BAT T CELLS RESPONSES TO SARS-COV-2 - PROJECT SUMMARY BATS ARE RESERVOIR HOSTS OF SEVERAL VIRUSES THAT CAUSE SIGNIFICANT DISEASE BURDEN IN HUMANS. IN NEARLY ALL OF THESE VIRUS-HOST RELATIONSHIPS, THE BATS SHOW NO SIGNS OF DISEASE AND TYPICALLY CLEAR INFECTIONS WITHIN A FEW WEEKS. SOME HAVE POSTULATED THAT BATS HAVE "DISEASE TOLERANCE" THAT ACCOUNTS FOR THEIR ABILITY TO HOST THESE VIRUSES. ALTHOUGH INNATE IMMUNITY OF BAT CELLS IN VITRO HAS BEEN WIDELY STUDIED, VIRTUALLY NOTHING IS KNOWN ABOUT THEIR ADAPTIVE IMMUNE RESPONSES TO THESE VIRUSES. THIS PROJECT WILL FOCUS ON CYTOTOXIC T CELL (CTL) RESPONSES OF JAMAICAN FRUIT BATS EXPERIMENTALLY INFECTED WITH SARS-COV-2, THE CAUSATIVE AGENT OF COVID-19. WE PREVIOUSLY SHOWED THAT JAMAICAN FRUIT BATS HAVE LOW SUSCEPTIBILITY TO SARS-COV-2, WITH NO EVIDENCE OF INFECTION IN THEIR LUNGS. HOWEVER, WE HAVE OVERCOME THIS LIMITATION BY USING AN ADENOVIRUS VECTOR FOR IN VIVO TRANSDUCTION OF BAT LUNG CELLS WITH HUMAN ACE2, WHICH SUBSEQUENTLY LEADS TO A ROBUST INFECTION OF THE LUNGS BUT WITHOUT MEANINGFUL DISEASE. USING THIS MODEL, WE PREVIOUSLY DETERMINED THAT CD4+ T CELLS FROM INFECTED BATS CAN BE IDENTIFIED BY FLOW CYTOMETRY AND WE WILL EXTEND THESE FINDINGS WITH CD8+ CTL STUDIES. THIS APPROACH WILL USE AUTOLOGOUS FIBROBLASTS FROM WING PUNCHES GENERATED IN ADVANCE TO PROVIDE MHC CLASS I-MATCHED ANTIGEN PRESENTING CELLS (APC) PRIOR TO INFECTION OF BATS WITH SARS-COV-2. THE APCS WILL THEN BE CULTURED IN VITRO WITH CD8+ SPLENOCYTES FROM INFECTED DONOR BATS AND PEPTIDE POOLS OF SARS-COV-2 SPIKE AND NUCLEOCAPSID ANTIGENS TO ACTIVATE ANTIGEN- SPECIFIC CTLS. THE CTLS WILL THEN BE USED FOR SINGLE-CELL RNA SEQUENCING ANALYSIS TO DETERMINE THEIR TRANSCRIPTOMIC PROFILES IN RESPONSE TO SARS-COV-2 ANTIGENIC STIMULATION. ADDITIONALLY WE WILL DEPLETE JAMAICAN FRUIT BATS OF CD8+ CTLS USING A MONOCLONAL ANTIBODY THAT WE HAVE DEVELOPED AND CHALLENGE THE BATS TO DETERMINE THE IN VIVO ROLE OF CTLS DURING SARS-COV-2 INFECTION. THIS MAY LEAD TO DISEASE, WHICH WOULD SHOW THAT, AT LEAST, IMMUNE TOLERANCE MAY NOT BE A MECHANISM THAT EXPLAINS WHY BATS CAN HARBOR THESE VIRUSES WITHOUT DISEASE. COLLECTIVELY, THESE STUDIES WILL BE THE FIRST TO EXAMINE CYTOTOXIC T CELL RESPONSES IN BATS AND WILL FILL IMPORTANT KNOWLEDGE GAPS ABOUT BAT ADAPTIVE IMMUNE RESPONSES TO VIRUSES.