PROJECT SUMMARY Approximately 40% of the world's population is at risk for illness caused by the four serotypes of dengue virus. Development of durable, safe, and efficacious tetravalent dengue vaccines is a global priority. Since incomplete immunity may predispose individuals to more severe disease via antibody-dependent enhancement, the goal of dengue vaccines is long-term simultaneous protection against all four serotypes. Given the weaknesses and risks observed following use of the only currently licensed dengue vaccine, it is critical to clarify components and mechanisms of durable four-serotype protection following vaccination. This proposal leverages the work of long- standing collaborative investigators involved in the development of the NIH live-attenuated tetravalent dengue vaccine. It builds on preliminary data from our monovalent and tetravalent vaccine studies, as well use of the human dengue virus challenge model for early indications of vaccine efficacy. Preliminary work suggests that protected vaccinees exhibit dengue-specific plasmablasts soon after vaccination, followed by neutralizing antibody responses targeted to all serotypes and cellular responses-including CD8+ T cell responses targeting dengue non-structural proteins. This proposal plans to evaluate data and specimens via four aims corresponding to specifically-designed vaccine and/or human viral challenge trials to iteratively expand and refine these observations for the immunologic characterization of durable protection. These trials include tetravalent vaccinations followed by short term (30d) or long term (>3 years) viral challenge; an incompletely protective tri- valent vaccine followed by missing-serotype challenge; and an endemic setting vaccine trial of naïve and previous dengue-experienced subjects followed for several years post-vaccination. We hypothesize that if homotypic antibodies all serotypes are not present, defined mechanisms may compensate for the missing serotype and to maintain protection from illness. We hope to demonstrate that in settings of incomplete or waning immunity, these mechanisms, such as cellular responses (CD8+ non-structural protein and CD4+ cytotoxic) or heterotypic antibodies to conserved epitopes, are necessary and effective even in the presence of enhancing antibodies. Overall, these immunologic evaluations will help answer critical and persistent questions about dengue vaccine risk and efficacy. Leveraged with highly controlled clinical studies these data should be broadly generalizable to the understanding of safe and durable immunity following tetravalent dengue vaccines.