1

Conformational Epitope-Specific Broadly Neutralizing Plasma Antibodies Obtained from 1

an HIV-1 Clade C Infected Elite Neutralizer Mediate Autologous Virus Escape through 2

Mutations in V1 Loop 3

4

Shilpa Patil11¶,

, Rajesh Kumar1¶,

, Suprit Deshpande1¶

, Sweety Samal1, Tripti Shrivastava

1, 5

Saikat Boliar1, Manish Bansal

1, Nakul Kumar Chaudhary

1, Aylur K. Srikrishnan

2, Kailapuri G. 6

Murugavel2, Suniti Solomon

2, Melissa Simek

3, Wayne C. Koff

3, Rajat Goyal

3, Bimal K. 7

Chakrabarti1, 3

, Jayanta Bhattacharya1, 3,

# 8

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1. HIV Vaccine Translational Research Laboratory, Translational Health Science and 10

Technology Institute, Faridabad, Haryana- 121001, India 11

2. Y.R. Gaitonde Research and Care Center, Chennai, India 12

3. International AIDS Vaccine Initiative, New York, USA 13

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Running title: HIV-1 clade C plasma confer cross clade neutralization 15

16

Key words: HIV-1, neutralizing antibody, envelope, plasma, clade C, protocol G, V1V2 loop 17

Abstract: 247 words 18

¶ Equal contribution 19

# Corresponding author 20

Tel: +91-01242867705 21

E-mail: JBhattacharya@iavi.org / JBhattacharya@thsti.res.in 22

23

JVI Accepted Manuscript Posted Online 13 January 2016J. Virol. doi:10.1128/JVI.03090-15Copyright © 2016, American Society for Microbiology. All Rights Reserved.

2

Abstract 24

Broadly neutralizing antibodies isolated from infected patients who are elite neutralizers 25

have identified targets on HIV-1 envelope (Env) glycoprotein that are vulnerable to antibody 26

neutralization; however, it is not known whether infection established by majority of the 27

circulating clade C strains in Indian patients elicit neutralizing antibody responses against any of 28

the known targets. In the present study, we examined the specificity of a broad and potent cross 29

neutralizing plasma obtained from an Indian elite neutralizer infected with HIV-1 clade C. This 30

plasma neutralized 53/57 (93%) HIV pseudoviruses prepared with Env from distinct HIV clades 31

of different geographical origin. Mapping studies using gp120 core protein, single residue 32

knockout mutants and chimeric viruses revealed that G37080 BCN plasma lacks specificities to 33

the CD4 binding site, gp41 membrane proximal external region, N160, N332 glycans as well as 34

R166 and K169 in V1-V3 region and are known predominant targets for BCN antibodies. 35

Depletion of G37080 plasma with soluble trimeric BG505-SOSIP.664 Env (but neither with 36

monomeric gp120 nor with clade C MPER peptides), resulted in significant reduction of virus 37

neutralization, suggesting that G37080 BCN antibodies mainly target epitopes on cleaved 38

trimeric Env. Further examination of autologous circulating Envs revealed association of 39

mutation of residues in V1 loop that contributed in neutralization resistance. In summary, we 40

report identification of plasma antibodies from a clade C infected elite neutralizer that mediates 41

neutralization breadth via epitopes on trimeric gp120 not yet reported and confer autologous 42

neutralization escape via mutation of residues in V1 loop. 43

44

3

Importance 45

46

A preventive vaccine to protect against HIV-1 is urgently needed. HIV-1 envelope 47

glycoproteins are targets of neutralizing antibodies and represent a key component for 48

immunogen design. Mapping of epitopes on viral envelopes vulnerable for immune evasion will 49

aid in defining targets of vaccine immunogens. We identified novel conformational epitopes on 50

viral envelope targeted by broadly cross neutralizing antibodies elicited in natural infection in an 51

elite neutralizer infected with HIV-1 clade C. Our data extend our knowledge on neutralizing 52

epitopes associated with virus escape and would potentially contribute in immunogen design and 53

antibody based prophylactic therapy. 54

55

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56

Introduction 57

58

Broadly neutralizing antibodies (BNAbs) target trimeric envelope glycoprotein (Env) spikes 59

of the Human Immunodeficiency Virus Type 1 (HIV-1). Characterization of the BNAbs has provided 60

key clues towards design and development of both prophylactic and therapeutic vaccines (7, 17, 30, 61

34, 35, 37). A small proportion of individuals chronically infected with HIV-1 develop BNAbs (5, 62

12, 21, 38, 52, 62, 66, 69) and isolation of several broad and potent neutralizing monoclonal 63

antibodies (bNAb) from such individuals with distinct molecular specificities to viral envelope (Env) 64

protein are reported (18, 31, 32, 64, 74, 76, 77, 79, 81). The cross neutralizing serum antibodies 65

obtained from such individuals (also referred to as ‘elite neutralizers’) with considerable breadth 66

target epitopes on structurally conserved regions of Env such as CD4 binding site (CD4bs) (11, 33, 67

65, 79), V1V2 including glycan moieties (39, 50, 74, 76), gp120-gp41 interface (3, 64) and the 68

membrane proximal external regions (MPER) (31, 45, 46, 83). Several studies have indicated that the 69

variable regions within the HIV-1 gp120 contain epitopes targeted by the autologous as well as 70

BNAbs (8, 14, 16, 28, 41, 59, 60, 73). Recently the V1V2 region has been linked to development 71

of broadly cross neutralizing (BCN) antibodies (16, 78) and the residues between 160 and 172 72

(notably R166S/K or K169A) in V1V2 have been demonstrated to be associated with virus 73

escape to autologous antibody response (16). Recent studies have further indicated that BCNAb 74

development in vivo is associated with antibody affinity maturation and co-evolution of virus 75

resulting in a considerable degree of somatic hypermutations (1, 13, 15, 16, 20, 29, 40, 63, 65, 75, 76, 76

80-82). Such information is crucial for design and development of suitable Env based immunogen 77

capable of eliciting broad and potent cross neutralizing antibodies through vaccination. 78

79

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While a number of studies on the molecular specificities of broadly neutralizing antibodies 80

obtained from African clade C infected individuals have been reported (2, 21-23, 25, 26, 41-44, 47, 81

51, 57, 58), knowledge on immune evasion in Indian clade C infected elite neutralizers is very 82

limited (53). 83

In the present study, we examined plasma samples obtained from two hundred asymptomatic 84

and anti-retroviral therapy (ART) naïve Indian HIV-infected donors and identified plasma with cross 85

neutralizing antibodies. The molecular specificities of plasma antibodies obtained from an HIV-1 86

clade C infected elite neutralizer was characterized in detail that displayed exceptional neutralization 87

breadth across clades of different geographical origins. Interestingly, we found that neutralization 88

breadth was associated with presence of unique epitopes on the trimeric gp120. 89

90

91

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92

Materials and Methods 93

94

Ethics statement. 95

The blood samples were collected under The IAVI Protocol G study from slow progressing anti-96

retroviral therapy (ART) naïve HIV-1 positive donors from Nellore District of the state of 97

Andhra Pradesh, Southern India by trained clinicians at the YRG Care hospital following 98

approval and clearance from the Institutional Review Board (IRB) and the Ethics Committee. 99

The serum and plasma samples collected were shipped to the HIV Vaccine Translational 100

Research Laboratory, Translational Health Science and Technology Institute for further 101

assessment and research on the neutralizing antibody response. 102

103

Plasmids, viruses, antibodies, proteins and cells. 104

Plasmids encoding HIV-1 envelopes representing distinct clades are shown in Table 1. 105

Monoclonal antibodies used in the study and TZM-bl cells were procured from the NIH AIDS 106

Research Reagents Reference program and from the IAVI Neutralizing Antibody Consortium 107

(NAC). 293T cells were purchased from the American Type Culture Collection (ATCC). 108

Plasmid DNA encoding BG505-SOSIP.664-D7324, its purified cleaved trimeric protein (55) and 109

pcDNA5-FRT BG505 Furin A (10) was kindly provided by Prof John Moore, Weill Cornell 110

Medical College, New York. Purified gp120 TripleMut core protein (19) was obtained from Prof 111

Richard Wyatt, The Scripps Research Institute through the IAVI Neutralizing Antibody 112

Consortium (NAC). HIV-2 7312A and its chimeric constructs were provided by Prof Lynn 113

Morris, NICD, Johannesburg. 114

115

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Purification of monomeric and trimeric Env proteins. 116

Codon optimized gp120 plasmid encoding clade C 4-2.J41 (4, 54) gp120 was cloned in pcDNA 117

3.1/V5-His-TOPO vector and transfected in 293T cells using polyethyleneimine (PEI). 118

Supernatants containing soluble gp120 were filtered through 0.45µm filter and subsequently 119

purified using Ni-NTA agarose matrix (Qiagen Inc.) by elution with phosphate buffered saline 120

(PBS) containing 300mM imidazole (pH 8.0). The purified monomeric gp120 protein was 121

extensively dialyzed with PBS (pH 7.4), concentrated using Amicon® Ultracentrifugal filers 122

(Millipore Inc.) with 30KDa cut off and stored in -80°C until further use. 123

The trimeric BG505-SOSIP.664 protein was purified using 293F cells essentially as 124

described by Sanders et al (61). Briefly, the 293F cells were transfected with plasmid DNA 125

encoding both BG505-SOSIP.664 gp140 envelope and furin (10). Supernatant containing soluble 126

BG505-SOSIP.664 gp140 was harvested 72 to 96 hours post transfection, filtered and passed 127

through a lectin agarose column obtained from Galanthus nivalis (Sigma Inc.). The 128

nonspecifically bound proteins were then washed in PBS (pH 7.4) supplemented with 0.5 M 129

NaCl. The bound proteins were then eluted using 0.5 M methyl alpha-D-manno-pyranoside, 130

extensively dialyzed with 1X PBS and concentrated. BG505-SOSIP.664 was further purified by 131

Sephadex G-200 size exclusion chromatography (AKTA, GE). Trimeric protein fractions were 132

collected, pooled, quality assessed by running in blue native polyacrylamide gel electrophoresis 133

(BN-PAGE) and favorably assessed for their ability to bind to only neutralizing and not to non-134

neutralizing and MPER directed monoclonal antibodies as described elsewhere (55) by ELISA. 135

136

Depletion of plasma antibodies by monomeric gp120 and trimeric gp140 Env proteins. 137

Purified soluble monomeric 4-2.J41 gp120 and trimeric BG505 SOSIP.664 proteins in addition 138

to the MPER peptide (C1C; encoding clade C sequence) (71) were used for depletion of plasma 139

8

antibodies, where purified proteins were covalently coupled to the MyOne Tosylactivated 140

Dynabeads (Life Technologies Inc.) according to the manufacturer’s protocol. Briefly, 30 mg of 141

beads were used to couple with 1 mg of both monomeric and trimeric Env proteins in coupling 142

buffer (0.1M NaBO4, 1M (NH4)2SO4; pH 9.4) overnight at 37ºC for 16-24 hrs. Proteins bound to 143

magnetic beads were separated from unbound using a DynaMag™ 15 magnet (Life 144

Technologies, Inc.). Beads bound to Env proteins were next incubated with blocking buffer [PBS 145

(pH 7.4), 0.1% bovine serum albumin (BSA; Sigma) and 0.05% Tween 20] at 37ºC to block the 146

unbound sites. The antigenic integrity of both 4-2.J41 monomeric gp120 and BG505-SOSIP.664 147

bound to the beads were assessed for their ability to bind VRC01 and 4E10 MAbs (for 148

monomeric gp120) and PGT121, F105 and 4E10 MAbs (for BG505-SOSIP.664) by flow 149

cytometry (FACS Canto, Becton and Dickinson, Inc.). 150

For depletion studies, G37080 plasma was diluted to 1:50 in DMEM containing 10% 151

Fetal Bovine Sera (FBS) and 500 µl of diluted plasma was incubated with 20µl of beads at room 152

temperature for 45 minutes. Unbound plasma antibodies were separated from ones those are 153

bound to protein coated beads using a DynaMag™ 15 magnet as described above. This step was 154

repeated 4-5 times for depletion of plasma antibodies by monomeric gp120 and 10-12 times in 155

case of BG505-SOSIP.664 coated beads respectively. As a negative control, G37080 plasma 156

antibodies were depleted with uncoated beads in parallel. In addition to ELISA, percent 157

depletion of G37080 plasma antibodies was assessed by examining the sequential decrease in 158

binding of protein coated beads with depleted plasma antibodies by FACS. PGT121 MAb was 159

taken as a positive control for checking depletion by BG505-SOSIP.664 trimeric Env. 160

161

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gp120 and gp140 ELISA 163

For gp120 ELISA, high binding polystyrene microtiter plate (Nunc, Inc.) was coated with 100μl 164

of monomeric 4-2.J41 gp120 (1μg/ml) in binding buffer comprising 0.1 M NaHCO3 (pH 8.6) 165

and incubated overnight at 4ºC. gp120 bound plate was washed once with 1X PBS (pH 7.4) and 166

blocked with 5% non-fat milk for 90 min at 37ºC. The plate was then washed three times with 167

1X PBS, followed by addition of 100μl of MAbs as well as the depleted and undepleted plasma 168

antibodies at different dilutions and incubated for 1hr at room temperature. The wells of the 169

ELISA plate were washed four times with PBS containing 0.1% Tween 20 (PBST) followed by 170

addition of 100µl of 1:3000 diluted HRP-conjugated anti-human IgG (Jackson Immunoresearch, 171

Inc.) and further incubated for 45 min at room temperature. Unbound conjugates were removed 172

by washing with PBST and color developed by addition of 100μl of 3, 3’, 5, 5’-173

tetramethylbenzidine (TMB) (Life Technologies, Inc.) substrate was added. Absorbance was 174

measured at 450 nm in a spectrophotometer. 175

Binding of antibodies to BG505-SOSIP.664-D7324 trimeric protein was assessed 176

essentially as described by Sanders et al (61) in a sandwich ELISA. Briefly, high binding 177

microtiter plate (Nunc, Inc.) was first coated with D7324 antibody at 10μg/ml (Aalto Bio 178

reagents, Dublin, Ireland) followed by blocking extra unbound sites with 5% non-fat milk for 90 179

min at 37ºC. 100 µl of BG505.664-D7324 trimeric protein (300ng/ml) was then added and 180

incubated for 45 mins at room temperature. The extent of binding of G37080 plasma antibodies 181

compared to known neutralizing monoclonal antibodies were assessed by addition of primary 182

and HRP-conjugated secondary anti-human antibody as described above. 183

184

185

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Neutralization assay. 186

Neutralization assays were carried out using TZM-bl cells as described before (54). Briefly, Env-187

pseudotyped viruses were incubated with varying dilutions of depleted plasma antibodies and 188

incubated for an hour at 37°C CO2 incubator under humidified condition and subsequently 1 X 189

104 TZM-bl cells were added into the mixture in presence of 25 μg/ml DEAE-dextran (Sigma, 190

Inc.). The plates were further incubated for 48 hours and the degree of virus neutralization was 191

assessed by measuring relative luminescence units (RLU) in a Luminometer (Victor X2, 192

PerkinElmer Inc.). 193

194

Amplification, cloning and mutagenesis of autologous HIV-1 envs. 195

Autologous complete env genes were obtained from G37080 plasma as described previously 196

with slight modification (54). Briefly, viral RNA were extracted using High Pure viral RNA kit 197

(Roche Inc.) following manufacturer’s protocol and cDNA prepared by RT-PCR using 198

Superscript-III first strand synthesis kit (Invitrogen Inc.). rev-gp160 env genes were amplified 199

using a Phusion hi fidelity DNA polymerase (New England Biolabs Inc.). The gp160 amplicons 200

were purified and ligated into pcDNA 3.1/V5-His-TOPO (Invitrogen Inc.) vector. Chimeric Envs 201

were prepared by overlapping PCR and point substitutions were made by Quikchange II kit 202

(Agilent technologies Inc.) following manufacturer’s protocol and as described previously (49). 203

204

Preparation of envelope pseudotyped viruses. 205

Pseudotyped viruses were prepared by co-transfection of envelope expressing plasmid with env-206

deleted HIV-1 backbone plasmid (pSG3ΔEnv) into 293T cells in 6-well tissue culture plates 207

using FuGENE6 Transfection kit (Promega Inc.). Cell supernatants containing pseudotyped 208

viruses were harvested 48 hours post-transfection and then stored at -80°C until further use. The 209

11

infectivity assays were done in TZM-bl cells (1 X 105cells/ml) containing DEAE-Dextran (25 210

μg/ml) in 96-well microtiter plates and the infectivity titers were determined by measuring the 211

luciferase activity using Britelite luciferase substrate (Perkin Elmer Inc.) with a Victor X2 212

Luminometer (Perkin Elmer Inc.). 213

214

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Results 215

216

Identification of an elite neutralizer with HIV-1 clade C infection whose plasma showed 217

exceptional neutralization breadth. 218

The present study under The IAVI Protocol G was designed (i) to screen and identify 219

plasma antibodies obtained from chronically infected Indian donors with HIV-1 clade C with 220

substantial breadth towards neutralizing cross clade HIV-1 primary variants and (ii) to elucidate 221

their molecular specificities associated with neutralization breadth. Our hypothesis was that the 222

genetic distinctness of clade C viruses of Indian and non-Indian origin, as well due to likely 223

differences in host genetics between populations with differences in their ancestral origin 224

associated with modulation of humoral immune responses, the specificities of antibodies 225

developed in vivo associated with neutralization breadth and potency would be different. 226

Through screening of two hundred plasma samples obtained from chronically-infected 227

ART naïve Indian patients against a panel of 57 pseudoviruses containing Envs of distinct clades 228

and geographical origins (Figure 1A), we identified one donor (G37080), whose plasma showed 229

exceptional neutralization breadth. Donor G37080 serum neutralized >90% of the 57 different 230

pseudoviruses tested with median ID50 value of 533.03 (Table 1, Figure 1B). 231

Follow up plasma sample from this donor (G37080) was subsequently obtained after 232

eight months to assess whether the neutralization breadth and potencies along with their 233

molecular specificities were retained and/or improved, expecting that during the course of 234

disease, breadth and potency of neutralizing antibodies broadens through somatic 235

hypermutations (70) and/or clonal selection processes. As shown in Figure 1B and Table 1, 236

follow up plasma antibodies of G37080 donor (referred to as Visit-2 samples) were found to 237

exhibit comparable neutralization breadth to that of visit 1 plasma. Overall, G37080 BCN plasma 238

13

was found to potently neutralize pseudoviruses containing Indian clade C Env with a 239

neutralization score of 2.5 (66). Furthermore, the neutralization sensitivity of Env-pseudotyped 240

viruses was found to be correlated with the serum IgG (data not shown), suggesting that the 241

broad neutralization was associated with IgG-specific response. Taken together, our data indicate 242

that a strong humoral immune response to HIV-1 was mounted in G37080 donor and was 243

maintained overtime. 244

245

Evidence that G37080 BCN plasma antibodies do not target epitopes in CD4bs, MPER and 246

known glycan and non-glycan residues in variable domains of Env 247

First, we examined whether the G37080 BCN plasma contains antibodies directed to 248

CD4bs on Env. Plasma samples obtained from both visits were pre-treated with 25µg/ml of 249

TripleMut core protein (19), which was a concentration that we found to inhibit neutralization of 250

25711-2.4 pseudovirus by VRC01 mAb by >95%. Pre-treated plasma was subsequently used to 251

neutralize pseudovirus 25711-2.4 Env, and as shown in Figure 2, no perturbation of G37080 252

neutralizing activity was observed against pseudovirus 25711-2.4. A similar observation was 253

made when these plasma antibodies were pre-treated with RSC3 core protein (79). In addition, 254

the G37080 BCN plasma antibodies were found to efficiently neutralize IgG1b12 and VRC01 255

resistant viruses (data not shown). Our data indicated that the G37080 BCN plasma antibodies do 256

not contain CD4bs directed neutralizing antibodies. 257

To elucidate whether the BCN plasma antibodies are directed to MPER in gp41, we used 258

HIV-2/HIV-1 chimeric viruses (24) that expressed minimal residues of HIV-1 MPER containing 259

epitopes required for MPER directed mAbs such as 2F5, 4E10, Z13e and 10E8. As shown in 260

Table 2, the G37080 BCN plasma from both the visits was found to show modest neutralization 261

of HIV-2 expressing HIV-1 clade C MPER (7312-C1C) with ID50 values of 306.42 and 371.02, 262

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respectively. We also found that depletion of G37080 plasma with a clade C MPER peptide 263

(C1C) completely abolished the sensitivity of 7312A-C1C virus to G37080 plasma (Table 3). 264

Our data suggest that although the G37080 BCN plasma neutralized 7312-C1C, presence of 265

MPER directed antibodies was not associated with neutralization breadth. 266

We next investigated whether the plasma antibodies of the donor G37080 target residues 267

in variable loops, particularly in V1V2 and V3 region that have been shown in several studies as 268

epitopes targeted by BCN antibodies on HIV-1 Env. First, we tested the extent of neutralization 269

by G37080 BCN plasma antibodies of Env pseudotyped viruses lacking glycans at 160 (N160) 270

and at 332 (N332) position in the V2 region and V3 base respectively, and also R166 and K169 271

in V2 region, which are major targets of recently identified broad and potent neutralizing 272

monoclonal antibodies. In order to test this, two clade C Envs (25711-2.4 and CAP239.G3) 273

containing N160A and N332A substitutions were tested and as shown in Table 2. Our data 274

indicate that the pseudoviruses containing Env expressing N160 or N332 substitutions have 275

identical sensitivities to G37080 plasma antibodies. Similar observations were found with 276

R166A and K169A in 93IN905 Env backbone. Taken together, our observations indicate that 277

G37080 BCN plasma antibodies did not utilize these residues in V2 and V3 regions for 278

neutralization breadth; which have been identified as important epitopes recognized by broadly 279

neutralizing antibodies elicited in clade C infection described before (16, 42, 78). 280

281

Association of neutralization breadth of G37080 plasma with recognition of conformational 282

epitopes on cleaved trimeric Env but not with that in monomeric gp120 or MPER. 283

In order to examine whether broad neutralization conferred by the G37080 plasma 284

antibodies was through recognition of epitopes on monomeric gp120 or cleaved near native Env 285

trimers, we tested binding of G37080 serum IgG to monomeric 4-2.J41 gp120 and soluble gp140 286

15

(BG505-SOSIP.664) by ELISA. We found that in addition to the monomeric 4-2.J41 gp120 287

(Figure 3A), G37080 serum polyclonal IgG was found to efficiently bind to the BG505 288

SOSIP.664-D7324 soluble trimeric Env (Figure 3B), indicating that the G37080 plasma 289

primarily contains neutralizing antibodies that targets epitopes on cleaved Env trimers. 290

We next examined whether binding of the G37080 plasma antibodies to epitopes on 291

cleaved BG505-SOSIP.664 trimeric envelope was associated with neutralization breadth. For 292

this, we tested the ability of G37080 plasma antibodies depleted with both monomeric and 293

trimeric Envs as well as with MPER peptides to neutralize a set of Env-pseudotyped viruses, 294

which were found to be sensitive to this particular plasma sample. Purified 4-2.J41 monomeric 295

gp120, BG505-SOSIP.664 trimeric gp140 and C1C MPER peptide bound to the magnetic beads 296

were used to deplete G37080 plasma antibodies as described in the ‘Materials and Methods’. The 297

depleted BCN G37080 antibodies were first assessed for their binding to 4-2.J41 gp120 298

monomers, BG505-SOSIP.664-D7324 and C1C peptide in comparison to undepleted plasma 299

antibodies by ELISA. As shown in Figure 3C and 3D, G37080 plasma depleted with monomeric 300

gp120 and trimeric gp140, respectively, had significantly reduced binding activity against 301

respective soluble proteins. Similar observation was made with MPER peptide (data not shown). 302

The depleted plasma antibodies were subsequently assessed for neutralization activity using a 303

panel of twelve Env pseudotyped viruses that were susceptible to untreated G37080 plasma 304

antibodies as mentioned above. As shown in Table 3, depletion with 4-2.J41gp120 monomer and 305

C1C peptide did not show any change in neutralization breadth of G37080 plasma antibodies, 306

while depletion with BG505-SOSIP.664 showed a significant reduction in virus neutralization. A 307

similar observations were made with the BG505-SOSIP.664 depleted PGT121 and C1C peptide 308

depleted 4E10 MAbs which lost the ability to efficiently neutralize Env-pseudotyped viruses 309

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(16055 and ZM233.6) and HIV-2/HIV-1 (7312A-C1C) chimeric virus compared to their 310

undepleted counterparts (data not shown); thus validating our data. Interestingly, C1C peptide 311

depleted G37080 plasma failed to neutralize HIV-2/HIV-1 (7312A-C1C) chimeric virus 312

indicating that the presence of residual traces of MPER directed antibodies (as shown in Table 2) 313

are not responsible for neutralization breadth. Furthermore, examination of chimeric Envs 314

prepared between the sensitive (25711-2.4) and resistant (16055-2.3 and CAP45.G3) Envs 315

indicated that the BCN G37080 plasma antibodies predominantly target epitopes in the V1V2 316

region (Table 4) in gp120. Our data clearly indicate a correlation between neutralization breadth 317

and binding of the G37080 BCN plasma antibodies to the conformational epitopes on cleaved 318

trimeric gp120, likely in the V1V2 region; however we do not rule out the possibility that this 319

BCN plasma targets other discontinuous epitopes in the gp120, but not in MPER. 320

321

Mutations in V1 region confer resistance of autologous viruses to the G37080 plasma 322

antibodies 323

In order to decipher the specificity of the G37080 plasma antibodies, we examined the 324

degree of susceptibility of pseudoviruses prepared using env genes amplified from 325

contemporaneous autologous G37080 plasma obtained at the baseline and follow ups visits. As 326

shown in Figure 4A, both the Envs obtained from visit 2 plasma (HVTR-PG80v2.eJ38 and 327

HVTR-PG80v2.eJ41) were found to be resistant to its contemporaneous plasma antibodies, 328

while Envs obtained from visit 1 plasma (HVTR-PG80v1.eJ7 and HVTR-PG80v1.eJ19) were 329

found to be modestly sensitive to visit 2 autologous G37080 plasma antibodies. To facilitate 330

mapping G37080 BCN antibody specificity, we prepared chimeric Envs between a sensitive 331

(HVTR-PG80v1.eJ7 and HVTR-PG80v1.eJ19) and the resistant (HVTR-PG80v2.eJ38) 332

17

autologous Envs by first swapping the V1V2 regions as their amino acid sequences differed 333

maximally in this region (Figure 4B). As shown in Table 4, substitution of V1V2 sequence of 334

HVTR-PG80v1.eJ7 and HVTR-PG80v1.eJ19 into HVTR-PG80v2.eJ38 conferred Env-335

pseudotyped viruses expressing HVTR-PG80v2.eJ38 Env with enhanced sensitivity to G37080 336

visit 2 plasma antibodies by >25 and >12-folds respectively. Conversely, the neutralization 337

susceptibilities of the Env-pseudotyped viruses expressing HVTR-PG80v1.eJ7 and HVTR-338

PG80v1.eJ19, which contained HVTR-PG80v2.eJ38 V1V2 sequence corresponding to visit 2 339

G37080 plasma, were found to be reduced by >45 and >23 folds respectively. We noted that 340

substitution of regions other than V1V2 loop in the autologous Env did not confer any change in 341

neutralization sensitivity (Table 4). To further narrow down residues in V1V2 loop, associated 342

with neutralization sensitivity and resistance of autologous Envs, chimeric Envs and point 343

mutants were prepared and tested for their degree of modulation in susceptibility to autologous 344

G37080 plasma obtained from second visit. As shown in Table 4, we found that the V1 sequence 345

but not the V2 sequence of the sensitive Envs (HVTR-PG80v1.eJ7 and HVTR-PG80v1.eJ19) 346

when transferred to the resistant HVTR-PG80v2.eJ38 Env, increased sensitivity to G37080 BCN 347

plasma antibodies by >50 and >37 folds respectively. In agreement with this result, V1 of 348

HVTR-PG80v2.eJ38 when transferred into the sensitive Envs stated above, increased 349

neutralization resistance by >27 and >28 folds respectively to the G37080 visit 2 BCN plasma 350

antibodies. We observed that removal of a glycan at the 140 position in V1 (T140D) in the 351

HVTR-PG80v1.eJ19 mediated enhanced sensitivity of this Env to G37080 plasma by 2.64 fold 352

(Table 4). Concurrent to this observation, we found that insertion of V1 region of PG80v1.eJ19 353

with T140D substitution in PG80v2.eJ38 Env exhibited enhanced susceptibility when compared 354

with that of PG80v2.eJ38 Env chimera containing PG80v1.eJ19 V1 loop as shown in Table 4. 355

18

Our data indicate that N138 glycan potentially mask the PG80v1.eJ19 Env from being efficiently 356

neutralized by the autologous plasma compared to that of its contemporaneous counterpart 357

PG80v1.eJ7 Env. Fine scanning of V1 regions of the autologous Envs further revealed that N133 358

glycan motif and P147 residues in the PG80v2.eJ38 Env played significant role in neutralization 359

resistance to G37080 BCN autologous plasma antibodies (Figure 4B). Interestingly, all the V1 360

chimeras as well as the point mutants showed comparable sensitivities to PG9 MAb as compared 361

to their wild types (Table 5), indicating that the shift in neutralization susceptibilities were not 362

due to change in Env conformation. Moreover, we noted that both the sensitive and the resistant 363

autologous Envs contain T332 in the V3 base, clearly indicating that absence of N332 was not 364

associated with resistance to autologous neutralization. Similar observations were made with 365

respect to N160, R166 and K169 amino acid residues further consolidating that the neutralization 366

conferred by G37080 BCN plasma antibodies was not associated with antibody targeting these 367

epitopes in autologous Envs and likely for all the Envs tested against G37080 plasma antibodies. 368

369

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Discussion 370

371

Identification of the molecular specificities of antibodies elicited in natural infection and 372

that mediate neutralization breadth and potency is key in design and development of suitable Env 373

based immunogen capable of eliciting similar antibody response upon vaccination. In the present 374

study, we characterized the molecular specificity of plasma antibodies obtained from an Indian 375

elite neutralizer (G37080) infected with HIV-1 clade C that displayed exceptional cross 376

neutralization of different clades of distinct geographical origins. The G37080 plasma was found 377

to contain the most broad and potent cross neutralizing antibodies amongst the two hundred 378

plasma samples obtained from Indian patients chronically infected with HIV-1. Plasma samples 379

collected from the G37080 donor at two time points at eight months apart showed similar 380

neutralization breadth with modest increase in potency in the follow up visit, indicating 381

association of sustained maturation of antibody producing B cells in this individual. 382

Since polyclonal plasma antibodies are not suitable for epitope mapping, we examined 383

the specificity of the G37080 BCN plasma by making use of mutant viruses with specific point 384

substitution of known neutralizing epitopes with non-specific amino acids and via depletion with 385

monomeric and trimeric Envs in addition to MPER peptide. The G37080 plasma antibodies did 386

not show dependence to the N160/K169 and N332 epitopes in V2 apex and V3 base respectively 387

Our data also is consistent with the target epitopes of the G37080 BCN antibodies being distinct 388

from those which are recognized by 2G12 (72), PGT121-128 (32) and PGT130-131, 135 (75) 389

(e.g., residues at the following positions: 295, 297, 301, 332, 334, 386, 388, 392, 394, 448, 450), 390

thus BCN G37080 antibodies appear to target a new epitope. Our data highlighting N332 391

independent development of neutralizing antibodies in a clade C infected donor G37080, also 392

differs from recent findings (27, 36, 43, 67) associating N332 with development of broad and 393

20

potent neutralizing antibody especially in clade C infection noted in African donors. Moreover, 394

recent studies indicating the role of K169 as a target of BCN antibodies obtained from a clade C 395

infected South African donor (42, 78) and the observation that vaccine-induced protection in the 396

RV144 vaccine trial was associated with antibodies targeting epitopes including K169 in V2 397

apex (39, 56) prompted us to examine whether broad neutralization of the G37080 plasma 398

antibodies was also dependent on K169 epitope. In our present study, the neutralization potency 399

of G37080 not only was unaffected by N160A/K169A knockout mutations but we also observed 400

that both sensitive and resistant autologous Envs obtained from both visits contain N160 and 401

K169 in the V2 region. Hence, owing to lack of association of neutralization breadth of the 402

G37080 BCN antibodies with N160, K169 and N332 dependences, our study further highlighted 403

that there is a likelihood of differences in development pathway of elicitation of broadly 404

neutralizing antibodies in individuals infected with HIV-1 clade C particularly those with 405

ethnically distinct. 406

Wibmer et al (78) recently demonstrated association between evolution of a broadly 407

neutralizing antibody response in a clade C infected donor with shifts in antibody specificities 408

from recognition of epitopes in V2 to the CD4bs. In the present study, the G37080 neutralizing 409

plasma antibodies obtained from both visits were found not to be absorbed out by the TripleMut 410

(9, 19) as well as the RSC3 (79) core proteins, which effectively absorb antibodies directed to the 411

CD4bs. This result indicates a lack of development of CD4bs directed neutralizing antibodies 412

during the disease course in G37080 donor. Additionally, absence of MPER directed antibodies 413

in G37080 plasma were found, although a negligible antibody titer (1:300 reciprocal dilutions) to 414

the HIV2/HIV1 (C1C) chimera was observed with both visit plasma samples. However, the 415

neutralization breadth of the G37080 plasma was not found to be associated with presence of 416

21

MPER directed antibody. Nonetheless, we do not rule out the possibility that in further course of 417

infection, this donor would possibly be able to develop MPER directed antibodies. 418

Recent studies have shown that neutralizing antibodies that targets conformational 419

epitopes binds exclusively to the cleaved near native trimeric Envs (6, 18, 48, 55). In the present 420

study, we found that absorption of G37080 plasma antibodies to soluble trimeric BG505-421

SOSIP.664 Env was associated with depletion of neutralizing activity in G37080 BCN plasma. 422

However, we do not rule out the possibility of presence of 39F, 19b and 14e like non-423

neutralizing antibodies that were reported to bind to BG505-SOSIP.664 trimeric Env (61). Our 424

findings indicate that the G37080 BCN antibodies target conformational epitopes in gp120. Our 425

observation also highlights that native like trimeric Envs such as BG505-SOSIP.664 can be 426

utilized in selecting antigen specific memory B cells as reported earlier (68) from G37080 donor 427

towards isolation of MAb correlating with broad neutralization displayed by the plasma 428

antibodies. 429

We made use of env clones obtained from autologous G37080 plasma from both the time 430

points to narrow down the fine specificity of the G37080 BCN plasma antibodies. By examining 431

chimeric Envs and mutant viruses we identified key residues in the V1 loop associated with 432

neutralization resistance. Interestingly, the Env chimera and mutant viruses showed comparable 433

susceptibility to PG9 MAb compared to their respective wild type Envs, indicating that they did 434

not alter Env conformation. We identified a glycan at the 133 position and a proline residue at 435

the 147 position within V1 loop of the resistant Env (PG80v2.eJ38) that were found to be 436

associated with neutralization escape, which indicated that these are contact sites for the G37080 437

BCN plasma antibodies. From our study we thus conclude that changes in V1 loop sequence are 438

associated with escape of autologous viruses to the BCN G37080 plasma. Additionally, 439

22

examination of degree of susceptibilities of pseudoviruses expressing chimeric heterologous 440

Envs to the G37080 plasma revealed that the BCN plasma antibodies predominantly target 441

epitopes in V1V2 region in gp120. However, we do not rule out the possibility of contribution of 442

other discontinuous epitopes in gp120 in mediating neutralization breadth. Isolation and 443

identification of monoclonal antibodies from this elite neutralizer donor (G37080) will help 444

precisely map specific epitope associated with neutralization breadth and potency. 445

In summary, we identified an HIV-1 infected elite neutralizer, whose plasma showed 446

exceptional neutralization breadth and provided evidence that it targets novel conformational 447

epitopes on trimeric Env predominantly in the V1V2 region not reported previously. Moreover, 448

neutralization resistance of the autologous Envs to G37080 plasma is associated with 449

substitutions of novel residues within V1 loop that form the key contact points of the BCN 450

plasma antibody. Identification of novel epitopes associated with broad neutralization of HIV-1, 451

in particular the majorly circulating clade C strains will significantly contribute in the efforts 452

towards effective immunogen design. 453

454

Funding information. 455

This study was made possible by the generous funding support of the American people through 456

the United States Agency for International Development (USAID) and support from the THSTI-457

IAVI HIV vaccine design grant through the Department of Biotechnology, Govt. of India; partly 458

by a grant from Department of Science and Technology, Govt. of India (DST/INT/SAFR/Mega-459

P3/2011 to Jayanta Bhattacharya) and partly by the DBT National Bioscience Research Award 460

grant (BT/HRD/NBA34/01/2012-13(iv) to Jayanta Bhattacharya). The funders had no role in 461

study design, data collection and interpretation, or the decision to submit the work for 462

publication. 463

23

464

Acknowledgements. 465

We thank all the Protocol G study participants registered with YRG Care, Chennai, and all the 466

research staffs at the Protocol G clinical center at the YRG Care, Chennai and all of the IAVI 467

Protocol G team members. We thank Dr. Albert Cupo, Prof. John P. Moore and the members the 468

SOSIP trimer HIVRAD team, Weill Cornell Medical College, New York for providing us with 469

BG505.SOSIP.664 plasmid DNA and purified protein. The following reagent was obtained 470

through the NIH AIDS Reagent Program, Division of AIDS, NIAID, NIH from Drs. John C. 471

Kappes and Xiaoyun Wu: pSG3Δenv. We thank Dr. David Montefiori, Prof Lynn Morris, Dr 472

Pascal Poignard, Dr. Richard Wyatt for making available many reagents used in our study. The 473

International AIDS Vaccine Initiative has filed a patent relating to the autologous HIV-1 clade C 474

envelope clones: U.S. Provisional Application no. 62/254,971, titled “HIV-1 clade C envelope 475

glycoproteins,” with inventors J. Bhattacharya, S. Deshpande, S. Patil, R. Kumar, B.K. 476

Chakrabarti. We sincerely thank Dr. Christopher Parks, IAVI Design and Development 477

Laboratory for providing valuable inputs in preparing the manuscript and we also thank Prof. G. 478

Balakrish Nair, Prof Sudhanshu Vrati, THSTI; Dr. Shreyasi Chatterjee and all the HVTR 479

laboratory members for support. IAVI's work was made possible by generous support from many 480

donors including: the Bill & Melinda Gates Foundation; the Ministry of Foreign Affairs of 481

Denmark; Irish Aid; the Ministry of Finance of Japan; the Ministry of Foreign Affairs of the 482

Netherlands; the Norwegian Agency for Development Cooperation (NORAD); the United 483

Kingdom Department for International Development (DFID); and the United States Agency for 484

International Development (USAID). The full list of IAVI donors is available at www.iavi.org. 485

The contents are the responsibility of the International AIDS Vaccine Initiative and do not 486

24

necessarily reflect the views of USAID or the United States Government. The contents of this 487

manuscript are the responsibility of IAVI and do not necessarily reflect the views of USAID or 488

the US Government. 489

490

491

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858

859

860

33

Table and Figure Legends: 861

862

Table 1. Neutralization breadth of Protocol G G37080 plasma samples collected at two different 863

points tested against 57 panel Env-pseudotyped viruses. 864

865

Table 2. Examination of known specificity of G37080 plasma antibodies obtained at both visits 866

to HIV Env. 867

868

Table 3. Degree of shift in sensitivity of the Env-pseudotyped viruses to G37080 BCN plasma 869

depleted with the soluble monomeric and trimeric Env proteins and a clade C MPER peptide 870

(C1C). 871

872

Table 4. Mapping specificities mediating neutralization resistance of the autologous and 873

heterologous Envs to G37080 BCN plasma. 874

875

Table 5. Sensitivity of wild type, chimera and point mutants of autologous Envs to PG9 MAb 876

877

Figure 1 A. Genetic divergence of amino acid sequences of 57 HIV-1 Env (gp160) used to 878

assess neutralization breadth and potency of G37080 BCN plasma. Maximum likelihood 879

bootstrapped consensus phylogenetic tree was constructed using Jones-Taylor-Thornton (JTT) 880

substitution model with 50 bootstrapped replicates in Mega 5.2 version. Bootstrapped values are 881

shown at the nodes of each branch. Hollow circles represent envelopes (16055-2.3 and 882

92TH021) resistant to neutralization by G37080 BCN plasma. B. Neutralization breadth of the 883

G37080 BCN plasma obtained at visit 1 and visit 2 were assessed against pseudotyped viruses 884

34

expressing HIV-1 Env representing different clades and origins. Neutralization titers (median 885

ID50 values) were obtained by titrating Env-pseudotyped viruses against G37080 plasma 886

samples. Values at top of each bar graph indicate number of viruses belonging to each 887

clade/origin tested. 888

889

Figure 2. Assessing dependence of G37080 BCN antibodies to CD4 binding site (CD4bs) region 890

of HIV-1 Env. G37080 BCN plasma samples and VRC01 MAb (concentrations that neutralized 891

25711-2.4 by >80%) pre-incubated with different concentrations, as indicated, with TripleMut 892

core (A) and RSC3 (B) proteins were examined for their ability to neutralize 25711-2.4 Env 893

pseudotyped virus in TZM-bl cell neutralization assay. Note that while VRC01 pre-absorbed 894

with both TripleMut and RSC3 proteins showed inhibition to neutralize 25711-2.4 in a dose-895

dependent manner, no such effect was observed with G37080 BCN plasma indicated absence of 896

CD4bs directed neutralizing antibodies. 897

898

Figure 3. Binding of G37080 BCN plasma IgG to 4-2.J41 monomeric gp120 (A) and BG505-899

SOSIP.664-D7324 cleaved trimeric gp140 (B) soluble proteins were assessed by ELISA. IgG 900

purified from HIV negative healthy donor and known MAbs were used as controls. Extent of 901

binding of the depleted and undepleted G37080 BCN plasma with magnetic beads coated with 4-902

2.J41 monomeric gp120 (C) and BG505-SOSIP.664 cleaved trimeric gp140 to their respective 903

proteins by ELISA. Note that binding to trimeric protein by ELISA was assessed by using 904

BG505-SOSIP.664 tagged with D7324 epitope to maintain native conformation of trimeric Env 905

as described before (61). 906

907

35

Figure 4. A. Neutralization susceptibility of autologous Envs to contemporaneous G37080 BCN 908

plasma and its follow up sample from the same donor. Neutralization titers (median ID50) were 909

obtained by titrating pseudotyped viruses expressing autologous Envs obtained from visit 1 and 910

follow up G37080 plasma to contemporaneous plasma antibodies. Note that both the Envs 911

obtained from follow up G37080 plasma (visit 2) were found to be resistant to contemporaneous 912

autologous plasma, while Envs obtained from visit 1 G37080 plasma were found to be sensitive 913

to follow up plasma antibodies. B. Alignment of V1V2 amino acid sequences of sensitive and 914

resistant autologous Envs obtained at both visits were done by using seqpublish available at HIV 915

Los Alamos database (www.hiv.lanl.gov). Key residues that mediate autologous neutralization 916

resistance are highlighted. 917

918

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 Envelope Accession No. Clade Visit-1 Visit-2

MuLV S53043.1 NA <20 <20 HIV-2 (7312A) JX235925.1 A <20 <20 16055-2.3 EF117268 C 23.69 25.95 16936_2.21 EF117270 C 293.39 358.14 25710-2.3 EF117271 C 887.84 1355.96 25711-2.4 EF117272 C 454.97 851.38 00836-2.5 EF117265 C 845.18 860.15 2-5.J3 GU945311.1 C 604.09 106.68 4.J22 EU908219.1 C 1557.8 848.91

India Clade C 4-2.J41 GU945316.2 C 2939.22 1308.11 3-5.J25 GU945314.1 C 3262.97 785.12 5-4.J16 GU945326.1 C 2201.58 969.68 5.J41 EU908221.1 C 3401.76 4851.8 7.J16 EU908222.1 C 418.44 288.59 7.J20 EU908223.1 C 838.77 765.86 11-3.J3 GU945330.1 C 3449.22 2279.27 11-5.J12 GU945333.1 C 1413.06 3081.77 LT-1.J1 JN400529 C 444.8 580.9 LT1.J3 JN400534 C 350.71 201.48 LT5.J3b JN400538 C 378.23 125.45 LT5.J7b JN400540 C 323.37 157.48 93IN905 AY669742.1 C 2389.65 8533 Median ID50 841.975 817.015 CAP45.G3 DQ435682.1 C 42.95 66.58 CAP84 EF203963.1 C 533.03 604.23 CAP88 EF203972.1 C 149.1 80.32 CAP239.G3 EF203983.1 C 642.06 520.14

Africa Clade C Du422.1 DQ411854.1 C 274.86 304.31 Du151.2 DQ411851.1 C 1485.89 355.89 DU156.12 DQ411852.1 C 1400.78 1573 DU172.17 DQ411853.1 C 672.8 195.63 ZM109F.PB4 AY424138.2 C 235.75 291.1 ZM197M.PB7 DQ388515.1 C 317.87 296.25 IAVIC22 -- C 1836.31 1592.26 Median ID50 533.03 304.31 JRFL U63632.1 B 229.36 122.15 PVO.4 AY835444.1 B 118.64 274.91 TRJO4551.58 AY835450 B 192.61 122.76 AC10.0.29 AY835446 B 721.69 963.76 QH0692.42 AY835439 B 199 187.2

Clade B REJO4549.67 AY835449 B 205.79 51.7 SC422661.8 AY835441.1 B 161.41 21.05 6535.3 AY835438 B 2238.72 1541.11 RHPA 4259.7 AY835447.1 B 584.69 491.46 HO61.14 EF210730 B 805.49 414.5 92BR020 AY669718.1 B 847.48 3508.16 JRCSF M38429.1 B 387.56 571.68 Median ID50 486.125 414.5 Q769.ENV.b9 AF407157.1 A 191.25 42.05 Q461.e2 AF407156 A 29.68 <20 Q842.d12 AF407160.1 A 851.19 1542.05

Clade A Q23.17 AF004885 A 1853.63 2328 Q259.d2.26 AF407152 A 3060.32 693.17 BG505 DQ208458.1 A 574.02 206.79 94UG103 AY669705.1 A 102.12 247.46 Median ID50 574.02 470.31 92TH021 AY669775.1 A/E 88.43 206.11 LT5.J12 FJ515876 B/C 341.4 130.44 CH038.12 EF042692 B/C 945.18 562.2

Others CH114.8 EF117264 B/C 343.17 314.47 CH120.6 EF117260 B/C 182.69 75.71 CRF 02AG_235 EU513195 A/G 445.06 319.86 191727_D1_12 HM215267.1 D 761.79 298.55 Median ID50 343.17 298.55

Table 2. Examination of specificity of G37080 plasma antibodies obtained at both visits to HIV Env

Visit 1 Plasma Visit 2 Plasma HIV-1 Env mutants Region Fold Decrease in

ID50* Fold Decrease in

ID50 HIV-1 25711-2.4 N160A

V2

1.02 <1 HIV-1 25711-2.4 R166A <1 <1 HIV-1 25711-2.4 K169E <1 <1 HIV-1 93IN905 R166A <1 <1 HIV-1 93IN905 K169A <1 <1

HIV-1 25711-2.4 N332A V3

1.52 <1 HIV-1 CAP239.G3 N332A 1.35 1.32

HIV-2/HIV-1 chimera Region of HIV-1 ID50 ID50

HIV-2 7312A HIV-2 WT <20 <20 HIV-2 7312A-C1C Clade C MPER 306.42 371.02

HIV-2 7312A-C3 2F5 epitope <20 <20 HIV-2 7312A-C4 4E10, Z13e1 & 10E8 epitopes 334.34 371.27 HIV-2 7312A-C6 4E10 minimal epitope <20 223.90 HIV-2 7312A-C7 2F5 minimal epitope <20 <20

* ID50 values refer to the reciprocal dilution that conferred 50% neutralization of viruses in a TZM-bl assay. Assays were done in duplicates and were repeated more than three times. WT refers to wild type; MPER refers to membrane proximal external region in gp41.

Table 3. Degree of shift in sensitivity of Env-pseudotyped viruses to G37080 BCN plasma

depleted with monomeric and trimeric Envs as well as a clade C MPER peptide (C1C)

Fold reduction in neutralization (ID50)

by depleted G37080 plasma Env-pseudotyped

Virus Panel gp140 Trimer

(BG505-SOSIP.664) gp120 Monomer

(4-2.J41) MPER

(C1C peptide)25710-2.3 > 10.30 1.3 0.83 25711-2.4 >8.52 1.4 1.44

3-5.J25 >7.85 0.9 0.84 4-2.J41 12.11 1.1 1.04

IAVI_C22 >15.92 1.2 1.18 92BR020 >35.08 1.1 1.34 93IN905 3.41 1.2 0.94

JRCSF >8.75 0.5 0.93 Q23.17 >23.28 1.0 0.98

Du156.12 >15.73 0.8 1.61 HVTR-PG80v1.eJ7 >10.03 0.9 1.12

HVTR-PG80v1.eJ19 >15.60 0.5 1.18 HIV-2 7312A-C1C - - >10

Fold reduction in virus neutralization was obtained by comparing the neutralization titer (ID50 values) of panel viruses against undepleted and depleted G37080 plasma. ID50 values are reciprocal dilutions at which the undepleted and depleted plasma conferred 50% neutralization of the Env-pseudotyped viruses in TZM-bl cells.

Table 4. Dissection of specificity for autologous neutralization resistance

* Fold changes in reciprocal dilution of plasma mediating 50% virus neutralization (ID50)

** Fold increase and decrease in neutralization titer (ID50 values)

Chimera & Point Mutants in

PG80v1.eJ7 Env backbone

Fold changes*

Effect**

V1V2 loop

PG80v2.eJ38 (V1V2) in v1.eJ7 45.75 decrease

PG80v2.eJ38 (V1) in v1.eJ7 27.97 decrease

PG80v2.eJ38 (V2) in v1.eJ7 1.35 no effect

Point mutations

PG80v1.eJ7 (D133N) 3.25 decrease

PG80v1.eJ7 (S143G) 0.87 no effect

PG80v1.eJ7 (D133N+S143G) 2.88 decrease

PG80v1.eJ7 (T147P) 8.21 decrease

Chimera & Point Mutants in

PG80v1.eJ19 Env backbone

Fold changes*

Effect**

V1V2 loop

PG80v2.eJ38(V1V2) in v1.eJ19 23.05 decrease

PG80v2.eJ38 (V1) in v1.eJ19 28.61 decrease

PG80v2.eJ38 (V2) in v1.eJ19 1.87 increase

Point mutations

PG80v1.eJ19 (D133N) 2.51 decrease

PG80v1.eJ19 (T139A) 0.99 no effect

PG80v1.eJ19 (T139A + T140D) 2.64 increase

PG80v1.eJ19 (N143G) 1.38 no effect

PG80v1.eJ19 (T139A + T140D + N143G) 2.16 increase

PG80v1.eJ19 (T145P) 3.24 decrease

Chimera & Point Mutants in

PG80v2.eJ38 Env backbone

Fold changes*

Effect**

V1V2 loop

PG80v1.eJ7 (V1V2) in v2.eJ38 26.56 increase

PG80v1.eJ7 (V1) in v2.eJ38 49.62 increase

PG80v1.eJ7 (V2) in v2.eJ38 1.07 no effect

PG80v1.eJ19(V1V2) in v2.eJ38 12.81 increase

PG80v1.eJ19 (V1) in v2.eJ38 37.60 increase

PG80v1.eJ19 (V2) in v2.eJ38 0.94 no effect

Other regions in gp120

PG80v1.eJ7 (V3C3) in v2.eJ38 0.84 no effect

PG80v1.eJ7 (V3C3V4C4) in v2.eJ38 0.90 no effect

PG80v1.eJ7 (C4V5C5) in v2.eJ38 1.08 no effect

PG80v1.eJ19 (V3C3) in PG80v2.eJ38 1.09 no effect

PG80v1.eJ19 (V3C3V4C4) in PG80v2.eJ38 1.15 no effect

PG80v1.eJ19 (C4V5C5) in PG80v2.eJ38 0.96 no effect

Point mutations

PG80v1.eJ19 V1 (T139A+T140D) in v2.eJ38 53.91 increase

PG80v2.eJ38 (N133D) 4.72 increase

PG80v2.eJ38 (G143S) 0.94 no effect

PG80v2.eJ38 (N133D+G143S) 3.63 increase

PG80v2.eJ38 (P147T) 4.11 increase

Heterologous Env chimera Fold changes* Effect**

V1V2 loop

16055-2.3 (25711-2.4 V1V2) 18.38 increase

25711-2.4 (16055-2.3 V1V2) 2.03 decrease

CAP45 (25711-2.4 V1V2) 16.84 increase

25711-2.4 (CAP45-V1V2) 10.54 decrease

Table 5. Sensitivity of wild type, chimera and point mutants of autologous Envs to PG9 MAb

Env chimera and mutants IC50 value PG80v1.eJ7 (wild type)

PG80v1.eJ19 (wild type)PG80v1.eJ19 (T139A + T140D)

PG80v2.eJ38 (wild type)PG80v1.eJ7 (V1) in v2.eJ38

PG80v1.eJ19 (V1) in v2.eJ38PG80v1.eJ19 (V1)(T139A+T140D) in v2.eJ38

PG80v2.eJ38 (N133D)PG80v2.eJ38 (P147T)

0.12 0.97 0.77 0.02 0.04 0.05 0.06 0.01 0.04