Induced pluripotent stem cell-based disease modeling and prospective immune therapy for coronavirus disease 2019

The emergence of the novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic poses a never before seen challenge to human health and the economy. Considering its clinical impact, with no streamlined therapeutic strategies in sight, it is crucial to understand the infection process of SARS-CoV-2. Our limited knowledge of the mechanisms underlying SARS-CoV-2 infection impedes the development of alternative therapeutics to address the pandemic. This aspect can be addressed by modeling SARS-CoV-2 infection in the human context to facilitate drug screening and discovery. Human induced pluripotent stem cell (iPSC)-derived lung epithelial cells and organoids recapitulating the features and functionality of the alveolar cell types can serve as an in vitro human model and screening platform for SARS-CoV-2. Recent studies suggest an immune system asynchrony leading to compromised function and a decreased proportion of specific immune cell types in coronavirus disease 2019 (COVID-19) patients. Replenishing these specific immune cells may serve as useful treatment modality against SARS-CoV-2 infection. Here the authors review protocols for deriving lung epithelial cells, alveolar organoids and specific immune cell types, such as T lymphocytes and natural killer cells, from iPSCs with the aim to aid investigators in making relevant in vitro models of SARS-CoV-2 along with the possibility derive immune cell types to treat COVID-19.     

Human Coronavirus HKU1 Infection of Primary Human Type II Alveolar Epithelial Cells: Cytopathic Effects and Innate Immune Response

Because they are the natural target for respiratory pathogens, primary human respiratory epithelial cells provide the ideal in vitro system for isolation and study of human respiratory viruses, which display a high degree of cell, tissue, and host specificity. Human coronavirus HKU1, first discovered in 2005, has a worldwide prevalence and is associated with both upper and lower respiratory tract disease in both children and adults. Research on HCoV-HKU1 has been difficult because of its inability to be cultured on continuous cell lines and only recently it was isolated from clinical specimens using primary human, ciliated airway epithelial cells. Here we demonstrate that HCoV-HKU1 can infect and be serially propagated in primary human alveolar type II cells at the air-liquid interface. We were not able to infect alveolar type I-like cells or alveolar macrophages. Type II alveolar cells infected with HCoV-HKU1 demonstrated formation of large syncytium. At 72 hours post inoculation, HCoV-HKU1 infection of type II cells induced increased levels of mRNAs encoding IL29,CXCL10, CCL5, and IL-6 with no significant increases in the levels of IFNβ. These studies demonstrate that type II cells are a target cell for HCoV-HKU1 infection in the lower respiratory tract, that type II alveolar cells are immune-competent in response to infection exhibiting a type III interferon and proinflammatory chemokine response, and that cell to cell spread may be a major factor for spread of infection. Furthermore, these studies demonstrate that human alveolar cells can be used to isolate and study novel human respiratory viruses that cause lower respiratory tract disease.     

Experimental Models for SARS-CoV-2 Infection

Severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-2) is a novel virus that causes coronavirus disease 2019 (COVID-19). To understand the identity, functional characteristics and therapeutic targets of the virus and the diseases, appropriate infection models that recapitulate the in vivo pathophysiology of the viral infection are necessary. This article reviews the various infection models, including Vero cells, human cell lines, organoids, and animal models, and discusses their advantages and disadvantages. This knowledge will be helpful for establishing an efficient system for defense against emerging infectious diseases.     

Single-cell RNA analysis on ACE2 expression provides insights into SARS-CoV-2 potential entry into the bloodstream and heart injury

Coronavirus disease-2019 (COVID-19) is a global pandemic with high infectivity and pathogenicity, accounting for tens of thousands of deaths worldwide. Recent studies have found that the pathogen of COVID-19, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), shares the same cell receptor angiotensin converting enzyme II (ACE2) as SARS-CoV. The pathological investigation of COVID-19 deaths showed that the lungs had characteristics of pulmonary fibrosis. However, how SARS-CoV-2 spreads from the lungs to other organs has not yet been determined. Here, we performed an unbiased evaluation of cell-type-specific expression of ACE2 in healthy and fibrotic lungs, as well as in normal and failed adult human hearts, using published single-cell RNA-seq data. We found that ACE2 expression in fibrotic lungs mainly locates in arterial vascular cells, which might provide a route for bloodstream spreading of SARS-CoV-2. Failed human hearts have a higher percentage of ACE2-expressing cardiomyocytes, and SARS-CoV-2 might attack cardiomyocytes through the bloodstream in patients with heart failure. Moreover, ACE2 was highly expressed in cells infected by respiratory syncytial virus or Middle East respiratory syndrome coronavirus and in mice treated by lipopolysaccharide. Our findings indicate that patients with pulmonary fibrosis, heart failure, and virus infection have a higher risk and are more susceptible to SARS-CoV-2 infection. The SARS-CoV-2 might attack other organs by getting into the bloodstream. This study provides new insights into SARS-CoV-2 blood entry and heart injury and might propose a therapeutic strategy to prevent patients from developing severe complications.     

Severe acute respiratory syndrome coronavirus infection of human ciliated airway epithelia: role of ciliated cells in viral spread in the conducting airways of the lungs

Severe acute respiratory syndrome coronavirus (SARS-CoV) emerged in 2002 as an important cause of severe lower respiratory tract infection in humans, and in vitro models of the lung are needed to elucidate cellular targets and the consequences of viral infection. The SARS-CoV receptor, human angiotensin 1-converting enzyme 2 (hACE2), was detected in ciliated airway epithelial cells of human airway tissues derived from nasal or tracheobronchial regions, suggesting that SARS-CoV may infect the proximal airways. To assess infectivity in an in vitro model of human ciliated airway epithelia (HAE) derived from nasal and tracheobronchial airway regions, we generated recombinant SARS-CoV by deletion of open reading frame 7a/7b (ORF7a/7b) and insertion of the green fluorescent protein (GFP), resulting in SARS-CoV GFP. SARS-CoV GFP replicated to titers similar to those of wild-type viruses in cell lines. SARS-CoV specifically infected HAE via the apical surface and replicated to titers of 10(7) PFU/ml by 48 h postinfection. Polyclonal antisera directed against hACE2 blocked virus infection and replication, suggesting that hACE2 is the primary receptor for SARS-CoV infection of HAE. SARS-CoV structural proteins and virions localized to ciliated epithelial cells. Infection was highly cytolytic, as infected ciliated cells were necrotic and shed over time onto the luminal surface of the epithelium. SARS-CoV GFP also replicated to a lesser extent in ciliated cell cultures derived from hamster or rhesus monkey airways. Efficient SARS-CoV infection of ciliated cells in HAE provides a useful in vitro model of human lung origin to study characteristics of SARS-CoV replication and pathogenesis.     

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.     

Perspectives on mesenchymal stem/progenitor cells and their derivates as potential therapies for lung damage caused by COVID-19

The new coronavirus, severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), which emerged in December 2019 in Wuhan, China, has reached worldwide pandemic proportions, causing coronavirus disease 2019 (COVID-19). The clinical manifestations of COVID-19 vary from an asymptomatic disease course to clinical symptoms of acute respiratory distress syndrome and severe pneumonia. The lungs are the primary organ affected by SARS-CoV-2, with a very slow turnover for renewal. SARS-CoV-2 enters the lungs via angiotensin-converting enzyme 2 receptors and induces an immune response with the accumulation of immunocompetent cells, causing a cytokine storm, which leads to target organ injury and subsequent dysfunction. To date, there is no effective antiviral therapy for COVID-19 patients, and therapeutic strategies are based on experience treating previously recognized coronaviruses. In search of new treatment modalities of COVID-19, cell-based therapy with mesenchymal stem cells (MSCs) and/or their secretome, such as soluble bioactive factors and extracellular vesicles, is considered supportive therapy for critically ill patients. Multipotent MSCs are able to differentiate into different types of cells of mesenchymal origin, including alveolar epithelial cells, lung epithelial cells, and vascular endothelial cells, which are severely damaged in the course of COVID-19 disease. Moreover, MSCs secrete a variety of bioactive factors that can be applied for respiratory tract regeneration in COVID-19 patients thanks to their trophic, anti-inflammatory, immunomodulatory, anti-apoptotic, pro-regenerative, and proangiogenic properties.     

Human Organoids as a Promising Platform for Fighting COVID-19

The coronavirus disease 2019 (COVID-19) global pandemic evoked by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has triggered a major public health problem with significant morbidity and mortality. Understanding the pathogenesis and molecular mechanisms underlying this novel virus is crucial for both fundamental research and clinical trials in order to devise effective therapies and vaccination regimens. Basic research on SARS-CoV-2 largely depends on ex vivo models that allow viral invasion and replication. Organoid models are now emerging as a valuable tool to investigate viral biology and disease progression, serving as an efficient platform to investigate potential therapies for COVID-19. Here, we summarize various human stem cell-derived organoid types employed in SARS-CoV-2 studies. We highlight key findings from these models, including cell tropisms and molecular mechanisms in viral infection. We also describe their use in identifying potential therapeutic agents against SARS-CoV-2. As more and more advanced organoids emerge, they will facilitate the understanding of disease pathogenesis for drug development in this dreaded pandemic.     

Roles of the gut microbiota in severe SARS-CoV-2 infection

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has spread worldwide. The pathophysiological mechanisms linking gut dysbiosis and severe SARS-CoV-2 infection are poorly understood, although gut microbiota disorders are related to severe SARS-CoV-2 infections. The roles of the gut microbiota in severe SARS-CoV-2 infection were compared with those in respiratory viral infection, which is an easily understood and enlightening analogy. Secondary bacterial infections caused by immune disorders and antibiotic abuse can lead to dysregulation of the gut microbiota in patients with respiratory viral infections. The gut microbiota can influence the progression of respiratory viral infections through metabolites and the immune response, which is known as the gut–lung axis. Angiotensin-converting enzyme 2 is expressed in both the lungs and the small intestine, which may be a bridge between the lung and the gut. Similarly, SARS-CoV-2 infection has been shown to disturb the gut microbiota, which may be the cause of cytokine storms. Bacteria in the gut, lung, and other tissues and respiratory viruses can be considered microecosystems and may exert overall effects on the host. By referencing respiratory viral infections, this review focused on the mechanisms involved in the interaction between SARS-CoV-2 infections and the gut microbiota and provides new strategies for the treatment or prevention of severe SARS-CoV-2 infections by improving gut microbial homeostasis.     

ACE2 receptor expression and severe acute respiratory syndrome coronavirus infection depend on differentiation of human airway epithelia

Studies of patients with severe acute respiratory syndrome (SARS) demonstrate that the respiratory tract is a major site of SARS-coronavirus (CoV) infection and disease morbidity. We studied host-pathogen interactions using native lung tissue and a model of well-differentiated cultures of primary human airway epithelia. Angiotensin converting enzyme 2 (ACE2), the receptor for both the SARS-CoV and the related human respiratory coronavirus NL63, was expressed in human airway epithelia as well as lung parenchyma. As assessed by immunofluorescence staining and membrane biotinylation, ACE2 protein was more abundantly expressed on the apical than the basolateral surface of polarized airway epithelia. Interestingly, ACE2 expression positively correlated with the differentiation state of epithelia. Undifferentiated cells expressing little ACE2 were poorly infected with SARS-CoV, while well-differentiated cells expressing more ACE2 were readily infected. Expression of ACE2 in poorly differentiated epithelia facilitated SARS spike (S) protein-pseudotyped virus entry. Consistent with the expression pattern of ACE2, the entry of SARS-CoV or a lentivirus pseudotyped with SARS-CoV S protein in differentiated epithelia was more efficient when applied to the apical surface. Furthermore, SARS-CoV replicated in polarized epithelia and preferentially exited via the apical surface. The results indicate that infection of human airway epithelia by SARS coronavirus correlates with the state of cell differentiation and ACE2 expression and localization. These findings have implications for understanding disease pathogenesis associated with SARS-CoV and NL63 infections.     

Drugs targeting various stages of the SARS-CoV-2 life cycle: Exploring promising drugs for the treatment of Covid-19

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a positive-sense, single-stranded RNA virus that causes the potentially lethal Covid-19 respiratory tract infection. It does so by binding to host cell angiotensin converting enzyme 2 (ACE2) receptors, leading to endocytosis with the receptor, and subsequently using the host cell’s machinery to replicate copies of itself and invade new cells. The extent of the spread of infection in the body is dependent on the pattern of ACE2 expression and overreaction of the immune system. Additionally, by inducing an imbalance in the renin-angiotensin-aldosterone system (RAAS) and the loss of ACE2 would favour the progression of inflammatory and thrombotic processes in the lungs. No drug or vaccine has yet been approved to treat human coronaviruses. Hundreds of clinical trials on existing approved drugs from different classes acting on a multitude of targets in the virus life cycle are ongoing to examine potential effectiveness for the prevention and treatment of the infection. This review summarizes the SARS-CoV-2 virus life cycle in the host cell and provides a biological and pathological point of view for repurposed and experimental drugs for this novel coronavirus. The viral life cycle provides potential targets for drug therapy.     

Modeling insights into SARS-CoV-2 respiratory tract infections prior to immune protection

Mechanistic insights into human respiratory tract (RT) infections from SARS-CoV-2 can inform public awareness as well as guide medical prevention and treatment for COVID-19 disease. Yet the complexity of the RT and the inability to access diverse regions pose fundamental roadblocks to evaluation of potential mechanisms for the onset and progression of infection (and transmission). We present a model that incorporates detailed RT anatomy and physiology, including airway geometry, physical dimensions, thicknesses of airway surface liquids (ASLs), and mucus layer transport by cilia. The model further incorporates SARS-CoV-2 diffusivity in ASLs and best-known data for epithelial cell infection probabilities, and, once infected, duration of eclipse and replication phases, and replication rate of infectious virions. We apply this baseline model in the absence of immune protection to explore immediate, short-term outcomes from novel SARS-CoV-2 depositions onto the air-ASL interface. For each RT location, we compute probability to clear versus infect; per infected cell, we compute dynamics of viral load and cell infection. Results reveal that nasal infections are highly likely within 1–2 days from minimal exposure, and alveolar pneumonia occurs only if infectious virions are deposited directly into alveolar ducts and sacs, not via retrograde propagation to the deep lung. Furthermore, to infect just 1% of the 140 m² of alveolar surface area within 1 week, either 10³ boluses each with 10⁶ infectious virions or 10⁶ aerosols with one infectious virion, all physically separated, must be directly deposited. These results strongly suggest that COVID-19 disease occurs in stages: a nasal/upper RT infection, followed by self-transmission of infection to the deep lung. Two mechanisms of self-transmission are persistent aspiration of infected nasal boluses that drain to the deep lung and repeated rupture of nasal aerosols from infected mucosal membranes by speaking, singing, or cheering that are partially inhaled, exhaled, and re-inhaled, to the deep lung.     

MUC1-C influences cell survival in lung adenocarcinoma Calu-3 cells after SARS-CoV-2 infection

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) induces coronavirus disease 2019 (COVID-19) and may increase the risk of adverse outcomes in lung cancer patients. In this study, we investigated the expression and function of mucin 1 (MUC1) after SARS-CoV-2 infection in the lung epithelial cancer cell line Calu-3. MUC1 is a major constituent of the mucus layer in the respiratory tract and contributes to pathogen defense. SARS-CoV-2 infection induced MUC1 C-terminal subunit (MUC1-C) expression in a STAT3 activation-dependent manner. Inhibition of MUC1-C signaling increased apoptosis-related protein levels and reduced proliferation-related protein levels; however, SARS-CoV-2 replication was not affected. Together, these results suggest that increased MUC1-C expression in response to SARS-CoV-2 infection may trigger the growth of lung cancer cells, and COVID-19 may be a risk factor for lung cancer patients.     

Nasal delivery of single-domain antibody improves symptoms of SARS-CoV-2 infection in an animal model

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) that causes the disease COVID-19 can lead to serious symptoms, such as severe pneumonia, in the elderly and those with underlying medical conditions. While vaccines are now available, they do not work for everyone and therapeutic drugs are still needed, particularly for treating life-threatening conditions. Here, we showed nasal delivery of a new, unmodified camelid single-domain antibody (VHH), termed K-874A, effectively inhibited SARS-CoV-2 titers in infected lungs of Syrian hamsters without causing weight loss and cytokine induction. In vitro studies demonstrated that K-874A neutralized SARS-CoV-2 in both VeroE6/TMPRSS2 and human lung-derived alveolar organoid cells. Unlike other drug candidates, K-874A blocks viral membrane fusion rather than viral attachment. Cryo-electron microscopy revealed K-874A bound between the receptor binding domain and N-terminal domain of the virus S protein. Further, infected cells treated with K-874A produced fewer virus progeny that were less infective. We propose that direct administration of K-874A to the lung could be a new treatment for preventing the reinfection of amplified virus in COVID-19 patients.     

Lethal infection of K18-hACE2 mice infected with severe acute respiratory syndrome coronavirus

The severe acute respiratory syndrome (SARS), caused by a novel coronavirus (SARS-CoV), resulted in substantial morbidity, mortality, and economic losses during the 2003 epidemic. While SARS-CoV infection has not recurred to a significant extent since 2003, it still remains a potential threat. Understanding of SARS and development of therapeutic approaches have been hampered by the absence of an animal model that mimics the human disease and is reproducible. Here we show that transgenic mice that express the SARS-CoV receptor (human angiotensin-converting enzyme 2 [hACE2]) in airway and other epithelia develop a rapidly lethal infection after intranasal inoculation with a human strain of the virus. Infection begins in airway epithelia, with subsequent alveolar involvement and extrapulmonary virus spread to the brain. Infection results in macrophage and lymphocyte infiltration in the lungs and upregulation of proinflammatory cytokines and chemokines in both the lung and the brain. This model of lethal infection with SARS-CoV should be useful for studies of pathogenesis and for the development of antiviral therapies.     

Inactivated rabies virus vectored SARS-CoV-2 vaccine prevents disease in a Syrian hamster model

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is an emergent coronavirus that has caused a worldwide pandemic. Although human disease is often asymptomatic, some develop severe illnesses such as pneumonia, respiratory failure, and death. There is an urgent need for a vaccine to prevent its rapid spread as asymptomatic infections accounting for up to 40% of transmission events. Here we further evaluated an inactivated rabies vectored SARS-CoV-2 S1 vaccine CORAVAX in a Syrian hamster model. CORAVAX adjuvanted with MPLA-AddaVax, a TRL4 agonist, induced high levels of neutralizing antibodies and generated a strong Th1-biased immune response. Vaccinated hamsters were protected from weight loss and viral replication in the lungs and nasal turbinates three days after challenge with SARS-CoV-2. CORAVAX also prevented lung disease, as indicated by the significant reduction in lung pathology. This study highlights CORAVAX as a safe, immunogenic, and efficacious vaccine that warrants further assessment in human trials.     

Tropism of and Innate Immune Responses to the Novel Human Betacoronavirus Lineage C Virus in Human Ex Vivo Respiratory Organ Cultures

Since April 2012, there have been 17 laboratory-confirmed human cases of respiratory disease associated with newly recognized human betacoronavirus lineage C virus EMC (HCoV-EMC), and 7 of them were fatal. The transmissibility and pathogenesis of HCoV-EMC remain poorly understood, and elucidating its cellular tropism in human respiratory tissues will provide mechanistic insights into the key cellular targets for virus propagation and spread. We utilized ex vivo cultures of human bronchial and lung tissue specimens to investigate the tissue tropism and virus replication kinetics following experimental infection with HCoV-EMC compared with those following infection with human coronavirus 229E (HCoV-229E) and severe acute respiratory syndrome coronavirus (SARS-CoV). The innate immune responses elicited by HCoV-EMC were also investigated. HCoV-EMC productively replicated in human bronchial and lung ex vivo organ cultures. While SARS-CoV productively replicated in lung tissue, replication in human bronchial tissue was limited. Immunohistochemistry revealed that HCoV-EMC infected nonciliated bronchial epithelium, bronchiolar epithelial cells, alveolar epithelial cells, and endothelial cells. Transmission electron microscopy showed virions within the cytoplasm of bronchial epithelial cells and budding virions from alveolar epithelial cells (type II). In contrast, there was minimal HCoV-229E infection in these tissues. HCoV-EMC failed to elicit strong type I or III interferon (IFN) or proinflammatory innate immune responses in ex vivo respiratory tissue cultures. Treatment of human lung tissue ex vivo organ cultures with type I IFNs (alpha and beta IFNs) at 1 h postinfection reduced the replication of HCoV-EMC, suggesting a potential therapeutic use of IFNs for treatment of human infection.     

Peroxisomes exhibit compromised structure and matrix protein content in SARS-CoV-2-infected cells

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a novel coronavirus that has triggered global health and economic crises. Here we report the effects of SARS-CoV-2 infection on peroxisomes of human cell lines Huh-7 and SK-N-SH. Peroxisomes undergo dramatic changes in morphology in SARS-CoV-2-infected cells. Rearrangement of peroxisomal membranes is followed by redistribution of peroxisomal matrix proteins to the cytosol, resulting in a dramatic decrease in the number of mature peroxisomes. The SARS-CoV-2 ORF14 protein was shown to interact physically with human PEX14, a peroxisomal membrane protein required for matrix protein import and peroxisome biogenesis. Given the important roles of peroxisomes in innate immunity, SARS-CoV-2 may directly target peroxisomes, resulting in loss of peroxisome structural integrity, matrix protein content and ability to function in antiviral signaling.     

SARS-CoV-2 Does Not Replicate in Aedes Mosquito Cells nor Present in Field-Caught Mosquitoes from Wuhan

With the rapid spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and growing fear, people have become very concerned about whether this novel coronavirus could be transmitted by mosquitoes. We evaluate the infectivity of SARS-CoV-2 in Aedes albopictus and Aedes aegypti derived cell lines. The results indicated that SARS-CoV-2 could not replicate in both C6/36, Sf9 and Aag2 cells. Further, no SARS-CoV-2 RNA was detected in the field collected Culex, and Anopheles mosquitoes during the months of April and May in Wuhan in 2020. Our findings highlight the restricted replication of SARS-CoV-2 in mosquito cells and field-caught mosquitoes.