A crucial missing component of current HIV vaccine candidates is an ability to elicit broadly neutralizing antibodies. In the proposed studies we combine vaccine design, next generation sequencing (NGS) bioinformatics, structural biology and mechanistic studies to overcome an important roadblock in HIV vaccine development, that of eliciting antibodies that recognize native conformations of gp41. The membrane proximal external region (MPER) of gp41 is a particularly desirable vaccine target as it is recognized by numerous broadly neutralizing antibodies, the most potent of which is 10E8. However, how antibodies are elicited to the MPER is virtually unknown. Meanwhile, preliminary data suggest that the 10E8 epitope involves interactions between subunits of the trimeric envelope spike. The structure and function of the MPER in its native conformations is also incompletely understood. We propose to immunize mice with three trimeric presentations of the MPER on stabilized native spikes, on liposomes and on nanoparticles. We will identify novel MPER antibodies to epitopes overlapping with 10E8 from immunized mice and from African donors. We will use NGS/bioinformatics analyses to deeply probe the B cell repertoires of the mice and humans to identify germline V-regions of MPER-specific antibodies. We will attempt to manipulate MPER specific B cell responses in mice through antigen modification. In collaboration we will study the EM structure of stabilized envelope spikes in complex with 10E8, while using a battery of assays to define the mechanism of neutralization by 10E8.