Characterization of structural features and diversity of variable-region determinants of related quaternary epitopes recognized by human and rhesus macaque monoclonal antibodies possessing unusually potent neutralizing activities.

A series of potently neutralizing monoclonal antibodies (MAbs) that target quaternary epitopes on the native Env trimer have recently been described. A common feature shared by these antibodies is the critical involvement of sites in both the V2 and V3 variable domains in antibody recognition. In this study the gp120 variable-region determinants were mapped for eight rhesus macaque monoclonal antibodies (RhMAbs) possessing potently neutralizing activity specific for a quaternary target in SF162 Env and compared to those originally identified for human MAb 2909. These studies showed that determinants for the epitopes defined by the RhMAbs differed in both the V2 (positions 160, 167, and 169) and V3 (positions 313 and 315) regions from 2909, and in a number of cases, from each other. Attempts to reconstitute expression of these epitopes on the cell surface by cotransfecting Envs containing either the V2 or the V3 determinant of the epitope were not successful, suggesting that these epitopes were expressed on individual protomers in a trimer-dependent manner. Several of the V2 positions found to be critical for expression of these quaternary epitopes also significantly affected exposure and neutralization sensitivity of targets in the V3 and CD4-binding domains. These results demonstrated a considerable diversity in the fine structure of this class of epitopes and further suggested a potentially important relationship between the expression of such quaternary epitopes and V1/V2-mediated masking of immunodominant epitopes.

Human immunodeficiency virus type 2 (HIV-2)/HIV-1 envelope chimeras detect high titers of broadly reactive HIV-1 V3-specific antibodies in human plasma.

Deciphering antibody specificities that constrain human immunodeficiency virus type 1 (HIV-1) envelope (Env) diversity, limit virus replication, and contribute to neutralization breadth and potency is an important goal of current HIV/AIDS vaccine research. Transplantation of discrete HIV-1 neutralizing epitopes into HIV-2 scaffolds may provide a sensitive, biologically functional context by which to quantify specific antibody reactivities even in complex sera. Here, we describe a novel HIV-2 proviral scaffold (pHIV-2(KR.X7)) into which we substituted the complete variable region 3 (V3) of the env gene of HIV-1(YU2) or HIV-1(Ccon) to yield the chimeric proviruses pHIV-2(KR.X7) YU2 V3 and pHIV-2(KR.X7) Ccon V3. These HIV-2/HIV-1 chimeras were replication competent and sensitive to selective pharmacological inhibitors of virus entry. V3 chimeric viruses were resistant to neutralization by HIV-1 monoclonal antibodies directed against the CD4 binding site, coreceptor binding site, and gp41 membrane proximal external region but exhibited striking sensitivity to HIV-1 V3-specific monoclonal antibodies, 447-52D and F425 B4e8 (50% inhibitory concentration of [IC(50)] <0.005 microg/ml for each). Plasma specimens from 11 HIV-1 clade B- and 10 HIV-1 clade C-infected subjects showed no neutralizing activity against HIV-2 but exhibited high-titer V3-specific neutralization against both HIV-2/HIV-1 V3 chimeras with IC(50) measurements ranging from 1:50 to greater than 1:40,000. Neutralization titers of B clade plasmas were as much as 1,000-fold lower when tested against the primary HIV-1(YU2) virus than with the HIV-2(KR.X7) YU2 V3 chimera, demonstrating highly effective shielding of V3 epitopes in the native Env trimer. This finding was replicated using a second primary HIV-1 strain (HIV-1(BORI)) and the corresponding HIV-2(KR.X7) BORI V3 chimera. We conclude that V3 is highly immunogenic in vivo, eliciting antibodies with substantial breadth of reactivity and neutralizing potential. These antibodies constrain HIV-1 Env to a structure(s) in which V3 epitopes are concealed prior to CD4 engagement but do not otherwise contribute to neutralization breadth and potency against most primary virus strains. Triggering of the viral spike to reveal V3 epitopes may be required if V3 immunogens are to be components of an effective HIV-1 vaccine.

Specific sequences commonly found in the V3 domain of HIV-1 subtype C isolates affect the overall conformation of native Env and induce a neutralization-resistant phenotype independent of V1/V2 masking.

Primary HIV-1 isolates are relatively resistant to neutralization by antibodies commonly induced after infection or vaccination. This is generally attributed to masking of sensitive epitopes by the V1/V2 domain and/or glycans situated at various positions in Env. Here we identified a novel masking effect mediated by subtype C-specific V3 sequences that contributes to the V1/V2-independent and glycan-independent neutralization resistance of chimeric and primary Envs to antibodies directed against multiple neutralization domains. Positions at several conserved charged and hydrophobic sites in the V3 crown and stem were also shown to affect neutralization phenotype. These results indicated that substitutions typically present in subtype C and related V3 sequences influence the overall conformation of native Env in a way that occludes multiple neutralization targets located both within and outside of the V3 domain, and may reflect an alternative mechanism for neutralization resistance that is particularly active in subtype C and related isolates.

Structure-guided design and immunological characterization of immunogens presenting the HIV-1 gp120 V3 loop on a CTB scaffold.

V3 loop is a major neutralizing determinant of the HIV-1 gp120. Using 3D structures of cholera toxin B subunit (CTB), complete V3 in the gp120 context, and V3 bound to a monoclonal antibody (mAb), we designed two V3-scaffold immunogen constructs (V3-CTB). The full-length V3-CTB presenting the complete V3 in a structural context mimicking gp120 was recognized by the large majority of our panel of 24 mAbs. The short V3-CTB presenting a V3 fragment in the conformation observed in the complex with the 447-52D Fab, exhibited high-affinity binding to this mAb. The immunogens were evaluated in rabbits using DNA-prime/protein-boost protocol. Boosting with the full-length V3-CTB induced high anti-V3 titers in sera that potently neutralize multiple HIV virus strains. The short V3-CTB was ineffective. The results suggest that very narrow antigenic profile of an immunogen is associated with poor Ab response. An immunogen with broader antigenic activity elicits robust Ab response.

High titer HIV-1 V3-specific antibodies with broad reactivity but low neutralizing potency in acute infection and following vaccination.

Identifying the earliest neutralizing antibody specificities that are elicited following infection or vaccination by HIV-1 is an important objective of current HIV/AIDS vaccine research. We have shown previously that transplantation of HIV-1 V3 epitopes into an HIV-2 envelope (Env) scaffold provides a sensitive and specific means to detect and quantify HIV-1 V3 epitope specific neutralizing antibodies (Nabs) in human sera. Here, we employ this HIV-2/HIV-1 V3 scaffolding strategy to study the kinetics of development and breadth of V3-specific Nabs in longitudinal sera from individuals acutely infected with clade C or clade B HIV-1 and in human subjects immunized with clade B HIV-1 immunogens. HIV-2/HIV-1 chimeras containing V3 sequences matched to virus type (HIV-2 or HIV-1), subtype (clade B or C), or strain (autologous or heterologous) were used as test reagents. We found that by 3-8 weeks post infection, 12 of 14 clade C subjects had a median IC(50) V3-specific Nab titer of 1:700 against chimeric viruses containing a heterologous clade C V3. By 5 months post-infection, all 14 subjects were positive for V3-specific Nabs with median titers of 1:8000 against heterologous clade C V3 and 1:1300 against clade B V3. Two acutely infected clade B patients developed heterologous clade B V3-specific Nabs at titers of 1:300 and 1:1800 by 13 weeks of infection and 1:5000 and 1:11000 by 7 months of infection. Titers were not different against chimeras containing autologous clade B V3 sequences. Each of 10 uninfected normal human volunteers who were immunized with clade B HIV-1 Env immunogens, but none of five sham immunized control subjects, developed V3-specific Nabs titers as high as 1:3000 (median 1:1300; range 1:700-1:3000). None of the HIV-1 infected or vaccinated subjects had antibodies that neutralized primary HIV-1 virus strains. These results indicate that high-titer, broadly reactive V3-specific antibodies are among the first to be elicited during acute and early HIV-1 infection and following vaccination but these antibodies lack neutralizing potency against primary HIV-1 viruses, which effectively shield V3 from antibody binding to the functional Env trimer.