SUMMARY Worldwide, over 3 billion people are at risk of infection and disease caused by dengue virus 1-4 (DENV1-4) and Zika virus (ZIKV), both potentially severe flaviviral diseases transmitted by Aedes mosquitoes. The devastating effects of endemic dengue across the tropics and subtropics are well documented. The recent Zika pandemic galvanized research as Zika swept across Latin America. Three years after the peak of the Zika pandemic, major dengue epidemics have started to re-occur; however, the future of flaviviral disease across areas with widespread ZIKV immunity is unknown. In this R01, we propose to develop new tools and address key knowledge gaps in flaviviral transmission and immunological interactions between DENV and ZIKV to understand how widespread ZIKV immunity impacts subsequent dengue disease and to inform evaluation of dengue and Zika clinical vaccine trials and post-licensure studies. Based on our serological, epidemiological, and clinical data to date, our overall hypothesis is that DENV1-4 and ZIKV are antigenically closely related and that immune interactions mutually affect transmission and disease severity. We will address this hypothesis with the ongoing Pediatric Dengue Cohort Study (PDCS, 2004-present), a community-based prospective cohort study in Managua, Nicaragua, following ~4,000 children, now in its 17th year. Samples from the PDCS, as well as companion studies in Managua, provide documented infection and disease data, as well as banked serum samples for over a decade before the arrival of ZIKV. The proposed study extends the cohort, ensuring that we are able to fully document the interactions of these viruses from the pre- to post-Zika eras. In Aim 1, we will develop innovative serologic tools based on glycan-fusion-loop-masked envelope proteins and new algorithms to distinguish DENV and ZIKV infection histories, critical for vaccination and epidemiological studies of dengue and Zika. We will then test our hypothesis that pre-existing ZIKV immunity can enhance disease severity caused by DENV3 but protect against DENV1. In Aim 2, we will measure changes in anti-DENV and anti-ZIKV antibody-mediated immunity over time, estimate annual changes in protective and enhancing population immunity to each virus, collect entomological data, and use modeling approaches to evaluate popula- tion susceptibility to DENV and ZIKV infection and the potential for future epidemics by incorporating immunolo- gical and entomological data. In Aim 3, we will identify determinants of protective and disease-enhancing anti- body-mediated immunity of prior DENV infection on Zika and prior ZIKV infection on dengue disease and severity. With support of expert collaborators, we will use state-of-the-art tools (e.g., new monoclonal antibodies, innovative flavivirus antigens, and antibody Fc profiling) to analyze specific infection histories and uncover potential immune correlates. Overall, this program will define new vaccine companion diagnostic assays, the dynamics of the antibody response to DENV and ZIKV, and correlates of protection and pathogenesis for dengue and Zika, which should be useful for the development and evaluation of dengue and Zika vaccines.

NARRATIVE Dengue and Zika are diseases of major importance worldwide, but surveillance, epidemic preparedness, and control are complicated by immunological cross-reactivity between dengue and Zika viruses (DENV and ZIKV), particularly in dengue-endemic Latin America after the explosive 2015-2016 Zika pandemic. It is critical for dengue and Zika vaccine evaluation and implementation, epidemic preparedness, and public health research to improve differential detection of prior infections by DENV and ZIKV and increase our understanding of how immunological cross-reactivity affects serological testing, seroprevalence estimation, viral transmission, and protection and/or enhancement of future flaviviral disease. Following upon our recent work showing that immunological asymmetries exist among the four DENV serotypes and ZIKV and that pre-existing immunity to ZIKV can enhance subsequent DENV2 infection and disease, this R01 will use new serological assays, diverse modeling approaches, and state-of-the-art methods for antibody analysis to study samples and data from the longest-running dengue/Zika cohort study and extend this unique Nicaraguan cohort into the next critical phase of flaviviral disease in the tropics and subtropics where infection and disease outcomes are modulated by widespread ZIKV immunity.