We have examined the exposure and conservation of antigenic epitopes on the surface envelope glycoproteins (gp120 and gp41) of 26 intact, native, primary human immunodeficiency virus type 1 (HIV-1) group M virions of clades A to H. For this, 47 monoclonal antibodies (MAbs) derived from HIV-1-infected patients were used which were directed at epitopes of gp120 (specifically V2, C2, V3, the CD4-binding domain [CD4bd], and C5) and epitopes of gp41 (clusters I and II). Of the five regions within gp120 examined, MAbs bound best to epitopes in the V3 and C5 regions. Only moderate to weak binding was observed by most MAbs to epitopes in the V2, C2, and CD4bd regions. Two anti-gp41 cluster I MAbs targeted to a region near the tip of the hydrophilic immunodominant domain bound strongly to >90% of isolates tested. On the other hand, binding of anti-gp41 cluster II MAbs was poor to moderate at best. Binding was dependent on conformational as well as linear structures on the envelope proteins of the virions. Further studies of neutralization demonstrated that MAbs that bound to virions did not always neutralize but all MAbs that neutralized bound to the homologous virus. This study demonstrates that epitopes in the V3 and C5 regions of gp120 and in the cluster I region of gp41 are well exposed on the surface of intact, native, primary HIV-1 isolates and that cross-reactive epitopes in these regions are shared by many viruses from clades A to H. However, only a limited number of MAbs to these epitopes on the surface of HIV-1 isolates can neutralize primary isolates.

Human anti-V3 monoclonal antibodies (mAbs) generated from HIV-1 infected individuals display diversity in the range of their cross-neutralization that may be related to their immunogenetic background. The study of the immunoglobulin (Ig) variable region gene usage of heavy chains have shown a preferential usage of the VH5-51 gene segment which was detected in 35% of 51 human anti-V3 mAbs. In contrast, human mAbs against other envelope regions of HIV-1 (anti-Env), including the CD4-binding domain, the CD4-induced epitope, and gp41 preferentially used the VH1-69 gene segment, and none of them used the VH5-51 gene. Furthermore, the usage of the VH4 family by anti-V3 mAbs was restricted to only one gene segment, VH4-59, while the VH3 gene family was used at a significantly lower frequency by all of the analyzed anti-HIV-1 mAbs. Multivariate analysis showed that usage of VH gene segments was significantly different between anti-V3 and anti-Env mAbs, and compared to antibodies from healthy subjects. In addition, the anti-V3 mAbs preferentially used the JH3 and D2-15 gene segments. The preferential usage of selected Ig gene segments and the characteristic pattern of Ig gene usage by anti-V3 mAbs can be related to the conserved structure of the V3 region.

Antibodies (Abs) against the V3 loop of the human immunodeficiency virus type 1 gp120 envelope glycoprotein were initially considered to mediate only type-specific neutralization of T-cell-line-adapted viruses. However, recent data show that cross-neutralizing V3 Abs also exist, and primary isolates can be efficiently neutralized with anti-V3 monoclonal Abs (MAbs). The neutralizing activities of anti-V3 polyclonal Abs and MAbs may, however, be limited due to antigenic variations of the V3 region, a lack of V3 exposure on the surface of intact virions, or Ab specificity. For clarification of this issue, a panel of 32 human anti-V3 MAbs were screened for neutralization of an SF162-pseudotyped virus in a luciferase assay. MAbs selected with a V3 fusion protein whose V3 region mimics the conformation of the native virus were significantly more potent than MAbs selected with V3 peptides. Seven MAbs were further tested for neutralizing activity against 13 clade B viruses in a single-round peripheral blood mononuclear cell assay. While there was a spectrum of virus sensitivities to the anti-V3 MAbs observed, 12 of the 13 viruses were neutralized by one or more of the anti-V3 MAbs. MAb binding to intact virions correlated significantly with binding to solubilized gp120s and with the potency of neutralization. These results demonstrate that the V3 loop is accessible on the native virus envelope, that the strength of binding of anti-V3 Abs correlates with the potency of neutralization, that V3 epitopes may be shared rather than type specific, and that Abs against the V3 loop, particularly those targeting conformational epitopes, can mediate the neutralization of primary isolates.The third variable domain (V3) of the human immunodeficiency virus type 1 (HIV-1) gp120 envelope glycoprotein is critical for the formation of syncytia and for virus entry into target cells (24,55). These functions are mediated by the interaction of the V3 loop with chemokine receptors and are maintained despite the sequence variation that characterizes this region of the virus envelope (18, 51). Indeed, contrary to its name, the V3 loop is characterized by a constant size of 30 to 35 amino acids, a conserved type II ␤-turn at its tip, a disulfide bond at its base, and a net positive charge (26, 28). Conserved features are also suggested by the structure of the V3 loop discerned by nuclear magnetic resonance studies (47, 52), and conserved elements in the V3 crown and stem are mandatory features for coreceptor interactions (9, 50). All of these structural constraints appear to be imposed by the required interaction of the V3 loop with the coreceptors for HIV-1, CXCR4 or CCR5, and suggest that this region of the virus envelope should induce antibodies (Abs) that are crossreactive among isolates and inhibitory to virus infectivity.Initial studies of anti-V3 Abs, induced by brief immunization protocols in animals and tested against a limited number of T-cell-line-adapted (TCLA) strains of the virus, suggested, however, that anti-V3 Abs were type spec...

During the past two decades, several epitopes that induce neutralizing antibodies (Abs) have been identified in the human immunodeficiency virus (HIV) envelope through studies of polyclonal and monoclonal Abs (MAbs). These epitopes include the V3 region defined with polyclonal Abs (30, 33) and several MAbs, such as 447-52D (16); the membrane-proximal external region in gp41 defined by MAbs 2F5 and 4E10 (6); the CD4-binding site on gp120 defined by MAb immunoglobulin G1b12 (IgG1b12) (7); and a glycan-rich region on gp120 defined by MAb 2G12 (37). With the exception of V3, none of these epitopes induce neutralizing Abs in the majority of infected humans. Thus, Abs to the membrane-proximal external region of gp41 (G. Shaw, H. Li, J. Decker, S. Allen, E. Hunter, E. Delaporte, M. Peters, B. Hahn, and F. Bibollet-Ruche, Abstr. AIDS Vaccine 2005, abstr. 29, 2005 (45), the CD4 binding site defined by IgG1b12 (25), and the designated carbohydrate moieties on gp120 (23, 37) are rare or absent from the sera of most HIV-infected individuals, and the epitope recognized by 2F5 (9,11,29) and the peptide mimotope for IgG1b12 (44) have failed to induce neutralizing Abs when used as experimental immunogens. Moreover, the recently described auto-reactive character of MAbs 2F5, 4E10, and IgG1b12, which recognize cardiolipin and/or double-stranded DNA, indicates that these epitopes may be problematic for the design of an anti-HIV vaccine (22). In contrast, the immunogenicity of the V3 region is reflected by the presence of anti-V3 Abs in the sera of essentially all HIV-infected individuals (38).Opinions about the V3 loop as an antigen for the induction of neutralizing Abs have changed over time. Early optimism related to the ability of anti-V3 MAbs to neutralize T-cell-lineadapted viruses was replaced by skepticism when it was suggested that the V3 of primary isolate JR-FL was "cryptic" (5). More recent data suggest that V3 is accessible on the surfaces of most virions (31) and that anti-V3 MAbs, such as 447-52D, can neutralize 62 to 92% of primary isolates that carry the epitope for which V3 is specific (3, 43). Nonetheless, recent studies have shown that V3 is masked in many viruses by the V1/V2 region (32) and/or by carbohydrate moieties on the envelope (39), both of which may contribute to the resistance of primary isolates (26,28). Moreover, it has been demonstrated in several studies that, despite the sequence variation in the V3 loop, many human anti-V3 Abs are cross-reactive (3,