SUMMARY The four dengue virus serotypes (DENV1-4) cause the most important mosquito-borne viral disease of humans, with several hundred million infections annually and almost 4 billion people worldwide at risk of infection. Yet, no treatment is currently approved for use in humans, and vaccine development and implementation has been problematic. The overall approach of Project 1 is to take advantage of unique Nicaraguan sample sets to address complex questions about DENV antibody and B cell immunology in a relevant clinical and epidemiological context. The overall hypothesis of this Project is that the magnitude and quality (i.e., repertoire and functionality) of B cell and antibody responses are shaped by distinct structural and antigenic characteristics of the four DENV serotypes in 1° infections and, via immunological imprinting, 2° infections that impact clinical and epidemiological outcome. Each aim derives from advances in the current P01. Carefully selected sequences of infections based on antigenic relatedness are analyzed using state-of-the- art immune profiling methods at the polyclonal and monoclonal level and mapped by antigenic cartography, antibody landscapes and antibody clustering visualizations, supported by rigorous biostatistical approaches, to generate a deeper understanding of DENV immunological interactions with implications for public health and vaccine development. The proposed research is possible due to the ongoing Pediatric Dengue Cohort Study (2004-present), a community-based prospective cohort study in Managua, Nicaragua, following ~4,000 children, now in its 20th year, and the Dengue Hospital-based Study (2005-present) in Managua, which together enable investigation of pre-infection samples and documented sequential DENV infections. This Project is highly synergistic with the other Projects and Cores in this P01 by sharing similar samples, methods and reagents (Projects 2, 3; Cores B, C, D) and comparing high-dimensional immunological profiling of the B cell/antibody response during DENV natural infections and vaccines (Project 2, Core C). Aim 1 will comprehensively define the structural and immunological features of primary DENV infections of all 4 serotypes. Aim 2 examines how immunological imprinting occurs from the 1° to the 2° DENV infection, leading to modulation of antibody and B cell responses. Results from cutting-edge immune profiling methods (Core B) will be analyzed by antigenic cartography, antibody landscapes, and serum clustering maps and compared by serotype sequence and to vaccine responses. How these varied antibody profiles affect infection and disease outcome are addressed in Aim 3, underpinned by specific hypotheses. Antibody repertoire will be defined at both polyclonal and monoclonal level, using orthogonal approaches. Overall, this project will advance the understanding of immunological imprinting in the complex DENV landscape and identify antibody/B cell correlates of protection and risk, which should be useful for development and evaluation of dengue vaccines.