Characterization and Epitope Mapping of Neutralizing Monoclonal Antibodies Produced by Immunization with Oligomeric Simian Immunodeficiency Virus Envelope Protein

Abstract

In an attempt to generate broadly cross-reactive, neutralizing monoclonal antibodies (MAbs) to simian immunodeficiency virus (SIV), we compared two immunization protocols using different preparations of oligomeric SIV envelope (Env) glycoproteins. In the first protocol, mice were immunized with soluble gp140 (sgp140) from CP-MAC, a laboratory-adapted variant of SIVmacBK28. Hybridomas were screened by enzymelinked immunosorbent assay, and a panel of 65 MAbs that recognized epitopes throughout the Env protein was generated. In general, these MAbs detected Env by Western blotting, were at least weakly positive in fluorescence-activated cell sorting (FACS) analysis of Env-expressing cells, and preferentially recognized monomeric Env protein. A subset of these antibodies directed toward the V1/V2 loop, the V3 loop, or nonlinear epitopes were capable of neutralizing CP-MAC, a closely related isolate (SIVmac1A11), and/or two more divergent strains (SIVsm⌬B670 CL3 and SIVsm543-3E). In the second protocol, mice were immunized with unfixed CP-MAC-infected cells and MAbs were screened for the ability to inhibit cell-cell fusion. In contrast to MAbs generated against sgp140, the seven MAbs produced using this protocol did not react with Env by Western blotting and were strongly positive by FACS analysis, and several reacted preferentially with oligomeric Env. All seven MAbs potently neutralized SIVmac1A11, and several neutralized SIVsm⌬B670 CL3 and/or SIVsm543-3E. MAbs that inhibited gp120 binding to CD4, CCR5, or both were identified in both groups. MAbs to the V3 loop and one MAb reactive with the V1/V2 loop interfered with CCR5 binding, indicating that these regions of Env play similar roles for SIV and human immunodeficiency virus. Remarkably, several of the MAbs generated against infected cells blocked CCR5 binding in a V3-independent manner, suggesting that they may recognize a region analogous to the conserved coreceptor binding site in gp120. Finally, all neutralizing MAbs blocked infection through the alternate coreceptor STRL33 much more efficiently than infection through CCR5, a finding that has important implications for SIV neutralization assays using CCR5-negative human T-cell lines.

Aiming to induce broadly reactive neutralizing antibody responses with HIV-1 vaccine candidates

“…The V1, V2 and V4 Env variable loops can also serve as targets for neutralizing antibodies to HIV-1 Env [97][98][99][100][101][102]. Similar to the V3 loop antibody repertoire, many of the V1, V2 and V4 specifities of induced antibodies are strain specific.…”

“…However, one goal of vaccine development is to find antibody specificities that can bind to native Env trimers and induce the exposure of critical epitopes on the V3 loop. V3 immunogens that reflect conserved functional regions of the V3 loop would then become viable vaccine targets.The V1, V2 and V4 Env variable loops can also serve as targets for neutralizing antibodies to HIV-1 Env [97][98][99][100][101][102]. Similar to the V3 loop antibody repertoire, many of the V1, V2 and V4 specifities of induced antibodies are strain specific.…”

Selective Expansion of HIV-1 Envelope Glycoprotein-Specific B Cell Subsets Recognizing Distinct Structural Elements Following Immunization

“…Inoculations were performed by intradermal injections of 50 g of gp140-GCN4 in IFA three times with 1-wk intervals, with a final boost 3 days before the hybridoma fusion was performed. Splenocytes were harvested and fused with SP2 myeloma cells, as described (46). Hybridomas were screened by ELISA for gp120 binding and cloned by limiting dilution.…”

Kinetic Rates of Antibody Binding Correlate with Neutralization Sensitivity of Variant Simian Immunodeficiency Virus Strains

“…One hypothesis is that neutralization sensitivity is related to the density of envelope spikes on the virion, requiring a threshold level of antibody binding to limit infectivity (27,(38)(39)(40). Alternatively, neutralization of virus by antibody has been associated with differences in the qualitative antibody binding properties, i.e., antibody affinity and epitope specificity (9,15,26,56). Previous studies have suggested a relationship between antibody affinity for oligomeric gp120 and neutralization of HIV-1 (19,44,46), and monoclonal antibodies (MAbs) that bind the V3 loop of diverse HIV-1 gp120 proteins with similar association rates exhibited marked differences in the dissociation rates that were predictive of neutralization capacity (48).…”