
2-5.J3 Env conferred enhanced susceptibility of 2-5.J3 Env to b12
MAb (Fig. 2, top figure). Thus, when the V2C2 region of the 4.J22
Env was substituted with that of 2–5.J3 Env [V1V2C2 (2–5.J3)], the
chimeric virus was shown to become sensitive. In the process of
fine mapping, we ruled out that the V1 and other regions besides
Fig. 1. Construction of chimeric HIV-1 clade C Envs. Env chimeras were prepared by swapping different fragments of the b12-sensitive (2-5.J3) and -resistant (4.J22) Envs. In
addition, specific point substitution of amino acid residues in both the b12-sensitive and -resistant Envs were made. The V2C2 sequence (including the six amino acid
residues modulating PG9/16 sensitivity) that modulated the susceptibility of Env-pseudotyped viruses to b12 MAb is highlighted. The dashes indicate identical amino acid
sequences while dots represent missing sequences.
S. Patil et al. / Virology 462-463 (2014) 266–272 267

3. V2C2 region (Fig. 1) played any role in altering b12 sensitivity.
Interestingly, apart from having difference in V2 loop lengths,
between 2-5.J3 and 4.J22 Envs, only six amino acid differences
were observed in the V2C2 region. To understand whether these
six residues (P183/D185/N186/N192 in V2 and V207/N215 in C2
regions) were responsible for enhanced susceptibility of 2-5.J3 Env
to b12 MAb, we substituted these residues in 2-5.J3 Env with their
corresponding ones from b12-resistant 4.J22 Env to give rise to
P183S/D185E/N186K/N192K/V207I/N215T [referred to as 2–5.J3 (4.
J22-V2C2/SEKKIT)] and examined their sensitivity to b12 MAb. As
shown in Fig. 2 (middle figure) 2-5.J3 (4.J22-V2C2/SEKKIT) dis-
played comparable b12 sensitivity as 2-5.J3 wild type Env, sug-
gesting that these residues did not play any role in b12
susceptibility and indicated that enhanced susceptibility of 2-5.J3
Env was due to the coordinated effects of residues in the V2
region.
We next examined whether increased sensitivity of 2-5.J3 Env
to b12 was due to increased shedding of gp120. Thus, pseudotyped
viruses expressing 2-5.J3 and V1V2C2 (2-5.J3) Envs were incu-
bated with b12 at a concentration that provided 50% neutraliza-
tion (0.3 mg/ml) and those expressing 4.J22 Env with 10 mg/ml at
37 1C in a CO2 incubator for different times (Fig. S1). The relative
gp120 shedding was assessed by measuring the infectivity of
pseudotyped virus-b12 complex at indicated times in TZM-bl cells.
We found that the reduction in infectivity was comparable
between the b12-sensitive and -resistant variants. Our data
indicated that enhanced sensitivity of 2-5.J3 Env was not due to
b12-induced gp120 shedding.
Substitution of 2-5.J3 V2C2 sequence in unrelated b12-resistant
primary clade C Envs displayed varied b12 and VRC01 susceptibilities
We next examined the ability of the 2-5.J3 V2C2 sequence to
alter b12 susceptibility of few unrelated primary clade C Envs. For
this, we selected four b12-resistant clade C Envs: 3-5.J25, 5.J41, 7.
J20 and 16055-2.3, and substituted their V2C2 with that of 2-5.J3
Env. As shown in Fig. 2 (bottom figure), all the four Envs contain-
ing 2–5.J3 V2C2 sequence differed in their degree of susceptibility
to b12 MAb. While 3-5.J25 and 16055-2.3 Envs containing 2-5.J3
V2C2 sequence displayed substantial and modest increase in b12
susceptibilities respectively compared to their corresponding wild
types, 7.J20 (2-5.J3 V2C2) showed evidence of marginal increase in
b12 susceptibility. Conversely, 2-5.J3 V2C2 sequence was not found
to influence 5.J41 Env in modulating its susceptibility to b12 MAb.
Interestingly, presence of aspartic acid at the position of 185
(D185) (Fig. 1) was not found to affect the neutralization of these
Fig. 2. Neutralization susceptibilities of wild type, chimeric and mutant pseudotyped viruses expressing 2-5.J3 and 4.J22 Envs (top and middle panel) and other unrelated
primary Envs expressing 2-5.J3 V2C2 sequence (bottom panel) to b12 MAb. The neutralization assays were carried out in duplicates in at least three independent time points.
S. Patil et al. / Virology 462-463 (2014) 266–272268

4. four Envs by b12 MAb, which is in contrast to the observation
reported very recently by Utachee et al. (2014).
Since b12 MAb recognizes residues in gp120 that overlaps
CD4bs, we next examined whether 2-5.J3 V2C2 sequence altered
Env sensitivities to VRC01 (which is another CD4bs recognizing
MAb), QS1420 (anti-CD4 MAb) and sCD4. As shown in Fig. 3, while
all the wild-type Envs and their chimeras (containing 2-5.J3 V2C2
sequence) showed comparable sensitivities to VRC01 MAb, sCD4
and QS1420 MAb, only 4.J22 and 16055-2.3 Envs containing 2-5.J3
V2C2 displayed modest reduction in their sensitivities to VRC01
MAb as compared to their respective wild types. Overall, our data
indicate that the effect of presence of 2-5.J3 V2C2 sequence on
susceptibility of Envs to b12 and VRC01 is context dependent and
likely would vary between Envs.
Determinants in V2C2 region modulated neutralization of env-
pseudotyped viruses by PG9/PG16 MAbs
Since the V2C2 sequences of 2-5.J3 and 4.J22 differ by six
residues (although 4.J22 has shorter V2 loop length compared to
2-5.J3 Env) as described above, we next examined whether these
residues play any role in modulating the sensitivities of these two
Envs to PG and PG16 MAbs as a small change in V2 loop was
previously reported to significantly modulate Env susceptibility to
Envs to PG9/16 MAbs which targets QNE in V2 loop (Doores and
Burton, 2010; Moore et al., 2011; Ringe et al., 2012). As shown in
Fig. 4, we found that introduction of S183/E185/K186/K192/I207/
T215 in 2-5.J3 Env conferred 2-5.J3 (4.J22-V2C2/SEKKIT) pseudo-
virus with a significant increase in susceptibility to PG9 and PG16
MAbs. Conversely, a significant reduction in the sensitivity of 4.J22
Env was observed when specific substitutions of P183/D185/N186/
N192/V207/N215 were made in 4.J22 Env. This effect was found to
be comparable when substitution of only P183/D185/N186/N192
was made in the V2 loop of the 4.J22 Env, suggesting these
minimal changes were sufficient in modulating the exposure of
PG9/16 epitopes that resulted in the reduction in sensitivity of 4.
J22 Env to PG9 and PG16 MAbs and further indicated that N186
and N192 abrogate access of QNE by PG9/16 MAbs. It is to be noted
in this context that substitution of 4.J22 V2C2 with that of 2-5.J3
Env displayed significant reduction in the susceptibility of
PG9/PG16 MAbs, which is likely due to the increase in V2 loop
length as described earlier.
Since an extra glycan residue at the position 192 is present in
the V2 loop of 2-5.J3 Env compared to the 4.J22 Env, we sought to
examine whether this glycan (N192) has any role in modulating
PG9/16 sensitivity. Hence, we substituted N192A in 2-5.J3 Env and
introduced a glycan (K192NSS) in 4.J22 Env and examined its
susceptibility to PG9 and PG16 MAbs. As shown in Fig. 4 (bottom
rows), introduction of glycan in 4.J22 Env (K192NSS) conferred 4.
J22 (K192NSS) Env-pseudotyped virus with significant reduction
in its susceptibility to PG9 and PG16 MAbs; on the contrary, N192A
substitution in 2-5.J3 Env showed marginal increment (though not
significant) in PG9 susceptibility but not to PG16 MAb. These
observations suggest that the presence of glycan at position 192 in
the V2 loop interferes with efficient recognition by PG9/16 MAbs
and likely is dependent on loop length.
In summary, in the present study, using chimeric Envs and
through site-directed mutagenesis, we demonstrated that V2C2
altered b12 susceptibility of HIV-1 clade C Envs in a context-
dependent manner. In addition, we identified minimal residues
in V2 loop that modulated Env susceptibility to PG9/PG16 MAbs.
The neutralization susceptibility of Envs to b12 and PG9/16 MAbs
was found to be associated with exposure of their corresponding
epitopes as determined as wash-out experiments (Fig. 5). We
ruled out that the presence of aspartic acid at the position 185
(D185) in V2 loop as recently demonstrated (Utachee et al., 2014)
is not absolutely required for enhanced susceptibility of Envs to
b12 MAb as 7.J20 wild-type Env although naturally containing
D185, it is resistant to b12 MAb. In addition, 5.J41 (2-5.J3-V2C2)
and 7.J20 (2-5.J3-V2C2) chimeric Envs expressing D185 did not
show any reasonable susceptibility to b12 MAbs. In this paper, we
also demonstrated that the presence of glycan residues at the
positions 186 (N186) and 192 (N192) significantly abrogated access
of PG9/16 MAbs to QNE in the V2 loop of the b12-sensitive 2-5.J3
Env. Our data was substantiated by the fact that N192A alone did
not show any change in improvement of access of PG9/16 MAbs in
2-5.J3 Env. However, in 4.J22 Env, addition of a glycan at the
position 192 (K192NSS) conferred significant reduction in access of
QNE by PG9/16 MAbs, indicating this effect was likely to be
associated with shorter V2 loop length.V1V2 domain in Env plays
a pivotal role in either recognizing the neutralizing antibodies or
Fig. 3. Neutralization susceptibilities of wild type and chimeric Env-pseudotyped viruses to VRC01 MAb (top panel) and to sCD4 and anti-CD4 MAb (QS4120) (bottom panel).
The neutralization assays were carried out in duplicates in at least three independent time points.
S. Patil et al. / Virology 462-463 (2014) 266–272 269

5. facilitating the escape of HIV-1 variants to the neutralizing anti-
bodies including autologous antibodies, possibly due to its con-
formation as well as glycosylation pattern (Overbaugh and Morris,
2012; Pinter et al., 2004; Rong et al., 2007). Historically, the V1V2
sequences have been shown to undergo changes earlier than that
of the other regions of the Env (Burns and Desrosiers, 1991;
Johnson et al., 1991; Laird et al., 2008; Moore et al., 2011; Moore
et al., 2009; Overbaugh and Rudensey, 1992; Overbaugh et al.,
1991). Our data suggest that the V2C2 region of the 2-5.J3 Env
possibly affected the spike apex region, which in turn influenced
Env to undergo conformational changes resulting in differential
modulation of exposure of epitopes targeted by b12 MAb. Because
conformational change is a complex phenomenon and is often
context dependent, this may possibly explain why despite contain-
ing the same 2-5.J3 V2C2 sequence, other Envs displayed distinct
susceptibilities to b12 MAb.
Materials and methods
HIV-1 Envs and antibodies
The HIV-1 clade C envelopes 2-5.J3, 3-5.J25 and 4.J22 obtained
from Indian patients with recent infection were described pre-
viously (Ringe et al., 2010). 7.J20 is also an HIV-1 clade C Env
cloned in pcDNA3.1D/V5-His-TOPO (Invitrogen Inc.) obtained from
an Indian patient with recent infection, while 16055-2.3 was
kindly provided by Dr. David Montefiori. b12 monoclonal antibody
was kindly provided by Dr. Dennis Burton, and was also obtained
from the NIH AIDS Research Reagent and Reference Program.
VRC01 and sCD4 were obtained from NIH AIDS Research Reagent
and Reference Program. PG9 and PG16 MAbs were kindly provided
by Dr. Wayne Koff, International AIDS Vaccine Initiative (IAVI),
New York. QS4120 MAb was purchased from Calbiochem Inc.
Construction of Env chimera and point mutations
Chimeric Env constructs were made (Fig. 1) by swapping different
variable and constant domains of b12-sensitive Env (2-5.J3) into
resistant Env (4.J22) by blunt ligation of PCR products using either
In-Fusion Dry Down PCR kit (Clontech Inc.) as described before
(Ringe and Bhattacharya, 2012) following the manufacturer's protocol
or by two rounds of overlapping PCR in which first-round amplicons
were used as primers in the second round. Site-directed mutagenesis
to substitute specific residues in V2 and C2 regions were carried out
using tailor-made primers by PCR using high fidelity polymerase,
Platinum Taq (Invitrogen Inc.) or Phusion high fidelity polymerase
(New England Biolabs Inc.). The purified PCR products were then
digested with DpnI joined with backbone plasmid by annealing in the
presence of pox DNA polymerase as described before (Ringe et al.,
2011).
Preparation of Env-pseudotyped viruses and infectivity assay
Env-pseudotyped viruses were prepared by co-transfection of
pSVIIIenv containing patient Envs and pSG3Δenv in 293T cells
using calcium phosphate (Promega, Inc.) as described before
(Ringe et al., 2010). The infectivity assay was carried out in TZM-
bl cells expressing CD4, CCR5 and CXCR4.
Neutralization assay
Neutralization assays were carried out using TZM-bl cells as
described before (Ringe et al., 2010). Env-pseudotyped viruses were
incubated with varying dilutions of plasma (heat-inactivated) and
varying concentrations of b12 and PG9/16 MAbs for 1 h at 37 1C, and
subsequently 1 Â 104
TZM-bl cells were added into the mixture in the
presence of 25 μg/ml DEAE dextran (Sigma Inc.). The plates were
further incubated in a CO2 incubator at 37 1C for two days and the
degree of virus neutralization was assessed by measuring relative
luminescence units (RLU) in a Luminometer (Victor 4, PerkinElmer
Inc.).
Virus-antibody washout assay
In order to assess the degree of exposure of neutralizing
epitopes recognized by b12 and PG9 MAbs, virus antibody wash-
out assay was carried out as essentially described by Chakrabarti
et al. (2011). Briefly, MAbs (b12 and PG9) were serially 5-fold
diluted in 250 ml of 10% DMEM media to which 250 ml of Env-
pseudoviruses were added such that the final concentrations of
Fig. 4. Effect of glycan modification and specific amino acid substitutions in 2–5.J3 and 4.J22 Envs towards modulation of their susceptibilities to PG9 and PG16 MAbs.
The point substitutions were made by site-directed mutagenesis. The data are representation of three independent experiments done in duplicates.
S. Patil et al. / Virology 462-463 (2014) 266–272270

6. antibodies became 50À0.08 mg/ml in a total volume of 500 ml,
while in the “no inhibitor” control, the same volume of media was
added instead of antibody. The reaction mixture was incubated for
30 min at 37 1C following which 250 ml of reaction mixture was
added in ultra-clear centrifuge tubes (Beckman Coulter, Inc.),
which was diluted to 4 ml by complete DMEM and centrifuged
at 35,000 rpm in an SW60 Ti swinging rotor (Beckman Optima-XE-
100, Inc.) for 2 h at 4 1C. The virus pellet was washed twice with
4 ml of media by centrifuging at 40,000 rpm for 30 min at 4 1C.
After the final wash, 250 ml of DMEM was added to the virus pellet
and suspended by gentle shaking at 4 1C for 30 min. 100 ml of the
suspended virus was used to infect 100 ml of TZM-b cells
(0.2 Â 106
/ml) in duplicate. From the remaining 250 ml of reaction
mixture, an equal volume of the antibody virus mixture was used
as a “no washout” control. Plates were incubated at 37 1C in a CO2
incubator for 2 days, following which the degree of virus neu-
tralization was assessed by measuring the reduction of infection in
TZM-bl cells.
GenBank accession number
The GenBank accession number of HIV-1 clade C Env, 7.J20 is
EU908223.1.
Acknowledgments
This work was supported in part by a grant from the Depart-
ment of Biotechnology, Government of India, to JB (BT/PR12853/
MED/29/141/2009) and by the intramural support from the IAVI-
THSTI HIV Vaccine Design Program. IAVI's work was made possible
by generous support from many donors 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
Department 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. This study was made
possible by the generous support of the American people through
USAID. The contents are the responsibility of the authors and do
not necessarily reflect the views of USAID or the United States
Government. We thank Dr. Dennis Burton, Scripps Research
Institute, La Jolla, California, and Dr. Wayne Koff, International
AIDS Vaccine Initiative, New York, for help with obtaining b12, PG9
and PG16 monoclonal antibodies, respectively. We sincerely
acknowledge the support of the members of the HVTR laboratory,
Prof. G Balakrish Nair, Prof. Sudhanshu Vrati, Dr. Rick King and Dr.
Rajat Goyal. We thank the Director, National AIDS Research
Institute, Pune, for making available the HIV-1 clade C envelope
clones.
Appendix A. Supporting information
Supplementary data associated with this article can be found in
the online version at http://dx.doi.org/10.1016/j.virol.2014.06.018.
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