Post on 24-Jan-2017
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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
9
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.
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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
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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
4
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
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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
162
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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
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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
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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
16
(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
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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
25
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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