SUMMARY – PROJECT 1 Neutralizing antibodies are a major component of vaccine-induced protective immunity. However, induction of broadly neutralizing antibody (bnAb) against the trimeric HIV-1 envelope (Env) spike protein gp160 continues to be a challenging goal. An effective vaccine must address the remarkable antigenic variability of gp160, its extensive glycan shield and/or conformational masking of conserved neutralizing target epitopes. Among bnAb targets, the membrane proximal external region (MPER) of gp41, the Env subunit critical for virus fusion, is attractive given its linear and highly conserved sequence as validated by the neutralization breadth of 4E10, 10E8 and DH511 bnAbs. Nonetheless, the poorly immunogenic nature of the MPER during the natural course of infection and the lack of neutralizing activities elicited by structure-based MPER immunogens demand an alternative immunogen design approach. The accessibility of the MPER is limited, being shielded by gp160 trimer ectodomain from above and the viral membrane from below. Furthermore, our recent MPER/liposome results suggest that the unrestricted approach angle afforded to the B cell receptor with current vaccine formulation is problematic, inducing a majority of polyclonal Abs as well as recombinant monoclonal Abs that lack Env reactivity. Therefore, MPER immunogens must mimic spatial occlusion while enforcing limited Ab accessibility to the MPER in a manner analogous to that imposed by the quaternary structural configuration of gp160 on the virion surface. In that regard, our recent analyses of the trimeric HIV-1 Env structure and molecular dynamics (MD) in nanodiscs provide compelling data for novel vaccine design considerations. With advanced messenger RNA lipid nanoparticle (mRNA-LNP) vaccine technology, Project 1 proposes to create novel mRNA-based immunogens that encode trimeric spike-MPER-TM (the MPER and adjacent transmembrane segment) chimeras. Therein the Env ectodomain is replaced by structurally suitable host self-proteins, focusing Ab responses solely against the conjoint membrane-arrayed viral MPER-TM epitopes. In Aim1, a pipeline involving automated computational searches of the PDB followed by MD and single-particle cryo-electron microscopy (cryoEM) structures in collaboration with Project 2 will generate multiple candidate immunogens. Optimization and further modifications of mRNA-LNP vaccines will be tested by 1) in vitro expression, 2) antigenicity and antigen presentation, and 3) in vivo immunogenicity studies. The latter use knock-in (KI) mice harboring introduced human Ig D3-3 and JH6 gene segments for long CDRH3 generation. In Aim 2, genetic, biochemical and functional characteristics of vaccine-elicited Abs at the single B cell level will be determined and compared with human bnAbs analyzed structurally in Project 2. The integrated analyses will offer iterative immunogen improvements to enhance the quantity and quality of anti-MPER Abs. In Aim 3, strategie...