PROJECT SUMMARY Human respiratory viruses not only contribute to substantial morbidity and mortality worldwide but also pose a massive pandemic threat. This broad range of viruses includes human coronavirus (hCoVs), influenza A, and human metapneumoviruses (HMPV). Vaccines are among the most powerful means for mitigating viral epidemics but require significant neutralizing antibody breadth to maximize the probability of effectiveness against unknown viral threats. While a number of components of a protective immune response could be targeted to form the basis of a broadly protective coronavirus vaccine, neutralizing antibodies are generally accepted to be a key component of protective immunity. Although development of effective first-generation SARS-CoV-2 vaccines that induced protective antibody responses against severe illness has proceeded with unprecedented speed and seasonal influenza A (IAV) vaccines continue to offer protection, their effectiveness against emergent seasonal variants (e.g., Omicron) and, importantly, against other potential zoonotic viruses is less likely. Indeed, risk of zoonotic spillover events of divergent CoVs, such as the recently documented cases of human transmission from a canine alpha-CoV and a porcine delta-CoV that led to flu-like symptoms in infected Haitian children, highlights the need for innovative approaches to identify countermeasures targeting highly-conserved sites on CoV spikes shared among the Orthocoronavirinae subfamily. Project 2 will prioritize the development of immunogens that elicit neutralizing antibodies that can be measured in vitro. We will synergize with other Project members to test as many immunization strategies as possible in human organoid models, where the multifaceted functions of antibodies, effector cells, and host factors can be used to inhibit viral replication in an exposed host. Specifically, this proposal will focus on three interrelated aims: 1) identify the structural correlates of broad and potent antibody neutralization against conserved viral epitopes in CoV-S and IAV-HA trimers, 2) design multivalent immunogens that stimulate cross-reactive immune responses in human organoid model systems, and 3) provide insights into the molecular mechanisms of feedback inhibition and immune imprinting that limit diversification of B cell responses. Overall, this proposal will employ immunology, bioinformatics, structural biology, protein engineering and immune system models to discover how to elicit antibodies capable of neutralizing a broad range of emerging pandemic threat RNA viruses. The results will inform our understanding of broadly-protective anti-CoV and anti-lAV immunity in recovered and vaccinated individuals and will inform ongoing and future vaccine efforts.