PROJECT 3: PROJECT SUMMARY Dengue virus (DENV) is the most prevalent mosquito–borne virus infecting humans in tropical and subtropical areas of the world. There are 4 DENV serotypes, namely DENV1, DENV2, DENV3 and DENV4 circulating in humans. Understanding the immunoprotective and immunopathogenic responses to dengue vaccines and responses to dengue virus (DENV) infection are critical to the development of a safe and effective dengue vaccine. To assess the innate and adaptive immune responses contributing to homo- and heterotypic immune responses to dengue vaccination and infection, we will analyze samples obtained from two clinical trials which evaluated the live attenuated dengue vaccine TV003 (LATV) or a trivalent admixture missing the DENV-2 component of TV003 (CIR287 and CIR300 respectively), against DENV-2 challenge. TV003 induced 100% protection against viremia and rash following DENV-2 challenge, the trivalent formulation induced only 20% protection against DENV-2 viremia. Using available samples from vaccine trials described above that are fully characterized serologically, we will analyze the cellular immune responses in PBMCs generated in those individuals over time, provided by the clinical core (Core B). With multiparametric immunological and genetic approaches performed by the immune phenotyping core (Core C) and genetic core (Core D), we will profile the innate and adaptive immune responses to tetra- and trivalent vaccine formulations (Aim 1) and to challenge with rDEN230, a live attenuated DENV-2 discarded as vaccine component (Aim 2). Additionally, we will use human tonsillar histocultures (HC) provided by core B to analyze the immune responses induced by different admixtures of the dengue vaccines (Aim 3). Tonsils have a well-defined spatial architecture and cellular microenvironments and can be readily dissociated for single cell assays. They also show important features to understand the generation of innate and adaptive immune responses, like the formation of germinal centers. These facts make them ideal for comparing cell suspension profiling assays such as Aurora flow cytometry with tissue imaging approaches like spatial transcriptomics (Core D). The understanding of the elements in the different vaccine formulations that confer immunogenicity and the contribution of the different serotypes to vaccine efficacy will be crucial for the development of efficacious vaccines. Our approach will provide important information on the generation of immune responses to vaccination and infections and the immune signatures induced by protective vaccines versus less protective ones as determined by the data management and analysis core (Core E). The analyses of the different cell populations present in PBMCs and in the tonsil HC will expand our knowledge on generation of innate and adaptive immune responses and will allow for the visualization of germinal center formation and other features important for the generation of protective immu...