Project Summary Flaviviruses are enveloped, single-stranded RNA viruses that cause epidemics of debilitating human disease on a global scale. The four serotypes of dengue virus (DENV) cause up to ~100 million infections per year including life-threatening shock syndrome. Zika virus (ZIKV) is linked to microcephaly and congenital malformations. Tick- borne flaviviruses (TBFV) are emergent threats that cause encephalitis, long-term neurological sequelae, and death. No specific therapy is available for any flavivirus. Flaviviruses adopt ensembles of structures in which certain antibody epitopes are exposed or hidden. Elucidating how antibodies recognize different structural ensembles of flavivirus virions can enhance our understanding of their functional properties including neutralizing activity. In this competitive renewal application, our long-standing collaborative team (Diamond, Kuhn, Fremont, Crowe, and Pierson) proposes to continue studying structural mechanisms of antibody recognition and neutralization of flaviviruses. We will define the functional properties of protective antibodies that neutralize DENV and ZIKV in a conformationally-restricted manner. We will use these antibodies to probe the spectrum of structures sampled by flaviviruses and how this influences epitope exposure. We also will perform new campaigns to identify neutralizing antibodies against ZIKV and the distantly-related tick-borne flaviviruses that recognize quaternary epitopes. We will use this information to engineer ZIKV subviral particles (prM-E; SVPs) that optimally enable binding of neutralizing antibodies including those quaternary epitopes spanning dimer rafts. Genetically modified ZIKV SVPs will be tested for improved immunogenicity and protection in mice. Overall, our experiments will provide new insight into the dynamic states of flaviviruses and the nature of accessible neutralizing antibody epitopes. These studies may facilitate the generation of novel sculpted antigens and immunogens with improved capacity to detect and elicit specific protective antibody responses against DENV, ZIKV, and likely other emerging flaviviruses.