ABSTRACT
There is an unmet need for preclinical models to understand the pathogenesis of human respiratory viruses and predict responsiveness to immunotherapies. Airway organoids can serve as an ex vivo human airway model to study respiratory viral pathogenesis; however, they rely on invasive techniques to obtain patient samples. Here, we report a noninvasive technique to generate human nose organoids (HNOs) as an alternative to biopsy-derived organoids. We made air-liquid interface (ALI) cultures from HNOs and assessed infection with two major human respiratory viruses, respiratory syncytial virus (RSV) and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Infected HNO-ALI cultures recapitulate aspects of RSV and SARS-CoV-2 infection, including viral shedding, ciliary damage, innate immune responses, and mucus hypersecretion. Next, we evaluated the feasibility of the HNO-ALI respiratory virus model system to test the efficacy of palivizumab to prevent RSV infection. Palivizumab was administered in the basolateral compartment (circulation), while viral infection occurred in the apical ciliated cells (airways), simulating the events in infants. In our model, palivizumab effectively prevented RSV infection in a concentration-dependent manner. Thus, the HNO-ALI model can serve as an alternative to lung organoids to study respiratory viruses and test therapeutics. IMPORTANCE Preclinical models that recapitulate aspects of human airway disease are essential for the advancement of novel therapeutics and vaccines. Here, we report a versatile airway organoid model, the human nose organoid (HNO), that recapitulates the complex interactions between the host and virus. HNOs are obtained using noninvasive procedures and show divergent responses to SARS-CoV-2 and RSV infection. SARS-CoV-2 induces severe damage to cilia and the epithelium, no interferon-λ response, and minimal mucus secretion. In striking contrast, RSV induces hypersecretion of mucus and a profound interferon-λ response with ciliary damage. We also demonstrated the usefulness of our ex vivo HNO model of RSV infection to test the efficacy of palivizumab, an FDA-approved monoclonal antibody to prevent severe RSV disease in high-risk infants. Our study reports a breakthrough in both the development of a novel nose organoid model and in our understanding of the host cellular response to RSV and SARS-CoV-2 infection.
ABSTRACT
Background: In-vitro models of differentiated primary human airway epithelial cells are a valuable tool to study severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. Through the use of these models, it has been shown that the expression of SARS-CoV-2 entry genes in human airway epithelia is influenced by various factors such as age, sex, smoking status, and pathogenic conditions. In this study, we aimed to determine the effects of cell culture conditions and donor demographic and clinical characteristics on the expression of SARS-CoV-2 entry genes including angiotensin converting enzyme 2 (ACE2), transmembrane serine protease 2 (TMPRSS2), cathepsin L (CTSL), and tyrosine protein kinase receptor UFO (AXL) in primary airway epithelial cells. Methods: Eleven lung cancer patients with or without chronic obstructive pulmonary disease (COPD) or asthma were recruited. Human bronchial epithelial cells (HBEC) or small airway epithelial cells (SAEC) isolated from submerged or air-liquid interface (ALI) cultures were analyzed by quantitative real-time PCR. We also tested for correlations with clinical data. Results: In ALI cultures, the expression of AXL was significantly higher in HBEC than in SAEC. In addition, the expression of ACE2, TMPRSS2, and CTSL was significantly increased in both HBEC and SAEC differentiated under ALI conditions compared with the submerged culture. Negligible association was found between the expression of SARS-CoV-2 entry genes in SAEC and the age, sex, smoking status, and complication of COPD, asthma or hypertension of the cell donors. Conclusion: These results demonstrate that the expression of SARS-CoV-2 entry genes in differentiated primary airway epithelial cells in-vitro is much more influenced by individual culture conditions than by specific characteristics of individual donors.
ABSTRACT
Air-liquid interface (ALI) culture of nasal epithelial cells is a valuable tool in the diagnosis and research of primary ciliary dyskinesia (PCD). Ex vivo samples often display secondary dyskinesia from cell damage during sampling, infection or inflammation confounding PCD diagnostic results. ALI culture enables regeneration of healthy cilia facilitating differentiation of primary from secondary ciliary dyskinesia. We describe a revised ALI culture method adopted from April 2018 across three collaborating PCD diagnostic sites, including current University Hospital Southampton COVID-19 risk mitigation measures, and present results. Two hundred and forty nasal epithelial cell samples were seeded for ALI culture and 199 (82.9%) were ciliated. Fifty-four of 83 (63.9%) ex vivo samples which were originally equivocal or insufficient provided diagnostic information following in vitro culture. Surplus basal epithelial cells from 181 nasal brushing samples were frozen in liquid nitrogen; 39 samples were ALI-cultured after cryostorage and all ciliated. The ciliary beat patterns of ex vivo samples (by high-speed video microscopy) were recapitulated, scanning electron microscopy demonstrated excellent ciliation, and cilia could be immuno-fluorescently labelled (anti-alpha-tubulin and anti-RSPH4a) in representative cases that were ALI-cultured after cryostorage. In summary, our ALI culture protocol provides high ciliation rates across three centres, minimising patient recall for repeat brushing biopsies and improving diagnostic certainty. Cryostorage of surplus diagnostic samples was successful, facilitating PCD research.