CORE B: Viral Assays Core

SUMMARY: Dengue viruses (DENV) are responsible for the most significant mosquito-borne viral disease in humans, causing over 100 million infections and ~50 million dengue cases annually worldwide. The complexity of the immune response to four related serotypes (DENV1-4) and lack of reliable correlates of protection have hindered efforts to develop/improve a safe, effective, and serotype-balanced DENV vaccine. The overall goal of the Program Project is to delineate the role of adaptive immunity in shaping clinical and immunological endpoints in the context of DENV natural infection and vaccination. The quality (antibody [Ab] breadth, diversity, and function) of humoral immune responses underpins the outcomes (protection or enhancement) of subsequent DENV infection. The Viruses and Assays Core B develops and provides advanced virological tools for high-throughput, sample- sparing platforms to comprehensively characterize the humoral immune profile of serum samples and monoclonal antibodies (mAbs) from Project 1, Project 2, Clinical, Data, Statistical Modelling Core C, and Antibodyomics Core D. Core B builds upon the state-of-the-art DENV reverse genetics system developed over the historical course of this program by the Baric laboratory (University of North Carolina, Chapel Hill). The key reagents and tools include DENV serotype and genotype panels, DENV chimeric domain swap panels, DENV Ab epitope removal and resurfacing panels, and DENV NanoLuc reporter panels. Additionally, a reagent-sparing, high-throughput NanoLuc reporter neutralization assay has been developed and is ready for deployment across the program. A novel assay has also been developed and tested that specifically measures the fraction of a serum response that encodes enhancing antibodies. The Tse Laboratory (St. Louis University), with expertise in saturation mutagenesis, will develop a Deep Mutational Scanning (DMS) platform for unbiased, high-throughput monoclonal antibody mapping and to dissect complex serum fingerprints with embedded epitope information for individual subjects. Thus, Core B will provide assays, reagents, and protocols to measure Ab binding profiles and neutralizing and enhancing Ab titers of selected sera for Project 1 and Project 2 as well as support hypothesis-driven questions about immune imprinting in DENV infection, vaccination, sequence of infection, and other relevant parameters. In collaboration with Core C, Core B will generate new antigenic cartography serum- antigen maps reflecting the endemic strains of the cohorts in Nicaragua (Project 1) and the Philippines (Project 2) and the NIH vaccine challenge study (Core C). These tools will be used to create serum signatures from longitudinal samples with unprecedented resolution to understand immunological imprinting for DENV infection.