SUMMARY. The long term goals of Project 3 of the Respiro, Rubula, Peribunya, Phenui (R2P2) ReVAMPP are to use a prototype pathogen approach to identify and validate generalizable vaccine strategies against peribunyaviruses, and advance therapeutically useful monoclonal antibodies (mAb). To achieve those goals, we selected Oropouche virus (OROV) as the prototype and will test our findings by extension into La Crosse virus (LACV). A central goal of this project is to understand how to stabilize the pre-fusion form of the class II fusogen Gc to determine whether principles successfully employed for class I fusogens and their advancement as vaccines can be leveraged to stabilize class II fusion machines. In doing so, we will generate foundational mechanistic knowledge regarding the structure and functions of the the attachment protein (Gn) and the fusion protein (Gc) in viral entry and as immunogens, that will also inform the optimal targets for therapeutic antibodies. In preliminary studies we identify viral entry factors including proteinaceous receptors in the LDL receptor superfamily, extending mechanistic parallels with studies of the Phenuiviridae in Project 4 of R2P2-ReVAMPP. In Aim 1, in conjunction with Core C Structure, we conduct a systematic structural and functional analysis of Gn and Gc to help inform those regions of Gn and Gc recognized by the human immune system capitalizing on the 2,500 monoclonal antibodies obtained from PBMC of convalescent humans generated in Aim 2 in conjunction with Core D Antibody. This knowledge will be leveraged to optimize the regions of Gn and Gc as immunogens. In aim 2, we interrogate the mechanism of action of those antibodies in vitro will be examined using a suite of assays to identify subsets that are neutralizing and define the precise steps at which they interfere with infection. Understanding the mechanism of neutralization will permit us to determine whether there are correlates between mechanism, genetic barriers to viral resistance, and optimal efficacy of antibodies in vivo. In Aim 3, we will iteratively test optimized immunogens as both protein subunit and mRNA-LNP vaccines and extend our work on VSV-vectored OROV vaccines, testing their efficacy in small animal challenge studies. This work will leverage the full power and expertise of Core E-Correlates to identify the correlates of protection against infection, and against disease. The close relationship between P3 and P4, ensures direct comparison of the extent to which a prototype peribunyavirus informs the related studies of phenuiviruses and vice-versa, bringing exceptionally strong synergy between the two projects that will accelerate go/no-go decision making.