Access of Antibody Molecules to the Conserved Coreceptor Binding Site on Glycoprotein gp120 Is Sterically Restricted on Primary Human Immunodeficiency Virus Type 1

Abstract

Anti-human immunodeficiency virus type 1 (HIV-1) antibodies whose binding to gp120 is enhanced by CD4 binding (CD4i antibodies) are generally considered nonneutralizing for primary HIV-1 isolates. However, a novel CD4i-specific Fab fragment, X5, has recently been found to neutralize a wide range of primary isolates.To investigate the precise nature of the extraordinary neutralizing ability of Fab X5, we evaluated the abilities of different forms (immunoglobulin G [IgG], Fab, and single-chain Fv) of X5 and other CD4i monoclonal antibodies to neutralize a range of primary HIV-1 isolates. Our results show that, for a number of isolates, the size of the neutralizing agent is inversely correlated with its ability to neutralize. Thus, the poor ability of CD4i-specific antibodies to neutralize primary isolates is due, at least in part, to steric factors that limit antibody access to the gp120 epitopes. Studies of temperature-regulated neutralization or fusion-arrested intermediates suggest that the steric effects are important in limiting the binding of IgG to the viral envelope glycoproteins after HIV-1 has engaged CD4 on the target cell membrane. The results identify hurdles in using CD4i epitopes as targets for antibody-mediated neutralization in vaccine design but also indicate that the CD4i regions could be efficiently targeted by small molecule entry inhibitors.Human immunodeficiency virus type 1 (HIV-1) entry into host cells is initiated by the binding of the gp120 subunit of the viral envelope glycoprotein (Env) complex to the host cell receptor (CD4) (8,20). This interaction induces conformational changes in gp120 resulting in the exposure of a conserved high-affinity binding site for the coreceptor (the chemokine receptors CCR5 or CXCR4) (46,47,54,56,59). A second obligatory binding step between the gp120-CD4 complex and the coreceptor is then thought to induce additional conformational changes that ultimately result in the fusion of viral and host cell membranes (9, 18).Neutralizing antibodies are believed to act, at least in part, by binding to the exposed Env surface and obstructing the initial interaction between a trimeric array of gp120 molecules on the virion surface and receptor molecules on the target cell (36,37,57). In response, HIV-1 has evolved a number of strategies to evade recognition by neutralizing antibodies, particularly those directed to the conserved CD4 and coreceptor binding sites of Env. The extent of protection of these sites from antibody recognition is limited by the necessity to preserve the accessibility for receptor interaction. In the case of the CD4bs this has led to the following structural features: (i) it is partially obscured from antibody recognition by the V1/V2 loop and associated carbohydrate structures; (ii) the flanking residues are variable and modified by glycosylation; (iii) it is recessed to an extent that limits direct access by an antibody variable region; (iv) clusters of residues within the CD4bs that do not directly interact with CD4 are subject to variation...

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

“…These findings distinguish the neutralization activity of D5-IgG1 from that of X5-IgG1, which showed poor activity against isolates other than HXB2 (23). Importantly, D5-IgG1 did not block infectivity of HIV pseudotyped with the vesicular stomatitis virus-G protein (Table 1), supporting the conclusion that the antiviral target of D5-IgG1 is the HIV envelope glycoprotein.…”

“…Previous reports found that the HIV-neutralizing activity of the X5 scFv was dramatically reduced upon conversion to an IgG, presumably because the larger IgG could not gain access to its binding site (23). Unlike X5, the human IgG1 form of D5 retained antiviral activity against HIV in both the HIVRP (Fig.…”

Neutralization of feline immunodeficiency virus by antibodies targeting the V5 loop of Env

“…A vaccine capable of protecting cats against infection with a wide range of virulent primary isolates remains a challenge, primarily because of the limited number of conserved neutralization epitopes that have been identified and the failure to present such epitopes appropriately in vaccines. Steric factors may render neutralization epitopes inaccessible to NAbs (Chiarantini et al, 1998;Labrijn et al, 2003), whilst most of the antibodies generated by vaccination may be directed against non-neutralizing epitopes (Dhillon et al, 2007;Richman et al, 2003). Accordingly, the identification of conserved determinants for neutralization will be an important advance in the design of the next generation of FIV vaccines.…”

“…A vaccine capable of protecting cats against infection with a wide range of virulent primary isolates remains a challenge, primarily because of the limited number of conserved neutralization epitopes that have been identified and the failure to present such epitopes appropriately in vaccines. Steric factors may render neutralization epitopes inaccessible to NAbs (Chiarantini et al, 1998;Labrijn et al, 2003), whilst most of the antibodies generated by vaccination may…”

“…A vaccine capable of protecting cats against infection with a wide range of virulent primary isolates remains a challenge, primarily because of the limited number of conserved neutralization epitopes that have been identified and the failure to present such epitopes appropriately in vaccines. Steric factors may render neutralization epitopes inaccessible to NAbs (Chiarantini et al, 1998;Labrijn et al, 2003), whilst most of the antibodies generated by vaccination mayThe GenBank/EMBL/DDBJ accession numbers for the novel FIV env sequences generated in this study are GU066862-GU066866.Supplementary tables showing oligonucleotides used for site-directed mutagenesis of V5 and GenBank numbers for new and reference sequences are available with the online version of this paper. be directed against non-neutralizing epitopes (Dhillon et al, 2007;Richman et al, 2003).…”