One of the primary goals of this P01 program project proposal is to define the functional and antigenic specificity of the memory humoral response before and after dengue virus (DENV) infection. Here, we will determine the molecular and structural basis for type-specific and cross-reactive human B cell responses to DENV infection. Aim 1 of Project 4 will study the epitopes and structures recognized by DENV3 type-specific potently neutralizing antibodies (Abs). The goal is to define a comprehensive antigenic map of neutralizing determinants on the DENV E protein, which will inform DENV vaccine design and testing efforts. In past work with investigators in this consortium, we have mapped many of the major antigenic determinants on DENV1 and DENV2. The protective determinants on DENV3 are less well studied. Fortunately, in preliminary experiments, in collaboration with Project 1 and Core C, we have generated a significant panel of potent DENV3-specific neutralizing monoclonal Abs (mAbs) from the Nicaraguan Pediatric Dengue Cohort Study (PDCS) samples. It is clear that many of these antibodies recognize novel epitopes that are not previously known. We will use these reagents to identify the molecular and structural basis for recognition using alanine scanning mutagenesis, hydrogen deuterium exchange mass spectrometry, cryo-EM and crystallography studies. If the antigenic maps appear incomplete, as determined by Project 1 in dengue-endemic populations, we will generate additional mAbs from the peripheral blood mononuclear cells (PBMCs) collected from children with documented repeat DENV3 infections, as well as DENV1 and DENV2 infections, in the PDCS to define the complete antigenic landscape. We hypothesize that most DENV3-specific Abs are directed to quaternary epitopes, including those near the DENV envelope domain I/II (EDI/II) hinge region, but that there is a diversity of binding poses and angles for recognition of this complex region. In Aim 2, we will define comprehensive antigenic maps for cross-reactive Abs that recognize and neutralize viruses of all 4 DENV serotypes. In preliminary studies, we have isolated broad and potent mAbs that have distinct profiles from those of the limited number of E dimer epitope (EDE) mAbs reported to date. The new mAbs suggest there are additional sites of vulnerability for broad and potent neutralizing responses that are not yet understood. Studies in this aim will define with biochemical, genetic and structural approaches the novel epitopes associated with highly neutralizing cross-reactive mAbs that differ from EDE epitopes after 2° DENV infection. Finally, in Aim 3, we will use emerging techniques in adaptive immune receptor next generation sequencing to interrogate the Ab variable gene repertoires in PDCS subjects. We will use deep sequencing of peripheral blood Ab gene repertoires in prior (pre-2° infection, pre-vaccination) PBMC samples from the same individuals. These studies will provide a global view ...