PROJECT SUMMARY– PROJECT 3: Mechanistic studies and comparisons of vaccines in preclinical models The two primary goals of the Project are to: (i) define the immunological parameters (i.e., CD4+ T cell help; innate immune response) that regulate the breadth of the antibody response in mechanistic studies in mice, and (ii) evaluate pan-sarbecovirus and pan-betacoronavirus vaccine candidates aimed at inducing broadly protective immune responses in nonhuman primates (NHPs) (with Core D, Nonhuman Primates, Villinger). In Aim 1, we will determine the mechanisms by which the innate immune system and T follicular cells regulate the breadth of antibody responses induced by broadly protective coronavirus vaccines designed in Project 2 (King). We will perform mechanistic studies in mice to determine the impact of CD4+ T cell help and specific DC subsets in regulating the breadth of vaccine-elicited Ab responses. This will yield insights about critical immunological parameters that determine antibody breadth and, looking ahead, provide a rational basis for the development of adjuvants that stimulate broad Ab responses. In Aim 2 we will conduct two studies in NHPs to evaluate pan-sarbecovirus and pan-betacoronavirus vaccine candidates from Project 2 and select lead candidates for further preclinical and clinical development. These studies will use clinically relevant adjuvants to ensure optimal translation to humans. We will assess the magnitude, breadth and durability of nAb responses induced by immunization with nanoparticle immunogens formulated with AS03 and 3M-052/alum, both of which have demonstrated superior adjuvant effects in terms of their capacity to promote high-magnitude and durable neutralizing antibody responses in NHPs. We will thus directly compare AS03 with 3M-052/alum for their capacity to enhance the breadth of nAb responses. We will work with Project 1 (Veesler) and Cores B (Virology, Baric), C (Viral Evolution, Bloom) and D to evaluate the immunogenicity and protective breadth of the responses elicited by each nanoparticle vaccine candidate and define the epitope specificities of cross-reactive responses. We will characterize the dynamics of the antigen-specific B cell response, assess plasmablast and plasma cell frequencies, and sort antigen-specific single B cells and isolate monoclonal antibodies from immunized NHPs. These mAbs will be produced by Core A (Protein Sciences, King) and analyzed in Project 1 and Cores B and C for their binding and neutralizing breadth, epitope specificity, and resistance to escape mutations.