Goo, LeslieStuart, Jackson2022-01-262022-01-262022-01-262021Stuart_washington_0250O_23706.pdfhttp://hdl.handle.net/1773/48308Thesis (Master's)--University of Washington, 2021Zika virus (ZIKV) and dengue virus (DENV) are two highly related mosquito-borne members of the genus Flavivirus that cause significant human morbidity and mortality. There is no licensed vaccine available for ZIKV and the sole vaccine available for DENV is suboptimal both in terms of its efficacy and safety. There is much interest in the development of a single vaccine effective against ZIKV and DENV as both viruses co-circulate due to shared vectors and vertebrate hosts, and due to the risk of antibody-dependent enhancement of subsequent infection by either virus. Two classes of broadly neutralizing antibodies (bnAbs) capable of potently neutralizing both viruses have recently been isolated: EDE-class and MZ4-class bnAbs. Through traditional epitope mapping methods, EDE bnAbs were found to bind to a conserved quaternary epitope found on the flavivirus envelope protein (E) while MZ4 bnAbs bind to a linker region found between domain I and domain III of E protein. However, these mapping methods are only capable of providing data on binding and structural footprints. In order to better understand the determinants of neutralization for these bnAbs, I utilized a high-throughput approach called deep mutational scanning (DMS) that is capable of providing amino acid-resolution data on the determinants of neutralization – data that will be useful for further investigation of these bnAbs as potential therapeutics and as the basis for a cross-protective vaccine. I demonstrate the successful rescue of a ZIKV E protein virus library, the identification of an inhibitory concentration of 99% for both bnAbs, and the initial attempts at performing a DMS selection assay.application/pdfen-USnoneVirologyPathobiologyThesis