Human Organotypic Airway and Lung Organoid Cells of Bronchiolar and Alveolar Differentiation Are Permissive to Infection by Influenza and SARS-CoV-2 Respiratory Virus

Abstract

The ongoing coronavirus disease 2019 (COVID-19) pandemic has led to the initiation of unprecedented research efforts to understand the pathogenesis mediated by Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2). More knowledge is needed regarding the cell type-specific cytopathology and its impact on cellular tropism. Furthermore, the impact of novel SARS-CoV-2 mutations on cellular tropism, alternative routes of entry, the impact of co-infections, and virus replication kinetics along the respiratory tract remains to be explored in improved models. Most applied virology models are not well suited to address the remaining questions, as they do not recapitulate the histoarchitecture and cellular composition of human respiratory tissues. The overall aim of this work was to establish from single biopsy specimens, a human adult stem cell-derived organoid model representing the upper respiratory airways and lungs and explore the applicability of this model to study respiratory virus infection. First, we characterized the organoid model with respect to growth pattern and histoarchitecture, cellular composition, and functional characteristics. Next, in situ expression of viral entry receptors, including influenza virus-relevant sialic acids and SARS-CoV-2 entry receptor ACE2 and TMPRSS2, were confirmed in organoids of bronchiolar and alveolar differentiation. We further showed successful infection by pseudotype influenza A H7N1 and H5N1 virus, and the ability of the model to support viral replication of influenza A H7N1 virus. Finally, successful infection and replication of a clinical isolate of SARS-CoV-2 were confirmed in the organoids by TCID50 assay and immunostaining to detect intracellular SARS-CoV-2 specific nucleocapsid and dsRNA. The prominent syncytia formation in organoid tissues following SARS-CoV-2 infection mimics the findings from infected human tissues in situ. We conclude that the human organotypic model described here may be particularly useful for virology studies to evaluate regional differences in the host response to infection. The model contains the various cell types along the respiratory tract, expresses respiratory virus entry factors, and supports successful infection and replication of influenza virus and SARS-CoV-2. Thus, the model may serve as a relevant and reliable tool in virology and aid in pandemic preparedness, and efficient evaluation of antiviral strategies.

In vitro high-content tissue models to address precision medicine challenges

“…These models have been suggested as a prominent platform to gain a fundamental understanding of viral infections such as COVID-19 ( Basanthakumar, 2021 ; de Melo et al, 2021 ; Deguchi et al, 2021 ; Harb et al, 2022 ; Kronemberger et al, 2021 ; Lv et al, 2021 ; Ramani et al, 2021 ; Sia et al, 2022 ; Sourimant et al, 2022 ; van der Vaart et al, 2021 ; Wang et al; Yu, 2021 ; Zech et al, 2021 ) and could be a good alternative to facilitate the development and discovery of new therapeutics and vaccines ( Clevers, 2020 ; Raimondi et al, 2020 ). To date, various types of organoid-enabled 3D models have been employed to study and understand SARS-CoV-2 infection such as the brain organoid ( Ramani et al, 2020 ), lung organoid ( Ekanger et al, 2022 ; Salahudeen et al, 2020 ; van der Vaart et al, 2021 ; Wang et al, 2021a ), intestine organoid ( Dickson, 2020 ), as well as liver ductal organoid and bile duct organoid ( Zhao et al, 2020a ) ( Fig. 3 ).…”

“…Scale bars are 100 μm. Reproduced with permission ( Ekanger et al, 2022 ). Copyright 2022, Frontiers Media S.A., Ekanger et al, Licensed under A Creative Commons Attribution License (CC BY).…”

“…Ekanger and coauthors established human adult stem cell-derived organoids as a model for the upper respiratory airways and lungs, and demonstrated that the model was permissive to infection by influenza and SARS-CoV-2 viruses ( Fig. 3 c) ( Ekanger et al, 2022 ).…”

NF-κB inhibitor alpha has a cross-variant role during SARS-CoV-2 infection in ACE2-overexpressing human airway organoids

“…These undifferentiated HAOs are tractable and easy to work with, while not being cancerously transformed and maintaining cellular diversity that resembles the airway epithelium. The undifferentiated organoid model has been used to study respiratory syncytial virus 17 and SARS-CoV-2 6 , 18 , 19 . However, work with the latter is plagued with variable, donor-dependent infection rates where most organoids carry less than 5% infected cells 18 , 19 , a major obstacle for further analysis (i.e., single-cell genomics).…”

“…The undifferentiated organoid model has been used to study respiratory syncytial virus 17 and SARS-CoV-2 6 , 18 , 19 . However, work with the latter is plagued with variable, donor-dependent infection rates where most organoids carry less than 5% infected cells 18 , 19 , a major obstacle for further analysis (i.e., single-cell genomics).…”