Integration of mononuclear phagocytes into the human gastrointestinal GOFlowChip for investigation of luminal antigen sampling

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Summary The emerging severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a highly infectious human respiratory tract pathogen that has the capacity to infect many different organ systems, including the gastrointestinal (GI) tract. The overall goal of this project is to elucidate the role of the GI mucosa in SARS- CoV-2 infection and transmission. Specifically, we seek to identify mucosal immune mechanisms involved in COVID-19 pathogenesis and compare epithelial responses to SARS-CoV-2 in the gut of humans and bats. Investigating bat cells is important, because bats are considered the original hosts for SARS-CoV-2, but do not develop overt disease upon infection. Our recently developed GOFlowChip platform, which integrates 3- dimensional GI organoids and immune cells on a millifluidic chip with luminal and basolateral flow capacity, is ideally suited to probe the mechanisms involved in SARS-CoV-2 infection of the gut mucosa. Here, we propose to utilize the GOFlowChip to elucidate mechanisms of viral infection, replication and spread involved in SARS-CoV-2 infection of the GI tract. Specifically, we seek to (1) Define how SARS-CoV-2 infection of the GI epithelium contributes to viral spread. (2) Determine to what extent GI mucosal immune mechanisms regulate SARS-CoV-2 infection and spreading in the GI tract. (3) Compare GI epithelial responses to SARS- CoV-2 between bats and humans. For Specific Aim 1, we will optimize the GOFlowChip for use under BSL-3 laboratory conditions, followed by SARS-CoV-2 infection experiments that will elucidate susceptibility and viral replication dynamics in different gut compartments. Specific Aim 2 will leverage our organoid-mononuclear phagocyte (MNP) co-culture system to elucidate whether MNP-dependent transport mechanisms impact viral invasion of the GI mucosa, and we will screen SARS-CoV-2 reactive patient sera for their ability to modulate gut infection. For Specific Aim 3, we will establish organoid lines from bat GI tissues in order to compare epithelial responses to SARS-CoV-2 in bats and humans using RNA sequencing in order to understand differences in viral pathogenicity between humans and bats. The proposed work is directly integrated with our existing project, because we will use out GOFlowChip to investigate how SARS-CoV-2 interacts with epithelial cells and immune system components to cross the GI barrier. Our research is technologically innovative, because we are using gut organoid-immune cell co-cultures in a fully contained tissue chip design to study a BSL-3 level pathogen. Our project is conceptually innovative, because it is the first to compare gut organoids from Jamaican fruit bats to human organoids for their ability to sustain SARS-CoV-2 infection and to mount cellular antiviral responses. The proposed research is significant because it will yield crucial information on the role of the GI mucosa in COVID-19 pathogenesis that may inform future strategies to identify viral carriers, prevent viral transmission, and design novel antiviral treatments and vaccines.