Abstract – Project 2 Animal betacoronaviruses (betaCoVs), such as those that gave rise to SARS-CoV-1, MERS, and now SARS- CoV-2, represent a significant, continuous threat to human health. Vaccines can provide pre-existing immunity to future zoonotic coronaviruses. A major limitation of the current effective vaccines to SARS-CoV-2 is that they likely will only protect SARS-CoV-2 isolates that are nearly identical to the Wuhan isolate used in the vaccines. Thus, the world remains vulnerable to SARS-CoV-2 mutational escape variants and from future animal betacoronavirus spillover events that have pandemic potential. Vaccine development that addresses this vulnerability is urgently needed. Project 2 will support this Program’s overall goal to develop vaccines that will prevent future betaCoV pandemics and SARS-CoV-2 escape variants by designing vaccine immunogens that elicit broadly neutralizing antibodies (bnAb) to provide protective immunity across the betaCoV genus. Structurally conserved neutralizing epitopes exist on betaCoV spike proteins, and we hypothesize that vaccines that elicit broadly protective antibodies to these sites can be developed. In preliminary studies, we have isolated cross-reactive neutralizing antibodies from SARS-CoV-1 or SARS-CoV-2-infected or recovered humans and used structural determination to precisely define their betaCoV bnAb epitopes. Our vaccine design strategy involves using computational and structural- based design techniques to develop immunogens that preferentially present these and additionally identified bnAb epitopes. Each immunogen design will be displayed on the surface of nanoparticles to enhance antigen avidity and antigen trafficking to germinal centers and follicular dendritic cells. We have demonstrated a proof-of-concept for our strategy using a multimerized SARS-CoV-2 receptor binding domain (RBD)- ferritin nanoparticle that induces high titers of antibodies that neutralize diverse human and animal Sarbecoviruses (group 2b) in immunized macaques. In Aim 1, we will isolate new antibodies to define additional bnAb epitopes and establish the full spectrum of regions on the surface of betaCoV S proteins that are capable of inducing bnAbs to animal betaCoVs in groups 2b and 2c. In Aim 2, we will utilize cutting-edge computational immunogen design methods to design broadly neutralizing epitope-focused immunogens. These immunogens have the spike protein fixed in conformations that expose neutralizing epitopes or are spike subunits that are truncated or resurfaced to remove irrelevant, non-neutralizing, or betaCoV strain-specific neutralizing epitopes while retaining broadly neutralizing epitopes. In Aim 3, we will immunize wild-type mice with these novel immunogens and evaluate their ability to induce human betaCoV bnAbs that can protect animals from viral challenge. Project 2 will impact the field by generating cross-reactive neutralizing antibodies that could be translated into therapeutics, elucidatin...