The epitopes of the V3 domain of the human immunodeficiency virus type 1 (HIV-1) gp120 glycoprotein have complex structures consisting of linear and conformational antigenic determinants. Anti-V3 antibodies (Abs) recognize both types of elements, but Abs which preferentially react to the conformational aspect of the epitopes may have more potent neutralizing activity against HIV-1, as recently suggested. To test this hypothesis, human anti-V3 monoclonal Abs (MAbs) were selected using a V3 fusion protein (V3-FP) which retains the conformation of the third variable region. The V3-FP consists of the V3 JR-CSF sequence inserted into a truncated form of murine leukemia virus gp70. Six human MAbs which recognize epitopes at the crown of the V3 loop were selected with the V3-FP. They were found to react more strongly with molecules displaying conformationally intact V3 than with linear V3 peptides. In a virus capture assay, these MAbs showed cross-clade binding to native, intact virions of clades A, B, C, D, and F. No binding was found to isolates from subtype E. The neutralizing activity of MAbs against primary isolates was determined in three assays: the GHOST cell assay, a phytohemagglutinin-stimulated peripheral blood mononuclear cell assay, and a luciferase assay. While these new MAbs displayed various degrees of activity, the pattern of cross-clade neutralization of clades A, B, and F was most pronounced. The neutralization of clades C and D viruses was weak and sporadic, and neutralization of clade E by these MAbs was not detected. Analysis by linear regression showed a highly significant correlation (P < 0.0001) between the strength of binding of these anti-V3 MAbs to intact virions and the percent neutralization. These studies demonstrate that human MAbs to conformation-sensitive epitopes of V3 display cross-clade reactivity in both binding to native, intact virions and neutralization of primary isolates.The third variable region (V3) of the gp120 envelope glycoprotein of human immunodeficiency virus type 1 (HIV-1) is critical for the infectivity of the virus, and certain single mutations in this region render the virus inactive (26,35). The sequence of the V3 loop plays a primary role in determining the cell tropism of the virus (8, 25), which is a function of its ability to differentially bind to the CCR5 and/or CXCR4 coreceptors on the target cell (22, 55). As a consequence of the key functions attributable to the V3 loop, anti-V3 antibodies (Abs) can be expected to block HIV-1 infectivity. Indeed, serum neutralization of T-cell line-adapted (TCLA) strains was found to be mediated by anti-V3 Abs (50, 65) and could be abolished by absorption with V3 peptides (2, 51) or by depletion of anti-V3 Abs (57). Moreover, monoclonal Abs (MAbs) against the V3 loop were shown to have potent neutralizing activity against TCLA strains, and this neutralization sensitivity to anti-V3 Abs led to the designation of the V3 loop as the principal neutralizing domain of the virus (27,47).Several studies, however, have weakened th...

The selection of human monoclonal antibodies (MAbs) specific for human immunodeficiency virus (HIV) type 1 by binding assays may fail to identify Abs to quaternary epitopes on the intact virions. The HIV neutralization assay was used for the selection of human MAb 2909, which potently neutralizes SF162 and recognizes an epitope on the virus surface but not on soluble proteins. Three regions of gp120, the V2 and V3 loops and the CD4 binding domain, contribute to the epitope recognized by MAb 2909. The existence of such a unique MAb, which defines a complex epitope formed by a quaternary structure, suggests that there may be other new neutralizing HIV epitopes to target with vaccines.

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.

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,

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...