Many human monoclonal antibodies (mAbs) to the envelope glycoproteins of HIV-1 have been produced but few are able to neutralize HIV-1 primary isolates. Because human anti-HIV neutralizing mAbs are thought to be the best reagents for passive immunization regimens to prevent and treat HIV infection, potent neutralizing mAbs need to be isolated, characterized, produced in quantity and tested in relevant in vitro and in vivo experiments. The work described in this proposal is designed to fulfil these needs. In AIM 1, we describe methods for producing heterohybridomas making human mAbs specific for epitopes on HIV virus particles, especially those on macrophage-tropic strains. (None of the available human mAbs were selected for reactivity with HIV virions, although Abs must react with virions to neutralize.) In AIM 2, we will develop new methods for quantifying HIV neutralization by treating viruses with mAbs and infecting various cell lines transfected with CD4, co-receptors (e.g., CCR5, CXCR4) and HIV LTR-driven reporter genes. The resulting assays will be reproducible and rapid and will permit the direct quantitation of neutralized infectious units, overcoming problems with most conventional HIV neutralization assays which measure neutralization indirectly. The new methods will be compared to currently used assays for relative sensitivity. In AIM 3, the new mAbs will be tested for their neutralizing activity against various primary isolates using the optimal new neutralization assay. Combinations of mAbs will also be tested to identify those that work in synergy. In AIM 4, cell lines producing the most potent mAbs will be provided to an industrial partner for scale-up. Then, in AIM 5, the efficacy of these mAbs (used alone and in combination) will be tested for preventing HIV infection in beta2-microglobulin knock-out NOD/LtSz-scid/scid mice reconstituted with human PBMC. These experiments will establish correlations between the in vitro and in vivo efficacy of these mAbs. They will, as well, permit study of break-through infections and the course of infection when sterilizing immunity is not achieved.