Project Summary The global pandemic caused by Severe Acute Respiratory Syndrome virus 2 (SARS-CoV-2) coronavirus (CoV) has highlighted the need to develop next-generation viral vaccines that offer protection against future Sarbecoviruses and Merbecoviruses of concern. One approach is to implement structure-based immunogen design strategies that elicit humoral responses to conserved sites on the surface-exposed spike (S) viral glycoprotein. Potential sites include the receptor binding domain (RBD) and the receptor binding motif (RBM) required for viral entry. Other conserved sites on S, outside the RBD, may also be targets of both neutralizing and non-neutralizing responses offering broad CoV protection. Project 1 will combine computational, structural, biophysical, and single B cell analyses to design S and RBD immunogens that elicit broad-spectrum humoral immunity against CoVs. Based on evolutionary analysis of CoV sequences, it is unlikely that a single antigen will confer broad enough immunity against the entire Betacoronavirus genus; we therefore anticipate generating a polyvalent S or RBD containing vaccine (with Project 3). Initially, we prioritize ACE2-receptor using CoVs as a proof-of-concept but envision that our design principles can be extended to other emerging CoVs where ACE2 is not the receptor. The Schmidt laboratory will implement structure-guided immunogen design strategies to focus immune responses towards conserved epitopes on the RBM and RBD. We will use heterologous CoV RBDs as molecular scaffolds to present selected ACE2-binding CoV RBMs and further immune focus using engineered glycans to mask non-conserved epitopes. The Scheuermann laboratory will use ancestral reconstructions to identify extant CoVs as additional S-based immunogens. The Ellebedy laboratory will isolate monoclonal antibodies (mAbs) from naturally infected, vaccinated, or naïve human subjects to define CoV cross- reactive, conserved epitopes on S recognized by neutralizing and non-neutralizing mAbs. We will define the breadth of mAb cross-reactivity, cross-neutralization, and effector functions (with Project 3). The Fremont laboratory will perform structural analyses by X-ray crystallography and cryo-electron microscopy (Fremont laboratory) of the isolated mAbs to define targeted epitopes on S; this information will aid in iterative immunogen optimization to elicit responses to these newly defined, cross-reactive epitopes. The output of Project 1 will be optimized RBD and S genes to transfer to Project 3 for vaccine formulation in VSV or ChAd vaccine platforms.