Recent studies using selected patients' sera with cross-clade neutralizing activity revealed that the specificity of at least one-third of the neutralizing activity remains uncharacterized. These results demonstrate the need to identify new epitopes which can guide efforts to develop a promising vaccine. We propose an innovative approach to produce human mAbs using a selected combination of techniques which are not being used together by any other group, i.e., the use of single IgG+ memory B cells, selected with virus-like particles (VLPs) from which recombinant monoclonal antibodies (mAbs) will be generated using highly efficient molecular techniques. A further innovation includes more efficient sorting and selection of B cells specific only for trimeric envelope (Env) proteins. The B cells will be derived from donors infected with diverse HIV-1 subtypes whose plasma Abs cross-neutralize Tier 2 viruses. We hypothesize that these selected volunteers produce neutralizing Abs to new as yet unidentified epitopes that are present on the native trimeric HIV-1 envelope and that reactivity to such epitopes will be detected using VLPs. SPECIFIC AIM 1. Production of recombinant mAbs from single B cells. The blood specimens will come from two well-established cohorts of infected subjects. Three PBMC samples will be provided by the Center for HIV/AIDS Vaccine Immunology (CHAVI) and 10 PBMCs samples will be obtained from Cameroonian subjects whose sera have been shown to mediate cross-clade neutralizing activity. The mAbs will be produced from single Env-specific B cells selected with GFP-tagged VLPs expressing trimeric Env proteins. The immunoglobulin variable genes will be amplified using RT-PCR, cloned into expression vectors, and the genes will be used for the transfection of 293T cells for mAb production. In total, PBMC specimens from 10-15 subjects will be studied; yielding 300-450 mAbs. SPECIFIC AIM 2. Characterization of various functional activities (neutralizing, ADCC and ADCVI) of new mAbs. The purified mAbs will be tested in functional assays for neutralization, ADCC and/or ADCVI activity. The neutralizing activity of mAbs will be screened against pseudoviruses and primary isolates in our lab and selected mAbs will be tested against a standard panel of pseudotyped viruses by a collaborator. New mAbs combining two or three inhibitory functions (neutralization, ADCC and/or ADCVI) will have priority for epitope mapping followed by those mAbs that cross-neutralize only and non-neutralizing mAbs with the Fc&#947;R-mediated activity. SPECIFIC AIM 3. Epitope mapping of new monoclonal Abs. Using VLPs for selection of Env-specific B cells will result in production of mAbs against various known epitopes and those which are present on Env trimers. A variety of mapping techniques will be used, including immunochemical and viral assays as well as crystallographic analysis. Mapping will be particularly focused on mAbs to quaternary and newly defined epitopes and that mediate double (or triple) functions. Epitopes of mAbs with potent, cross-neutralizing and varied activities will serve as templates for the future design of immunogens to induce protective Abs.