Characterization and epitope mapping of neutralizing monoclonal antibodiesproduced by immunization with oligomeric simian immunodeficiency virus envelope protein

In an attempt to generate broadly cross-reactive, neutralizing monoclonal a ntibodies (MAbs) to simian immunodeficiency virus (SIV), we compared two im munization protocols using different preparations of oligomeric SIV envelop e (Env) glycoproteins, In the first protocol, mice were immunized with solu ble gp140 (sgp140) from CP-MAC, a laboratory-adapted variant of SIVmacBK28. Hybridomas were screened by enzyme-linked immunosorbent assay, and a panel of 65 MAbs that recognized epitopes throughout the Env protein was generat ed. In general, these MAbs detected Env by Western blotting, were at least weakly positive in fluorescence-activated cell sorting (FACS) analysis of E nv-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 Delta B670 C L3 and SIVsm543-3E), In the second protocol, mice were immunized with unfix ed 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 M Abs produced using this protocol did not react with Env by Western blotting and were strongly positive by FACS analysis, and several reacted preferent ially with oligomeric Env. All seven MAbs potently neutralized SIVmac1A11, and several neutralized SIVsm Delta B670 CL3 and/or SIVsm543-3E. MAbs that inhibited gp120 binding to CD4, CCR5, or both were identified in both group s. 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 g enerated against infected cells blocked CCR5 binding in a V3-independent ma nner, suggesting that they may recognize a region analogous to the conserve d coreceptor binding site in gp120. Finally, all neutralizing MAbs blocked infection through the alternate coreceptor STRL33 much more efficiently tha n infection through CCR5, a finding that has important implications for SIV neutralization assays using CCR5-negative human T-cell lines.