Project Summary Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the virus responsible for the global coronavirus disease 2019 (COVID-19) pandemic. Elucidating the mechanisms underlying the adaptive immune response to SARS-CoV-2 is crucial for predicting vaccine efficacy and assessing the risk of reinfection. Neutralizing antibodies against SARS-CoV-2 have been studied extensively and are clearly protective, but may be short-lived and are not elicited in all infected individuals. Mounting evidence indicates that T cells play a vital role in the clearance of SARS-CoV-2 and in formation of long-term memory responses to this virus. Extensive structural information is now available on neutralizing antibodies from COVID-19 convalescent patients (CPs) bound to the SARS-CoV-2 spike trimer or RBD (>330 PDB entries), resulting in a comprehensive picture of the B cell response to this virus. By contrast, very little structural information is available for SARS-CoV-2-specific TCRs bound to their peptide–MHC (pMHC) targets (4 PDB entries), despite the increasingly appreciated role of T cell-mediated immunity in combatting COVID-19. Our goal is to address this major unmet need by obtaining atomic-level information on SARS-CoV-2 recognition by MHC class I-restricted TCRs from COVID-19 CPs. We will focus on CD8+ T cell epitopes because cytotoxic CD8+ T cells participate directly in viral clearance. Our Specific Aims are: Aim 1: Determine crystal structures of TCR–pMHC complexes. We will target for X-ray crystallographic analysis TCRs specific for epitopes that elicit high levels of TCR expansion in COVID-19 CPs, as well as TCRs associated with protection. To demonstrate feasibility, we have already determined structures of a public and a private TCR from COVID-19 CPs in complex with HLA-A2 and two SARS-CoV-2 spike (S) epitopes, YLQ and RLQ. We will extend these studies to three additional S epitopes (FVF, FQF, RLN) and three nucleocapsid (N) epitopes (LLL, LQL, SPR), which elicit strong T cell responses, and are presented by HLA-A2 or HLA-B7 MHCs. We have diverse panels of TCRs targeting each of these CD8+ T cell epitopes. Aim 2: Analysis of TCR–pMHC structures. TCR–pMHC complex structures determined in Aim 1 will be analyzed using advanced computational structural biology tools to delineate key features of their recognition, as well as the predicted targeting of epitope variants from SARS-CoV-2 and related coronaviruses. These studies will yield foundational knowledge about TCR recognition of SARS-CoV-2 epitopes, about the generation of clonal diversity in epitope-specific TCR repertoires, and about the ways mutations in these epitopes may enable the virus to escape immune surveillance.