| CD4 and a family of coreceptors provide necessary cellular protein attachments and/or interactions for entry of human and simian immunodeficiency viruses (HIV and SIV). This project characterized a panel of monoclonal antibodies that neutralized replication of HIV and SIV strains. Antibody binding studies mapped the panel of eight antibodies specifically to CXCR4, the major T cell-tropic coreceptor for HIV. Detailed molecular analyses of the CXCR4-specific antibodies became relevant to dissecting the interactions of CXCR4-tropic viruses. Human CXCR2/CXCR4 chimeras showed the necessity of extracellular loops 1 and 2 for antibody recognition, while rat/human and feline/human CXCR4 chimeras revealed reliance upon extracellular loops 2 and 3. Site-directed mutagenesis of extracellular loop 2 yielded human-feline reciprocal mutants where binding of four of the antibodies was isolated to the amino-terminal side of loop 2. Specifically, one monoclonal antibody, 12G5, required both Ser178 and Asp182 for recognition in a feline background, while antibodies 44702, 44708, and 44712 showed reactivity with only Ser178 present in the feline background. Antibodies 44701, 44716, 44717, and 44718 cross-reacted with both human and feline CXCR4. Inhibitor analysis with the CXCR4-specific antibodies were conducted with HIV and SIV infections of lymphoid cell lines. All antibodies inhibited SIV/cp-mac induced cell-cell fusion, and seven antibodies (excluding 44702) demonstrated does-dependent neutralization of SIV/cp-mac in measurements of cell-free virus production. HIV-2/vcp demonstrated CD4-independent entry via CXCR4, and showed marked neutralization by seven of the eight CXCR4-specific antibodies. HIV-1 strains IIIb and 89.6 both showed substantial reduction by feline-specific antibodies (44701, 44717, and 44718) but only marginal effects by the remaining antibodies at highest concentrations. Subsequent studies analyzed the ability of the antibodies to block ligand-mediated activation through stromal derived factor-1alpha (SDF-1alpha), and revealed a similar pattern of strong inhibitory activity by feline-specific antibodies (excepting 44716). The data from the mapping studies, the ligand inhibitions, and the viral neutralizations are consistent with the most potent antibodies recognizing a wider variety of CXCR4 conformational states. These results reveal the ability of CXCR4-specific antibodies to inhibit virus entry as well as CXCR4-mediated signaling, and suggest potential therapeutic applications. |
