Organoid-based investigations of human respiratory viruses and virus-host interaction : rhinovirus C and SARS-CoV-2

Abstract

A comprehensive understanding of respiratory viruses and their interplay with host cells requires a robust and physiologically relevant in vitro system. We have established the human respiratory organoids from adult stem cells (ASCs) in the primary lung tissues, or epithelial cells noninvasively procured from the nasal cavity of individual donors. The derived organoids, including three-dimensional (3D) nasal and lung organoids, can be long-term expanded for over half a year. We induced differentiation and generated mature nasal, airway, and alveolar organoids that faithfully mimic the genetic and functional characteristics of the native respiratory epithelia, including cellular composition, epithelial function, and the susceptibility/host response to respiratory viruses. In this study, we have leveraged respiratory organoids to propagate RV-C and investigated virus biology and virus-host interaction of RV-C and SARS-CoV-2. Standard cell lines are typically refractory to RV-C infection, which substantially hinders our understanding of this common respiratory pathogen. We developed an organoid-based system to reproducibly propagate RV-C and characterize virus-host interactions in airway and nasal organoids. We demonstrated that human airway organoids sustained serial RV-C passage with CYT387-mediated immunosuppression, while nasal organoids, which closely simulate the upper airway epithelium, achieved this without immunosuppression. Nasal organoids were more susceptible to RV-C than airway organoids. Upon RV-C infection, we observed a more robust innate immune response in airway organoids than in nasal organoids, which was reproduced in a Poly(I:C) stimulation assay. Treatment with anti-CDHR3 antibodies and antivirals significantly reduced RV-C replication in organoids. Additionally, we established an organoid-based immunofluorescence assay (IFA) to titrate RV-C infectious particles. The high transmissibility of SARS-CoV-2 Omicron variants was generally ascribed to immune escape. We hypothesized that the Omicron variants may have acquired increased replicative fitness in human respiratory epithelial cells; immune escape may not be the sole driver of the escalating transmissibility. We evaluated the replicative fitness of BA.5 and earlier variants in physiologically active respiratory organoids. BA.5 exhibited dramatically increased replicative capacity and infectivity than B.1.1.529 and the ancestral wildtype (WT) strain in human nasal and airway organoids. The BA.5 spike pseudovirus showed significantly higher entry efficiency than those carrying WT or B.1.1.529 spikes. We observed prominent syncytium formation in nasal and airway organoids infected by BA.5, whereas it was absent in WT- and B.1.1.529-infected organoids. BA.5 spike-triggered syncytium formation was verified by lentiviral overexpression of the spike protein in nasal organoids. Furthermore, BA.5 replicated modestly in alveolar organoids, with significantly lower titers than B.1.1.529 and WT, which might account for its benign clinical manifestation. Collectively, the human respiratory organoids serve as a transformative tool for studying respiratory viruses in a physiologically relevant context. We demonstrated the unique strength of respiratory organoids in successfully propagating RV-C and revealing critical insights into virus-host interactions. Additionally, we uncovered the enhanced replicative fitness of BA.5 and its capability to induce syncytium formation, which constituted the biological mechanisms underlying its elevated transmissibility. The physiologically relevant respiratory organoids have shown immense potential as robust and novel tools to drive progress in virological research and therapeutic innovation.