The identification of a few rarely made antibodies to different HIV-1 envelope epitopes which are capable of neutralizing a broad range of HIV-1 isolates in vitro and controlling SHIV infection in vivo has provided hope for achieving an antibody-based HIV-1 vaccine strategy. One of these rare antibodies, 2F5, has particularly potent and broad neutralizing activity and is directed against an attractive antibody-based vaccine target, the MPER epitope of the HIV-1 gp41 region. 2F5 has unusually long charged CDR3s and exhibits polyreactivity, raising the possibility that antibodies like 2F5 cannot be elicited because the B cells from which they originate have reactivity with host antigens. Using a gene-targeting approach, we have generated mice (2F5 VH) with directed expression of the original 2F5 heavy chain (HC) variable region into the mouse HC locus, and made the important observation that 2F5 HC-expressing B cells are primarily deleted in the bone marrow at the pre-B to immature transition. Despite this profound defect, a small, but detactable population of B cells are present in the periphery which retain the 2F5 HC. In this application, we propose to comprehensively extend our characterization of 2F5 VH mice, first to test the hypothesis that most of their remaining peripheral B cells have lost autoreactivity, either by LC editing, anergy or modification of the 2F5 HC. Using a systematic array of genetic, immunization, and pharmacological approaches to modulate self-tolerance, we will then test the hypothesis that these mice can be pushed into producing high titers of broadly neutralizing antibodies. Finally, we will extend/refine our studies in 2F5 VH mice by using a series of back-mutated 2F5 VH mice (expressing 2F5 VH regions that are either germline or have different selected back-mutations), to examine the ability of the 2F5 precursor B cell repertoire, when shaped by immunization-driven affinity maturation, to generate 2F5-like neutralizing antibodies. PUBLIC HEALTH RELEVANCE: The development of a protective antibody-based HIV-1 vaccine remains an enormous challenge. In this study, we propose to use a series of mouse strains we have engineered to express different versions of the broadly and potently neutralizing HIV-1 antibody 2F5 in order to: 1) determine how the processes of self-tolerance and affinity maturation impact the ability of B cells to make neutralizing antibodies, and 2) to examine if appropriate manipulation of these processes can elicit B cells to make such antibodies. These experiments should be helpful in formulating novel strategies for properly eliciting protective HIV-1 antibodies in patients.