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A human monoclonal antibody neutralizes diverse HIV-1 isolates by binding a critical gp41 epitope.

ABSTRACT: HIV-1 entry into cells is mediated by the envelope glycoprotein receptor-binding (gp120) and membrane fusion-promoting (gp41) subunits. The gp41 heptad repeat 1 (HR1) domain is the molecular target of the fusion-inhibitor drug enfuvirtide (T20). The HR1 sequence is highly conserved and therefore considered an attractive target for vaccine development, but it is unknown whether antibodies can access HR1. Herein, we use gp41-based peptides to select a human antibody, 5H/I1-BMV-D5 (D5), that binds to HR1 and inhibits the assembly of fusion intermediates in vitro. D5 inhibits the replication of diverse HIV-1 clinical isolates and therefore represents a previously unknown example of a crossneutralizing IgG selected by binding to designed antigens. NMR studies and functional analyses map the D5-binding site to a previously identified hydrophobic pocket situated in the HR1 groove. This hydrophobic pocket was proposed as a drug target and subsequently identified as a common binding site for peptide and peptidomimetic fusion inhibitors. The finding that the D5 fusion-inhibitory antibody shares the same binding site suggests that the hydrophobic pocket is a "hot spot" for fusion inhibition and an ideal target on which to focus a vaccine-elicited antibody response. Our data provide a structural framework for the design of new immunogens and therapeutic antibodies with crossneutralizing potential.

SUBMITTER: Miller MD

PROVIDER: S-EPMC1253587 | biostudies-literature | 2005 Oct

REPOSITORIES: biostudies-literature

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A human monoclonal antibody neutralizes diverse HIV-1 isolates by binding a critical gp41 epitope.

Miller Michael D MD Geleziunas Romas R Bianchi Elisabetta E Lennard Simon S Hrin Renee R Zhang Hangchun H Lu Meiqing M An Zhiqiang Z Ingallinella Paolo P Finotto Marco M Mattu Marco M Finnefrock Adam C AC Bramhill David D Cook James J Eckert Debra M DM Hampton Richard R Patel Mayuri M Jarantow Stephen S Joyce Joseph J Ciliberto Gennaro G Cortese Riccardo R Lu Ping P Strohl William W Schleif William W McElhaugh Michael M Lane Steven S Lloyd Christopher C Lowe David D Osbourn Jane J Vaughan Tristan T Emini Emilio E Barbato Gaetano G Kim Peter S PS Hazuda Daria J DJ Shiver John W JW Pessi Antonello A

Proceedings of the National Academy of Sciences of the United States of America 20051003 41

HIV-1 entry into cells is mediated by the envelope glycoprotein receptor-binding (gp120) and membrane fusion-promoting (gp41) subunits. The gp41 heptad repeat 1 (HR1) domain is the molecular target of the fusion-inhibitor drug enfuvirtide (T20). The HR1 sequence is highly conserved and therefore considered an attractive target for vaccine development, but it is unknown whether antibodies can access HR1. Herein, we use gp41-based peptides to select a human antibody, 5H/I1-BMV-D5 (D5), that binds ...[more]

PMID: 16203977

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Project description:Previous studies in our laboratory characterized a panel of highly mutated HIV-specific conformational epitope-targeting antibodies (Abs) from a panel of HIV-infected long-term nonprogressors (LTNPs). Despite binding HIV envelope protein and having a high number of somatic amino acid mutations, these Abs had poor neutralizing activity. Because of the evidence of antigen-driven selection and the long CDR3 region (21 amino acids [aa]), we further characterized the epitope targeting of monoclonal Ab (MAb) 76-Q3-2C6 (2C6). We confirmed that 2C6 binds preferentially to trimeric envelope and recognizes the clades A, B, and C SOSIP trimers. 2C6 binds gp140 constructs of clades A, B, C, and D, suggesting a conserved binding site that we localized to the ectodomain of gp41. Ab competition with MAb 50-69 suggested this epitope localizes near aa 579 to 613 (referenced to HXB2 gp160). Peptide library scanning showed consistent binding in this region but to only a single peptide. Lack of overlapping peptide binding supported a nonlinear epitope structure. The significance of this site is supported by 2C6 having Ab-dependent cell cytotoxicity (ADCC) against envelope proteins from two clades. Using 2C6 and variants, alanine scanning mutagenesis identified three amino acids (aa 592, 595, and 596) in the overlapping region of the previously identified peptide. Additional amino acids at sites 524 and 579 were also identified, helping explain its conformational requirement. The fact that different amino acids were included in the epitope depending on the targeted protein supports the conclusion that 2C6 targets a native conformational epitope. When we mapped these amino acids on the trimerized structure, they spanned across oligomers, supporting the notion that the epitope targeted by 2C6 lies in a recessed pocket between two gp41 oligomers. A complete understanding of the epitope specificity of ADCC-mediating Abs is essential for developing effective immunization strategies that optimize protection by these Abs.IMPORTANCE This paper further defines the function and area of the HIV trimeric envelope protein targeted by the monoclonal antibody 2C6. 2C6 binding is influenced by amino acid mutations across two separate gp41 sections of the envelope trimer. This epitope is recognized on multiple clades (variant groups of circulating viruses) of gp41, gp140 trimers, and SOSIP trimers. For the clades tested, 2C6 has robust ADCC. As the target of 2C6 is available in the major clades of HIV and has robust ADCC activity, further definition and appreciation of targeting of antibodies similar to 2C6 during vaccine development should be considered.

Project description:UnlabelledSoluble forms of trimeric HIV-1 envelope glycoprotein (Env) have long been sought as immunogens and as reagents for analysis of Env structure and function. Isolation of trimers that mimic native Env, derived from diverse viruses, however, represents a major challenge. Thus far, the most promising native-like (NL) structures have been obtained by engineering trimer-stabilizing mutations, termed SOSIP, into truncated Env sequences. However, the abundances of NL trimeric conformers vary among Envs, necessitating purification by monoclonal antibodies (MAbs) like PGT145, which target specific epitopes. To surmount this inherent limitation, we developed an approach that uses lectin affinity chromatography, ion-exchange chromatography, hydrophobic-interaction chromatography (HIC), and size exclusion chromatography (SEC) to isolate NL trimers from nonnative Env species. We validated this method with SOSIP trimers from HIV-1 clades A and B. Analyses by SEC, blue native PAGE, SDS-PAGE, and dynamic light scattering indicated that the resulting material was homogeneous (>95% pure), fully cleaved, and of the appropriate molecular weight and size for SOSIP trimers. Negative-stain electron microscopy further demonstrated that our preparations were composed of NL trimeric structures. By hydrogen/deuterium-exchange mass spectrometry, these HIC-pure trimers exhibited structural organization consistent with NL trimers and inconsistent with profiles seen in nonnative Envs. Screened for antigenicity, some Envs, like BS208.b1 and KNH1144 T162A, did not present the glycan/quaternary structure-dependent epitope for PGT145 binding, suggesting that these SOSIPs would be challenging to isolate by existing MAb affinity methods. By selecting based on biochemical rather than antigenic properties, our method offers an epitope-independent alternative to MAbs for isolation of NL Env trimers.ImportanceThe production and purification of diverse soluble Env trimers that maintain native-like (NL) structure present technical challenges that must be overcome in order to advance vaccine development and provide reagents for HIV research. Low levels of NL trimer expression amid heterogeneous Env conformers, even with the addition of stabilizing mutations, have presented a major challenge. In addition, it has been difficult to separate the NL trimers from these heterogeneous mixtures. While MAbs with specificity for quaternary NL trimer epitopes have provided one approach to purifying the desirable species, such methods are dependent on the Env displaying the proper epitope. In addition, MAb affinity chromatography can be expensive, the necessary MAb may be in limited supply, and large-scale purification may not be feasible. Our method based on biochemical separation techniques offers an epitope-independent approach to purification of NL trimers with general application to diverse Envs.

Dataset Information

A human monoclonal antibody neutralizes diverse HIV-1 isolates by binding a critical gp41 epitope.

Publications

A human monoclonal antibody neutralizes diverse HIV-1 isolates by binding a critical gp41 epitope.

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