PROJECT SUMMARY HIV-1 spreads mainly through mucosal exposures. Dominant in mucosal secretions, IgA has long been the class of antibodies desired at the portal of entrance to block infection. However, due to a paucity of IgA responses to HIV-1, compared to IgG, previous antibody isolation efforts and antibody repertoire analyses have focused on IgG+ B cells and missed IgA+ B cells. With a novel vesicular stomatitis virus (VSV)-based platform, we displayed the membrane-embedded HIV-1 envelope (Env) trimer to probe Env-specific memory B cells and isolate HIV-1 broadly neutralizing antibodies (bNAbs) from infected individuals. During this process, we included IgA antibody repertoire analyses and identified two HIV-1 bNAb lineages that class-switched to both IgG and IgA, thus for the first time identified bona fide IgA bNAbs produced during HIV-1 natural infection. Additionally, we have isolated two Env-directed IgA monoclonal antibodies (mAbs) that exhibited partial virus neutralization and potent antibody-dependent cellular phagocytosis (ADCP) function to eliminate HIV-1-infected cells. As recent VRC01 prevention trials showed no overall efficacy and only 75% efficacy to VRC01-sensitive strains, it calls for improved bNAb-mediated prevention. Given the unique properties of mucosal SIgA, such as dimerization, high stability, and resistance to enzymatic degradation, we aim to test whether IgA (dimer) bNAb passive infusion is better than IgG to block infection. Supported by these scientific premises and the need to improve bNAb- mediated prevention, we propose to identify additional HIV-1 IgA bNAbs and ADCP IgA mAbs from peripheral blood of clade-B and non-clade-B infected individuals, including those followed longitudinally, and from breast milk cells of HIV clade C infected mothers from the BAN cohort (Aim 1), then structurally define and characterize the IgA targeted epitopes (Aim 2), and finally test a representative IgA bNAb and ADCP IgA, using an IgG bNAb as benchmark, for protection efficacy in a rhesus macaque SHIV mucosal challenge model (Aim 3). We aim to test the hypothesis that 1) significant IgA bNAbs and ADCP responses are elicited in systemic and mucosal compartments during HIV-1 infection; 2) IgA bNAbs and ADCP IgAs may target epitopes distinct from those of previously known IgG bNAbs; 3) IgA bNAb is comparable to or better than its IgG bNAb counterpart to protect against SHIV mucosal challenge, and ADCP IgA may also protect from SHIV mucosal challenge. If successful, the project will unveil and validate the potential antiviral functions of IgA to fight against HIV-1.