Project 2: Abstract Rubulaviruses are respiratory pathogens with a high level of transmissibility (R0 = 4-7). MuV is an ideal prototype pathogen to study paramyxoviruses because we have a human vaccine that elicits protective humoral immunity directed to undefined target(s). P2-Rubulaviruses dissects the structure and functions of the F and HN glycoproteins in entry and as antigens. The goal is to identify a general strategy for stabilization of the pre-fusion form of the F glycoprotein and move this and the HN glycoprotein forward, either individually or in complex as vaccine candidates using established in vivo models. In Aim 1 rational antigen design builds upon existing head domain structure of HN adding regions of the stalk and generates new structures of stabilized, pre-fusion F glycoprotein. This is augmented by cryoelectron tomography (Cryo-ET) to study the interactions of the native F/HN complex on the virion. The immunogenicity of individual and glycoprotein pairs will be assessed in vivo to iteratively inform design, and the results tested in divergent rubulaviruses. In Aim 2 we isolate single B cells from vaccines and characterize the mAbs to further inform immunogen design. The mechanism of action of the mAbs will be assessed in vitro using a combination of assays including virus-cell and cell-cell fusion. Their individual and synergistic activities will be determined and ability to protect animals from infection and disease evaluated in a cotton rat respiratory system model. Genetic barriers to escape from neutralization will be defined to reveal how rapidly resistance mutations are acquired and their location(s) in the glycoproteins. Extension of this work by immunization of a humanized mouse with bat mumps (BatMuV) will extend the findings to related rubulaviruses considered to represent “pre-spillover” pathogens poised with pandemic potential. Lead immunogens are evaluated in Aim 3 for in vivo for efficacy as soluble-adjuvated proteins, mRNAs and within the context of a replication-competent rVSV-expressing both glycoproteins. Identification of correlates of protection will facilitate iterative analysis of next generation immunogens and may identify an optimal platform. The P2-Rubulaviruses project has exceptionally strong synergy with P1-Respiroviruses which will rapidly enable go/no-go decision making and contribute to down-selection and prioritization of mAbs and immunogens for a pathway to industrial translation. Similarly, the cross-talk between the data obtained from these two projects using the three vaccine platforms will be useful to drive existing and develop new collaborations with partners in industry.