Project Summary/Abstract Zika virus (ZIKV) is an emerging infectious pathogen and has been classified as an international public emergency by the World Health Organization due to its rapid spread around the world and its association with devastating neurological complications including Guillain-Barr syndrome and infant microcephaly. Thus, the development of therapeutic drugs and prophylactic vaccines is of high priority. Recent studies point to the antigenic relationship between Zika and dengue viruses, with implications for vaccines and therapeutics since evidence suggests that cross-reactive antibodies elicited by ZIKV or dengue virus (DENV) infection can mediate heterologous antibody-dependent enhancement (ADE) leading to more severe or fatal disease. Until now, human ZIKV-reactive antibodies have been isolated by single cell cloning of peripheral B cells only. In contrast, the epitopes targeted by the actual circulating antibodies in convalescent patients, especially those serum antibodies that are most abundant and thus playing a dominant role in the polyclonal response, including protective and pathogenic ones, remain experimentally unverified. Our central hypothesis is that the ZIKV serological antibody repertoire is statistically distinguishable from the cognate BCR repertoire, in terms of the frequency of specific and cross-reactive antibodies, in terms of DENV infection history, and in functional properties such as in vitro neutralization and ADE activity. Thus, the aims of this collaborative project are to (i) inform on the complexity and molecular features of the serum antibody repertoire elicited by infection, (ii) identify and evaluate the persistence of ZIKV-specific and ZIKV/DENV cross-reactive antibodies in serum, (iii) isolate and characterize monoclonal antibodies identified from serum for neutralization and ADE activities, and (iv) determine the new epitopes targeted by the antibodies. Our multidisciplinary approach will include a set of original tools for analyzing adaptive immune responses?developed, published, and protected by two issued US patents and currently available only in our laboratories at UT Austin?to comprehensively identify, at high throughput, high-affinity human cross-reactive serum antibodies and delineate the molecular nature of cross- reactive antibody generation, and characterize the identified monoclonal antibodies via the reagents and approaches developed by the Baric laboratory at UNC. Collectively our results have the potential to provide structure-based rationales for the design of future ZIKV-specific vaccines.