Heavy Chain-Only IgG2b Llama Antibody Effects Near-Pan HIV-1 Neutralization by Recognizing a CD4-Induced Epitope That Includes Elements of Coreceptor- and CD4-Binding Sites

Abstract

The conserved HIV-1 site of coreceptor binding is protected from antibody-directed neutralization by conformational and steric restrictions. While inaccessible to most human antibodies, the coreceptor site has been shown to be accessed by antibody fragments. In this study, we used X-ray crystallography, surface plasmon resonance, and pseudovirus neutralization to characterize the gp120-envelope glycoprotein recognition and HIV-1 neutralization of a heavy chain-only llama antibody, named JM4. We describe full-length IgG2b and IgG3 versions of JM4 that target the coreceptor-binding site and potently neutralize over 95% of circulating HIV-1 isolates. Contrary to established trends that show improved access to the coreceptor-binding region by smaller antibody fragments, the single-domain (VHH) version of JM4 neutralized less well than the full-length IgG2b version of JM4. The crystal structure at 2.1-Å resolution of VHH JM4 bound to HIV-1 YU2 gp120 stabilized in the CD4-bound state by the CD4-mimetic miniprotein, M48U1, revealed a JM4 epitope that combined regions of coreceptor recognition (including the gp120 bridging sheet, V3 loop, and ␤19 strand) with gp120 structural elements involved in recognition of CD4 such as the CD4-binding loop. The structure of JM4 with gp120 thus defines a novel CD4-induced site of vulnerability involving elements of both coreceptor-and CD4-binding sites. The potently neutralizing JM4 IgG2b antibody that targets this newly defined site of vulnerability adds to the expanding repertoire of broadly neutralizing antibodies that effectively neutralize HIV-1 and thereby potentially provides a new template for vaccine development and target for HIV-1 therapy.

Drift of the HIV-1 Envelope Glycoprotein gp120 toward Increased Neutralization Resistance over the Course of the Epidemic: a Comprehensive Study Using the Most Potent and Broadly Neutralizing Monoclonal Antibodies

“…HIV-1 variants from CP were more resistant to the llama-derived antibody JM4sdAb than the variants that were transmitted 2 decades earlier (median IC 50 s of 1.720, 1.930, and 3.065 g/ml for HP, IP, and CP, respectively; P ϭ 0.002). This bNAb reacts with a CD4-induced epitope, and this reaction involves elements of both the coreceptor-and CD4-binding sites (37,39). Although not significant, a similar trend was observed for the human bNAb 8ANC195, which targets a newly defined glycan-dependent epitope adjacent to the CD4-binding site and spanning gp120-gp41 interface (17).…”

“…A progressively increasing resistance to neutralization was observed over calendar time, for both human sera and the bNAbs b12, VRC01, VRC03, NIH45-46 G54W , PG9, PG16, PGT121, PGT128, and PGT145 (33). Since then, bNAbs with increased potency or targeting different epitopes have been generated, including bNAbs improved by structure-based gene modifications (37)(38)(39)(40)(41). Therefore, we extended the analyses to a panel of new bNAbs described: PG9-iMab, PG16-iMab, 10E8, 3BNC117, NIH45-46m2, NIH45-46m7, 10-1074, JM4sdAb, 8ANC195, and PG9-16-RSH (Table 1).…”

Antigen recognition by single-domain antibodies: structural latitudes and constraints

“…Probably the clearest examples of epitopes that are more favorable for binding by sdAbs than conventional antibodies can be found in the envelope glycoprotein trimer of HIV-1: heterologous cross-strain neutralization is extraordinarily difficult to achieve by conventional antibodies, requiring months of chronic infection and multiple rounds of somatic mutation and selection, yet cross-neutralizing camelid heavy chain-only antibodies directed against the CD4-binding site 72-75 and CD4-induced sites 76-78 can be easily elicited by routine immunizations with recombinant protein antigens. Similar examples can be found for other viral pathogens.…”

“…No studies have reported hapten-specific V NAR s, and only one study has described a carbohydrate-specific V H H directed against Neisseira meningitidis lipopolysaccharide; 120 at least two camelid V H Hs have been described that bind to glycopeptide epitopes. 68,76 No sdAbs of any type have been described that convincingly bind lipids or nucleic acids. Together, the consensus of the data is that it is probably difficult, but not impossible, for sdAb paratopes to accommodate haptens and that three CDRs are sufficient to form the binding pockets and grooves required for such interactions, although potential involvement of solubility-enhancing FR2 residues at the former V L interface in pocket formation may impose restrictions on hapten-binding specificities.…”

Intracellular Expression of Camelid Single-Domain Antibodies Specific for Influenza Virus Nucleoprotein Uncovers Distinct Features of Its Nuclear Localization

“…These properties allow the VHHs to be expressed in the cytosol of eukaryotic cells with retention of the antigen binding capabilities. This in turn permits the specific targeting of host or viral proteins recognized by VHHs, thus enabling possible perturbation of target protein function (26)(27)(28)(29)(30)(31)(32); for a review, see reference 33). VHHs are therefore unique tools for analysis of essential proteins encoded by RNA viruses in living cells.…”