Conformational Epitope-Specific Broadly Neutralizing PlasmaAntibodies Obtained from an HIV-1 Clade C-Infected EliteNeutralizer Mediate Autologous Virus Escape through Mutations inthe V1 Loop

Shilpa Patil,a Rajesh Kumar,a Suprit Deshpande,a Sweety Samal,a Tripti Shrivastava,a Saikat Boliar,a Manish Bansal,a

Nakul Kumar Chaudhary,a Aylur K. Srikrishnan,b Kailapuri G. Murugavel,b Suniti Solomon,b Melissa Simek,c Wayne C. Koff,c

Rajat Goyal,c Bimal K. Chakrabarti,a,c Jayanta Bhattacharyaa,c

HIV Vaccine Translational Research Laboratory, Translational Health Science and Technology Institute, Faridabad, Haryana, Indiaa; Y. R. Gaitonde Research and Care Center,Chennai, Indiab; International AIDS Vaccine Initiative, New York, New York, USAc

ABSTRACT

Broadly neutralizing antibodies isolated from infected patients who are elite neutralizers have identified targets on HIV-1 enve-lope (Env) glycoprotein that are vulnerable to antibody neutralization; however, it is not known whether infection established bythe majority of the circulating clade C strains in Indian patients elicit neutralizing antibody responses against any of the knowntargets. In the present study, we examined the specificity of a broad and potent cross-neutralizing plasma obtained from an In-dian elite neutralizer infected with HIV-1 clade C. This plasma neutralized 53/57 (93%) HIV pseudoviruses prepared with Envfrom distinct HIV clades of different geographical origins. Mapping studies using gp120 core protein, single-residue knockoutmutants, and chimeric viruses revealed that G37080 broadly cross-neutralizing (BCN) plasma lacks specificities to the CD4 bind-ing site, gp41 membrane-proximal external region, N160 and N332 glycans, and R166 and K169 in the V1-V3 region and areknown predominant targets for BCN antibodies. Depletion of G37080 plasma with soluble trimeric BG505-SOSIP.664 Env (butwith neither monomeric gp120 nor clade C membrane-proximal external region peptides) resulted in significant reduction ofvirus neutralization, suggesting that G37080 BCN antibodies mainly target epitopes on cleaved trimeric Env. Further examina-tion of autologous circulating Envs revealed the association of mutation of residues in the V1 loop that contributed to neutral-ization resistance. In summary, we report the identification of plasma antibodies from a clade C-infected elite neutralizer thatmediate neutralization breadth via epitopes on trimeric gp120 not yet reported and confer autologous neutralization escape viamutation of residues in the V1 loop.

IMPORTANCE

A preventive vaccine to protect against HIV-1 is urgently needed. HIV-1 envelope glycoproteins are targets of neutralizing anti-bodies and represent a key component for immunogen design. The mapping of epitopes on viral envelopes vulnerable to im-mune evasion will aid in defining targets of vaccine immunogens. We identified novel conformational epitopes on the viral enve-lope targeted by broadly cross-neutralizing antibodies elicited in natural infection in an elite neutralizer infected with HIV-1clade C. Our data extend our knowledge on neutralizing epitopes associated with virus escape and potentially contribute to im-munogen design and antibody-based prophylactic therapy.

Broadly neutralizing antibodies (BNAbs) target trimeric enve-lope glycoprotein (Env) spikes of human immunodeficiency

virus type 1 (HIV-1). Characterization of the BNAbs has providedkey clues toward the design and development of both prophylacticand therapeutic vaccines (1–6). A small proportion of individualschronically infected with HIV-1 develop BNAbs (7–14), and theisolation of several broad and potent neutralizing monoclonal an-tibodies (MAbs) from such individuals with distinct molecularspecificities to viral envelope protein has been reported (15–23).The cross-neutralizing serum antibodies obtained from such in-dividuals (also referred to as elite neutralizers), which have con-siderable breadth, target epitopes on structurally conserved re-gions of Env such as the CD4 binding site (CD4bs) (22, 24–26),V1V2, including glycan moieties (19, 20, 27, 28), the gp120-gp41interface (18, 29), and the membrane-proximal external regions(MPER) (16, 30–32). Several studies have indicated that the vari-able regions within HIV-1 gp120 contain epitopes targeted byautologous antibodies as well as BNAbs (33–40). Recently the

V1V2 region has been linked to the development of broadly cross-neutralizing (BCN) antibodies (35, 41), and the residues between160 and 172 (notably R166S/K or K169A) in V1V2 have beendemonstrated to be associated with virus escape from autologous

Received 9 December 2015 Accepted 7 January 2016

Accepted manuscript posted online 13 January 2016

Citation Patil S, Kumar R, Deshpande S, Samal S, Shrivastava T, Boliar S, Bansal M,Chaudhary NK, Srikrishnan AK, Murugavel KG, Solomon S, Simek M, Koff WC, GoyalR, Chakrabarti BK, Bhattacharya J. 2016. Conformational epitope-specific broadlyneutralizing plasma antibodies obtained from an HIV-1 clade C-infected eliteneutralizer mediate autologous virus escape through mutations in the V1 loop.J Virol 90:3446 –3457. doi:10.1128/JVI.03090-15.

Editor: W. I. Sundquist, University of Utah

Address correspondence to Jayanta Bhattacharya, JBhattacharya@iavi.org.

S.P., R.K., and S.D. contributed equally to this work.

Copyright © 2016, American Society for Microbiology. All Rights Reserved.

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antibody response (35). Recent studies have further indicated thatBCNAb development in vivo is associated with antibody affinitymaturation and coevolution of virus, resulting in a considerabledegree of somatic hypermutations (19, 20, 23, 26, 35, 42–50). Suchinformation is crucial for the design and development of suitableEnv-based immunogens capable of eliciting broad and potentcross-neutralizing antibodies through vaccination.

While a number of studies on the molecular specificities ofbroadly neutralizing antibodies obtained from African clade C-in-fected individuals have been reported (9, 37, 51–62), knowledgeon immune evasion in Indian clade C-infected elite neutralizers isvery limited (63).

In the present study, we examined plasma samples obtainedfrom two hundred asymptomatic and antiretroviral therapy(ART) naive Indian HIV-infected donors and identified plasmawith cross-neutralizing antibodies. The molecular specificities ofplasma antibodies obtained from an HIV-1 clade C-infected eliteneutralizer was characterized in detail that displayed exceptionalneutralization 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 trimericgp120.

MATERIALS AND METHODSEthics statement. The blood samples were collected under the IAVI Pro-tocol G study from slow-progressing ART naive HIV-1-positive donorsfrom Nellore District of the state of Andhra Pradesh, southern India, bytrained 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 HIVVaccine Translational Research Laboratory, Translational Health Scienceand Technology Institute, for further assessment and research on the neu-tralizing antibody response.

Plasmids, viruses, antibodies, proteins, and cells. Plasmids encodingHIV-1 envelopes representing distinct clades are shown in Table 1. Mono-clonal antibodies used in the study and TZM-bl cells were procured fromthe NIH AIDS Research and Reagents Reference program and from theIAVI Neutralizing Antibody Consortium (NAC). 293T cells were pur-chased from the American Type Culture Collection (ATCC). PlasmidDNA encoding BG505-SOSIP.664-D7324, its purified cleaved trimericprotein (64), and pcDNA5-FRT BG505 furin A (65) were kindly providedby John Moore, Weill Cornell Medical College, New York. Purified gp120TripleMut core protein (66) was obtained from Richard Wyatt, TheScripps 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 clonedin pcDNA 3.1/V5-His-TOPO vector and transfected into 293T cells usingpolyethyleneimine (PEI). Supernatants containing soluble gp120 were fil-tered through 0.45-�m-pore-size filters and subsequently purified usingnickel-nitrilotriacetic acid (Ni-NTA) agarose matrix (Qiagen Inc.) by elutionwith phosphate-buffered saline (PBS) containing 300 mM imidazole (pH8.0). The purified monomeric gp120 protein was extensively dialyzed withPBS (pH 7.4), concentrated using Amicon ultracentrifugal filters (MilliporeInc.) with a 30-kDa cutoff, and stored at �80°C until further use.

The trimeric BG505-SOSIP.664 protein was purified using 293Fcells essentially as described by Sanders et al. (69). Briefly, the 293Fcells 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 fromGalanthus nivalis (Sigma Inc.). The nonspecifically bound proteins then

were washed in PBS (pH 7.4) supplemented with 0.5 M NaCl. The boundproteins then were eluted using 0.5 M methyl �-D-mannopyranoside,extensively dialyzed with 1� PBS, and concentrated. BG505-SOSIP.664was further purified by Sephadex G-200 size exclusion chromatography(AKTA; GE). Trimeric protein fractions were collected and pooled, theirquality was assessed by running in blue native polyacrylamide gel electro-

TABLE 1 Neutralization breadth of Protocol G G37080 plasma samplescollected 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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phoresis (BN-PAGE), and they were favorably assessed for their ability tobind to only neutralizing and not to nonneutralizing and MPER-directedmonoclonal antibodies as described elsewhere (64) by enzyme-linked im-munosorbent assay (ELISA).

Depletion of plasma antibodies by monomeric gp120 and trimericgp140 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 plasmaantibodies 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 ofboth monomeric and trimeric Env proteins in coupling buffer [0.1 MNaBO4, 1 M (NH4)2SO4; pH 9.4] overnight at 37°C for 16 to 24 h. Proteinsbound to magnetic beads were separated from unbound proteins using aDynaMag 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 blockthe unbound sites. The antigenic integrity of both 4-2.J41 monomericgp120 and BG505-SOSIP.664 bound to the beads was assessed for theirability to bind VRC01 and 4E10 MAbs (for monomeric gp120) andPGT121, 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 beadsat room temperature for 45 min. Unbound plasma antibodies were sepa-rated from those that were bound to protein-coated beads using aDynaMag 15 magnet as described above. This step was repeated 4 to 5times for the depletion of plasma antibodies by monomeric gp120 and 10to 12 times in the case of BG505-SOSIP.664-coated beads. As a negativecontrol, G37080 plasma antibodies were depleted with uncoated beads inparallel. In addition to ELISA, the percent depletion of G37080 plasmaantibodies was assessed by examining the sequential decrease in bindingof 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 monomeric4-2.J41 gp120 (1 �g/ml) in binding buffer containing 0.1 M NaHCO3 (pH8.6) and incubated overnight at 4°C. The gp120-plate was washed oncewith 1� PBS (pH 7.4) and blocked with 5% nonfat milk for 90 min at37°C. The plate then was washed three times with 1� PBS followed by theaddition of 100 �l of MAbs, as well as the depleted and undepleted plasmaantibodies 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 of1:3,000-diluted horseradish peroxidase (HRP)-conjugated anti-humanIgG (Jackson ImmunoResearch, Inc.), and further incubated for 45 min atroom temperature. Unbound conjugates were removed by washing withPBST, 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 proteinwas assessed essentially as described by Sanders et al. (69) in a sandwichELISA. Briefly, a high-binding microtiter plate (Nunc, Inc.) first wascoated with D7324 antibody at 10 �g/ml (Aalto Bio Reagents, Dublin,Ireland) followed by blocking extra unbound sites with 5% nonfat milkfor 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 roomtemperature. The extent of binding of G37080 plasma antibodies com-pared to that of known neutralizing monoclonal antibodies was assessedby the addition of primary and HRP-conjugated secondary anti-humanantibody as described above.

Neutralization assay. Neutralization assays were carried out usingTZM-bl cells as described before (68). Briefly, Env-pseudotyped viruses

were incubated with various dilutions of depleted plasma antibodies andincubated for an hour at 37°C in a CO2 incubator under humidified con-ditions, and subsequently 1 � 104 TZM-bl cells were added to the mixturein the presence of 25 �g/ml DEAE-dextran (Sigma, Inc.). The plates werefurther incubated for 48 h, and the degree of virus neutralization wasassessed by measuring relative luminescence units (RLU) in a luminom-eter (Victor X2; PerkinElmer Inc.).

Amplification, cloning, and mutagenesis of autologous HIV-1 envgenes. Autologous complete env genes were obtained from G37080plasma as described previously, with slight modifications (68). Briefly,viral RNA was extracted using a high-pure viral RNA kit (Roche Inc.) byfollowing manufacturer’s protocol, and cDNA was prepared by reversetranscription-PCR (RT-PCR) using a Superscript III first-strand synthesiskit (Invitrogen Inc.). rev-gp160 env genes were amplified using a Phusionhigh-fidelity DNA polymerase (New England BioLabs Inc.). The gp160amplicons 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 andas described previously (71).

Preparation of envelope-pseudotyped viruses. Pseudotyped viruseswere prepared by the cotransfection of envelope-expressing plasmid withan env-deleted HIV-1 backbone plasmid (pSG3�Env) into 293T cells in6-well tissue culture plates using a FuGENE6 transfection kit (PromegaInc.). Cell supernatants containing pseudotyped viruses were harvested 48h posttransfection and then stored at �80°C until further use. The infec-tivity assays were done in TZM-bl cells (1 � 105cells/ml) containingDEAE-dextran (25 �g/ml) in 96-well microtiter plates, and the infectivitytiters were determined by measuring the luciferase activity using Briteliteluciferase substrate (PerkinElmer Inc.) with a Victor X2 luminometer(PerkinElmer Inc.).

RESULTSIdentification 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) toscreen and identify plasma antibodies obtained from Indian do-nors chronically infected with HIV-1 clade C, with substantialbreadth toward neutralizing cross-clade HIV-1 primary variants, and(ii) to elucidate the molecular specificities associated with neutraliza-tion breadth. Our hypothesis was that given the genetic distinctness ofclade C viruses of Indian and non-Indian origin, as well the likelydifferences in host genetics between populations and their ancestralorigins associated with the modulation of humoral immune re-sponses, the specificities of antibodies developed in vivo associatedwith neutralization breadth and potency would be different.

Through screening of two hundred plasma samples obtainedfrom chronically infected ART naive Indian patients against apanel of 57 pseudoviruses containing Envs of distinct clades andgeographical origins (Fig. 1A), we identified one donor (G37080)whose plasma showed exceptional neutralization breadth. DonorG37080 serum neutralized �90% of the 57 different pseudovi-ruses tested, with a median 50% inhibitory dose (ID50) value of533.03 (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 molecularspecificities, were retained and/or improved, as we expected thatduring 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 thevisit 2 samples) were found to exhibit neutralization breadth com-

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parable to that of visit 1 plasma. Overall, G37080 BCN plasma wasfound to potently neutralize pseudoviruses containing Indianclade C Env with a neutralization score of 2.5 (13). Furthermore,the neutralization sensitivity of Env-pseudotyped viruses wasfound 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 stronghumoral immune response to HIV-1 was mounted in donorG37080 and was maintained over time.

Evidence that G37080 BCN plasma antibodies do not targetepitopes in CD4bs, MPER, and known glycan and nonglycanresidues in variable domains of Env. We first examined whetherthe G37080 BCN plasma contains antibodies directed to theCD4bs on Env. Plasma samples obtained from both visits werepretreated with 25 �g/ml of TripleMut core protein (66), whichwas a concentration that we found to inhibit the neutralization of25711-2.4 pseudovirus by VRC01 MAb by �95%. Pretreatedplasma 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. Asimilar observation was made when these plasma antibodies werepretreated with RSC3 core protein (22). In addition, the G37080BCN plasma antibodies were found to efficiently neutralizeIgG1b12- and VRC01-resistant viruses (data not shown). Ourdata indicated that the G37080 BCN plasma antibodies do notcontain CD4bs-directed neutralizing antibodies.

To elucidate whether the BCN plasma antibodies are directedto MPER in gp41, we used HIV-2/HIV-1 chimeric viruses (73)that expressed minimal residues of HIV-1 MPER containingepitopes required for MPER-directed MAbs, such as 2F5, 4E10,Z13e, and 10E8. As shown in Table 2, the G37080 BCN plasmafrom both visits was found to show modest neutralization ofHIV-2 expressing HIV-1 clade C MPER (7312-C1C), with ID50

values of 306.42 and 371.02, respectively. We also found that thedepletion of G37080 plasma with a clade C MPER peptide (C1C)completely abolished the sensitivity of 7312A-C1C virus to

FIG 1 (A) Genetic divergence of amino acid sequences of 57 HIV-1 Env (gp160) pseudoviruses used to assess neutralization breadth and potency of G37080 BCNplasma. The maximum likelihood bootstrapped consensus phylogenetic tree was constructed using the Jones-Taylor-Thornton (JTT) substitution model with50 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 againstpseudotyped viruses expressing HIV-1 Env representing different clades and origins. Neutralization titers (median ID50 values) were obtained by titratingEnv-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 �80%) preincubated with different concentrations, as indicated, with TripleMut core (A) and RSC3 (B) proteinswere examined for their ability to neutralize 25711-2.4 Env pseudotyped virus in a TZM-bl cell neutralization assay. Note that while VRC01 preabsorbed withboth TripleMut and RSC3 proteins showed inhibited neutralization of 25711-2.4 in a dose-dependent manner, no such effect was observed with G37080 BCNplasma, indicating the absence of CD4bs-directed neutralizing antibodies.

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G37080 plasma (Table 3). Our data suggest that although theG37080 BCN plasma neutralized 7312-C1C, the presence ofMPER-directed antibodies was not associated with neutralizationbreadth.

We next investigated whether the plasma antibodies of the do-nor G37080 target residues in variable loops, particularly in V1V2and V3 regions, which have been shown in several studies to beepitopes targeted by BCN antibodies on HIV-1 Env. We firsttested the extent of neutralization by G37080 BCN plasma anti-bodies of Env-pseudotyped viruses lacking glycans at positions160 (N160) and 332 (N332) in the V2 region and V3 base, respec-tively, and also R166 and K169 in the V2 region, which are majortargets 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. Similarobservations were found with R166A and K169A in the 93IN905Env backbone. Taken together, our observations indicate thatG37080 BCN plasma antibodies did not utilize these residues inV2 and V3 regions for neutralization breadth; these have beenidentified as important epitopes recognized by broadly neutraliz-ing antibodies elicited in clade C infection as described before (35,41, 56).

Association of neutralization breadth of G37080 plasma withrecognition of conformational epitopes on cleaved trimeric Envbut not with that in monomeric gp120 or MPER. In order toexamine whether broad neutralization conferred by the G37080plasma antibodies was through the recognition of epitopes onmonomeric gp120 or cleaved near-native Env trimers, we testedthe binding of G37080 serum IgG to monomeric 4-2.J41 gp120and soluble gp140 (BG505-SOSIP.664) by ELISA. We found that

in addition to the monomeric 4-2.J41 gp120 (Fig. 3A), G37080serum polyclonal IgG was found to efficiently bind to the BG505SOSIP.664-D7324 soluble trimeric Env (Fig. 3B), indicating thatthe G37080 plasma primarily contains neutralizing antibodiesthat 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 testedthe ability of G37080 plasma antibodies depleted of both mono-meric and trimeric Envs, as well as of MPER peptides, to neutralizea set of Env-pseudotyped viruses, which were found to be sensitiveto this particular plasma sample. Purified 4-2.J41 monomericgp120, BG505-SOSIP.664 trimeric gp140, and C1C MPER pep-tide bound to the magnetic beads were used to deplete G37080plasma antibodies as described in Materials and Methods. Thedepleted BCN G37080 antibodies first were assessed for theirbinding to 4-2.J41 gp120 monomers, BG505-SOSIP.664-D7324,and C1C peptide and compared to undepleted plasma antibodiesby ELISA. As shown in Fig. 3C and D, G37080 plasma depletedwith monomeric gp120 and trimeric gp140, respectively, had sig-nificantly reduced binding activity against the respective solubleproteins. Similar observation was made with MPER peptide (datanot shown). The depleted plasma antibodies subsequently wereassessed for neutralization activity using a panel of 12 Env-pseu-dotyped viruses that were susceptible to untreated G37080 plasmaantibodies as mentioned above. As shown in Table 3, depletionwith 4-2.J41gp120 monomer and C1C peptide did not show anychange in neutralization breadth of G37080 plasma antibodies,while depletion with BG505-SOSIP.664 showed a significant re-duction in virus neutralization. Similar observations were madewith the BG505-SOSIP.664-depleted PGT121 and C1C peptide-depleted 4E10 MAbs, which lost the ability to efficiently neutralizeEnv-pseudotyped viruses (16055 and ZM233.6) and HIV-2/HIV-1 (7312A-C1C) chimeric virus compared to their undepletedcounterparts (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 toG37080 BCN plasma depleted with monomeric and trimeric Envs andC1C peptide

Env-pseudotypedvirus

Fold reduction in neutralization (ID50)a

gp140 trimer(BG505-SOSIP.664)

gp120 monomer(4-2.J41)

MPER (C1Cpeptide)

25710-2.3 �10.30 1.3 0.8325711-2.4 �8.52 1.4 1.443-5.J25 �7.85 0.9 0.844-2.J41 12.11 1.1 1.04IAVI_C22 �15.92 1.2 1.1892BR020 �35.08 1.1 1.3493IN905 3.41 1.2 0.94JRCSF �8.75 0.5 0.93Q23.17 �23.28 1.0 0.98Du156.12 �15.73 0.8 1.61HVTR-PG80v1.eJ7 �10.03 0.9 1.12HVTR-PG80v1.eJ19 �15.60 0.5 1.18HIV-2 7312A-C1C �10a Fold reduction in virus neutralization was obtained by comparing the neutralizationtiter (ID50 values) of panel viruses against undepleted and depleted G37080 plasma.ID50 values are reciprocal dilutions at which the undepleted and depleted plasmaconferred 50% neutralization of the Env-pseudotyped viruses in TZM-bl cells.

TABLE 2 Examination of specificity of G37080 plasma antibodiesobtained at both visits to HIV Env

HIV type Region

Fold decrease inID50

a for plasmaat visit:

1 2

HIV-1 Env mutantsHIV-1 25711-2.4 N160A V2 1.02 �1HIV-1 25711-2.4 R166A �1 �1HIV-1 25711-2.4 K169E �1 �1HIV-1 93IN905 R166A �1 �1HIV-1 93IN905 K169A �1 �1HIV-1 25711-2.4 N332A V3 1.52 �1HIV-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 �20 �20HIV-2 7312A-C1C Clade C MPER 306.42 371.02HIV-2 7312A-C3 2F5 epitope �20 �20HIV-2 7312A-C4 4E10, Z13e1, and

10E8 epitopes334.34 371.27

HIV-2 7312A-C6 4E10 minimal epitope �20 223.90HIV-2 7312A-C7 2F5 minimal epitope �20 �20

a ID50 values refer to the reciprocal dilution that conferred 50% neutralization ofviruses in a TZM-bl assay. Assays were done in duplicate and were repeated morethan three times.

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HIV-1 (7312A-C1C) chimeric virus, indicating that the presenceof residual traces of MPER-directed antibodies (as shown inTable 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 BCNG37080 plasma antibodies predominantly target epitopes in theV1V2 region (Table 4) in gp120. Our data clearly indicate a cor-relation between neutralization breadth and binding of theG37080 BCN plasma antibodies to the conformational epitopeson cleaved trimeric gp120, likely in the V1V2 region; however, wedo not rule out the possibility that this BCN plasma targets otherdiscontinuous 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 thespecificity of the G37080 plasma antibodies, we examined the de-gree of susceptibility of pseudoviruses prepared using env genesamplified 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 resistantto its contemporaneous plasma antibodies, while Env proteinsobtained 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 G37080BCN antibody specificity, we prepared chimeric Envs between

a sensitive (HVTR-PG80v1.eJ7 and HVTR-PG80v1.eJ19) and aresistant (HVTR-PG80v2.eJ38) autologous Env by first swap-ping the V1V2 regions, as their amino acid sequences differedmaximally in this region (Fig. 4B). As shown in Table 4, theinsertion of the V1V2 sequences of HVTR-PG80v1.eJ7 andHVTR-PG80v1.eJ19 into HVTR-PG80v2.eJ38 conferred Env-pseudotyped viruses expressing HVTR-PG80v2.eJ38 Env withsensitivity to G37080 visit 2 plasma antibodies enhanced by�25- and �12-fold, respectively. Conversely, the neutraliza-tion susceptibilities of the Env-pseudotyped viruses expressingHVTR-PG80v1.eJ7 and HVTR-PG80v1.eJ19, which containedHVTR-PG80v2.eJ38 V1V2 sequence corresponding to visit 2G37080 plasma, were found to be reduced by �45- and �23-fold, respectively. We noted that alterations of regions otherthan the V1V2 loop in the autologous Env did not confer anychange in neutralization sensitivity (Table 4). To further spec-ify residues in the V1V2 loop associated with neutralizationsensitivity and resistance of autologous Envs, chimeric Envsand point mutants were prepared and tested for their degree ofmodulation in susceptibility to autologous G37080 plasma ob-tained from the second visit. As shown in Table 4, we found thatthe 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 �50 and �37-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 wasassessed by ELISA. IgG purified from HIV-negative healthy donor and known MAbs were used as controls. The extent of binding of the depleted and undepletedG37080 BCN plasma with magnetic beads coated with 4-2.J41 monomeric gp120 (C) and BG505-SOSIP.664 cleaved trimeric gp140 to their respective proteinsby ELISA. Note that binding to trimeric protein by ELISA was assessed by using BG505-SOSIP.664 tagged with the D7324 epitope to maintain the nativeconformation of trimeric Env as described before (69).

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Env. In agreement with this result, V1 of HVTR-PG80v2.eJ38,when transferred into the sensitive Envs described above, in-creased neutralization resistance by �27- and �28-fold, respec-tively, to the G37080 visit 2 BCN plasma antibodies. We observedthat the removal of a glycan at the 138 position in V1 (T140D) inHVTR-PG80v1.eJ19 mediated the enhanced sensitivity of this Envto G37080 plasma by 2.64-fold (Table 4). Concurrent with thisobservation, we found that the alteration of the V1 region ofPG80v1.eJ19 with the T140D substitution in PG80v2.eJ38 Envexhibited enhanced susceptibility compared to that of thePG80v2.eJ38 Env chimera containing the PG80v1.eJ19 V1 loop, asshown in Table 4. Our data indicate that N138 glycan masks thePG80v1.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 P147residues 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 thepoint mutants showed sensitivities to PG9 MAb comparable tothose of their wild types (Table 5), indicating that the shifts inneutralization susceptibilities were not due to changes in Env con-formation. Moreover, we noted that both the sensitive and theresistant autologous Envs contain T332 in the V3 base, clearlyindicating that the absence of N332 was not associated with resis-tance to autologous neutralization. Similar observations weremade with respect to N160, R166, and K169 amino acid residues,further consolidating that the neutralization conferred by G37080BCN plasma antibodies was not associated with antibody target-ing these epitopes in autologous Envs, and is likely the case for allof 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 breadthand potency is key in the design and development of suitable Env-based immunogens capable of eliciting similar antibody responsesupon vaccination. In the present study, we characterized the mo-lecular specificity of plasma antibodies obtained from an Indianelite neutralizer (G37080) infected with HIV-1 clade C that dis-played exceptional cross-neutralization of different clades of distinctgeographical origins. The G37080 plasma was found to contain themost broad and potent cross-neutralizing antibodies among the twohundred plasma samples obtained from Indian patients chronicallyinfected 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 epitopemapping, we examined the specificity of the G37080 BCN plasmaby making use of mutant viruses with specific point substitutionsof known neutralizing epitopes with nonspecific amino acids andvia depletion with monomeric and trimeric Envs in addition toMPER peptide. The G37080 plasma antibodies did not show de-pendence on the N160/K169 and N332 epitopes in the V2 apexand V3 base, respectively. Our data also are consistent with thetarget epitopes of the G37080 BCN antibodies being distinct fromthose which are recognized by 2G12 (74), PGT121-128 (17), andPGT130-131 and PGT135 (19) (e.g., residues at the following po-

TABLE 4 Dissection of specificity for autologous neutralizationresistance

Chimera and point mutant

Neutralization potency(ID50)

Foldchangea Effectb

PG80v1.eJ7 Env backboneV1V2 loop

PG80v2.eJ38 (V1V2) in v1.eJ7 45.75 DecreasePG80v2.eJ38 (V1) in v1.eJ7 27.97 DecreasePG80v2.eJ38 (V2) in v1.eJ7 1.35 No effect

Point mutationsPG80v1.eJ7 (D133N) 3.25 DecreasePG80v1.eJ7 (S143G) 0.87 No effectPG80v1.eJ7 (D133N S143G) 2.88 DecreasePG80v1.eJ7 (T147P) 8.21 Decrease

PG80v1.eJ19 Env backboneV1V2 loop

PG80v2.eJ38(V1V2) in v1.eJ19 23.05 DecreasePG80v2.eJ38 (V1) in v1.eJ19 28.61 DecreasePG80v2.eJ38 (V2) in v1.eJ19 1.87 Increase

Point mutationsPG80v1.eJ19 (D133N) 2.51 DecreasePG80v1.eJ19 (T139A) 0.99 No effectPG80v1.eJ19 (T139A T140D) 2.64 IncreasePG80v1.eJ19 (N143G) 1.38 No effectPG80v1.eJ19 (T139A T140D N143G) 2.16 IncreasePG80v1.eJ19 (T145P) 3.24 Decrease

PG80v2.eJ38 Env backboneV1V2 loop

PG80v1.eJ7 (V1V2) in v2.eJ38 26.56 IncreasePG80v1.eJ7 (V1) in v2.eJ38 49.62 IncreasePG80v1.eJ7 (V2) in v2.eJ38 1.07 No effectPG80v1.eJ19(V1V2) in v2.eJ38 12.81 IncreasePG80v1.eJ19 (V1) in v2.eJ38 37.60 IncreasePG80v1.eJ19 (V2) in v2.eJ38 0.94 No effect

Other regions in gp120PG80v1.eJ7 (V3C3) in v2.eJ38 0.84 No effectPG80v1.eJ7 (V3C3V4C4) in v2.eJ38 0.90 No effectPG80v1.eJ7 (C4V5C5) in v2.eJ38 1.08 No effectPG80v1.eJ19 (V3C3) in PG80v2.eJ38 1.09 No effectPG80v1.eJ19 (V3C3V4C4) in PG80v2.eJ38 1.15 No effectPG80v1.eJ19 (C4V5C5) in PG80v2.eJ38 0.96 No effect

Point mutationsPG80v1.eJ19 V1 (T139AT140D) in

v2.eJ3853.91 Increase

PG80v2.eJ38 (N133D) 4.72 IncreasePG80v2.eJ38 (G143S) 0.94 No effectPG80v2.eJ38 (N133DG143S) 3.63 IncreasePG80v2.eJ38 (P147T) 4.11 Increase

Heterologous Env chimeraV1V2 loop

16055-2.3 (25711-2.4 V1V2) 18.38 Increase25711-2.4 (16055-2.3 V1V2) 2.03 DecreaseCAP45 (25711-2.4 V1V2) 16.84 Increase25711-2.4 (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).

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sitions: 295, 297, 301, 332, 334, 386, 388, 392, 394, 448, and 450);thus, BCN G37080 antibodies appear to target a new epitope. Ourdata highlighting the N332-independent development of neutral-izing antibodies in clade C-infected donor G37080 also differ fromrecent findings (57, 75–77) associating N332 with the develop-ment of broad and potent neutralizing antibody, especially inclade C infection in African donors. Moreover, recent studies in-dicating the role of K169 as a target of BCN antibodies obtainedfrom a clade C-infected South African donor (41, 56) and theobservation that vaccine-induced protection in the RV144 vaccinetrial was associated with antibodies targeting epitopes, includingK169 in the V2 apex (27, 78), prompted us to examine whether

broad neutralization of the G37080 plasma antibodies also wasdependent on the K169 epitope. In the present study, the neutral-ization potency of G37080 was unaffected by N160A/K169Aknockout mutations, and we also observed that both sensitive andresistant autologous Envs obtained from both visits contain N160and K169 in the V2 region. Hence, owing to the lack of associationof neutralization breadth of the G37080 BCN antibodies withN160, K169, and N332 dependencies, our study further high-lighted that there is a likelihood of differences in the developmentpathway 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 ina clade C-infected donor with shifts in antibody specificities fromthe recognition of epitopes in V2 to the CD4bs. In the presentstudy, the G37080 neutralizing plasma antibodies obtained fromboth 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 ofMPER-directed antibodies from G37080 plasma was found, al-though a negligible antibody titer (1:300 reciprocal dilutions) tothe 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 G37080plasma to contemporaneous plasma antibodies. Note that both of the Envs obtained from follow-up G37080 plasma (visit 2) were found to be resistant tocontemporaneous autologous plasma, while Envs obtained from visit 1 G37080 plasma were found to be sensitive to follow-up plasma antibodies. (B) Alignmentof V1V2 amino acid sequences of sensitive and resistant autologous Envs obtained at both visits was done by using seqpublish, available at the HIV Los Alamosdatabase (www.hiv.lanl.gov). Key residues that mediate autologous neutralization resistance are highlighted.

TABLE 5 Sensitivity of wild type, chimera, and point mutants ofautologous Envs to PG9 MAb

Env chimera and mutant ID50

PG80v1.eJ7 (wild type) 0.12PG80v1.eJ19 (wild type) 0.97PG80v1.eJ19 (T139A T140D) 0.77PG80v2.eJ38 (wild type) 0.02PG80v1.eJ7 (V1) in v2.eJ38 0.04PG80v1.eJ19 (V1) in v2.eJ38 0.05PG80v1.eJ19 (V1) (T139A T140D) in v2.eJ38 0.06PG80v2.eJ38 (N133D) 0.01PG80v2.eJ38 (P147T) 0.04

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ples from both visits. However, the neutralization breadth of theG37080 plasma was not found to be associated with the presenceof MPER-directed antibody. Nonetheless, we do not rule out thepossibility that in the further course of infection, this donor maybe able to develop MPER-directed antibodies.

Recent studies have shown that neutralizing antibodies that targetconformational epitopes bind exclusively to the cleaved near-nativetrimeric Envs (15, 64, 80, 81). In the present study, we found that theabsorption 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 nonneutralizingantibodies that were reported to bind to BG505-SOSIP.664 trimericEnv (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, canbe utilized in selecting antigen-specific memory B cells, as reportedearlier (82), from donor G37080 toward isolation of MAb correlatingwith broad neutralization displayed by the plasma antibodies.

We made use of env clones obtained from autologous G37080plasma from both time points to refine the fine specificity of theG37080 BCN plasma antibodies. By examining chimeric Envs andmutant viruses, we identified key residues in the V1 loop associ-ated with neutralization resistance. Interestingly, the Env chimeraand mutant viruses showed susceptibility to PG9 MAb compara-ble to that of their respective wild-type Envs, indicating that theydid not alter Env conformation. We identified a glycan at the 133position and a proline residue at the 147 position within the V1loop of the resistant Env (PG80v2.eJ38) that were found to beassociated with neutralization escape, which indicated that theseare contact sites for the G37080 BCN plasma antibodies. Thus,from our study we conclude that changes in V1 loop sequence areassociated with the escape of autologous viruses to the BCNG37080 plasma. Additionally, an examination of the degree ofsusceptibilities of pseudoviruses expressing chimeric heterolo-gous Envs to the G37080 plasma revealed that the BCN plasmaantibodies predominantly target epitopes in the V1V2 region ingp120. However, we do not rule out the possibility of the contri-bution of other discontinuous epitopes in gp120 in mediatingneutralization breadth. The isolation and identification of mono-clonal antibodies from this elite neutralizer donor (G37080) willhelp precisely map specific epitopes associated with neutralizationbreadth and potency.

In summary, we identified an HIV-1-infected elite neutralizerwhose plasma showed exceptional neutralization breadth, and weprovided evidence that it targets novel conformational epitopeson trimeric Env, predominantly in the V1V2 region, not reportedpreviously. Moreover, the neutralization resistance of the autolo-gous Envs to G37080 plasma is associated with substitutions ofnovel residues within the V1 loop that form the key contact pointsof the BCN plasma antibody. The identification of novel epitopesassociated with broad neutralization of HIV-1, in particular themajor circulating clade C strains, will significantly contribute toefforts toward effective immunogen design.

ACKNOWLEDGMENTS

We thank all of the Protocol G study participants registered with YRGCare, Chennai, all of the research staff members at the Protocol G clinicalcenter 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 SOSIPtrimer HIVRAD team, Weill Cornell Medical College, New York, for pro-viding us with BG505.SOSIP.664 plasmid DNA and purified protein. Wethank David Montefiori, Lynn Morris, Pascal Poignard, and Richard Wy-att for making available many reagents used in our study. The followingreagent was obtained through the NIH AIDS Reagent Program, Divisionof 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 ofForeign Affairs of Denmark, Irish Aid, the Ministry of Finance of Japan,the Ministry of Foreign Affairs of the Netherlands, the Norwegian Agencyfor Development Cooperation (NORAD), the United Kingdom Depart-ment for International Development (DFID), and the United StatesAgency for International Development (USAID). The full list of IAVIdonors is available at www.iavi.org. The contents are the responsibility ofthe International AIDS Vaccine Initiative and do not necessarily reflectthe views of USAID or the United States Government.

The International AIDS Vaccine Initiative has filed a patent relating tothe autologous HIV-1 clade C envelope clones (J. Bhattacharya, S. Desh-pande, S. Patil, R. Kumar, and B. K. Chakrabarti, U.S. patent application62/254,971).

FUNDING INFORMATIONThis study was made possible by the generous support of the Americanpeople through the United States Agency for International Development(USAID) through the IAVI, support from a THSTI-IAVI HIV VaccineDesign Program grant through the Department of Biotechnology, Gov-ernment of India, in part by a grant from the Department of Science andTechnology, Government of India (DST/INT/SAFR/Mega-P3/2011 toJ.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 studydesign, data collection and interpretation, or the decision to submit thework for publication.

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