Intestinal organoid modeling of SARS-CoV-2-stimulated innate and adaptive immunity

ABSTRACT The COVID-19 pandemic, engendered by the novel coronavirus SARS-CoV-2, is a grave threat to public health, with lung infection and respiratory failure. However, the intestine is also targeted by SARS-CoV-2, as many patients present with GI symptoms and the SARS-CoV-2 receptor angiotensin-converting enzyme 2 (ACE2) is abundantly expressed by intestinal epithelium. COVID-19 mortality is strongly associated with systemic inflammation as in "cytokine storm", fostering attempts at therapeutic immunomodulation, and raising a critical need for in vitro human systems modeling SARS-CoV-2-induced immune cell interactions with intestinal epithelium. Conventional 3D organoid cultures ("enteroids") allow intestinal epithelial SARS-CoV-2 infection but unfortunately omit immune cells. Here we generate a holistic intestinal in vitro model of SARS-CoV-2 infection using air-liquid interface (ALI) organoids containing both epithelium and infiltrating immune cells en bloc without artificial reconstitution. The complex intestinal immune system of ALI intestinal organoids contains various innate and adaptive immune cells, highly diverse T cell receptor (TCR) and B cell receptor (BCR) repertoire, and plasma B cell-derived antibody transcripts. Importantly, immune components in these ALI organoids respond efficiently to epithelial damage and ALI intestinal organoids are highly susceptible to bacterial and viral infections. Here, we utilize this unique ALI organoid technology with integrated immune components to explore sequelae of SARS-CoV-2 infection. Aim 1 establishes and optimizes BSL3 SARS-CoV-2 infection of ALI intestinal organoids, exploiting a novel eversion method to relocate the apical aspect of ACE2-expressing cells to the external surface, allowing survey of SARS-CoV-2 infection of different regions of small intestine and colon. The time course of tissue-resident SARS-CoV-2-induced cross-talk between intestinal epithelium and immune cells is unknown, as hindered by lack of human experimental systems. Thus, Aim 2 performs a scRNA- seq and CyTOF study of SARS-CoV-2-induced immune responses within ALI organoids to (1) create a network model of the temporal propagation of immunity and bidirectional communication between epithelium and immune cells and (2) perform therapeutic testing against nodal vulnerabilities, correlating against clinical status (naïve, convalescent, immunized, cross-reactive coronavirus). Current epithelial organoid systems also do not allow study of adaptive immunity. Aim 3 thus recapitulates SARS-CoV-2 adaptive immune responses by co-culturing ALI intestinal organoids with newly developed human lymph node organoids. Within such SARS-CoV-2-infected co-cultures we correlate adaptive immune responses with clinical status including prior cross-reactive coronavirus infection and SARS-CoV-2 naïve, convalescent and ultimately immunized states. Overall, we leverage collaboration from Mark Davis (human LN culture) and Catherine Blish and Scott Boyd (SARS-CoV-2) to create human in vitro systems modeling SARS-CoV-2-induced innate and adaptive immunity, with relevance for pathogenesis investigations and therapeutics testing.