SUMMARY – PROJECT 2 There are only a handful of sites on the HIV-1 envelope protein (Env) that represent regions of vulnerability for elicitation of broadly neutralizing antibodies (bnAbs). Four of these sites are associated with the gp120 subunit while one of the sites resides in the membrane-proximal external region (MPER) of gp41. We previously showed that the MPER is a conserved lipid-immersed, structural element consisting of two helices separated by a hinge with tandem joints that facilitate viral hemifusion and fusion. MPER-specific bnAbs manifest extraordinary HIV-1 strain and clade neutralization breadth, exerting anti-viral effect by disturbing gp41 conformational change required for viral entry into host cells. The MPER is poorly immunogenic, however, either as a component of the intact gp160 trimeric spike or in the context of an isolated gp41 subunit immunogen. Our recent cryo-EM and molecular dynamics (MD) studies of membrane-embedded Env trimers reveal that each MPER protomer radiates toward the 3-fold axis, generating an occluded membrane-associated tripod structure beneath the upright Env spike. Notwithstanding, spontaneous spike tilting exposes a 20-Å slot for an Fab arm to insert in a restricted vectorial space. By creating mRNA-based chimeric vaccines, replacing the Env ectodomain with a trimeric host self-protein protomer linked to a viral MPER-TM segment as detailed in Project 1, misguiding and sequence-variable viral structural elements are replaced by a non-immunogenic structure. MPER immunogenicity should then no longer be subdominant. Aim 1 will express these MPER immunogens, incorporate them into nanodiscs and compare their structure and dynamics, by themselves and in complex with Fabs of interest, with those of gp160 in nanodiscs. Aim 2 will clone and characterize human-derived MPER-specific IgG1 and IgG3 bnAbs from patient biobanked samples. Serial longitudinal serological and single memory B-cell analyses will allow for recombinant monoclonal antibody (rmAb) cloning, thereby defining MPER epitope locales, somatic hypermutation (SHM), neutralization breadth/potency, affinity, CDRH3 length and lipid binding/polyreactivity, with explicit comparison of IgG1 and IgG3 isotypes and their aggregate molecular evolution. Aim 3 shall perform X-ray crystallographic studies using Fabs generated in Aim 2 in complex with MPER epitopes, comparing the features of these bnAbs with others defined previously in patients as well as those elicited through vaccination in Project 1. Structural and MD studies of germline and mature IgG1 and IgG3 bnAbs shall reveal whether CH1-CL interdomain motions, elbow angle flexibility, and/or antibody-to-framework distance are similar or different and impacted by hinge length. We anticipate that the additional dexterity afforded by the long IgG3 hinge will allow for facile entry of their Fab arms to approach the MPER, augmenting the ability of unmutated common ancestors (UCAs) and their progeny with mi...