SUMMARY – OVERALL While there is an urgent need for vaccines eliciting broadly neutralizing antibodies (bnAbs) against the highly mutable HIV-1 retrovirus to stem its global spread, this goal remains elusive. Antibodies produced against trimeric gp160 sites of vulnerability during natural infection drive retroviral mutation further, diversifying quasi- species in individuals. One apparent exception is the conserved membrane-proximal external region (MPER) site, critical for hemifusion/fusion and which is stealth, largely immersed in lipid and only transiently revealed during spontaneous ectodomain tilting. Liposome-arrayed MPER segments induce specific antibodies in mice but without neutralizing activity, explained by the restricted antibody access to the native MPER residing in a narrow “crawlspace” between the viral membrane below and the base of the gp120 and gp41 protomers of the trimeric gp160 envelop. Biomaterial formulation of polymer “steric clouds” on liposomes or alternative origami- based approaches on nanodiscs to limit access to the MPER, akin to that mandated by the trimer three-fold axis, were technically cumbersome and confounded by the uncertainty of MPER topology. Our recent analysis of the structure and dynamics of nanodisc-embedded trimeric HIV-1 spike protein provides compelling data for alternative design. In addition, synthetic RNA lipid nanoparticle (LNP) encapsulation technology obviates vaccine challenges with hydrophobic proteins such as the MPER and adjacent transmembrane segment (MPER-TM). This P01 comprises two projects. Project 1 shall determine the immunogenicity of RNA vaccines encoding MPER-TM trimers fused to structurally suitable self-proteins to focus antibody responses against the conjoint membrane arrayed viral MPER-TM, with the self-proteins imposing steric restriction comparable to that exerted by the gp160 ectodomain on the native MPER. Serological, single B cell, recombinant mAb methods, neutralization assays and Ig bioinformatic analyses are applied. Automated computational searches of the pdb informed by single-particle cryo-electron microscopy (cryo-EM) structures and using RNA technology reveal antigenicity and immunogenicity of construct design for planned immunization studies in normal as well as knock- in mice harboring human Ig D3-3 and JH6 gene segments for long CDRH3 loop generation. Project 2 will perform cryo-EM and X-ray crystallographic studies to compare existing and additional HIV-1 patient-derived bnAbs obtained from biobanked serial samples (Osier, IAVI) with those that are vaccine elicited, in the context of nanodisc-arrayed MPER immunogens and native spike protein. We shall investigate Ig requirements of somatic hypermutation (SHM), Fab molecular dynamics (MD) and co-evolution of antibody approach angles and affinities of bnAbs of IgG1 and IgG3 isotypes. Projects 1 & 2 shall be performed by Kim/Weissman (DFCI/UPenn) and Reinherz (DFCI), respectively, with four Technology Components: ...