Project Summary Exposure to one flavivirus can elicit immune responses that cross-react with genetically related viruses, in complex relationships with a variety of impacts on subsequent flavivirus infections. The best-characterized example of this is within the dengue virus (DENV) serocomplex. Pre-existing immunity to one of the 4 DENV serotypes, DENV-1, for instance, can increase the risk of severe disease upon infection with a different sero- type in what is termed antibody-dependent enhancement (ADE). Interestingly, emerging evidence suggests that immunological cross-reactivity among flaviviruses is not always reciprocal—that is, pre-existing immunity to virus A may protect against disease associated with virus B, while pre-existing immunity to virus B may increase the risk of disease upon infection with virus A. For example, we and others have shown in hu- man cohorts that pre-existing immunity to DENV reduces the risk of disease associated with Zika virus (ZIKV) infection, while immunity to ZIKV enhances the risk of disease associated with certain DENV serotypes. Thus, the degree to which pre-existing flavivirus immunity is cross-protective, enhancing, or neutral may depend on the order in which the host has encountered different flaviviruses previously. We hypothesize that differences in the antigenic sites recognized by antibodies and/or antibody func- tional “quality” are major determinants of non-reciprocal flavivirus immunity. To address this hypothesis, we have assembled a unique team of investigators with expertise in virology, immunology, epidemiology, and nonhuman primate models, with access to samples and data from our 19-year Pediatric Dengue Cohort Study in Nicaragua, the longest continuous such study in the arbovirus field. We will integrate studies of flavivirus immunological cross-reactivity in nonhuman primate models and human cohorts to evaluate the impact of flavivirus exposure on virus replication dynamics, antibody repertoire diversi- ty, neutralization titer, and Fc effector function. We will also leverage our unique cohort of children in Nicaragua with known flavivirus infection histories to examine how the order of exposure to DENV serotypes and to ZIKV shapes cross-reactive antibody profiles, specifically, the capacity to respond to Spondweni virus (SPONV), a model emerging flavivirus and the closest known relative to ZIKV. We include SPONV here due to our prelimi- nary data indicating that cross-reactive immunity between ZIKV and SPONV in macaques is strikingly non-re- ciprocal. Macaques with ZIKV immunity were completely protected against SPONV challenge, while SPONV immunity provided no protection against ZIKV. This study will provide tractable model systems in which to identify how the order of flavivirus exposure impacts immune responses and infection outcomes. Our findings will have broad implications for how we assess the risk of emerging viruses and disease in flavivirus-exposed populations and design next-ge...