Evolution of an HIV glycan-dependent broadly neutralizing antibody epitope through immune escape Penny L. Moore1,2, Elin S. Gray1 Constantinos Kurt Wibmer1,2, Jinal N. Bhiman1,2, Molati Nonyane1, Tandile Hermanus1, Daniel Sheward3, Shringkhala Bajimaya4, Nancy L. Tumba1, Melissa-Rose Abrahams2, Lihua Ping5, Nobubelo Ngandu3, Quarraisha Abdool Karim6, Salim S. Abdool Karim6, Ronald I Swanstrom5, Michael S. Seaman4, Carolyn Williamson2 and Lynn Morris1,2* and the CAPRISA 002 study team 1Centre for HIV and STIs, National Institute for Communicable Diseases of the National Health Laboratory Services, Johannesburg, South Africa, 2University of the Witwatersrand, Johannesburg, South Africa; 3Institute of Infectious Disease and Molecular Medicine, University of Cape Town, Cape Town, South Africa; 4Division of Viral Pathogenesis, Department of Medicine, Harvard Medical School, Beth Israel Deaconess Medical Center, Boston, MA 02215 5University of North Carolina at Chapel Hill, North Carolina, USA; 6Centre for the AIDS Programme of Research in South Africa (CAPRISA), University of KwaZulu Natal, Durban, South Africa. 4 Figures and 2 Supplementary Figures *Corresponding author: Prof Lynn Morris National Institute for Communicable Diseases of the NHLS, Private Bag X4, Sandringham 2131, Johannesburg, South Africa. email: lynnm@nicd.ac.za; phone: +2711 386 6332; fax +2711 386 6453 Short title: Key neutralizing epitope absent on HIV subtype C transmitted/founder viruses Neutralizing antibodies are likely to play a crucial role in a preventative HIV-1 vaccine. Although efforts to elicit broadly cross-neutralizing (BCN) antibodies by vaccination have been unsuccessful1-3, approximately a quarter of infected individuals naturally develop these antibodies after many years4-7. How such antibodies arise, and the role of viral evolution in shaping these responses is unknown. Here we show in two individuals who developed BCN antibodies dependent on the glycan at residue 332, that this glycan was absent on the infecting virus. However, this BCN epitope evolved within 6 months, through immune escape from earlier strain-specific antibodies that resulted in a shift of a glycan to position 332. Both acute viruses that lacked the 332 glycan were resistant to N332-dependent BCN monoclonal antibody PGT1288, while escaped variants that acquired this glycan were sensitive. Analysis of large sequence datasets showed the 332 glycan to be selectively under-represented in subtype C transmitted/founder viruses compared to chronic viruses, which corresponded with resistance to PGT128. These findings highlight the dynamic interplay between early antibodies and viral escape in driving the evolution of conserved epitopes, which may have implications for vaccination strategies aimed at eliciting BCN antibodies. Although the role of glycans in shielding neutralizing epitopes has long been known9,10, it has only recently become clear that many BCN responses directly target glycans, including that at position 332 in the C3 region of the gp120 subunit of the envelope protein8,11-17. The recent isolation of mAbs that target this glycan, which are the most potent yet described, has focused increased attention on this epitope. These mAbs (PGT121-123, 125-128, 130-131 and 135-137) bind to glycans on gp120, and neutralize effectively across all subtypes, with the broadest, PGT128, neutralizing >70% of global viruses8. Crystal structures of PGT127 and PGT128 show that they penetrate the glycan shield, recognizing high mannose glycans at residues 301 and 332, in addition to a short ß-strand in the C-terminus of the V3 loop18. The conserved nature of these residues and the high potency of this class of mAbs suggest that this region may be an important vaccine target. This is further supported by data showing that this epitope is immunogenic with N332-dependent BCN antibodies often found in infected subjects who develop neutralization breadth8,13-16. However, as with other BCN antibodies, the factors that favor the emergence of N332-dependent BCN antibodies remains unclear. Here we hypothesized that the evolution of viral populations, which are under significant immune and fitness selection pressures, drives the development of neutralizing antibodies to target conserved epitopes. From a cohort of 79 subtype C infected individuals followed from acute infection, we focused on 2 of the 3 participants who developed N332-dependent BCN antibodies. CAP177 produced antibodies by three years post-infection capable of neutralizing 88% of a large multi-subtype panel of 225 heterologous primary viruses (Lacerda, Moore et al, submitted). The second individual, CAP314, neutralized 46% of 41 heterologous viruses after 2 years of infection (Supplementary Table 1). In experiments to map the target of the BCN antibodies, removal of the glycan at position 332 in sensitive heterologous viruses resulted in almost complete loss of neutralization sensitivity from the time when BCN responses were first detected and thereafter (Fig. 1a and b), suggesting that BCN antibodies in both individuals recognized a 332 glycan-dependent epitope in the C3 region similar to the PGT128 mAb. Single genome amplification and sequencing were used to sample the envelopes of circulating viral populations at the earliest available time point (for CAP177 this was 2 weeks post-infection, enabling inference of the transmitted/founder virus19, and for CAP314 it was 3 months post-infection). In both cases, these acute viruses lacked the N-linked glycan at position 332, although almost all sequences contained an intact glycosylation site at position 334 (Fig. 1c and d). By 6 months of infection, a glycan at position 332 evolved in both CAP177 (10/11 sequences) and CAP314 (13/13 sequences) through a N334T or N334S substitution, which also resulted in the destruction of the neighboring glycan at position 334 (Fig. 1c and d). By 12 months post-infection, the glycan was fixed in both cases. Therefore, for both CAP177 and CAP314, who developed N332-dependent BCN antibodies by 2-3 years of infection, the glycan at residue 332 that formed the basis of the BCN epitope was absent at the earliest time point, but evolved by 6 months of infection. The 332 glycan lies within the C3 region which is highly immunogenic in HIV-1 subtype C, and is often targeted by early strain-specific antibodies20. Indeed we have previously shown that strain-specific neutralizing antibodies in CAP177 target the C3 region21. Since viral escape from neutralizing antibodies often involves glycan re-arrangements10 , we postulated that the shift of a glycan from position 334 to 332 in CAP177 and CAP314 may have been the result of neutralization escape from early strain-specific antibodies. To test this, we cloned the acute virus, and a representative 6-month clone from CAP177 and CAP314. We also mutated the asparagine at position 334 to a serine to create acute viruses containing the 332 glycan (and deleting that at 334) (Fig. 2a and b). All 3 viruses from both individuals were tested for sensitivity to autologous plasma from 6 months post-infection. Autologous neutralizing responses were detected against the acute viruses whereas the 6-month clones were resistant to the contemporaneous serum, as expected (Fig. 2c and d). Introduction of the glycan at position 332 resulted in reduced neutralization sensitivity (Fig. 2c and d), suggesting that the 332 glycan evolved to afford escape from early strain-specific antibodies. The absence of the glycan at position 332 on acute viruses from CAP177 and CAP314 suggested that they might be resistant to the PGT121, PGT128 and PGT135 mAbs that depend on this glycan. Furthermore, we postulated that later viruses that evolved the 332 glycan would acquire sensitivity to these mAbs. Neutralization experiments showed that with one exception, the CAP177 and CAP314 acute clones were resistant to these mAbs (Fig. 3). In contrast, the 6-month clones from both subjects were highly sensitive to both PGT121 and PGT128 (with IC50 <0.06 ug/ml). The CAP177 acute clone which showed moderate sensitivity to PGT121 (0.21 ug/ml), was nevertheless 20-fold more resistant compared to the later clone. Neither 6-month clone was sensitive to PGT135 (Fig. 3), which in addition to the 332 glycan, also depends on the glycans at positions 295 and 392 that are less common among subtype C viruses22-26, including CAP177 and CAP314. Both N334S mutants (acute clones with a glycan introduced at position 332) were sensitive to PGT121 and PGT128 confirming that the glycan at position 332 conferred sensitivity to these mAbs. Thus, while shifting a glycan from position 334 to 332 allowed the virus to escape autologous neutralizing antibodies, this created a new neutralizing antibody target that may have provided the antigenic stimulus to elicit BCN antibodies targeting the 332 glycan. We assessed whether this pattern of selection of the 332 glycan was evident at a population level using more than 7,300 SGA-derived gp160 envelope sequences from a large number (n=304) of acute and chronic subtype B27 and subtype C19 infections (supplementary methods). In subtype C, the 332 glycan was significantly less common among transmitted/founder viruses (45/68, 66%) compared to chronic viruses (52/62, 84%, P = 0.0265, Fig. 4a). Although we observed the same trend in subtype B sequences, this was not statistically significant (P = 0.1232). There was no difference in the frequency of the 301 glycan either within or between subtypes (Fig. 4b). Taken together, these results suggest that the pattern of evolution we describe here for CAP177 and CAP314 may be relatively common, and that there might be a fitness advantage for viruses lacking the 332 glycan during transmission or early viral outgrowth. The observation that the N332 glycan is particularly underrepresented in subtype C may also explain why the C3 region is so often a target of autologous neutralizing antibodies in this subtype20,28,29, with the absence of a glycan potentially increasing the accessibility of this region. Phenotypic analysis of 101 transmitted/founder subtype C viruses supported the genotypic analysis with 46% of viruses showing resistance to PGT128 neutralization (Fig. 4c). Resistance was strongly correlated with the absence of the 332 glycan (p<0,0001) (Fig. 4d), although some viruses that contained the glycan were also resistant, consistent with the fact that additional residues are needed to form this epitope8. Although this virus panel was tested only against PGT128, resistance to this monoclonal antibody generally extends to other 332dependent PGT monoclonal antibodies8. These data suggest that N332-dependent antibodies present either through passive immunotherapy or vaccination might be only partially effective in preventing subtype C infections. Subtype-restricted efficacy suggests that combinations of mAbs targeting different epitopes might need be tailored to match circulating viral subtypes23,25,30 . The epitope defined by dependence on the 332 glycan is one of four BCN antibody epitope clusters on the HIV-1 envelope identified to date. While these epitopes are highly conserved, they rarely elicit BCN responses in humans. Nevertheless, antibodies targeting the N332 epitope, together with those that target the PG9/16 epitope17 (defined by the glycan at position 160 in V2) develop more commonly in infected people compared to CD4 binding site or membrane proximal region directed BCN antibodies11-13. The data presented here raise the hypothesis that, at least in the case of N322-directed antibodies, viral evolution may facilitate their elicitation. Whether or not the absence of the N332 glycan on the transmitted/founder virus contributes to the delayed development of BCN antibodies is also worth considering, although clearly other factors are involved. In a study of a single SHIV-infected macaque31 potent and broad N332-dependent BCN antibodies developed rapidly, within 9 months. Additional studies are needed to ascertain if BCN antibodies targeting glycans are generally more easily induced following vaccination. If so, the relative absence of the 332 glycan argues against the use of HIV-1 subtype C transmitted/founder viruses as sole immunogens. Rather designing immunogens based on sequential viral variants that mimic but accelerate the evolutionary processes that occur during natural infection, might prove more useful in eliciting such BCN antibodies32. Acknowledgments We thank the participants in the CAPRISA 002 and 004 cohorts, and the clinical and laboratory staff at CAPRISA for providing specimens. We are grateful to Drs Dennis Burton and Wayne Koff of IAVI for providing the PGT monoclonal antibodies, and to Dr S. Gnanakaran for providing transmitted/founder and chronic subtype B sequences. We thank Dr. Ziyaad Valley-Omar and Nonkululeko Ndabambi for generating some of the envelope sequences. We are grateful to Drs Beatrice Hahn and Jerome Kim who contributed transmitted/founder subtype C clones to the VIMC Standard Virus Panel Consortium. This work was funded by CAPRISA, CHAVI and the South African HIV/AIDS Research and Innovation Platform (SHARP) of the Department of Science and Technology (DST). CAPRISA was supported by the National Institute of Allergy and Infectious Diseases (NIAID), National Institutes of Health (NIH), U.S. Department of Health and Human Services (Grant U19 AI51794). M.S.S. is funded by the Bill and Melinda Gates Foundation, Vaccine Immune Monitoring Consortium grant # 1032144. P.M. and E.G. were supported by the Columbia University-Southern African Fogarty AIDS International Training and Research Program (AITRP) through the Fogarty International Center, National Institutes of Health (grant # 5 D43 TW000231). P.M. is a Wellcome Trust Intermediate Fellow in Public Health and Tropical Medicine (Grant 089933/Z/09/Z). Author contributions P.L.M. designed the study, performed experiments, analyzed data and wrote the manuscript; E.S.G, C.K.W, J.N.B, T.H. and N.T performed neutralization experiments and analyzed data; C.K.W and M.N generated single genome sequences; D.S and N.N performed part of the sequence analyses; M-R.A., L-H.P., R.S. and C.W contributed the subtype C acute and chronic sequences; S.B. and M.S designed and performed the neutralization experiments using the panel of transmitted/founder viruses; S.S.A.K., Q.A.K. and C.W. established the CAPRISA cohorts and contributed samples and data for these subjects; and L.M. designed the study and wrote the manuscript. Figure legends: Figure 1. CAP177 and CAP314 BCN antibodies are dependent on the glycan at position 332, which is absent from the acute virus but evolves by 6 months post-infection. (a) CAP177 and (b) CAP314 serial plasma neutralization of three heterologous viruses (Q23, Du156 and TRO.11 for CAP177 and Q23, Du156 and CAP200 for CAP314) was reduced or abrogated by the removal of the N332 glycan. Multiple HIV-1 gp160 envelope sequences were obtained from (c) CAP177 and (d) CAP314 by single genome amplification of plasma viral RNA, and direct sequencing. The alignment shows part of the C3 region at 2 weeks or 3 months (acute viruses), 6 months and 12 months post-infection. Gray shading indicates the presence of a predicted N-linked glycan and dashes indicate sequence identity compared to the reference sequence. HxB2 numbering is used throughout. Figure 2. The glycan at position 332 mediates neutralization escape in CAP177 and CAP314. (a) Partial sequence alignment showing the CAP177 acute virus (CAP177.2wks), a representative 6-month clone (CAP177.6mo) and the acute virus mutated to introduce a glycan at position 332, which deleted the glycan at position 334 (CAP177.2wks N334S). (b) Partial sequence alignment showing the CAP314 acute virus (CAP314.3mo), a representative 6 month clone (CAP314.6mo) and the mutated virus (CAP314.3mo N334S). (c) Sensitivity of CAP177 envelope clones and mutants to autologous neutralization by CAP177 serum from 6 months p.i. and (d) Sensitivity of CAP314 envelope clones and mutants to autologous neutralization by CAP314 serum from 6 month p.i. Figure 3. Sensitivity of CAP177 and CAP314 clones to neutralization by PGT monoclonal antibodies. The acute, 6 month and N334S mutant viruses from both CAP177 and CAP314 were tested against the N332-dependent mAbs, PGT121, PGT128 and PGT135 mAbs in a TZM-bl neutralization assay. Neutralization titers reflect the IC50 concentration (ug/ml). Red shading indicates IC50 < 0.1 ug/ml, orange shading indicates IC50 between 0.1 and 1 ug/ml and blue shading indicates neutralization resistance, with IC50 > 10 ug/ml. TRO.11, a subtype B virus containing the glycan at residue 332 was included as a positive control. Figure 4. The glycan at 332 is underrepresented in subtype C transmitted/founder viruses, which are frequently resistant to the 332-dependent BCN monoclonal antibody PGT128. Comparison of the frequency of the glycans at position (a) 332 and (b) 301, which contribute to the BCN epitope defined by PGT128. For subtype B, a published dataset of 2,715 acute sequences from 88 subjects, and 2,122 sequences from 86 chronic infections was used27. For the subtype C analysis, we used 1,371 envelope sequences from 68 subjects in acute/early infection described elsewhere19. The subtype C chronic dataset included 1,111 sequences from 62 subjects with chronic infections (Ping et al, submitted). For each subject, the consensus sequence was generated, and the frequency of the 332 glycan, was assessed. Significance was assessed using Fisher’s exact test. c) Neutralization sensitivity of 101 transmitted/founder viruses to neutralization by the PGT128 mAb. Transmitted/founder envelope sequences were inferred from single genome amplified and sequenced envelope amplicons derived from plasma from acutely HIV infected subjects33 and cloned into a mammalian expression vector. Clones were generated as part of the Vaccine Immune Monitoring Core Standard Virus Panel Consortium. Envelope clones were transfected into 293T cells with the HIV backbone construct pSG.Env to produce envelope-pseudotyped particles, and neutralization assays were performed as described in supplementary methods. Of 101 transmitted/founder viruses, 46 (46%) were resistant to neutralization by PGT128 at the highest concentration tested (10 ug/ml); d) Neutralization resistance of subtype C transmitted/founder viruses correlated strongly with the absence of the 332 glycan. Of 30 viruses in which the 332 glycan was absent, only three exhibited neutralization sensitivity. Of these, two contained the glycan at position 295 which is very rare in subtype C viruses25, but structurally proximal to the 332 glycan and shown by mutagenesis to affect the PGT128 epitope18. Supplementary Figure 1. Neutralization breadth exhibited by CAP314 serum at 2 years post-infection. Serum was tested against 25 subtype C viruses, 11 subtype B viruses and 5 subtype A viruses, of which 19 were neutralized (46% of the total panel), showing a slight preferential neutralization of subtype C and A viruses compared to subtype B viruses. Neutralization titers are shown in gray and reflect an ID50 (the concentration of plasma that reduces viral infectivity by 50%). Supplementary Figure 2. PGT128 neutralization of subtype C transmitted/founder viruses. PGT128 was tested against a panel of 101 transmitted/founder subtype C viruses, with 55 viruses exhibiting neutralization sensitivity. The presence of intact glycosylation motifs at positions 301 and 332 are indicated for each virus. Neutralization is shown as IC50 (µg/ml). Gray shading indicates viruses (n=3) without the glycan at 332 that exhibit sensitivity to PGT128. Blue shading indicates neutralization resistance (IC50 > 10 ug/ml), yellow shading shows and IC50 between 1 and 10 ug/ml, orange shading IC50 of 0.1 to 1 10 ug/ml and red shading an IC50 < 0.1 ug/ml. 1 Online Materials and Methods 2 3 Participants. CAP177 was part of the CAPRISA 002 Acute Infection study, a cohort of 245 4 high risk HIV negative women which was established in 2004 in Durban, South Africa for follow-up and subsequent identification of HIV seroconversion34. She was one of 7 women in 6 this cohort of 40 who developed neutralization breadth and one of only 2 who developed 7 332N-directed BCN antibodies14. The second individual, CAP314, was enrolled in the 8 CAPRISA 004 trial, a two-arm, double-blind, randomized, placebo-controlled trial, conducted 9 from May 2007 to March 2010 in order to assess the effectiveness and safety of Tenofovir Gel for the prevention of HIV infection in women 35. Monitoring BCN antibodies in this cohort 11 of 39 women showed she was one of 3 who developed breadth by 2 years of infection, 2 of 12 whom targeted a 332N epitope. The CAPRISA 002 Acute Infection study was reviewed and 13 approved by the research ethics committees of the University of KwaZulu-Natal (E013/04), 14 the University of Cape Town (025/2004), and the University of the Witwatersrand (MM040202). The CAPRISA 004 trial was approved by the University of KwaZulu-Natal’s 16 Biomedical Research Ethics Committee (E111/06), Family Health International’s Protection of 17 Human Subjects Committee (#9946) and the South African Medicines Control Council 18 (#20060835). Both participants provided written informed consent. 19 Single Genome Amplification and Sequencing. HIV-1 RNA was isolated from plasma from 21 CAP177 and CAP314 using the Qiagen Viral RNA kit, and reverse transcribed to cDNA using 22 Superscript III Reverse Transcriptase (Invitrogen, CA). The env genes were amplified from 23 single genome templates 33 and amplicons were directly sequenced using the ABI PRISM Big 24 Dye Terminator Cycle Sequencing Ready Reaction kit (Applied Biosystems, Foster City, CA) and resolved on an ABI 3100 automated genetic analyzer. The full-length env sequences 26 were assembled and edited using Sequencher v.4.0 software (Genecodes, Ann Arbor, MI). 27 The number of potential N-linked glycosylation sites (PNGs) was determined using N28 glycosite (http:/www.hiv.lanl.gov/content/hiv-db/GLYCOSITE/glycosite.html). Multiple 29 sequence alignments were performed using Clustal X (ver. 1.83) and edited with BioEdit (ver. 5.0.9) Sequence alignments were visualized using Highlighter for Amino Acid Sequences 31 v1.1.0 (beta). Selected amplicons were cloned into the expression vector pcDNA 3.1 32 (directional) (Invitrogen) by re-amplification of SGA first-round products using Phusion 33 enzyme (Finn Enzymes) with the EnvM primer36 and directional primer, EnvAdir20. Cloned env 34 genes were sequenced to confirm that they exactly matched the sequenced amplicon 36 Site-directed mutagenesis. Sensitive heterologous viruses Q23 and Du156 were mutated at 37 key residues (332-334) by site-directed mutagenesis using the Stratagene QuickChange II kit 38 (Stratagene) as described by the manufacturer. Mutations were confirmed by sequencing. 39 Similarly, autologous clones derived from CAP177 and CAP314 were mutated to shift the glycan from position 334 to 332, and mutants verified by sequencing. Neutralization assays. The JC53bl-13 cell line, engineered by J. Kappes and X. 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J Virol 81, 6187-6196 (2007). Q23 0 50 100 150 200 0 200 400 600 800 weeks p.i. ID50 Du156 0 50 100 150 200 100 200 300 400 weeks p.i. ID50 TRO.11 0 50 100 150 200 0 100 200 300 400 weeks p.i. ID50 Q23 0 25 50 75 100 125 150 0 200 400 600 weeks p.i. ID50 Du156 0 25 50 75 100 125 150 100 200 300 400 weeks p.i. ID50 CAP200 0 50 100 150 0 50 100 150 200 weeks p.i. ID50 CAP314 Fig. 1 332 334 339 | | | CAP177.2wks.A12 INGIIGDIRQAYCNINGSEWNSTLEQ CAP177.2wks.A6 -------------------------- CAP177.2wks.A2 -------------------------- CAP177.2wks.A4 -------------------------- CAP177.2wks.A11 -------------------------- CAP177.2wks.A13 -------------------------- CAP177.2wks.B12 -------------------------- CAP177.2wks.B13 -------------------------- CAP177.2wks.TB1 -------------------------- CAP177.2wks.TB3 -------------------------- CAP177.2wks.TB6 -------------------------- CAP177.2wks.A3 -------------------------- CAP177.2wks.A15 -------------------------- CAP177.2wks.TB2 -------------------------- CAP177.2wks.A1 -------------------------- CAP177.2wks.TB11 -------------------------- CAP177.2wks.A5 -------------------------- CAP177.2wks.A14 -------------------------- CAP177.2wks.TB9 -------------------------- CAP177.2wks.A10 -------------------------- CAP177.6mo.cH7 ---------------S---------K CAP177.6mo.cG8 ---------------S---------K CAP177.6mo.cA7 ---------------S---------K CAP177.6mo.cD7 --D------------S---------K CAP177.6mo.cB8 --D------------S---------K CAP177.6mo.cF7 ---------------T--K------- CAP177.6mo.cH8 ---------------T--K------- CAP177.6mo.cB7 ---------------T--K------- CAP177.6mo.cG7 ---------------T--K------- CAP177.6mo.cE7 ------------------------KCAP177.6mo. cH9 ---------------S---------- CAP177.12mo.c44 ---------------TS-K------- CAP177.12mo.c4 --D------------T--K------- CAP177.12mo.c11 --D------------S--D------K CAP177.12mo.c26 --D------------S--G------K CAP177.12mo.c13 --D------------S--G------K CAP177.12mo.c31 --D--------H---S--K------K CAP177.12mo.c7 --D---N--------S--D------K CAP177.12mo.c36 --D---N--------S--D------K CAP177.12mo.c28 --D------------T--K-D----K CAP177.12mo.c46 --D------------T--K-D----K CAP177.12mo.c32 -----------H---T--K---M--- CAP177.12mo.c1 ---------------T--K------- CAP177 6 months 2 weeks 6 months 3 months a CAP177 b CAP314 c d 332 334 339 | | | CAP314.3mo.6 TNGIIGDIRQAYCNINSTQWNKTLEG CAP314.3mo.17 -------------------------- CAP314.3mo.14 -------------------------- CAP314.3mo.24 -------------------------- CAP314.3mo.9 -----------------M-------- CAP314.3mo.7 -------------------------- CAP314.3mo.15 -------------------------- CAP314.3mo.18 -------------------------- CAP314.3mo.19 -------------------------- CAP314.3mo.20 -------------------------- CAP314.3mo.21 -------------------------- CAP314.3mo.22 -------------------------- CAP314.3mo.23 -------------------------- CAP314.3mo.13 -------------------------- CAP314.3mo.25 -------------------------- CAP314.6mo.7A ---------------S---------- CAP314.6mo.7D ---------------S---------- CAP314.6mo.7E --D------------S---------- CAP314.6mo.8B --D------------S---------- CAP314.6mo.8F ---------------S---------- CAP314.6mo.9D ---------------S---------- CAP314.6mo.10D A--------------S---------- CAP314.6mo.11C --D------------S---------- CAP314.6mo.11H --D------------S---------- CAP314.6mo.7F A--------------S---------- CAP314.6mo.9B ---------------S---------- CAP314.6mo.10H ---------------S---------- CAP314.6mo.12B ---------------S---------- CAP314.12mo.1 --D--------H---S---------- CAP314.12mo.2 --D------------S---------- CAP314.12mo.3 --D--------H---S---------- CAP314.12mo.4 --D--------H---S---------- CAP314.12mo.5 --D--------H---S---------- CAP314.12mo.7 -----------H---S---------- CAP314.12mo.8 --D--------H---S---------- CAP314.12mo.9 --D--------H---S---------- CAP314.12mo.12 --D--------H---S---------- 12 months 12 months Wild type N332A mutant Fig. 2 a. b. c. d. 332 334 339 | | | CAP314.3mo TNGIIGDIRQAYCNINSTQWNKTLEG CAP314.6mo --D------------S---------- CAP314.3mo N334S ---------------S---------- 332 334 339 | | | CAP177.2wks INGIIGDIRQAYCNINGSEWNSTLEQ CAP177.6mo --D------------S---------K CAP177.2wks N334S ---------------S---------- CAP177 2wk 6mo 2wk N334S 0 200 400 600 800 Titer CAP314 3mo 6mo 3mo N334S 0 2,000 4,000 6,000 8,000 10,000 Titer Fig. 3 mAb (IC50 titers PNG 332 PGT121 PGT128 PGT135 CAP177.2wk - 0.21 >10 >10 CAP177.6mo + 0.01 0.06 >10 CAP177.2wk N334S + 0.04 0.71 >10 CAP314.3mo - >10 >10 >10 CAP314.6mo + 0.03 0.03 >10 CAP314.3mo N334S + 0.03 0.07 >10 TRO.11 (control) + 0.03 0.06 0.16 Neutralization of CAP177 and CAP314 pseudoviruses by members of the PGT family of mAbs PNG at 332 Acute Chronic Acute Chronic 50 60 70 80 90 100 Subtype B Subtype C P = 0.1232 *P = 0.0265 (n=88) (n=86) (n=68) (n=62) % consensus sequences PNG at 301 Acute Chronic Acute Chronic 50 60 70 80 90 100 Subtype B Subtype C P = ns# P = 1.000 (n=91) (n=86) (n=68) (n=62) % consensus sequences PNG 332 + PNG 332 - 0.0001 0.001 0.01 0.1 1 10 100 (n=70) (n=31) P <0,0001 Sensitivity of Subtype C t/f viruses to PGT128 (n=101) PGT128 IC50 Titer (ug/ml) Correlation between PGT128 sensitivity and the glycan at 332 IC50 > 10 (n=46) 1 > IC50 < 10 (n=4) IC50 < 0.1 (n=39) 0.1 > IC50 < 1 (n=12) Figure 4. a. b. c. d.