JVI Accepted Manuscript Posted Online 13 January 2016 J. Virol. doi:10.1128/JVI.03090-15 Copyright © 2016, American Society for Microbiology. All Rights Reserved.
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Conformational Epitope-Specific Broadly Neutralizing Plasma Antibodies Obtained from
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an HIV-1 Clade C Infected Elite Neutralizer Mediate Autologous Virus Escape through
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Mutations in V1 Loop
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Shilpa Patil11¶, , Rajesh Kumar1¶, , Suprit Deshpande1¶, Sweety Samal1, Tripti Shrivastava1,
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Saikat Boliar1, Manish Bansal1, Nakul Kumar Chaudhary1, Aylur K. Srikrishnan2, Kailapuri G.
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Murugavel2, Suniti Solomon2, Melissa Simek3, Wayne C. Koff 3, Rajat Goyal3, Bimal K.
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Chakrabarti1, 3, Jayanta Bhattacharya1, 3, #
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1. HIV Vaccine Translational Research Laboratory, Translational Health Science and
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Technology Institute, Faridabad, Haryana- 121001, India
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2. Y.R. Gaitonde Research and Care Center, Chennai, India
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3. International AIDS Vaccine Initiative, New York, USA
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Running title: HIV-1 clade C plasma confer cross clade neutralization
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Key words: HIV-1, neutralizing antibody, envelope, plasma, clade C, protocol G, V1V2 loop
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Abstract: 247 words
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¶ Equal contribution
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# Corresponding author
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Tel: +91-01242867705
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E-mail:
[email protected] /
[email protected]
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1
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Abstract
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Broadly neutralizing antibodies isolated from infected patients who are elite neutralizers
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have identified targets on HIV-1 envelope (Env) glycoprotein that are vulnerable to antibody
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neutralization; however, it is not known whether infection established by majority of the
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circulating clade C strains in Indian patients elicit neutralizing antibody responses against any of
29
the known targets. In the present study, we examined the specificity of a broad and potent cross
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neutralizing plasma obtained from an Indian elite neutralizer infected with HIV-1 clade C. This
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plasma neutralized 53/57 (93%) HIV pseudoviruses prepared with Env from distinct HIV clades
32
of different geographical origin. Mapping studies using gp120 core protein, single residue
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knockout mutants and chimeric viruses revealed that G37080 BCN plasma lacks specificities to
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the CD4 binding site, gp41 membrane proximal external region, N160, N332 glycans as well as
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R166 and K169 in V1-V3 region and are known predominant targets for BCN antibodies.
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Depletion of G37080 plasma with soluble trimeric BG505-SOSIP.664 Env (but neither with
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monomeric gp120 nor with clade C MPER peptides), resulted in significant reduction of virus
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neutralization, suggesting that G37080 BCN antibodies mainly target epitopes on cleaved
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trimeric Env. Further examination of autologous circulating Envs revealed association of
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mutation of residues in V1 loop that contributed in neutralization resistance. In summary, we
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report identification of plasma antibodies from a clade C infected elite neutralizer that mediates
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neutralization breadth via epitopes on trimeric gp120 not yet reported and confer autologous
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neutralization escape via mutation of residues in V1 loop.
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2
45
Importance
46 47
A preventive vaccine to protect against HIV-1 is urgently needed. HIV-1 envelope
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glycoproteins are targets of neutralizing antibodies and represent a key component for
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immunogen design. Mapping of epitopes on viral envelopes vulnerable for immune evasion will
50
aid in defining targets of vaccine immunogens. We identified novel conformational epitopes on
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viral envelope targeted by broadly cross neutralizing antibodies elicited in natural infection in an
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elite neutralizer infected with HIV-1 clade C. Our data extend our knowledge on neutralizing
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epitopes associated with virus escape and would potentially contribute in immunogen design and
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antibody based prophylactic therapy.
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3
56 57
Introduction
58 59
Broadly neutralizing antibodies (BNAbs) target trimeric envelope glycoprotein (Env) spikes
60
of the Human Immunodeficiency Virus Type 1 (HIV-1). Characterization of the BNAbs has provided
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key clues towards design and development of both prophylactic and therapeutic vaccines (7, 17, 30,
62
34, 35, 37). A small proportion of individuals chronically infected with HIV-1 develop BNAbs (5,
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12, 21, 38, 52, 62, 66, 69) and isolation of several broad and potent neutralizing monoclonal
64
antibodies (bNAb) from such individuals with distinct molecular specificities to viral envelope (Env)
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protein are reported (18, 31, 32, 64, 74, 76, 77, 79, 81). The cross neutralizing serum antibodies
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obtained from such individuals (also referred to as ‘elite neutralizers’) with considerable breadth
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target epitopes on structurally conserved regions of Env such as CD4 binding site (CD4bs) (11, 33,
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65, 79), V1V2 including glycan moieties (39, 50, 74, 76), gp120-gp41 interface (3, 64) and the
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membrane proximal external regions (MPER) (31, 45, 46, 83). Several studies have indicated that the
70
variable regions within the HIV-1 gp120 contain epitopes targeted by the autologous as well as
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BNAbs (8, 14, 16, 28, 41, 59, 60, 73). Recently the V1V2 region has been linked to development
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of broadly cross neutralizing (BCN) antibodies (16, 78) and the residues between 160 and 172
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(notably R166S/K or K169A) in V1V2 have been demonstrated to be associated with virus
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escape to autologous antibody response (16). Recent studies have further indicated that BCNAb
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development in vivo is associated with antibody affinity maturation and co-evolution of virus
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resulting in a considerable degree of somatic hypermutations (1, 13, 15, 16, 20, 29, 40, 63, 65, 75, 76,
77
80-82). Such information is crucial for design and development of suitable Env based immunogen
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capable of eliciting broad and potent cross neutralizing antibodies through vaccination.
79 4
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While a number of studies on the molecular specificities of broadly neutralizing antibodies
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obtained from African clade C infected individuals have been reported (2, 21-23, 25, 26, 41-44, 47,
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51, 57, 58), knowledge on immune evasion in Indian clade C infected elite neutralizers is very
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limited (53).
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In the present study, we examined plasma samples obtained from two hundred asymptomatic
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and anti-retroviral therapy (ART) naïve Indian HIV-infected donors and identified plasma with cross
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neutralizing antibodies. The molecular specificities of plasma antibodies obtained from an HIV-1
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clade C infected elite neutralizer was characterized in detail that displayed exceptional neutralization
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breadth across clades of different geographical origins. Interestingly, we found that neutralization
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breadth was associated with presence of unique epitopes on the trimeric gp120.
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Materials and Methods
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Ethics statement.
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The blood samples were collected under The IAVI Protocol G study from slow progressing anti-
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retroviral therapy (ART) naïve HIV-1 positive donors from Nellore District of the state of
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Andhra Pradesh, Southern India by trained clinicians at the YRG Care hospital following
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approval and clearance from the Institutional Review Board (IRB) and the Ethics Committee.
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The serum and plasma samples collected were shipped to the HIV Vaccine Translational
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Research Laboratory, Translational Health Science and Technology Institute for further
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assessment and research on the neutralizing antibody response.
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Plasmids, viruses, antibodies, proteins and cells.
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Plasmids encoding HIV-1 envelopes representing distinct clades are shown in Table 1.
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Monoclonal antibodies used in the study and TZM-bl cells were procured from the NIH AIDS
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Research Reagents Reference program and from the IAVI Neutralizing Antibody Consortium
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(NAC). 293T cells were purchased from the American Type Culture Collection (ATCC).
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Plasmid DNA encoding BG505-SOSIP.664-D7324, its purified cleaved trimeric protein (55) and
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pcDNA5-FRT BG505 Furin A (10) was kindly provided by Prof John Moore, Weill Cornell
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Medical College, New York. Purified gp120 TripleMut core protein (19) was obtained from Prof
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Richard Wyatt, The Scripps Research Institute through the IAVI Neutralizing Antibody
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Consortium (NAC). HIV-2 7312A and its chimeric constructs were provided by Prof Lynn
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Morris, NICD, Johannesburg.
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Purification of monomeric and trimeric Env proteins.
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Codon optimized gp120 plasmid encoding clade C 4-2.J41 (4, 54) gp120 was cloned in pcDNA
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3.1/V5-His-TOPO vector and transfected in 293T cells using polyethyleneimine (PEI).
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Supernatants containing soluble gp120 were filtered through 0.45µm filter and subsequently
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purified using Ni-NTA agarose matrix (Qiagen Inc.) by elution with phosphate buffered saline
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(PBS) containing 300mM imidazole (pH 8.0). The purified monomeric gp120 protein was
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extensively dialyzed with PBS (pH 7.4), concentrated using Amicon® Ultracentrifugal filers
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(Millipore Inc.) with 30KDa cut off and stored in -80°C until further use.
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The trimeric BG505-SOSIP.664 protein was purified using 293F cells essentially as
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described by Sanders et al (61). Briefly, the 293F cells were transfected with plasmid DNA
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encoding both BG505-SOSIP.664 gp140 envelope and furin (10). Supernatant containing soluble
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BG505-SOSIP.664 gp140 was harvested 72 to 96 hours post transfection, filtered and passed
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through a lectin agarose column obtained from Galanthus nivalis (Sigma Inc.). The
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nonspecifically bound proteins were then washed in PBS (pH 7.4) supplemented with 0.5 M
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NaCl. The bound proteins were then eluted using 0.5 M methyl alpha-D-manno-pyranoside,
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extensively dialyzed with 1X PBS and concentrated. BG505-SOSIP.664 was further purified by
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Sephadex G-200 size exclusion chromatography (AKTA, GE). Trimeric protein fractions were
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collected, pooled, quality assessed by running in blue native polyacrylamide gel electrophoresis
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(BN-PAGE) and favorably assessed for their ability to bind to only neutralizing and not to non-
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neutralizing and MPER directed monoclonal antibodies as described elsewhere (55) by ELISA.
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Depletion of plasma antibodies by monomeric gp120 and trimeric gp140 Env proteins.
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Purified soluble monomeric 4-2.J41 gp120 and trimeric BG505 SOSIP.664 proteins in addition
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to the MPER peptide (C1C; encoding clade C sequence) (71) were used for depletion of plasma 7
140
antibodies, where purified proteins were covalently coupled to the MyOne Tosylactivated
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Dynabeads (Life Technologies Inc.) according to the manufacturer’s protocol. Briefly, 30 mg of
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beads were used to couple with 1 mg of both monomeric and trimeric Env proteins in coupling
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buffer (0.1M NaBO4, 1M (NH4)2SO4; pH 9.4) overnight at 37ºC for 16-24 hrs. Proteins bound to
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magnetic beads were separated from unbound using a DynaMag™ 15 magnet (Life
145
Technologies, Inc.). Beads bound to Env proteins were next incubated with blocking buffer [PBS
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(pH 7.4), 0.1% bovine serum albumin (BSA; Sigma) and 0.05% Tween 20] at 37ºC to block the
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unbound sites. The antigenic integrity of both 4-2.J41 monomeric gp120 and BG505-SOSIP.664
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bound to the beads were assessed for their ability to bind VRC01 and 4E10 MAbs (for
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monomeric gp120) and PGT121, F105 and 4E10 MAbs (for BG505-SOSIP.664) by flow
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cytometry (FACS Canto, Becton and Dickinson, Inc.).
151
For depletion studies, G37080 plasma was diluted to 1:50 in DMEM containing 10%
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Fetal Bovine Sera (FBS) and 500 µl of diluted plasma was incubated with 20µl of beads at room
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temperature for 45 minutes. Unbound plasma antibodies were separated from ones those are
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bound to protein coated beads using a DynaMag™ 15 magnet as described above. This step was
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repeated 4-5 times for depletion of plasma antibodies by monomeric gp120 and 10-12 times in
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case of BG505-SOSIP.664 coated beads respectively. As a negative control, G37080 plasma
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antibodies were depleted with uncoated beads in parallel. In addition to ELISA, percent
158
depletion of G37080 plasma antibodies was assessed by examining the sequential decrease in
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binding of protein coated beads with depleted plasma antibodies by FACS. PGT121 MAb was
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taken as a positive control for checking depletion by BG505-SOSIP.664 trimeric Env.
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gp120 and gp140 ELISA
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For gp120 ELISA, high binding polystyrene microtiter plate (Nunc, Inc.) was coated with 100μl
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of monomeric 4-2.J41 gp120 (1μg/ml) in binding buffer comprising 0.1 M NaHCO3 (pH 8.6)
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and incubated overnight at 4ºC. gp120 bound plate was washed once with 1X PBS (pH 7.4) and
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blocked with 5% non-fat milk for 90 min at 37ºC. The plate was then washed three times with
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1X PBS, followed by addition of 100μl of MAbs as well as the depleted and undepleted plasma
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antibodies at different dilutions and incubated for 1hr at room temperature. The wells of the
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ELISA plate were washed four times with PBS containing 0.1% Tween 20 (PBST) followed by
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addition of 100µl of 1:3000 diluted HRP-conjugated anti-human IgG (Jackson Immunoresearch,
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Inc.) and further incubated for 45 min at room temperature. Unbound conjugates were removed
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by washing with PBST and color developed by addition of 100μl of 3, 3’, 5, 5’-
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tetramethylbenzidine (TMB) (Life Technologies, Inc.) substrate was added. Absorbance was
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measured at 450 nm in a spectrophotometer.
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Binding of antibodies to BG505-SOSIP.664-D7324 trimeric protein was assessed
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essentially as described by Sanders et al (61) in a sandwich ELISA. Briefly, high binding
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microtiter plate (Nunc, Inc.) was first coated with D7324 antibody at 10μg/ml (Aalto Bio
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reagents, Dublin, Ireland) followed by blocking extra unbound sites with 5% non-fat milk for 90
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min at 37ºC. 100 µl of BG505.664-D7324 trimeric protein (300ng/ml) was then added and
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incubated for 45 mins at room temperature. The extent of binding of G37080 plasma antibodies
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compared to known neutralizing monoclonal antibodies were assessed by addition of primary
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and HRP-conjugated secondary anti-human antibody as described above.
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Neutralization assay.
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Neutralization assays were carried out using TZM-bl cells as described before (54). Briefly, Env-
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pseudotyped viruses were incubated with varying dilutions of depleted plasma antibodies and
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incubated for an hour at 37°C CO2 incubator under humidified condition and subsequently 1 X
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104 TZM-bl cells were added into the mixture in presence of 25 μg/ml DEAE-dextran (Sigma,
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Inc.). The plates were further incubated for 48 hours and the degree of virus neutralization was
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assessed by measuring relative luminescence units (RLU) in a Luminometer (Victor X2,
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PerkinElmer Inc.).
194 195
Amplification, cloning and mutagenesis of autologous HIV-1 envs.
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Autologous complete env genes were obtained from G37080 plasma as described previously
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with slight modification (54). Briefly, viral RNA were extracted using High Pure viral RNA kit
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(Roche Inc.) following manufacturer’s protocol and cDNA prepared by RT-PCR using
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Superscript-III first strand synthesis kit (Invitrogen Inc.). rev-gp160 env genes were amplified
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using a Phusion hi fidelity DNA polymerase (New England Biolabs Inc.). The gp160 amplicons
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were purified and ligated into pcDNA 3.1/V5-His-TOPO (Invitrogen Inc.) vector. Chimeric Envs
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were prepared by overlapping PCR and point substitutions were made by Quikchange II kit
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(Agilent technologies Inc.) following manufacturer’s protocol and as described previously (49).
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Preparation of envelope pseudotyped viruses.
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Pseudotyped viruses were prepared by co-transfection of envelope expressing plasmid with env-
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deleted HIV-1 backbone plasmid (pSG3ΔEnv) into 293T cells in 6-well tissue culture plates
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using FuGENE6 Transfection kit (Promega Inc.). Cell supernatants containing pseudotyped
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viruses were harvested 48 hours post-transfection and then stored at -80°C until further use. The 10
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infectivity assays were done in TZM-bl cells (1 X 105cells/ml) containing DEAE-Dextran (25
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μg/ml) in 96-well microtiter plates and the infectivity titers were determined by measuring the
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luciferase activity using Britelite luciferase substrate (Perkin Elmer Inc.) with a Victor X2
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Luminometer (Perkin Elmer Inc.).
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Results
216 217
Identification of an elite neutralizer with HIV-1 clade C infection whose plasma showed
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exceptional neutralization breadth.
219
The present study under The IAVI Protocol G was designed (i) to screen and identify
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plasma antibodies obtained from chronically infected Indian donors with HIV-1 clade C with
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substantial breadth towards neutralizing cross clade HIV-1 primary variants and (ii) to elucidate
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their molecular specificities associated with neutralization breadth. Our hypothesis was that the
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genetic distinctness of clade C viruses of Indian and non-Indian origin, as well due to likely
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differences in host genetics between populations with differences in their ancestral origin
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associated with modulation of humoral immune responses, the specificities of antibodies
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developed in vivo associated with neutralization breadth and potency would be different.
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Through screening of two hundred plasma samples obtained from chronically-infected
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ART naïve Indian patients against a panel of 57 pseudoviruses containing Envs of distinct clades
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and geographical origins (Figure 1A), we identified one donor (G37080), whose plasma showed
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exceptional neutralization breadth. Donor G37080 serum neutralized >90% of the 57 different
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pseudoviruses tested with median ID50 value of 533.03 (Table 1, Figure 1B).
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Follow up plasma sample from this donor (G37080) was subsequently obtained after
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eight months to assess whether the neutralization breadth and potencies along with their
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molecular specificities were retained and/or improved, expecting that during the course of
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disease, breadth and potency of neutralizing antibodies broadens through somatic
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hypermutations (70) and/or clonal selection processes. As shown in Figure 1B and Table 1,
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follow up plasma antibodies of G37080 donor (referred to as Visit-2 samples) were found to
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exhibit comparable neutralization breadth to that of visit 1 plasma. Overall, G37080 BCN plasma
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was found to potently neutralize pseudoviruses containing Indian clade C Env with a
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neutralization score of 2.5 (66). Furthermore, the neutralization sensitivity of Env-pseudotyped
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viruses was found to be correlated with the serum IgG (data not shown), suggesting that the
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broad neutralization was associated with IgG-specific response. Taken together, our data indicate
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that a strong humoral immune response to HIV-1 was mounted in G37080 donor and was
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maintained overtime.
245 246
Evidence that G37080 BCN plasma antibodies do not target epitopes in CD4bs, MPER and
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known glycan and non-glycan residues in variable domains of Env
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First, we examined whether the G37080 BCN plasma contains antibodies directed to
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CD4bs on Env. Plasma samples obtained from both visits were pre-treated with 25µg/ml of
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TripleMut core protein (19), which was a concentration that we found to inhibit neutralization of
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25711-2.4 pseudovirus by VRC01 mAb by >95%. Pre-treated plasma was subsequently used to
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neutralize pseudovirus 25711-2.4 Env, and as shown in Figure 2, no perturbation of G37080
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neutralizing activity was observed against pseudovirus 25711-2.4. A similar observation was
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made when these plasma antibodies were pre-treated with RSC3 core protein (79). In addition,
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the G37080 BCN plasma antibodies were found to efficiently neutralize IgG1b12 and VRC01
256
resistant viruses (data not shown). Our data indicated that the G37080 BCN plasma antibodies do
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not contain CD4bs directed neutralizing antibodies.
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To elucidate whether the BCN plasma antibodies are directed to MPER in gp41, we used
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HIV-2/HIV-1 chimeric viruses (24) that expressed minimal residues of HIV-1 MPER containing
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epitopes required for MPER directed mAbs such as 2F5, 4E10, Z13e and 10E8. As shown in
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Table 2, the G37080 BCN plasma from both the visits was found to show modest neutralization
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of HIV-2 expressing HIV-1 clade C MPER (7312-C1C) with ID50 values of 306.42 and 371.02, 13
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respectively. We also found that depletion of G37080 plasma with a clade C MPER peptide
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(C1C) completely abolished the sensitivity of 7312A-C1C virus to G37080 plasma (Table 3).
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Our data suggest that although the G37080 BCN plasma neutralized 7312-C1C, presence of
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MPER directed antibodies was not associated with neutralization breadth.
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We next investigated whether the plasma antibodies of the donor G37080 target residues
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in variable loops, particularly in V1V2 and V3 region that have been shown in several studies as
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epitopes targeted by BCN antibodies on HIV-1 Env. First, we tested the extent of neutralization
270
by G37080 BCN plasma antibodies of Env pseudotyped viruses lacking glycans at 160 (N160)
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and at 332 (N332) position in the V2 region and V3 base respectively, and also R166 and K169
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in V2 region, which are major targets of recently identified broad and potent neutralizing
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monoclonal antibodies. In order to test this, two clade C Envs (25711-2.4 and CAP239.G3)
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containing N160A and N332A substitutions were tested and as shown in Table 2. Our data
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indicate that the pseudoviruses containing Env expressing N160 or N332 substitutions have
276
identical sensitivities to G37080 plasma antibodies. Similar observations were found with
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R166A and K169A in 93IN905 Env backbone. Taken together, our observations indicate that
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G37080 BCN plasma antibodies did not utilize these residues in V2 and V3 regions for
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neutralization breadth; which have been identified as important epitopes recognized by broadly
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neutralizing antibodies elicited in clade C infection described before (16, 42, 78).
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Association of neutralization breadth of G37080 plasma with recognition of conformational
283
epitopes on cleaved trimeric Env but not with that in monomeric gp120 or MPER.
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In order to examine whether broad neutralization conferred by the G37080 plasma
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antibodies was through recognition of epitopes on monomeric gp120 or cleaved near native Env
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trimers, we tested binding of G37080 serum IgG to monomeric 4-2.J41 gp120 and soluble gp140 14
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(BG505-SOSIP.664) by ELISA. We found that in addition to the monomeric 4-2.J41 gp120
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(Figure 3A), G37080 serum polyclonal IgG was found to efficiently bind to the BG505
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SOSIP.664-D7324 soluble trimeric Env (Figure 3B), indicating that the G37080 plasma
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primarily contains neutralizing antibodies that targets epitopes on cleaved Env trimers.
291
We next examined whether binding of the G37080 plasma antibodies to epitopes on
292
cleaved BG505-SOSIP.664 trimeric envelope was associated with neutralization breadth. For
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this, we tested the ability of G37080 plasma antibodies depleted with both monomeric and
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trimeric Envs as well as with MPER peptides to neutralize a set of Env-pseudotyped viruses,
295
which were found to be sensitive to this particular plasma sample. Purified 4-2.J41 monomeric
296
gp120, BG505-SOSIP.664 trimeric gp140 and C1C MPER peptide bound to the magnetic beads
297
were used to deplete G37080 plasma antibodies as described in the ‘Materials and Methods’. The
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depleted BCN G37080 antibodies were first assessed for their binding to 4-2.J41 gp120
299
monomers, BG505-SOSIP.664-D7324 and C1C peptide in comparison to undepleted plasma
300
antibodies by ELISA. As shown in Figure 3C and 3D, G37080 plasma depleted with monomeric
301
gp120 and trimeric gp140, respectively, had significantly reduced binding activity against
302
respective soluble proteins. Similar observation was made with MPER peptide (data not shown).
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The depleted plasma antibodies were subsequently assessed for neutralization activity using a
304
panel of twelve Env pseudotyped viruses that were susceptible to untreated G37080 plasma
305
antibodies as mentioned above. As shown in Table 3, depletion with 4-2.J41gp120 monomer and
306
C1C peptide did not show any change in neutralization breadth of G37080 plasma antibodies,
307
while depletion with BG505-SOSIP.664 showed a significant reduction in virus neutralization. A
308
similar observations were made with the BG505-SOSIP.664 depleted PGT121 and C1C peptide
309
depleted 4E10 MAbs which lost the ability to efficiently neutralize Env-pseudotyped viruses 15
310
(16055 and ZM233.6) and HIV-2/HIV-1 (7312A-C1C) chimeric virus compared to their
311
undepleted counterparts (data not shown); thus validating our data. Interestingly, C1C peptide
312
depleted G37080 plasma failed to neutralize HIV-2/HIV-1 (7312A-C1C) chimeric virus
313
indicating that the presence of residual traces of MPER directed antibodies (as shown in Table 2)
314
are not responsible for neutralization breadth. Furthermore, examination of chimeric Envs
315
prepared between the sensitive (25711-2.4) and resistant (16055-2.3 and CAP45.G3) Envs
316
indicated that the BCN G37080 plasma antibodies predominantly target epitopes in the V1V2
317
region (Table 4) in gp120. Our data clearly indicate a correlation between neutralization breadth
318
and binding of the G37080 BCN plasma antibodies to the conformational epitopes on cleaved
319
trimeric gp120, likely in the V1V2 region; however we do not rule out the possibility that this
320
BCN plasma targets other discontinuous epitopes in the gp120, but not in MPER.
321 322
Mutations in V1 region confer resistance of autologous viruses to the G37080 plasma
323
antibodies
324
In order to decipher the specificity of the G37080 plasma antibodies, we examined the
325
degree of susceptibility of pseudoviruses prepared using env genes amplified from
326
contemporaneous autologous G37080 plasma obtained at the baseline and follow ups visits. As
327
shown in Figure 4A, both the Envs obtained from visit 2 plasma (HVTR-PG80v2.eJ38 and
328
HVTR-PG80v2.eJ41) were found to be resistant to its contemporaneous plasma antibodies,
329
while Envs obtained from visit 1 plasma (HVTR-PG80v1.eJ7 and HVTR-PG80v1.eJ19) were
330
found to be modestly sensitive to visit 2 autologous G37080 plasma antibodies. To facilitate
331
mapping G37080 BCN antibody specificity, we prepared chimeric Envs between a sensitive
332
(HVTR-PG80v1.eJ7 and HVTR-PG80v1.eJ19) and the resistant (HVTR-PG80v2.eJ38)
16
333
autologous Envs by first swapping the V1V2 regions as their amino acid sequences differed
334
maximally in this region (Figure 4B). As shown in Table 4, substitution of V1V2 sequence of
335
HVTR-PG80v1.eJ7 and HVTR-PG80v1.eJ19 into HVTR-PG80v2.eJ38 conferred Env-
336
pseudotyped viruses expressing HVTR-PG80v2.eJ38 Env with enhanced sensitivity to G37080
337
visit 2 plasma antibodies by >25 and >12-folds respectively. Conversely, the neutralization
338
susceptibilities of the Env-pseudotyped viruses expressing HVTR-PG80v1.eJ7 and HVTR-
339
PG80v1.eJ19, which contained HVTR-PG80v2.eJ38 V1V2 sequence corresponding to visit 2
340
G37080 plasma, were found to be reduced by >45 and >23 folds respectively. We noted that
341
substitution of regions other than V1V2 loop in the autologous Env did not confer any change in
342
neutralization sensitivity (Table 4). To further narrow down residues in V1V2 loop, associated
343
with neutralization sensitivity and resistance of autologous Envs, chimeric Envs and point
344
mutants were prepared and tested for their degree of modulation in susceptibility to autologous
345
G37080 plasma obtained from second visit. As shown in Table 4, we found that the V1 sequence
346
but not the V2 sequence of the sensitive Envs (HVTR-PG80v1.eJ7 and HVTR-PG80v1.eJ19)
347
when transferred to the resistant HVTR-PG80v2.eJ38 Env, increased sensitivity to G37080 BCN
348
plasma antibodies by >50 and >37 folds respectively. In agreement with this result, V1 of
349
HVTR-PG80v2.eJ38 when transferred into the sensitive Envs stated above, increased
350
neutralization resistance by >27 and >28 folds respectively to the G37080 visit 2 BCN plasma
351
antibodies. We observed that removal of a glycan at the 140 position in V1 (T140D) in the
352
HVTR-PG80v1.eJ19 mediated enhanced sensitivity of this Env to G37080 plasma by 2.64 fold
353
(Table 4). Concurrent to this observation, we found that insertion of V1 region of PG80v1.eJ19
354
with T140D substitution in PG80v2.eJ38 Env exhibited enhanced susceptibility when compared
355
with that of PG80v2.eJ38 Env chimera containing PG80v1.eJ19 V1 loop as shown in Table 4.
17
356
Our data indicate that N138 glycan potentially mask the PG80v1.eJ19 Env from being efficiently
357
neutralized by the autologous plasma compared to that of its contemporaneous counterpart
358
PG80v1.eJ7 Env. Fine scanning of V1 regions of the autologous Envs further revealed that N133
359
glycan motif and P147 residues in the PG80v2.eJ38 Env played significant role in neutralization
360
resistance to G37080 BCN autologous plasma antibodies (Figure 4B). Interestingly, all the V1
361
chimeras as well as the point mutants showed comparable sensitivities to PG9 MAb as compared
362
to their wild types (Table 5), indicating that the shift in neutralization susceptibilities were not
363
due to change in Env conformation. Moreover, we noted that both the sensitive and the resistant
364
autologous Envs contain T332 in the V3 base, clearly indicating that absence of N332 was not
365
associated with resistance to autologous neutralization. Similar observations were made with
366
respect to N160, R166 and K169 amino acid residues further consolidating that the neutralization
367
conferred by G37080 BCN plasma antibodies was not associated with antibody targeting these
368
epitopes in autologous Envs and likely for all the Envs tested against G37080 plasma antibodies.
369
18
370
Discussion
371 372
Identification of the molecular specificities of antibodies elicited in natural infection and
373
that mediate neutralization breadth and potency is key in design and development of suitable Env
374
based immunogen capable of eliciting similar antibody response upon vaccination. In the present
375
study, we characterized the molecular specificity of plasma antibodies obtained from an Indian
376
elite neutralizer (G37080) infected with HIV-1 clade C that displayed exceptional cross
377
neutralization of different clades of distinct geographical origins. The G37080 plasma was found
378
to contain the most broad and potent cross neutralizing antibodies amongst the two hundred
379
plasma samples obtained from Indian patients chronically infected with HIV-1. Plasma samples
380
collected from the G37080 donor at two time points at eight months apart showed similar
381
neutralization breadth with modest increase in potency in the follow up visit, indicating
382
association of sustained maturation of antibody producing B cells in this individual.
383
Since polyclonal plasma antibodies are not suitable for epitope mapping, we examined
384
the specificity of the G37080 BCN plasma by making use of mutant viruses with specific point
385
substitution of known neutralizing epitopes with non-specific amino acids and via depletion with
386
monomeric and trimeric Envs in addition to MPER peptide. The G37080 plasma antibodies did
387
not show dependence to the N160/K169 and N332 epitopes in V2 apex and V3 base respectively
388
Our data also is consistent with the target epitopes of the G37080 BCN antibodies being distinct
389
from those which are recognized by 2G12 (72), PGT121-128 (32) and PGT130-131, 135 (75)
390
(e.g., residues at the following positions: 295, 297, 301, 332, 334, 386, 388, 392, 394, 448, 450),
391
thus BCN G37080 antibodies appear to target a new epitope. Our data highlighting N332
392
independent development of neutralizing antibodies in a clade C infected donor G37080, also
393
differs from recent findings (27, 36, 43, 67) associating N332 with development of broad and 19
394
potent neutralizing antibody especially in clade C infection noted in African donors. Moreover,
395
recent studies indicating the role of K169 as a target of BCN antibodies obtained from a clade C
396
infected South African donor (42, 78) and the observation that vaccine-induced protection in the
397
RV144 vaccine trial was associated with antibodies targeting epitopes including K169 in V2
398
apex (39, 56) prompted us to examine whether broad neutralization of the G37080 plasma
399
antibodies was also dependent on K169 epitope. In our present study, the neutralization potency
400
of G37080 not only was unaffected by N160A/K169A knockout mutations but we also observed
401
that both sensitive and resistant autologous Envs obtained from both visits contain N160 and
402
K169 in the V2 region. Hence, owing to lack of association of neutralization breadth of the
403
G37080 BCN antibodies with N160, K169 and N332 dependences, our study further highlighted
404
that there is a likelihood of differences in development pathway of elicitation of broadly
405
neutralizing antibodies in individuals infected with HIV-1 clade C particularly those with
406
ethnically distinct.
407
Wibmer et al (78) recently demonstrated association between evolution of a broadly
408
neutralizing antibody response in a clade C infected donor with shifts in antibody specificities
409
from recognition of epitopes in V2 to the CD4bs. In the present study, the G37080 neutralizing
410
plasma antibodies obtained from both visits were found not to be absorbed out by the TripleMut
411
(9, 19) as well as the RSC3 (79) core proteins, which effectively absorb antibodies directed to the
412
CD4bs. This result indicates a lack of development of CD4bs directed neutralizing antibodies
413
during the disease course in G37080 donor. Additionally, absence of MPER directed antibodies
414
in G37080 plasma were found, although a negligible antibody titer (1:300 reciprocal dilutions) to
415
the HIV2/HIV1 (C1C) chimera was observed with both visit plasma samples. However, the
416
neutralization breadth of the G37080 plasma was not found to be associated with presence of
20
417
MPER directed antibody. Nonetheless, we do not rule out the possibility that in further course of
418
infection, this donor would possibly be able to develop MPER directed antibodies.
419
Recent studies have shown that neutralizing antibodies that targets conformational
420
epitopes binds exclusively to the cleaved near native trimeric Envs (6, 18, 48, 55). In the present
421
study, we found that absorption of G37080 plasma antibodies to soluble trimeric BG505-
422
SOSIP.664 Env was associated with depletion of neutralizing activity in G37080 BCN plasma.
423
However, we do not rule out the possibility of presence of 39F, 19b and 14e like non-
424
neutralizing antibodies that were reported to bind to BG505-SOSIP.664 trimeric Env (61). Our
425
findings indicate that the G37080 BCN antibodies target conformational epitopes in gp120. Our
426
observation also highlights that native like trimeric Envs such as BG505-SOSIP.664 can be
427
utilized in selecting antigen specific memory B cells as reported earlier (68) from G37080 donor
428
towards isolation of MAb correlating with broad neutralization displayed by the plasma
429
antibodies.
430
We made use of env clones obtained from autologous G37080 plasma from both the time
431
points to narrow down the fine specificity of the G37080 BCN plasma antibodies. By examining
432
chimeric Envs and mutant viruses we identified key residues in the V1 loop associated with
433
neutralization resistance. Interestingly, the Env chimera and mutant viruses showed comparable
434
susceptibility to PG9 MAb compared to their respective wild type Envs, indicating that they did
435
not alter Env conformation. We identified a glycan at the 133 position and a proline residue at
436
the 147 position within V1 loop of the resistant Env (PG80v2.eJ38) that were found to be
437
associated with neutralization escape, which indicated that these are contact sites for the G37080
438
BCN plasma antibodies. From our study we thus conclude that changes in V1 loop sequence are
439
associated with escape of autologous viruses to the BCN G37080 plasma. Additionally,
21
440
examination of degree of susceptibilities of pseudoviruses expressing chimeric heterologous
441
Envs to the G37080 plasma revealed that the BCN plasma antibodies predominantly target
442
epitopes in V1V2 region in gp120. However, we do not rule out the possibility of contribution of
443
other discontinuous epitopes in gp120 in mediating neutralization breadth. Isolation and
444
identification of monoclonal antibodies from this elite neutralizer donor (G37080) will help
445
precisely map specific epitope associated with neutralization breadth and potency.
446
In summary, we identified an HIV-1 infected elite neutralizer, whose plasma showed
447
exceptional neutralization breadth and provided evidence that it targets novel conformational
448
epitopes on trimeric Env predominantly in the V1V2 region not reported previously. Moreover,
449
neutralization resistance of the autologous Envs to G37080 plasma is associated with
450
substitutions of novel residues within V1 loop that form the key contact points of the BCN
451
plasma antibody. Identification of novel epitopes associated with broad neutralization of HIV-1,
452
in particular the majorly circulating clade C strains will significantly contribute in the efforts
453
towards effective immunogen design.
454 455
Funding information.
456
This study was made possible by the generous funding support of the American people through
457
the United States Agency for International Development (USAID) and support from the THSTI-
458
IAVI HIV vaccine design grant through the Department of Biotechnology, Govt. of India; partly
459
by a grant from Department of Science and Technology, Govt. of India (DST/INT/SAFR/Mega-
460
P3/2011 to Jayanta Bhattacharya) and partly by the DBT National Bioscience Research Award
461
grant (BT/HRD/NBA34/01/2012-13(iv) to Jayanta Bhattacharya). The funders had no role in
462
study design, data collection and interpretation, or the decision to submit the work for
463
publication. 22
464 465
Acknowledgements.
466
We thank all the Protocol G study participants registered with YRG Care, Chennai, and all the
467
research staffs at the Protocol G clinical center at the YRG Care, Chennai and all of the IAVI
468
Protocol G team members. We thank Dr. Albert Cupo, Prof. John P. Moore and the members the
469
SOSIP trimer HIVRAD team, Weill Cornell Medical College, New York for providing us with
470
BG505.SOSIP.664 plasmid DNA and purified protein. The following reagent was obtained
471
through the NIH AIDS Reagent Program, Division of AIDS, NIAID, NIH from Drs. John C.
472
Kappes and Xiaoyun Wu: pSG3Δenv. We thank Dr. David Montefiori, Prof Lynn Morris, Dr
473
Pascal Poignard, Dr. Richard Wyatt for making available many reagents used in our study. The
474
International AIDS Vaccine Initiative has filed a patent relating to the autologous HIV-1 clade C
475
envelope clones: U.S. Provisional Application no. 62/254,971, titled “HIV-1 clade C envelope
476
glycoproteins,” with inventors J. Bhattacharya, S. Deshpande, S. Patil, R. Kumar, B.K.
477
Chakrabarti. We sincerely thank Dr. Christopher Parks, IAVI Design and Development
478
Laboratory for providing valuable inputs in preparing the manuscript and we also thank Prof. G.
479
Balakrish Nair, Prof Sudhanshu Vrati, THSTI; Dr. Shreyasi Chatterjee and all the HVTR
480
laboratory members for support. IAVI's work was made possible by generous support from many
481
donors including: the Bill & Melinda Gates Foundation; the Ministry of Foreign Affairs of
482
Denmark; Irish Aid; the Ministry of Finance of Japan; the Ministry of Foreign Affairs of the
483
Netherlands; the Norwegian Agency for Development Cooperation (NORAD); the United
484
Kingdom Department for International Development (DFID); and the United States Agency for
485
International Development (USAID). The full list of IAVI donors is available at www.iavi.org.
486
The contents are the responsibility of the International AIDS Vaccine Initiative and do not 23
487
necessarily reflect the views of USAID or the United States Government. The contents of this
488
manuscript are the responsibility of IAVI and do not necessarily reflect the views of USAID or
489
the US Government.
490 491
24
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858 859 860
32
861
Table and Figure Legends:
862 863
Table 1. Neutralization breadth of Protocol G G37080 plasma samples collected at two different
864
points tested against 57 panel Env-pseudotyped viruses.
865 866
Table 2. Examination of known specificity of G37080 plasma antibodies obtained at both visits
867
to HIV Env.
868 869
Table 3. Degree of shift in sensitivity of the Env-pseudotyped viruses to G37080 BCN plasma
870
depleted with the soluble monomeric and trimeric Env proteins and a clade C MPER peptide
871
(C1C).
872 873
Table 4. Mapping specificities mediating neutralization resistance of the autologous and
874
heterologous Envs to G37080 BCN plasma.
875 876
Table 5. Sensitivity of wild type, chimera and point mutants of autologous Envs to PG9 MAb
877 878
Figure 1 A. Genetic divergence of amino acid sequences of 57 HIV-1 Env (gp160) used to
879
assess neutralization breadth and potency of G37080 BCN plasma. Maximum likelihood
880
bootstrapped consensus phylogenetic tree was constructed using Jones-Taylor-Thornton (JTT)
881
substitution model with 50 bootstrapped replicates in Mega 5.2 version. Bootstrapped values are
882
shown at the nodes of each branch. Hollow circles represent envelopes (16055-2.3 and
883
92TH021) resistant to neutralization by G37080 BCN plasma. B. Neutralization breadth of the
884
G37080 BCN plasma obtained at visit 1 and visit 2 were assessed against pseudotyped viruses 33
885
expressing HIV-1 Env representing different clades and origins. Neutralization titers (median
886
ID50 values) were obtained by titrating Env-pseudotyped viruses against G37080 plasma
887
samples. Values at top of each bar graph indicate number of viruses belonging to each
888
clade/origin tested.
889 890
Figure 2. Assessing dependence of G37080 BCN antibodies to CD4 binding site (CD4bs) region
891
of HIV-1 Env. G37080 BCN plasma samples and VRC01 MAb (concentrations that neutralized
892
25711-2.4 by >80%) pre-incubated with different concentrations, as indicated, with TripleMut
893
core (A) and RSC3 (B) proteins were examined for their ability to neutralize 25711-2.4 Env
894
pseudotyped virus in TZM-bl cell neutralization assay. Note that while VRC01 pre-absorbed
895
with both TripleMut and RSC3 proteins showed inhibition to neutralize 25711-2.4 in a dose-
896
dependent manner, no such effect was observed with G37080 BCN plasma indicated absence of
897
CD4bs directed neutralizing antibodies.
898 899
Figure 3. Binding of G37080 BCN plasma IgG to 4-2.J41 monomeric gp120 (A) and BG505-
900
SOSIP.664-D7324 cleaved trimeric gp140 (B) soluble proteins were assessed by ELISA. IgG
901
purified from HIV negative healthy donor and known MAbs were used as controls. Extent of
902
binding of the depleted and undepleted G37080 BCN plasma with magnetic beads coated with 4-
903
2.J41 monomeric gp120 (C) and BG505-SOSIP.664 cleaved trimeric gp140 to their respective
904
proteins by ELISA. Note that binding to trimeric protein by ELISA was assessed by using
905
BG505-SOSIP.664 tagged with D7324 epitope to maintain native conformation of trimeric Env
906
as described before (61).
907
34
908
Figure 4. A. Neutralization susceptibility of autologous Envs to contemporaneous G37080 BCN
909
plasma and its follow up sample from the same donor. Neutralization titers (median ID50) were
910
obtained by titrating pseudotyped viruses expressing autologous Envs obtained from visit 1 and
911
follow up G37080 plasma to contemporaneous plasma antibodies. Note that both the Envs
912
obtained from follow up G37080 plasma (visit 2) were found to be resistant to contemporaneous
913
autologous plasma, while Envs obtained from visit 1 G37080 plasma were found to be sensitive
914
to follow up plasma antibodies. B. Alignment of V1V2 amino acid sequences of sensitive and
915
resistant autologous Envs obtained at both visits were done by using seqpublish available at HIV
916
Los Alamos database (www.hiv.lanl.gov). Key residues that mediate autologous neutralization
917
resistance are highlighted.
918
35
Table 1. Neutralization breadth of Protocol G G37080 plasma samples collected at two different time points tested against 57 panel Env-pseudotyped viruses
G37080 Plasma
India Clade C
Africa Clade C
Clade B
Clade A
Others
Envelope MuLV HIV-2 (7312A) 16055-2.3 16936_2.21 25710-2.3 25711-2.4 00836-2.5 2-5.J3 4.J22 4-2.J41 3-5.J25 5-4.J16 5.J41 7.J16 7.J20 11-3.J3 11-5.J12 LT-1.J1 LT1.J3 LT5.J3b LT5.J7b 93IN905 Median ID50 CAP45.G3 CAP84 CAP88 CAP239.G3 Du422.1 Du151.2 DU156.12 DU172.17 ZM109F.PB4 ZM197M.PB7 IAVIC22 Median ID50 JRFL PVO.4 TRJO4551.58 AC10.0.29 QH0692.42 REJO4549.67 SC422661.8 6535.3 RHPA 4259.7 HO61.14 92BR020 JRCSF Median ID50 Q769.ENV.b9 Q461.e2 Q842.d12 Q23.17 Q259.d2.26 BG505 94UG103 Median ID50 92TH021 LT5.J12 CH038.12 CH114.8 CH120.6 CRF 02AG_235 191727_D1_12 Median ID50
Accession No. S53043.1 JX235925.1 EF117268 EF117270 EF117271 EF117272 EF117265 GU945311.1 EU908219.1 GU945316.2 GU945314.1 GU945326.1 EU908221.1 EU908222.1 EU908223.1 GU945330.1 GU945333.1 JN400529 JN400534 JN400538 JN400540 AY669742.1
Clade NA A C C C C C C C C C C C C C C C C C C C C
DQ435682.1 EF203963.1 EF203972.1 EF203983.1 DQ411854.1 DQ411851.1 DQ411852.1 DQ411853.1 AY424138.2 DQ388515.1 --
C C C C C C C C C C C
U63632.1 AY835444.1 AY835450 AY835446 AY835439 AY835449 AY835441.1 AY835438 AY835447.1 EF210730 AY669718.1 M38429.1
B B B B B B B B B B B B
AF407157.1 AF407156 AF407160.1 AF004885 AF407152 DQ208458.1 AY669705.1
A A A A A A A
AY669775.1 FJ515876 EF042692 EF117264 EF117260 EU513195 HM215267.1
A/E B/C B/C B/C B/C A/G D
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