Stanford/UNC Biomimetic U19 Research Center PROJECT SUMMARY/ABSTRACT – CENTER OVERVIEW Infectious diseases continue to pervasively afflict global health and socioeconomic stability despite substantial prevention and treatment initiatives. Respiratory and gastrointestinal pathogens rank amongst the most intractable infectious diseases, particularly notable for recurrent waves of zoonotic coronaviruses and the recent COVID-19 pandemic, engendered by SARS-CoV-2. Overall, an urgent need exists for improved in vitro experimental models of human disease to study pathogenesis and to validate therapeutics. The central mission of the Stanford/UNC Biomimetic U19 Research Center is thus to deploy novel 3-dimensional organoid culture models to elucidate the biology and therapy of respiratory and gastrointestinal infectious pathogens. Our application is a renewal of our prior Stanford NAMSED U19 Research Center and is comprised of two Cores and three research Projects, leveraging complementary and synergistic expertise of our investigators at Stanford University and the University of North Carolina. The Center continues to be led by the Multi-PIs, Calvin Kuo and Manuel Amieva, who also co-lead Core A (Administrative Core). Core B (Organoid Core) is led by Calvin Kuo and provides novel capabilities for lung and GI organoid culture, gene editing and multiplexed screening. The three Projects extensively utilize organoid biomimetics for exploration of GI and respiratory pathogens. Project 1, (PI, Manuel Amieva) investigates H. pylori and Salmonella colonization, competition and invasion in the GI tract, while Project 2 (PI, Harry Greenberg) investigates rotavirus host range, neutralization, and M cell interactions in enteric biomimetics. Project 3 (PI, Ralph Baric) is a new addition and extensively uses organoids to model SARS-CoV-2, other closely related epidemic and pre-epidemic emerging coronaviruses and 1918 H1N1 influenza to reveal common and unique host networks associated with severe pulmonary outcomes. The activities of the Stanford/UNC Biomimetic U19 Research Center reside within three overarching Aims. In Aim 1, our Center performs organoid modeling of the epithelium-pathogen interface to investigate pathogenesis, susceptibility and host range restriction. This employs robust reverse genetics and CRISPR screens to systematically manipulate host versus viral/bacterial compartments, within novel apical-basal polarity modulated distal lung/alveolar, nasal sinus, stomach and intestinal organoid systems. Aim 2 defines how SARS- CoV-2, pre-epidemic coronaviruses, rotavirus and Salmonella can perturb reciprocal cross-talk between tissue epithelium and resident immune cells. This exploits a unique 3D air-liquid interface organoid method preserving GI and lung epithelium en bloc with diverse endogenous infiltrating immune cell types without artificial reconstitution. Lastly, Aim 3 performs organoid-based evaluation of therapeutic candidates against SARS-CoV- 2...