PMCCPMCCPMCC

Search tips
Search criteria 

Advanced

 
Logo of wtpaEurope PMCEurope PMC Funders GroupSubmit a Manuscript
 
Nat Med. Author manuscript; available in PMC Nov 9, 2012.
Published in final edited form as:
Published online Oct 21, 2012. doi:  10.1038/nm.2985
PMCID: PMC3494733
EMSID: EMS50330
Evolution of an HIV glycan–dependent broadly neutralizing antibody epitope through immune escape
Penny L Moore,1,2 Elin S Gray,1 C Kurt Wibmer,1,2 Jinal N Bhiman,1,2 Molati Nonyane,1 Daniel J Sheward,3 Tandile Hermanus,1 Shringkhala Bajimaya,4 Nancy L Tumba,1 Melissa-Rose Abrahams,3 Bronwen E Lambson,1 Nthabeleng Ranchobe,1 Lihua Ping,5 Nobubelo Ngandu,3 Quarraisha Abdool Karim,6 Salim S Abdool Karim,6 Ronald I Swanstrom,5 Michael S Seaman,4 Carolyn Williamson,3 and Lynn Morris1,2
1Centre for HIV and STI, National Institute for Communicable Diseases of the National Health Laboratory Service, Johannesburg, South Africa
2University of the Witwatersrand, Johannesburg, South Africa
3Institute of Infectious Diseases 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, Massachusetts, USA
5University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, USA
6Centre for the AIDS Programme of Research in South Africa (CAPRISA), University of KwaZulu Natal, Durban, South Africa
Correspondence should be addressed to L.M. (lynnm/at/nicd.ac.za).
Neutralizing antibodies are likely to play a crucial part in a preventative HIV-1 vaccine. Although efforts to elicit broadly cross-neutralizing (BCN) antibodies by vaccination have been unsuccessful1-3, a minority of individuals naturally develop these antibodies after many years of infection4-7. How such antibodies arise, and the role of viral evolution in shaping these responses, is unknown. Here we show, in two HIV-1–infected individuals who developed BCN antibodies targeting the glycan at Asn332 on the gp120 envelope, that this glycan was absent on the initial 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 viruses that lacked the glycan at amino acid 332 were resistant to the Asn332-dependent BCN monoclonal antibody PGT128 (ref. 8), whereas escaped variants that acquired this glycan were sensitive. Analysis of large sequence and neutralization data sets showed the 332 glycan to be significantly underrepresented in transmitted subtype C viruses compared to chronic viruses, with the absence of this glycan corresponding with resistance to PGT128. These findings highlight the dynamic interplay between early antibodies and viral escape in driving the evolution of conserved BCN antibody epitopes.
Although the role of glycans in shielding neutralizing epitopes has long been known9-11, it has only recently become clear that many BCN responses directly target glycans, including the one at position 332 in the C3 region of the gp120 subunit of the HIV-1 envelope protein8,12-18. The recent isolation of monoclonal antibodies (mAbs) that target this glycan, which are the most potent yet described, has focused attention on this epitope8. These mAbs (PGT121–PGT123, PGT125–PGT128, PGT130, PGT131 and PGT135–PGT137) neutralize effectively across all HIV-1 subtypes, with the broadest, PGT128, neutralizing >70% of viruses tested8. Crystal structures of PGT127 and PGT128 have shown that these mAbs penetrate the glycan shield, recognizing high-mannose glycans at amino acids 301 and 332, in addition to a short β-strand in the C terminus of the V3 loop19. The conserved nature of these amino acids and the high potency of this class of mAbs suggest that this region may be an important vaccine target. Furthermore, this epitope is immunogenic, as Asn332-dependent BCN antibodies are often found in infected subjects who develop neutralization breadth8,14-17. However, as with other BCN antibodies, the factors that favor the emergence of Asn332-dependent BCN antibodies remain unclear. Here we hypothesize that the evolution of viral populations, which are under considerable immune and fitness selection pressures, creates BCN antibody epitopes essential for the development of neutralization breadth.
From a cohort of 79 HIV-1 subtype C-infected women studied starting at the point of acute infection, we focused on two participants who developed Asn332-dependent BCN antibodies. Subject CAP177 produced antibodies by 3 years after infection that were capable of neutralizing 88% of a large multisubtype panel of 225 heterologous viruses (M. Lacerda, P.L.M., N. N., M.S.S., E.S.G. et al., unpublished data). The second individual, CAP314, neutralized 46% of 41 heterologous viruses after only 2 years of infection (Supplementary Fig. 1). Plasmas from CAP177 and CAP314 were unable to neutralize heterologous viruses that lacked the glycan at position 332 from the time when BCN responses were first detected and thereafter (Fig. 1a), confirming that their BCN activity depended on the 332 glycan, similar to the PGT128 mAb.
Figure 1
Figure 1
Characterizing the antibody specificities and viral populations in CAP177 and CAP314. (a) Serial plasma neutralization of three heterologous viruses (Q23, Du156 and TRO.11 for CAP177 and Q23, Du156 and CAP200 for CAP314) compared to mutant viruses where (more ...)
Single-genome amplification and gp160 sequencing from plasma viral RNA (Online Methods) was used to determine the amino acid sequence of the envelopes of circulating viral populations at multiple time points. This included the earliest available plasma samples, which were taken 2 weeks after infection for CAP177 (enabling inference of the transmitted/founder virus20) and 3 months after infection for CAP314. In both cases, these acute viruses lacked the predicted N-linked glycan at amino acid 332, although almost all sequences contained an intact glycosylation site at position 334 (Fig. 1b,c). By 5–6 months of infection, a glycan at position 332 evolved in both CAP177 and CAP314 through an N334T or N334S substitution, which also resulted in the destruction of the neighboring glycan at position 334. By 12–15 months, when BCN antibodies became detectable, the 332 glycan was present in all sequences; however, at around 2 years the glycan reverted to position 334, most likely as a consequence of viral escape from the Asn332-dependent BCN response (Fig. 1b,c). Therefore, for both CAP177 and CAP314, the glycan that formed the basis of the BCN epitope was not present on the infecting virus but evolved shortly thereafter, coincident with the appearance of strain-specific antibodies (Fig. 1c).
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 antibodies21. Indeed, we have previously shown that in CAP177 early neutralizing antibodies targeted the C3 region22. As viral escape often involves glycan rearrangements10, 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 virus from CAP177 and CAP314 and inserted the 332 glycan into the acute clones by mutating the asparagine at position 334 to a serine (which also deleted the glycan at 334). We tested all three viruses from both individuals for sensitivity to autologous plasma from 6 months after infection. We detected autologous neutralizing responses against the acute viruses, whereas the 6-month clones were resistant to contemporaneous plasma, as expected (Fig. 2a,b). Introduction of the glycan at position 332 resulted in almost complete resistance to neutralization by plasma from CAP177 and reduced neutralization sensitivity to plasma from CAP314, suggesting that the 332 glycan evolved to afford escape from early strain-specific antibodies.
Figure 2
Figure 2
The glycan at position 332 mediates neutralization escape in CAP177 and CAP314. (a) Partial sequence alignment showing the CAP177 acute virus (CAP177 2 weeks), a representative 6-month clone (CAP177 6 months) and the acute virus mutated to contain a glycan (more ...)
The absence of the glycan at position 332 on the acute viruses from CAP177 and CAP314 suggested that they might be resistant to BCN mAbs that depend on this glycan, such as PGT128. Neutralization experiments showed that CAP177 and CAP314 acute clones were resistant to PGT128, whereas the 6-month clones that contained the 332 glycan were highly sensitive, with a half-maximum inhibitory concentration (IC50) <0.06 μg ml−1 (Table 1). We obtained similar data with a related mAb, PGT121, although the CAP177 acute clone showed moderate sensitivity (0.21 μg ml−1) but was nevertheless 20-fold more resistant compared to the 6-month clone. Both N334S mutants (acute clones with a glycan introduced at position 332) were sensitive to PGT121 and PGT128, confirming the role of the 332 glycan in conferring sensitivity to these mAbs. None of the CAP177 or CAP314 clones was sensitive to the mAbs PGT135 or 2G12 (Table 1), which depend on additional glycans at other amino acids, that are uncommon among subtype C viruses23-27. Thus, whereas shifting a glycan from position 334 to 332 allowed the virus to escape autologous neutralizing antibodies, this created a new neutralizing antibody epitope that provided the antigenic stimulus to elicit BCN antibodies targeting the 332 glycan.
Table 1
Table 1
Neutralization of CAP177 and CAP314 pseudoviruses by BCN monoclonal antibodies
We assessed whether this pattern of selection of the 332 glycan was evident at a population level using more than 7,300 single-genome amplification—derived gp160 envelope sequences from acute and chronic HIV-1 infections (Online Methods)20,28. Sequences with evidence of dual infection were excluded. For each subject, the consensus sequence was generated and the frequency of the 332 glycan was determined. We assessed significance with Fisher’s exact test. In subtype C, the 332 glycan was significantly less common among transmitted/founder viruses (45/68, 66%) compared to unmatched chronic viruses (52/62, 84%, P = 0.0166, Fig. 3a). To ensure that this was not due to adaption of HIV to neutralizing antibodies over the course of the epidemic time29, we performed the same analysis using a smaller data set of 502 matched sequences from 20 individuals, with similar results (P = 0.0457, Fig. 3a). Although we observed the same trend in subtype B sequences, it was not statistically significant (Fig. 3a). Taken together, these results suggest that the pattern of evolution we describe for CAP177 and CAP314 may be relatively common and that the absence of the 332 glycan on subtype C viruses may provide an advantage during transmission or early viral outgrowth.
Figure 3
Figure 3
The glycan at residue 332 is underrepresented in subtype C transmitted/founder viruses, which are also frequently resistant to the PGT128 mAb. (a) Comparison of the frequency of the 332 glycan among 1,371 envelope sequences from 68 subjects with HIV-1 (more ...)
We analyzed the phenotypes of 101 transmitted/founder subtype C viruses using envelope clones generated as part of the Vaccine Immune Monitoring Core Standard Virus Panel Consortium. For this, transmitted/founder envelope sequences were inferred from singlegenome amplified and sequenced envelope amplicons derived from plasma from acutely HIV-infected subjects30 and cloned into a mammalian expression vector. Envelope clones were transfected into 293T cells with the HIV backbone construct pSG3ΔEnv to produce envelope pseudotyped particles, and neutralization assays were performed in TZM-bl cells as described in the Online Methods. Phenotypic analysis supported the genotypic analysis, with a high proportion (46%) of viruses resistant to PGT128 neutralization at the highest concentration tested (10 μg ml−1) (Fig. 3b and Supplementary Fig. 2). Resistance strongly correlated with the absence of the 332 glycan (P < 0.0001) (Fig. 3c), although some viruses that contained the glycan were also resistant, consistent with the fact that additional residues are needed to form this epitope8. Of 31 viruses in which the glycan at position 332 was absent, only three showed neutralization sensitivity. Of these, two contained the glycan at position 295, which is very rare in subtype C viruses26 but structurally proximal to the 332 glycan and shown by mutagenesis to affect the PGT128 epitope19. Although this virus panel was tested only against PGT128, resistance to this mAb generally extends to other Asn332-dependent PGT mAbs8. These data suggest that Asn332-dependent antibodies present either through passive immunotherapy or vaccination might be only partially effective in preventing subtype C infections and that combinations of antibodies targeting different epitopes may need to be tailored to match circulating viral variants24,26,31.
In addition to the Asn332-dependent epitope, a second BCN antibody epitope that includes the glycans at amino acid positions 156 and 160 in the V2 region has been defined by the mAbs PG9, PG16 (ref. 18) and PGT145 (ref. 8). To assess whether epitope evolution was also associated with the appearance of Asn160-dependent BCN antibodies, we studied two individuals, CAP8 and CAP256, who developed this specificity15 (Supplementary Fig. 3a). In both cases, the glycan at amino acid 160 was absent from envelope sequences at transmission but appeared at 6 and 3 months in CAP8 and CAP256, respectively, suggesting a pattern of evolution similar to that observed with 332 glycan (Supplementary Fig. 3b). Furthermore, we found acute viruses to be resistant to BCN mAbs PG9, PG16 and PGT145, whereas later clones that contained the 160 glycan became sensitive to PG9 and PGT145, although not to PG16 (Supplementary Fig. 4). However, although insertion of the 160 glycan by site-directed mutagenesis into the resistant acute clones rendered them sensitive to PG9 and PGT145, unlike the case with the CAP177 and CAP314 mutants containing the 332 glycan, this single change did not mediate neutralization escape. It was also not possible to show selection of the 160 glycan using large sequence data sets, as it is highly conserved in both acute and chronic sequences (Supplementary Fig. 5).
Despite the fact that BCN epitopes are highly conserved, they only occasionally elicit BCN responses in humans. Those targeting epitopes centered around the 332 or 160 glycans develop more frequently in infected people compared to BCN antibodies directed at the CD4 binding site or membrane-proximal region12-14. The data presented here raise the hypothesis that, at least in the case of Asn322-directed antibodies, viral evolution may facilitate their elicitation. However, the absence of these glycans on the acute virus is not a prerequisite for the development of breadth, nor does the evolution of a BCN epitope guarantee that these antibodies will arise. Indeed, within this cohort, examples of both scenarios exist (Supplementary Fig. 6a,b). Furthermore, in a study of a SHIV-infected macaque32 where the 332 glycan was present at transmission, potent and broad Asn332-dependent BCN antibodies developed within 9 months. In HIV-1 subtype C viruses, where the absence of the 332 glycan is favored at transmission, subsequent immune pressure exerted by strain-specific neutralizing antibodies results in the evolution of the 332 glycan. This process provides a mechanism for the evolution of BCN epitopes, with neutralization escape driving viral convergence toward glycan motifs that are highly conserved and serve as targets for BCN antibodies.
Participants
CAP177, CAP8 and CAP256 were part of the CAPRISA 002 Acute Infection study, a cohort of 245 high-risk, HIV-negative women that was established in 2004 in Durban, South Africa, for follow-up and subsequent identification of HIV seroconversion33. These three HIV-infected subjects were among the seven women in this cohort who developed neutralization breadth15. The fourth individual, CAP314, was enrolled in the CAPRISA 004 trial, a two-arm, double-blind, randomized, placebo-controlled trial, conducted from May 2007 to March 2010, to assess the effectiveness and safety of tenofovir gel for the prevention of HIV infection in women34. Monitoring BCN antibodies in this cohort of 39 women showed that she was one of three who developed breadth by 2 years of infection, two of whom targeted an Asn332-dependent epitope. The CAPRISA 002 Acute Infection study was reviewed and approved by the research ethics committees of the University of KwaZulu-Natal (E013/04), 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 Biomedical Research Ethics Committee (E111/06), Family Health International’s Protection of Human Subjects Committee (9946) and the South African Medicines Control Council (20060835). All participants provided written informed consent.
Single-genome amplification and sequencing
HIV-1 RNA was isolated from plasma using the Qiagen QIAamp Viral RNA kit and reverse transcribed to cDNA using SuperScript III Reverse Transcriptase (Invitrogen, CA). The envelope genes were amplified from single-genome templates30, and amplicons were directly sequenced using the ABI PRISM Big 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 were assembled and edited using Sequencher version 4.0 software (Genecodes, Ann Arbor, MI). The number of potential N-linked glycosylation sites was determined using N-glycosite (http://www.hiv.lanl.gov/content/sequence/GLYCOSITE/glycosite.html/). Multiple sequence alignments were performed using Clustal X (version 1.83) and edited with BioEdit (version 5.0.9) Sequence alignments were visualized using Highlighter for Amino Acid Sequences version 1.1.0 (beta). Selected amplicons were cloned into the expression vector pcDNA 3.1 (directional) (Invitrogen) by reamplification of SGA first-round products using Phusion enzyme (Finn Enzymes) with the EnvM primer35 and the directional primer EnvAdir21. Cloned env genes were sequenced to confirm that they exactly matched the sequenced amplicon.
Site-directed mutagenesis
Sensitive heterologous viruses Q23, Du156, TRO.11, CAP200 and ConC were mutated at key residues (332–334 or 160–162) by site-directed mutagenesis using the Stratagene QuickChange II kit (Stratagene) as described by the manufacturer. Mutations were confirmed by sequencing. Autologous clones derived from CAP177 and CAP314 were mutated to shift the glycan from position 334 to 332, and mutants were verified by sequencing. Similarly autologous clones from CAP8 and CAP256 were mutated to introduce the glycan at position 160, and mutants were verified by sequencing.
Neutralization assays
The JC53bl-13 (TZM-bl) cell line was obtained from the NIH AIDS Research and Reference Reagent Program. 293T cells were obtained from G. Shaw. Both cell lines were cultured in DMEM (Gibco BRL Life Technologies) containing 10% heat-inactivated FBS and 50 μg/ml gentamicin (Sigma). Cell monolayers were disrupted at confluency by treatment with 0.25% trypsin in 1mM EDTA. Env-pseudotyped viruses were obtained by co-transfecting the Env plasmid with pSG3ΔEnv10 using FuGENE transfection reagent (Roche) as previously described36. Neutralization was measured as described36 by a reduction in luciferase gene expression after single-round infection of JC53bl-13 cells with Env-pseudotyped viruses. Titers were calculated as the reciprocal plasma dilution (ID50) causing 50% reduction of relative light units (RLU).
Supplementary Material
Supplementary
ACKNOWLEDGMENTS
We thank the participants in the CAPRISA 002 and 004 cohorts and the clinical and laboratory staff at CAPRISA for managing the cohort and providing specimens. 293T cells were obtained from G. Shaw (University of Alabama, Birmingham). We are grateful to D. Burton and W. Koff of the International AIDS Vaccine Initiative for providing the PGT monoclonal antibodies and to S. Gnanakaran (Los Alamos National Laboratories, Los Alamos, New Mexico) for providing transmitted/founder and chronic subtype B sequences. We thank Z. Valley-Omar and N. Ndabambi for generating some envelope sequences. We are grateful to B. Hahn and J. Kim, who contributed transmitted/founder subtype C clones to the Vaccine Immune Monitoring Core Standard Virus Panel Consortium. This work was funded by CAPRISA, the Centre for HIV/AIDS Vaccine Immunology, the South African HIV/AIDS Research and Innovation Platform of the South African Department of Science and Technology and by a US National Institutes of Health grant (number AI088610). CAPRISA was initially supported by the US National Institute of Allergy and Infectious Diseases, US National Institutes of Health, US Department of Health and Human Services grant U19 AI51794. M.S.S. is funded by the Bill & Melinda Gates Foundation, Vaccine Immune Monitoring Consortium of the Collaboration for AIDS Vaccine Discovery (grant 1032144). P.L.M. and E.S.G. were supported by the Columbia University Southern African Fogarty AIDS International Training and Research Program through the Fogarty International Center, National Institutes of Health (grant 5 D43 TW000231). P.L.M. is a Wellcome Trust Intermediate Fellow in Public Health and Tropical Medicine (grant 089933/Z/09/Z).
Footnotes
Note: Supplementary information is available in the online version of the paper.
AUTHOR CONTRIBUTIONS
P.L.M. and E.S.G. designed the study, performed experiments, analyzed data and wrote the manuscript; E.S.G., C.K.W., J.N.B., T.H. and N.L.T. performed neutralization experiments and analyzed data; E.S.G., C.K.W., D.J.S., M.N., B.E.L. and N.R. generated single-genome sequences; D.J.S. and N.N. performed part of the sequence analyses; M.-R.A., L.P., R.I.S. and C.W. contributed the subtype C acute and chronic sequences; S.B. and M.S.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.
COMPETING FINANCIAL INTERESTS
The authors declare no competing financial interests.
1. Haynes BF, Montefiori DC. Aiming to induce broadly reactive neutralizing antibody responses with HIV-1 vaccine candidates. Expert Rev. Vaccines. 2006;5:347–363. [PMC free article] [PubMed]
2. Hu SL, Stamatatos L. Prospects of HIV Env modification as an approach to HIV vaccine design. Curr. HIV Res. 2007;5:507–513. [PubMed]
3. Phogat S, Wyatt R. Rational modifications of HIV-1 envelope glycoproteins for immunogen design. Curr. Pharm. Des. 2007;13:213–227. [PubMed]
4. Li Y, et al. Analysis of neutralization specificities in polyclonal sera derived from human immunodeficiency virus type 1–infected individuals. J. Virol. 2009;83:1045–1059. [PMC free article] [PubMed]
5. Sather DN, et al. Factors associated with the development of cross-reactive neutralizing antibodies during human immunodeficiency virus type 1 infection. J. Virol. 2009;83:757–769. [PMC free article] [PubMed]
6. Simek MD, et al. Human immunodeficiency virus type 1 elite neutralizers: individuals with broad and potent neutralizing activity identified by using a high-throughput neutralization assay together with an analytical selection algorithm. J. Virol. 2009;83:7337–7348. [PMC free article] [PubMed]
7. Stamatatos L, Morris L, Burton DR, Mascola JR. Neutralizing antibodies generated during natural HIV-1 infection: good news for an HIV-1 vaccine? Nat. Med. 2009;15:866–870. [PubMed]
8. Walker LM, et al. Broad neutralization coverage of HIV by multiple highly potent antibodies. Nature. 2011;477:466–470. [PMC free article] [PubMed]
9. Richman DD, Wrin T, Little SJ, Petropoulos CJ. Rapid evolution of the neutralizing antibody response to HIV type 1 infection. Proc. Natl. Acad. Sci. USA. 2003;100:4144–4149. [PubMed]
10. Wei X, et al. Antibody neutralization and escape by HIV-1. Nature. 2003;422:307–312. [PubMed]
11. Frost SD, et al. Neutralizing antibody responses drive the evolution of human immunodeficiency virus type 1 envelope during recent HIV infection. Proc. Natl. Acad. Sci. USA. 2005;102:18514–18519. [PubMed]
12. Gray ES, et al. Antibody specificities associated with neutralization breadth in plasma from HIV-1 subtype C infected blood donors. J. Virol. 2009;83:8925–8937. [PMC free article] [PubMed]
13. Tomaras GD, et al. Polyclonal B cell responses to conserved neutralization epitopes in a subset of HIV-1–infected individuals. J. Virol. 2011;85:11502–11519. [PMC free article] [PubMed]
14. Walker LM, et al. A limited number of antibody specificities mediate broad and potent serum neutralization in selected HIV-1 infected individuals. PLoS Pathog. 2010;6:e1001028. [PMC free article] [PubMed]
15. Gray ES, et al. HIV-1 neutralization breadth develops incrementally over 4 years and is associated with CD4+ T cell decline and high viral load during acute infection. J. Virol. 2011;85:4828–4840. [PMC free article] [PubMed]
16. Nandi A, et al. Epitopes for broad and potent neutralizing antibody responses during chronic infection with human immunodeficiency virus type 1. Virology. 2010;396:339–348. [PMC free article] [PubMed]
17. Tang H, et al. epitopes immediately below the base of the V3 loop of gp120 as targets for the initial autologous neutralizing antibody response in two HIV-1 subtype B–infected individuals. J. Virol. 2011;85:9286–9299. [PMC free article] [PubMed]
18. Walker LM, et al. Broad and potent neutralizing antibodies from an African donor reveal a new HIV-1 vaccine target. Science. 2009;326:285–289. [PMC free article] [PubMed]
19. Pejchal R, et al. A potent and broad neutralizing antibody recognizes and penetrates the HIV glycan shield. Science. 2011;334:1097–1103. [PMC free article] [PubMed]
20. Abrahams MR, et al. Quantitating the multiplicity of infection with HIV-1 subtype C reveals a non-Poisson distribution of transmitted variants. J. Virol. 2009;83:3556–3567. [PMC free article] [PubMed]
21. Moore PL, et al. The C3–V4 region is a major target of autologous neutralizing antibodies in human immunodeficiency virus type 1 subtype C infection. J. Virol. 2008;82:1860–1869. [PMC free article] [PubMed]
22. Moore PL, et al. Limited neutralizing antibody specificities drive neutralization escape in early HIV-1 subtype C infection. PLoS Pathog. 2009;5:e1000598. [PMC free article] [PubMed]
23. Gray ES, Moore PL, Pantophlet RA, Morris L. N-linked glycan modifications in gp120 of human immunodeficiency virus type 1 subtype C render partial sensitivity to 2G12 antibody neutralization. J. Virol. 2007;81:10769–10776. [PMC free article] [PubMed]
24. Binley JM, et al. Comprehensive cross-clade neutralization analysis of a panel of anti-human immunodeficiency virus type 1 monoclonal antibodies. J. Virol. 2004;78:13232–13252. [PMC free article] [PubMed]
25. Bures R, et al. Regional clustering of shared neutralization determinants on primary isolates of clade C human immunodeficiency virus type 1 from South Africa. J. Virol. 2002;76:2233–2244. [PMC free article] [PubMed]
26. Gray ES, Meyers T, Gray G, Montefiori DC, Morris L. Insensitivity of paediatric HIV-1 subtype C viruses to broadly neutralising monoclonal antibodies raised against subtype B. PLoS Med. 2006;3:e255. [PMC free article] [PubMed]
27. Zhang M, et al. Tracking global patterns of N-linked glycosylation site variation in highly variable viral glycoproteins: HIV, SIV, and HCV envelopes and influenza hemagglutinin. Glycobiology. 2004;14:1229–1246. [PubMed]
28. Gnanakaran S, et al. Recurrent signature patterns in HIV-1 B clade envelope glycoproteins associated with either early or chronic infections. PLoS Pathog. 2011;7:e1002209. [PMC free article] [PubMed]
29. Bunnik EM, et al. Adaptation of HIV-1 envelope gp120 to humoral immunity at a population level. Nat. Med. 2010;16:995–997. [PubMed]
30. Salazar-Gonzalez JF, et al. Deciphering human immunodeficiency virus type 1 transmission and early envelope diversification by single-genome amplification and sequencing. J. Virol. 2008;82:3952–3970. [PMC free article] [PubMed]
31. Gorny MK, Zolla-Pazner S. Immunoprophylaxis against mother-to-child transmission of HIV-1. PLoS Med. 2006;3:e259. [PMC free article] [PubMed]
32. Walker LM, et al. Rapid development of glycan-specific, broad, and potent anti-HIV-1 gp120 neutralizing antibodies in an R5 SIV/HIV chimeric virus infected macaque. Proc. Natl. Acad. Sci. USA. 2011;108:20125–20129. [PubMed]
33. van Loggerenberg F, et al. Establishing a cohort at high risk of HIV infection in South Africa: challenges and experiences of the CAPRISA 002 acute infection study. PLoS ONE. 2008;3:e1954. [PMC free article] [PubMed]
34. Abdool Karim Q, et al. Effectiveness and safety of tenofovir gel, an antiretroviral microbicide, for the prevention of HIV infection in women. Science. 2010;329:1168–1174. [PMC free article] [PubMed]
35. Gao F, et al. Molecular cloning and analysis of functional envelope genes from human immunodeficiency virus type 1 sequence subtypes A through G. The WHO and NIAID Networks for HIV Isolation and Characterization. J. Virol. 1996;70:1651–1667. [PMC free article] [PubMed]
36. Gray ES, et al. Neutralizing antibody responses in acute human immunodeficiency virus type 1 subtype C infection. J. Virol. 2007;81:6187–6196. [PMC free article] [PubMed]