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NIH-Led Scientists Discover HIV Antibody that Binds to Novel Target on Virus - "Broad and potent HIV-1 neutralization by a human antibody that binds the gp41-gp120 interface"
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WHAT:
An NIH-led team of scientists has discovered a new vulnerability in the armor of HIV that a vaccine, other preventive regimen or treatment could exploit. The site straddles two proteins, gp41 and gp120, that jut out of the virus and augments other known places where broadly neutralizing antibodies (bNAbs) bind to HIV. This newly identified site on the viral spike is where a new antibody found by the scientists in an HIV-infected person binds to the virus. Called 35O22, the antibody prevents 62 percent of known HIV strains from infecting cells in the laboratory and is extremely potent, meaning even a relatively small amount of it can neutralize the virus.
Following their discoveries, the scientists found that 35O22-like antibodies were common in a group of HIV-infected people whose blood contained antibodies that potently neutralized a broad array of HIV strains. According to the researchers, this suggests that it might be easier for a vaccine to elicit 35O22 than some other known bNAbs, which are less common.
Since 35O22 binds only to forms of the viral spike that closely resemble those that naturally appear on HIV, the scientists believe a vaccine that elicits 35O22-like antibodies would need to mimic the natural shape of the spike as closely as possible. This would require a different approach than that used in many previous experimental HIV vaccines, which have included just parts of the viral spike rather than a structure that looks like the entire native viral spike.
In addition, the researchers report, the HIV strains that 35O22 neutralizes complement strains neutralized by other bNAbs. This suggests that eliciting or combining 35O22 with a few other bNAbs in a vaccine or a prevention or treatment regimen could likely neutralize the vast majority of HIV strains found around the globe, according to the scientists.
ARTICLE:
J Huang et al. Broad and potent HIV-1 neutralization by a human antibody that binds the gp41-120 interface. Nature DOI: 10.1038/nature13601 (2014).
WHO:
Anthony S. Fauci, M.D., director of the National Institute of Allergy and Infectious Diseases, part of NIH, is available for comment. Mark Connors, M.D., chief of the HIV-Specific Immunity Section of the NIAID Laboratory of Immunoregulation and the principal investigator of the study, also is available for interviews.
CONTACT:
To schedule interviews, please contact Laura S. Leifman, (301) 402-1663, niaidnews@niaid.nih.gov.
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Broad and potent HIV-1 neutralization by a human antibody that binds the gp41-gp120 interface
Nature
03 September 2014
"Our findings concerning the 35O22 antibody and its specificity have a number of implications for the use of antibodies in HIV therapy, prophylaxis and efforts to stimulate HIV-specific antibodies with vaccines. Its novel binding site and spectrum of activity against HIV strains suggest that it could complement other antibodies used in passive immunotherapy or prophylaxis. In addition, the antibody is extremely potent, indicating that its activity in vivo may therefore persist even at low concentrations. Perhaps most importantly, the novel epitope bound by the 35O22 antibody represents a new site of vulnerability that could potentially be targeted by HIV vaccines. The high prevalence of 35O22-like neutralizing activity in HIV-infected cohorts increases the likelihood that production of similar antibodies could be induced by vaccination. In addition, the highly specific recognition by 35O22 of BG505 SOSIP.664 suggests that this soluble, cleaved trimer antigenically resembles the native Env trimer at the gp120-gp41 interface. Given the binding characteristics of 35O22, these results underscore the possibility that immunogens structurally similar to the native trimer are required for elicitation of such antibodies27."
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Broad and potent HIV-1 neutralization by a human antibody that binds the gp41-gp120 interface
Nature
03 September 2014
Jinghe Huang1, Byong H. Kang1, Marie Pancera2, Jeong Hyun Lee3,4, Tommy Tong5, Yu Feng4, Ivelin S. Georgiev2, Gwo-Yu Chuang2, Aliaksandr Druz2, Nicole A. Doria-Rose2, Leo Laub1, Kwinten Sliepen6, Marit J. van Gils6, Alba Torrents de la Pena6, Ronald Derking6, Per-Johan Klasse7, Stephen A. Migueles1, Robert T. Bailer2, Munir Alam8, Pavel Pugach7, Barton F. Haynes8, Richard T. Wyatt4, Rogier W. Sanders6,7, James M. Binley5, Andrew B.Ward3,4, John R. Mascola2, Peter D. Kwong2 & Mark Connors1
1HIV-Specific Immunity Section, Laboratory of Immunoregulation, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland 20892, USA. 2Vaccine Research Center, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland 20892, USA. 3Department of Integrative Structural and Computational Biology, The Scripps Research Institute, La Jolla, California 92037, USA. 4The Scripps Center for HIV/AIDS Vaccine Immunology and Immunogen Discovery, The Scripps Research Institute, La Jolla, California 92037, USA. 5San Diego Biomedical Research Institute, San Diego, California 92121, USA. 6Department of Medical Microbiology, Academic Medical Center, University of Amsterdam, Amsterdam 1100 DD, The Netherlands. 7Department of Microbiology and Immunology, Weill Medical College of Cornell University, New York, New York 10065, USA. 8Duke Human Vaccine Institute, Duke University, Durham, North Carolina 27710, USA.
The isolation of human monoclonal antibodies is providing important insights into the specificities that underlie broad neutralization of HIV-1 (reviewed in ref. 1). Here we report a broad and extremely potent HIV-specific monoclonal antibody, termed 35O22, which binds a novel HIV-1 envelope glycoprotein (Env) epitope. 35O22 neutralized 62% of 181 pseudoviruses with a half-maximum inhibitory concentration (IC50) <50 μg ml-1. The median IC50 of neutralized viruses was 0.033 μg ml-1, among the most potent thus far described. 35O22 did not bind monomeric forms of Env tested, but did bind the trimeric BG505 SOSIP.664. Mutagenesis and a reconstruction by negative-stain electron microscopy of the Fab in complex with trimer revealed that it bound to a conserved epitope, which stretched across gp120 and gp41. The specificity of 35O22 represents a novel site of vulnerability on HIV Env, which serum analysis indicates to be commonly elicited by natural infection. Binding to this new site of vulnerability may thus be an important complement to current monoclonal-antibody-based approaches to immunotherapies, prophylaxis and vaccine design.
Induction of a potent neutralizing antibody response capable of recognizing highly diverse isolates of HIV-1 is one of the most important goals of HIV vaccine research. This represents a considerable challenge given the extraordinary antigenic variability of the Env surface glycoprotein. However, approximately 20% of the HIV-infected population does develop a humoral immune response capable of recognizing highly diverse strains2, 3, 4, 5, 6. In the past several years improved patient cohorts2, 3, 4, 5, 6, HIV-specific B-cell isolation7, 8, 9, and IgG cloning techniques10, 11 have permitted extraordinary progress in the isolation of broadly neutralizing monoclonal antibodies (bNabs) from these individuals. Thus far, these primarily fall into four categories based on the position of their epitopes on the Env protein, a trimer of gp120 and gp41 heterodimers that is the target of neutralizing antibodies. These sites include the CD4-binding site on gp120 (refs 8, 12) (of which VRC01 is an example), the glycan-containing regions of V1V2 on gp120 (of which PG9 and PG16 are examples), the V3 region centred on the N332 glycan of gp120 (refs 7, 13) (of which PGT121 is an example) and the membrane-proximal external region (MPER) on gp41 (of which 10E8 is an example)14, 15. It remains unclear to what extent these four categories represent the prevalent and immunodominant sites of Env vulnerability through which broad neutralizing responses are mediated, or whether additional specificities exist16, 17, 18, 19.
Here we report the isolation of a broad and potently neutralizing HIV-specific monoclonal antibody, 35O22, that binds a novel epitope. The neutralizing activity of 35O22 is highly complementary to the activities of other known bNabs. We used mutagenesis, crystallography and electron microscopy to define the Env site targeted by 35O22. Our results indicate that 35O22 neutralization occurs by a novel mode of trimer recognition along a conserved face on contiguous areas of gp41 and gp120.
For a better understanding of the specificities that underlie broadly neutralizing antibody responses we applied a technique to identify human monoclonal antibodies of interest from peripheral blood B cells without previous knowledge of the target specificity9. IgG+ B cells of a donor (N152) with broad and potent neutralizing serum and from whom recently described 10E8 antibody was cloned20 were sorted and expanded. The supernatants of B-cell microcultures were screened for neutralizing activity and IgG genes from positive wells were cloned and re-expressed. In addition to the 10E8 antibody, eight clonal family variants of an additional antibody with neutralization activity were found, among which the 35O22 antibody was the most potent and broad (Supplementary Table 1a, b). This antibody was derived from IGHV-1-18*02 and IGLV-2-14*02 germline genes, and was highly somatically mutated in variable genes of both heavy chain (35%) and λ light chain (24%) compared to germ line. The 35O22 antibody possessed a heavy-chain complementarity-determining 3 region (CDR H3) composed of 14 amino acids (Fig. 1a and Supplementary Table 2) and an insertion of 8 amino acids in framework 3 (FR3). High levels of somatic mutation and FR3 insertions are features of other HIV-specific bNabs7, 8, 9, 12, 13, 21, 22. Autoreactivity or polyreactivity are properties of several HIV-specific antibodies23, 24 that could limit their use in therapies or prophylaxis. However, 35O22 bound HEP-2 epithelial cells only modestly (Extended Data Fig. 1a) and did not bind a panel of autoantigens (Extended Data Fig. 1b, c). Against a large panel of pseudoviruses, 35O22 neutralized 62% of 181 isolates with an IC50 <50 μg ml-1 (Fig. 1b and Supplementary Table 3). In numerous cases where the IC50 of 10E8 was >1 μg ml-1, that of 35O22 was 100 to 1,000-fold lower (Supplementary Tables 1b and 3), indicating that their activities were highly complementary. It is likely that 35O22-like antibodies account for much of the breadth and potency of the N152 patient serum against clades A and B (Supplementary Table 3), whereas 10E8-like antibodies may account for much of the breadth against clade C isolates. Overall, the median IC50 of 35O22 for sensitive viruses was 0.033 μg ml-1, which is among the most potent thus far described (Fig. 1b).
The neutralizing spectrum of 35O22 was then compared to those of other bNabs. The IC50 of 35O22 against a panel of diverse isolates did not correlate with those of the bNabs VRC01, 10E8, PG9 and PGT121 (Extended Data Fig. 2a). In addition, a neutralization-based clustering analysis revealed that 35O22 clustered separately from other bNabs (Extended Data Fig. 2b). Furthermore, 35O22 did not compete with other known bNabs when bound to virus-like particles (VLPs) (Extended Data Fig. 2c). Its neutralization of many pseudoviruses did not exceed 80% even at high concentrations (Extended Data Fig. 3a) and its potency increased when pseudoviruses were produced in the presence of the glycosidase inhibitors NB-DNJ or kifunensine, consistent with recognition of high mannose (Extended Data Fig. 3b)25. However, neutralization was unaffected by mutation of N-linked glycosylation sites critical for binding of known bNabs (Extended Data Fig. 4a-c)7, 13. Taken together, these data suggested that 35O22 binds glycans, but its specificity differed from all previously characterized bNabs.
Mutation of four predicted sites of N-linked glycosylation on HIVJRCSF (HIV-1/clade B) Env diminished neutralization potency-N88A, N230A, N241A and N625A (Fig. 2a and Supplementary Table 4). This result suggested that 35O22 recognized elements of both gp120 and gp41, a property that may be consistent with several recently isolated antibodies16, 17, 18, 19. When mutations were introduced in the five residues on either side of these four sites, the V89A, T90A, K227A, T232A and S243A mutations each diminished neutralization (Supplementary Table 4). With the exception of V89A and K227A, it is likely that the impact of each of these mutations was to disrupt the Asn-X-Ser/Thr glycotransferase sequon. The T627A mutation had no impact, suggesting that a glycan may not be present at 625 and 35O22 may make a protein contact at this position. Overall, similar results were obtained using replication-competent HIVLAI (HIV-1/clade B) viruses (Supplementary Table 5). Examination of the sequences of resistant pseudoviruses within clade C did not reveal a clear pattern of variation at each of the positions found to affect 35O22 neutralization or within the glycosylation sequon. It is therefore possible that the resistance of clade C viruses is mediated by other factors such as variations in glycosylation pattern or conformation.
35O22 did not bind to a panel of soluble recombinant Env proteins (Extended Data Fig. 5a-c), suggesting that these do not have the appropriate conformation or glycosylation for binding. However, the 35O22 antibody did bind a recently described stabilized, cleaved, soluble trimer, BG505 SOSIP.664 (Fig. 2b)26. Despite a plateau in neutralization below 50% (Extended Data Fig. 3a) and lacking glycans at positions 230 and 241, binding to BG505 SOSIP.664 trimer had numerous characteristics consistent with its activity against the HIVJRCSF pseudovirus. 35O22 binding was increased to trimer produced in cells treated with kifunensine or cells deficient in glycan processing (Fig. 2b), and diminished by mutations at positions 88 and 625 (Fig. 2c). 35O22 did bind to BG505 SOSIP trimer lacking the furin cleavage site (BG505 SOSIP.SEKS). In surface plasmon resonance (SPR) experiments, 35O22 also bound to immobilized BG505 SOSIP.664 with high affinity (dissociation constant (Kd) = 5.6 nM) (Fig. 2d). Binding was markedly lower to the gp120-gp41ECTO protomer and no binding was detected to the gp120 monomer (Fig. 2e). 35O22 bound the uncleaved BG505 SOSIP.SEKS but no binding was observed to the uncleaved form lacking the SOSIP mutations (Fig. 2f). These observations, combined with the lack of binding of 35O22 to all other soluble forms of Env tested, suggested that this antibody requires a trimeric structure for binding its epitope on gp120 and gp41 (ref. 27).
To provide an atomic-level understanding of the structure of the 35O22 antibody, we crystallized the Fab of 35O22. Crystals were obtained that diffracted to 1.55 A resolution (Supplementary Table 6). Overall the structure of 35O22 Fab revealed a relatively flat antigen-combining site, lacking long protruding loops, and flanked by the complementarity-determining region 1 on the light chain (CDR L1) and the 8-amino-acid insertion in FR3 of the heavy chain (Fig. 3a). The surface of the antigen-combining site was heavily altered by somatic mutation, and two pairs of cysteines introduced by somatic mutation in CDR L1 and L3 formed disulphide bonds (Fig. 3a and Extended Data Fig. 6a).
We next sought to determine the structure of the antibody-antigen complex. The ability of 35O22 to bind the BG505 SOSIP.664 trimer permitted imaging of the antibody-antigen complex by negative stain electron microscopy (EM). The reconstruction of these images showed that three 35O22 Fabs bound to the trimers at sites close to the predicted viral membrane (Fig. 3b and Extended Data Fig. 6b). Superposition of the negative stain reconstruction of the soluble BG505 SOSIP.664 with 35O22 Fab onto the BaL EM tomogram of the viral spike (Extended Data Fig. 6b) suggested that the viral membrane is in close contact to the 35O22 Fab light chain. Residues Tyr 68 and Trp 69 in the light chain and FR3 tyrosines at residues 65 and 72 form potential surfaces of membrane association. The 35O22 heavy chain was in close proximity to the four sites observed to contribute to the 35O22 epitope in mutagenesis experiments (N88, N230, N241 and N625). The CDR H3 was predicted to interact with N625 and CDR H2 with N88. The 8-amino-acid insertion in the framework 3 heavy chain is located close to residues 88-90 on gp120. Reversion of this insertion to germline markedly diminished neutralization against most pseudoviruses in our panel (Supplementary Table 7). 35O22 binds a surface on the Env spike that is distinct from two other antibodies, 8ANC195 and PGT151, reported to bind gp120 and gp41 (Extended Data Fig. 7)16, 17, 18.
Analysis of the 35O22 site of vulnerability (Extended Data Fig. 8a-c) indicated that it is highly conserved. The glycans predicted at positions 88, 241 and 625 were found to be among the most highly conserved N-linked glycosylation sequons of 4,265 HIV-1 sequences in the Los Alamos database (Supplementary Table 8). Despite this high level of conservation, analysis of the Env gene of the patient's plasma virus showed that the predicted amino acid sequence varied at the critical 35O22 contacts. In addition to the previously published 10E8 escape mutations W680R and K683Q, an N230Q is predicted in one sequence, N241D in half of the sequences, and an N624D and N625Q in all sequences (Extended Data Fig. 9a). When these mutations were introduced into HIVJRCSF pseudoviruses, there was a drop in neutralization with the greatest effect caused by the N625Q mutation found in all of the plasma sequences (Extended Data Fig. 9b). These data suggest that the autologous virus has escaped neutralization by 35O22.
To gain insight into the prevalence of the specificity of 35O22, we added the 35O22 neutralization fingerprint to the ten that we had previously identified (Extended Data Fig. 8d, e)28. Notably, 13 of the sera (38%) showed significant 35O22 neutralization signals (>0.2). This level of prevalence was substantially higher than for the V1V2-directed response (typified by the PG9 antibody) or that of the 8ANC195 antibody. However, it was lower than the prevalence of responses to the MPER (50% prevalence), the CD4-binding site (53% prevalence), or the V3 glycan site (82% prevalence). The neutralizing activity of sera was also measured against HIVJRCSF pseudoviruses bearing N88A, N230A, N241A or N625A mutations (Extended Data Table 1). These mutations caused a greater than fivefold increase in ID50 (50% inhibitory dilution) in more than half of donors, with a high level of concordance between the impact of each of these mutations within a given serum. These results suggested that 35O22 is unlikely to be the product of a unique B-cell repertoire or infecting virus, but rather arises commonly among patients that develop HIV-specific neutralizing antibodies.
To achieve a better understanding of the mechanism of 35O22's activity, we examined the timing of its binding and neutralization during virus fusion. Given the proximity of the 35O22 epitope to the membrane, it was important to perform these experiments in the context of Env expressed on cells or virions. In these settings, MPER-specific antibodies have limited access to the native trimer and bind best after the conformational changes induced by CD4 attachment29. Binding of 35O22 to Env expressed on the cell surface was low and similar to the MPER antibody 2F5 (Fig. 4a). 35O22 binding to VLPs was weak compared to that of VRC01 but similar to that of 10E8 (Fig. 4b). 35O22 binding was slightly inhibited by soluble CD4 (sCD4) binding, suggesting the 35O22 epitope is not induced by sCD4 under these experimental conditions. 35O22 neutralization was partially eliminated by washing of pseudovirions before infection, a result consistent with limited access to Env on free virions (Fig. 4c)29. However, if 35O22 was incubated with VLPs, permitted to bind to target cells, and then after 2 h the cells were washed, there was little impact on neutralization compared with the leave in format, similar to all other antibodies except the MPER 10E8 antibody (Fig. 4d). Similar to 2G12 and 10E8, 35O22 activity was relatively high in the post-CD4 format (see Methods), consistent with previous work showing that virus-sCD4 complexes are more sensitive to neutralization than virus alone30. In a post-CD4/CCR5 assay, only 10E8 neutralized virus, consistent with previous observations (Fig. 4d)30. Taken together with the structural data, these results suggest that in the context of a lipid membrane, 35O22 binds Env poorly before CD4 attachment. However, after trimer attachment to CD4, 35O22 may bind to an early intermediate that exposes the 35O22 epitope possibly by raising the Env spike within the viral membrane (Fig. 4e).
Our findings concerning the 35O22 antibody and its specificity have a number of implications for the use of antibodies in HIV therapy, prophylaxis and efforts to stimulate HIV-specific antibodies with vaccines. Its novel binding site and spectrum of activity against HIV strains suggest that it could complement other antibodies used in passive immunotherapy or prophylaxis. In addition, the antibody is extremely potent, indicating that its activity in vivo may therefore persist even at low concentrations. Perhaps most importantly, the novel epitope bound by the 35O22 antibody represents a new site of vulnerability that could potentially be targeted by HIV vaccines. The high prevalence of 35O22-like neutralizing activity in HIV-infected cohorts increases the likelihood that production of similar antibodies could be induced by vaccination. In addition, the highly specific recognition by 35O22 of BG505 SOSIP.664 suggests that this soluble, cleaved trimer antigenically resembles the native Env trimer at the gp120-gp41 interface. Given the binding characteristics of 35O22, these results underscore the possibility that immunogens structurally similar to the native trimer are required for elicitation of such antibodies27.
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