Apical-Out Human Airway Organoids Modeling SARS-CoV-2 Infection.

The respiratory epithelium, particularly the airway epithelium, is the primary infection site for respiratory pathogens. The apical surface of epithelial cells is constantly exposed to external stimuli including invading pathogens. Efforts have been made to establish organoid cultures to recapitulate the human respiratory tract. However, a robust and simple model with an easily accessible apical surface would benefit respiratory research. Here, we report the generation and characterization of apical-out airway organoids from the long-term expandable lung organoids that we previously established. The apical-out airway organoids morphologically and functionally recapitulated the human airway epithelium at a comparable level to the apical-in airway organoids. Moreover, apical-out airway organoids sustained productive and multicycle replication of SARS-CoV-2, and accurately recapitulated the higher infectivity and replicative fitness of the Omicron variants BA.5 and B.1.1.529 and an ancestral virus. In conclusion, we established a physiologically relevant and convenient apical-out airway organoid model for studying respiratory biology and diseases.

SARS-CoV-2 infection of airway organoids reveals conserved use of Tetraspanin-8 by Ancestral, Delta, and Omicron variants.

Ancestral SARS coronavirus-2 (SARS-CoV-2) and variants of concern (VOC) caused a global pandemic with a spectrum of disease severity. The mechanistic explaining variations related to airway epithelium are relatively understudied. Here, we biobanked airway organoids (AO) by preserving stem cell function. We optimized viral infection with H1N1/PR8 and comprehensively characterized epithelial responses to SARS-CoV-2 infection in phenotypically stable AO from 20 different subjects. We discovered Tetraspanin-8 (TSPAN8) as a facilitator of SARS-CoV-2 infection. TSPAN8 facilitates SARS-CoV-2 infection rates independently of ACE2-Spike interaction. In head-to-head comparisons with Ancestral SARS-CoV-2, Delta and Omicron VOC displayed lower overall infection rates of AO but triggered changes in epithelial response. All variants shared highest tropism for ciliated and goblet cells. TSPAN8-blocking antibodies diminish SARS-CoV-2 infection and may spur novel avenues for COVID-19 therapy.

Evaluation of Broad Anti-Coronavirus Activity of Autophagy-Related Compounds Using Human Airway Organoids.

To deal with the broad spectrum of coronaviruses, including severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), that threaten human health, it is essential to not only drugs develop that target viral proteins but also consider drugs that target host proteins/cellular processes to protect them from being hijacked for viral infection and replication. To this end, it has been reported that autophagy is deeply involved in coronavirus infection. In this study, we used airway organoids to screen a chemical library of autophagic modulators to identify compounds that could potentially be used to fight against infections by a broad range of coronaviruses. Among the 80 autophagy-related compounds tested, cycloheximide and thapsigargin reduced SARS-CoV-2 infection efficiency in a dose-dependent manner. Cycloheximide treatment reduced the infection efficiency of not only six SARS-CoV-2 variants but also human coronavirus (HCoV)-229E and HCoV-OC43. Cycloheximide treatment also reversed viral infection-induced innate immune responses. However, even low-dose (1 µM) cycloheximide treatment altered the expression profile of ribosomal RNAs; thus, side effects such as inhibition of protein synthesis in host cells must be considered. These results suggest that cycloheximide has broad-spectrum anti-coronavirus activity in vitro and warrants further investigation.

The Human Nose Organoid Respiratory Virus Model: an Ex Vivo Human Challenge Model To Study Respiratory Syncytial Virus (RSV) and Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) Pathogenesis and Evaluate Therapeutics.

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.

Avoiding culture shock with the SARS-CoV-2 spike protein.

When culturing SARS-CoV-2 in the laboratory it is vital to avoid deletions in the gene for the spike protein that could affect the interpretation of experiments.

Generation of self-replicating airway organoids from the cave nectar bat Eonycteris spelaea as a model system for studying host-pathogen interactions in the bat airway epithelium.

Bats are reservoir hosts for various zoonotic viruses with pandemic potential in humans and livestock. In vitro systems for studying bat host-pathogen interactions are of significant interest. Here, we establish protocols to generate bat airway organoids (AOs) and airway epithelial cells differentiated at the air-liquid interface (ALI-AECs) from tracheal tissues of the cave-nectar bat Eonycteris spelaea. In particular, we describe steps which enable laboratories that do not have access to live bats to perform extended experimental work upon procuring an initial batch of bat primary airway tissue. Complete mucociliary differentiation required treatment with IL-13. E. spelaea ALI-AECs supported productive infection with PRV3M, an orthoreovirus for which Pteropodid bats are considered the reservoir species. However, these ALI-AECs did not support SARS-CoV-2 infection, despite E. spelaea ACE2 receptor being capable of mediating SARS-CoV-2 spike pseudovirus entry. This work provides critical model systems for assessing bat species specific virus susceptibility and the reservoir likelihood for emerging infectious agents.

Recapitulating infection, thermal sensitivity and antiviral treatment of seasonal coronaviruses in human airway organoids.

BACKGROUND: Human seasonal coronaviruses usually cause mild upper-respiratory tract infection, but severe complications can occur in specific populations. Research into seasonal coronaviruses is limited and robust experimental models are largely lacking. This study aims to establish human airway organoids (hAOs)-based systems for seasonal coronavirus infection and to demonstrate their applications in studying virus-host interactions and therapeutic development. METHODS: The infections of seasonal coronaviruses 229E, OC43 and NL63 in 3D cultured hAOs with undifferentiated or differentiated phenotypes were tested. The kinetics of virus replication and production was profiled at 33 °C and 37 °C. Genome-wide transcriptome analysis by RNA sequencing was performed in hAOs under various conditions. The antiviral activity of molnupiravir and remdesivir, two approved medications for treating COVID19, was tested. FINDINGS: HAOs efficiently support the replication and infectious virus production of seasonal coronaviruses 229E, OC43 and NL63. Interestingly, seasonal coronaviruses replicate much more efficiently at 33 °C compared to 37 °C, resulting in over 10-fold higher levels of viral replication. Genome-wide transcriptomic analyses revealed distinct patterns of infection-triggered host responses at 33 °C compared to 37 °C temperature. Treatment of molnupiravir and remdesivir dose-dependently inhibited the replication of 229E, OC43 and NL63 in hAOs. INTERPRETATION: HAOs are capable of modeling 229E, OC43 and NL63 infections. The intriguing finding that lower temperature resembling that in the upper respiratory tract favors viral replication may help to better understand the pathogenesis and transmissibility of seasonal coronaviruses. HAOs-based innovative models shall facilitate the research and therapeutic development against seasonal coronavirus infections. FUNDING: This research is supported by funding of a VIDI grant (No. 91719300) from the Netherlands Organization for Scientific Research and the Dutch Cancer Society Young Investigator Grant (10140) to Q.P., and the ZonMw COVID project (114025011) from the Netherlands Organization for Health Research and Development to R.R.

Human Nasal Organoids Model SARS-CoV-2 Upper Respiratory Infection and Recapitulate the Differential Infectivity of Emerging Variants.

The human upper respiratory tract, specifically the nasopharyngeal epithelium, is the entry portal and primary infection site of respiratory viruses. Productive infection of SARS-CoV-2 in the nasal epithelium constitutes the cellular basis of viral pathogenesis and transmissibility. Yet a robust and well-characterized in vitro model of the nasal epithelium remained elusive. Here we report an organoid culture system of the nasal epithelium. We derived nasal organoids from easily accessible nasal epithelial cells with a perfect establishment rate. The derived nasal organoids were consecutively passaged for over 6 months. We then established differentiation protocols to generate 3-dimensional differentiated nasal organoids and organoid monolayers of 2-dimensional format that faithfully simulate the nasal epithelium. Moreover, when differentiated under a slightly acidic pH, the nasal organoid monolayers represented the optimal correlate of the native nasal epithelium for modeling the high infectivity of SARS-CoV-2, superior to all existing organoid models. Notably, the differentiated nasal organoid monolayers accurately recapitulated higher infectivity and replicative fitness of the Omicron variant than the prior variants. SARS-CoV-2, especially the more transmissible Delta and Omicron variants, destroyed ciliated cells and disassembled tight junctions, thereby facilitating virus spread and transmission. In conclusion, we establish a robust organoid culture system of the human nasal epithelium for modeling upper respiratory infections and provide a physiologically-relevant model for assessing the infectivity of SARS-CoV-2 emerging variants. IMPORTANCE An in vitro model of the nasal epithelium is imperative for understanding cell biology and virus-host interaction in the human upper respiratory tract. Here we report an organoid culture system of the nasal epithelium. Nasal organoids were derived from readily accessible nasal epithelial cells with perfect efficiency and stably expanded for more than 6 months. The long-term expandable nasal organoids were induced maturation into differentiated nasal organoids that morphologically and functionally simulate the nasal epithelium. The differentiated nasal organoids adequately recapitulated the higher infectivity and replicative fitness of SARS-CoV-2 emerging variants than the ancestral strain and revealed viral pathogenesis such as ciliary damage and tight junction disruption. Overall, we established a human nasal organoid culture system that enables a highly efficient reconstruction and stable expansion of the human nasal epithelium in culture plates, thus providing a facile and robust tool in the toolbox of microbiologists.

Long-Term Expanding Porcine Airway Organoids Provide Insights into the Pathogenesis and Innate Immunity of Porcine Respiratory Coronavirus Infection.

Respiratory coronaviruses cause serious health threats to humans and animals. Porcine respiratory coronavirus (PRCoV), a natural transmissible gastroenteritis virus (TGEV) mutant with partial spike deletion, causes mild respiratory disease and is an interesting animal respiratory coronavirus model for human respiratory coronaviruses. However, the absence of robust ex vivo models of porcine airway epithelium hinders an understanding of the pathogenesis of PRCoV infection. Here, we generated long-term porcine airway organoids (AOs) derived from basal epithelial cells, which recapitulate the in vivo airway complicated epithelial cellularity. Both 3D and 2D AOs are permissive for PRCoV infection. Unlike TGEV, which established successful infection in both AOs and intestinal organoids, PRCoV was strongly amplified only in AOs, not intestinal organoids. Furthermore, PRCoV infection in AOs mounted vigorous early type I and III interferon (IFN) responses and upregulated the expression of overzealous inflammatory genes, including pattern recognition receptors (PRRs) and proinflammatory cytokines. Collectively, these data demonstrate that stem-derived porcine AOs can serve as a promising disease model for PRCoV infection and provide a valuable tool to study porcine respiratory infection. IMPORTANCE Porcine respiratory CoV (PRCoV), a natural mutant of TGEV, shows striking pathogenetic similarities to human respiratory CoV infection and provides an interesting animal model for human respiratory CoVs, including SARS-CoV-2. The lack of an in vitro model recapitulating the complicated cellularity and structure of the porcine respiratory tract is a major roadblock for the study of PRCoV infection. Here, we developed long-term 3D airway organoids (AOs) and further established 2D AO monolayer cultures. The resultant 3D and 2D AOs are permissive for PRCoV infection. Notably, PRCoV mediated pronounced IFN and inflammatory responses in AOs, which recapitulated the inflammatory responses associated with PRCoV in vivo infection. Therefore, porcine AOs can be utilized to characterize the pathogenesis of PRCoV and, more broadly, can serve as a universal platform for porcine respiratory infection.

Generation and Effect Testing of a SARS-CoV-2 RBD-Targeted Polyclonal Therapeutic Antibody Based on a 2-D Airway Organoid Screening System.

Coronavirus disease 2019 (COVID-19) is an acute respiratory infectious disease caused by infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The US FDA has approved several therapeutics and vaccines worldwide through the emergency use authorization in response to the rapid spread of COVID-19. Nevertheless, the efficacies of these treatments are being challenged by viral escape mutations. There is an urgent need to develop effective treatments protecting against SARS-CoV-2 infection and to establish a stable effect-screening model to test potential drugs. Polyclonal antibodies (pAbs) have an intrinsic advantage in such developments because they can target rapidly mutating viral strains as a result of the complexity of their binding epitopes. In this study, we generated anti-receptor-binding domain (anti-RBD) pAbs from rabbit serum and tested their safety and efficacy in response to SARS-CoV-2 infection both in vivo and ex vivo. Primary human bronchial epithelial two-dimensional (2-D) organoids were cultured and differentiated to a mature morphology and subsequently employed for SARS-CoV-2 infection and drug screening. The pAbs protected the airway organoids from viral infection and tissue damage. Potential side effects were tested in mouse models for both inhalation and vein injection. The pAbs displayed effective viral neutralization effects without significant side effects. Thus, the use of animal immune serum-derived pAbs might be a potential therapy for protection against SARS-CoV-2 infection, with the strategy developed to produce these pAbs providing new insight into the treatment of respiratory tract infections, especially for infections with viruses undergoing rapid mutation.

Risk Assessment for Highly Pathogenic Avian Influenza A(H5N6/H5N8) Clade 2.3.4.4 Viruses.

The numerous global outbreaks and continuous reassortments of highly pathogenic avian influenza (HPAI) A(H5N6/H5N8) clade 2.3.4.4 viruses in birds pose a major risk to the public health. We investigated the tropism and innate host responses of 5 recent HPAI A(H5N6/H5N8) avian isolates of clades 2.3.4.4b, e, and h in human airway organoids and primary human alveolar epithelial cells. The HPAI A(H5N6/H5N8) avian isolates replicated productively but with lower competence than the influenza A(H1N1)pdm09, HPAI A(H5N1), and HPAI A(H5N6) isolates from humans in both or either models. They showed differential cellular tropism in human airway organoids; some infected all 4 major epithelial cell types: ciliated cells, club cells, goblet cells, and basal cells. Our results suggest zoonotic potential but low transmissibility of the HPAI A(H5N6/H5N8) avian isolates among humans. These viruses induced low levels of proinflammatory cytokines/chemokines, which are unlikely to contribute to the pathogenesis of severe disease.

Human airway cells prevent SARS-CoV-2 multibasic cleavage site cell culture adaptation.

Virus propagation methods generally use transformed cell lines to grow viruses from clinical specimens, which may force viruses to rapidly adapt to cell culture conditions, a process facilitated by high viral mutation rates. Upon propagation in VeroE6 cells, SARS-CoV-2 may mutate or delete the multibasic cleavage site (MBCS) in the spike protein. Previously, we showed that the MBCS facilitates serine protease-mediated entry into human airway cells (Mykytyn et al., 2021). Here, we report that propagating SARS-CoV-2 on the human airway cell line Calu-3 - that expresses serine proteases - prevents cell culture adaptations in the MBCS and directly adjacent to the MBCS (S686G). Similar results were obtained using a human airway organoid-based culture system for SARS-CoV-2 propagation. Thus, in-depth knowledge on the biology of a virus can be used to establish methods to prevent cell culture adaptation.

SARS-CoV-2 entry into human airway organoids is serine protease-mediated and facilitated by the multibasic cleavage site.

Coronavirus entry is mediated by the spike protein that binds the receptor and mediates fusion after cleavage by host proteases. The proteases that mediate entry differ between cell lines, and it is currently unclear which proteases are relevant in vivo. A remarkable feature of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike is the presence of a multibasic cleavage site (MBCS), which is absent in the SARS-CoV spike. Here, we report that the SARS-CoV-2 spike MBCS increases infectivity on human airway organoids (hAOs). Compared with SARS-CoV, SARS-CoV-2 entered faster into Calu-3 cells and, more frequently, formed syncytia in hAOs. Moreover, the MBCS increased entry speed and plasma membrane serine protease usage relative to cathepsin-mediated endosomal entry. Blocking serine proteases, but not cathepsins, effectively inhibited SARS-CoV-2 entry and replication in hAOs. Our findings demonstrate that SARS-CoV-2 enters relevant airway cells using serine proteases, and suggest that the MBCS is an adaptation to this viral entry strategy.

An organoid-derived bronchioalveolar model for SARS-CoV-2 infection of human alveolar type II-like cells.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes coronavirus disease 2019 (COVID-19), which may result in acute respiratory distress syndrome (ARDS), multiorgan failure, and death. The alveolar epithelium is a major target of the virus, but representative models to study virus host interactions in more detail are currently lacking. Here, we describe a human 2D air-liquid interface culture system which was characterized by confocal and electron microscopy and single-cell mRNA expression analysis. In this model, alveolar cells, but also basal cells and rare neuroendocrine cells, are grown from 3D self-renewing fetal lung bud tip organoids. These cultures were readily infected by SARS-CoV-2 with mainly surfactant protein C-positive alveolar type II-like cells being targeted. Consequently, significant viral titers were detected and mRNA expression analysis revealed induction of type I/III interferon response program. Treatment of these cultures with a low dose of interferon lambda 1 reduced viral replication. Hence, these cultures represent an experimental model for SARS-CoV-2 infection and can be applied for drug screens.