PROJECT SUMMARY Influenza and SARS-CoV-2 are respiratory viruses that represent a continual threat to the health of many Americans, and will likely account for 100,000 deaths and many times that number of hospitalizations in the US alone each year. The available vaccines are inadequate, with the recent flu vaccine estimated to be only 40% effective and while the RNA vaccines for SARS-CoV-2 have been remarkable at preventing severe illness or death, they do not prevent reinfection in many people. Many with pre-existing conditions or immune deficiencies (e.g., obesity, diabetes) are vulnerable, which leads to suppressed response to vaccine or increased risk of severe outcome upon infection. Thus, the need to understand and improve both vaccines and the response to them is urgent. The Stanford CCHI has been a leader in understanding influenza vaccination and infection, and very quickly developed complementary expertise during the pandemic, making great strides in both understanding SARS-CoV- 2 infection and vaccine responses, and also in developing new technologies that promise even greater advances. These range from Dr. Wang’s insights in the effects of antibody glycosylation and lung inflammation, to Dr. Barnes seminal work on the structures of antibodies to SARS-CoV-2 antigens. Work that Dr. Barnes will carry further by designing and testing novel flu and SARS-Cov-2 antigen constructs. Dr. Khatri has also developed bioinformatic methods that have revealed conserved gene signatures regarding the immune response to viruses, and which he will use to define the cells and mechanisms that underlie these signatures. The clinical core under Dr. Chinthrajah will recruit and vaccinate obese and diabetic patients at risk for severe COVID and Influenza illness, and the results will be analyzed by Dr. Wang and also by the Human Immune Monitoring core under Dr. Maecker. Dr. Davis will head the Technology Development project which will continue his development of immune organoids and the use of spleen organoids particularly to characterize both existing and novel vaccine responses, and also attempt to reconstruct immune responses to vaccination and infection using skin and lung organoids from the same donors. Organoid responses to vaccination or infection will be validated against data in human subjects using our collection of influenza and SARS-CoV-2 specific T and B cell probes, as well as through TCR and BCR sequence analysis. Organoid processing and banking will be handled by the clinical core and all projects will make use of this resource, which will be especially useful in testing particular immunogens for their ability to make the desired antibodies, or the use of gene editing to identify key loci mediating particular effects. Immune organoids and the networks we propose to create will give us the ability to test hypotheses and define mechanisms in an entirely human system. The overall theme of this submission is to continue our efforts to understand In...