The development of a successful HIV vaccine will require induction of neutralizing antibodies that can protect from infection by a broad range of HIV isolates. Although antibody responses can be generated as a result of vaccination or infection, these antibodies often recognize only immunodominant, highly variable epitopes that do not confer neutralizing protection. Broadly neutralizing antibodies (BNA) that are capable of neutralizing HIV across clades have been isolated from HIV patients that sustain high CD4 numbers, and low viral titers, over many years. Several of the BNA isolated from this long-term non-progressor (LTNP) population are also reactive to self-antigens, suggesting a relationship with autoreactive B cell development. The B cell source of HIV BNA is unclear. We propose to identify the cellular source of HIV BNA by precisely identifying specific subsets of human B cells using multicolor flow cytometry and to interrogate them with high-throughput technology capable of detecting presumably rare BNA-producing B cells. We will also determine if autoreactive B cells are enriched for HIV BNA. B cells will be isolated from healthy patients and those with HIV and evaluated for the ability to produce HIV BNA. Molecular mimics will be generated against antibodies that recognize the CD4bs of gp120 and any newly identified BNA, that will serve as probes to examine the BNA development and maintenance in HIV patients. The results will be important to define the B cell subsets to be targeted and the type of antigen and adjuvant strategy to stimulate these subsets to become long-lived plasma cells able to sustain BNA production. The development of an effective vaccine to prevent HIV infection will require a better understanding of how to generate antibodies that will neutralize the virus. We propose to precisely identify the B cells that produce protective HIV neutralizing antibodies in order to determine how they arise, are maintained, and how vaccines can be modeled to induce them.