Project Summary/Abstract – Project 3: Peribunyaviridae Pathogenesis and Vaccine Testing The orthobunyaviruses (OBVs) of Peribunyviridae cause disease in humans manifesting with fever, nausea, fatigue, and viral encephalitis, as well as long-term neurological complications. Identifying receptors and immune factors mediating infection will highlight additional avenues of therapeutic development to work in tandem with vaccination efficacy approaches. As these viruses are increasing in prevalence and vector range, their emergent potential is high, and there is a critical need to better understand their pathogenesis and develop safe and effective vaccines against OBVs. As part of this U19, this project will focus on identifying additional vaccine targets while validating protein-adjuvant and mRNA vaccines developed in Projects 1 and 2. Importantly, these studies will define both acute and long-term correlates of protection. The long-term goals are to 1) validate immunogenic, multi-platform vaccines for OBVs, targeting LACV, OROV, and CVV as representative viruses; 2) delineate mechanisms of OBV pathogenesis; and 3), define the role vaccines play in preventing sequelae. This will test the hypothesis that 1) differential cytokine expression drives early and late stage OBV disease, 2) vaccination prevents both acute and long-term effects of infection, and 3) OBVs exploit conserved receptors for entry into the CNS. The first aim will define host immune responses to OBV infection in vivo. To determine early and late-stage host immune responses to OBV infection, we will assess innate and adaptive immunity in humanized immuno-competent and -compromised mouse models after infection with LACV, OROV, or CVV. Serum cytokine levels, regulation of immune signaling genes, and histopathology will be assessed. This will define key immunological mechanisms of protection against OBV infection and highlight avenues for additional therapeutic development. The second aim will assess short and long-term protection against OBV infection with adjuvanted subunit and mRNA vaccines in vivo. Adjuvanted subunit vaccines and mRNA vaccines targeting Gc/Gn and NP antigens will be tested for protection against short- and long-term OBV outcomes in animal models to identify lead vaccine candidates. MRI and behavioral analyses will be applied to understand vaccine protection against disease sequelae. The third aim will identify conserved host cell receptors for OBV attachment and entry mediating CNS tropism. The Antibody Generation, Epitope Mapping, and Machine Learning Core will provide computational interaction screening, after which candidates will be verified in permissive CNS cells and mouse models. Promising receptors will be blocked with inhibitors and knocked out by CRISPR/Cas9 with subsequent infectivity measured in mouse and brain co-culture systems. Receptor deficient mice will also be evaluated for changes in viral replication. Mouse models of severe infection will be treate...