Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the etiological agent of Coronavirus Disease 2019 (COVID-19). There is a dire need for novel effective antivirals to treat COVID-19, as the only approved direct-acting antiviral to date is remdesivir, targeting the viral polymerase complex. A potential alternate target in the viral life cycle is the main SARS-CoV-2 protease 3CLpro (Mpro). The drug candidate PF-00835231 is the active compound of the first anti-3CLpro regimen in clinical trials. Here, we perform a comparative analysis of PF-00835231, the pre-clinical 3CLpro inhibitor GC-376, and the polymerase inhibitor remdesivir, in alveolar basal epithelial cells modified to express ACE2 (A549+ACE2 cells). We find PF-00835231 with at least similar or higher potency than remdesivir or GC-376. A time-of-drug-addition approach delineates the timing of early SARS-CoV-2 life cycle steps in A549+ACE2 cells and validates PF-00835231's early time of action. In a model of the human polarized airway epithelium, both PF-00835231 and remdesivir potently inhibit SARS-CoV-2 at low micromolar concentrations. Finally, we show that the efflux transporter P-glycoprotein, which was previously suggested to diminish PF-00835231's efficacy based on experiments in monkey kidney Vero E6 cells, does not negatively impact PF-00835231 efficacy in either A549+ACE2 cells or human polarized airway epithelial cultures. Thus, our study provides in vitro evidence for the potential of PF-00835231 as an effective SARS-CoV-2 antiviral and addresses concerns that emerged based on prior studies in non-human in vitro models.Importance:The arsenal of SARS-CoV-2 specific antiviral drugs is extremely limited. Only one direct-acting antiviral drug is currently approved, the viral polymerase inhibitor remdesivir, and it has limited efficacy. Thus, there is a substantial need to develop additional antiviral compounds with minimal side effects and alternate viral targets. One such alternate target is its main protease, 3CLpro (Mpro), an essential component of the SARS-CoV-2 life cycle processing the viral polyprotein into the components of the viral polymerase complex. In this study, we characterize a novel antiviral drug, PF-00835231, which is the active component of the first-in-class 3CLpro-targeting regimen in clinical trials. Using 3D in vitro models of the human airway epithelium, we demonstrate the antiviral potential of PF-00835231 for inhibition of SARS-CoV-2.

https://journals.asm.org/doi/pdf/10.1128/JVI.01819-20

A comparative analysis of SARS-CoV-2 antivirals characterizes 3CL pro inhibitor PF-00835231 as a potential new treatment for COVID-19.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the etiological agent of Coronavirus Disease 2019 (COVID-19), a pandemic that has claimed over 700,000 human lives. The only SARS-CoV-2 antiviral, for emergency use, is remdesivir, targeting the viral polymerase complex. PF-00835231 is a pre-clinical lead compound with an alternate target, the main SARS-CoV-2 protease 3CLpro (Mpro). Here, we perform a comparative analysis of PF-00835231 and remdesivir in A549+ACE2 cells, using isolates of two major SARS-CoV-2 clades. PF-00835231 is antiviral for both clades, and, in this assay, statistically more potent than remdesivir. A time-of-drug-addition approach delineates the timing of early SARS-CoV-2 life cycle steps and validates PF-00835231’s time of action. Both PF-00835231 and remdesivir potently inhibit SARS-CoV-2 in human polarized airway epithelial cultures. Thus, our study provides in vitro evidence for the potential of PF-00835231 as an effective antiviral for SARS-CoV-2, addresses concerns from non-human in vitro models, and supports further studies with this compound.

/pdf/comparative-study-of-a-3clpro-inhibitor-and-remdesivir-4awf55ysnr.pdf

Comparative study of a 3CLpro inhibitor and remdesivir against both major SARS-CoV-2 clades in human airway models

Respiratory tract infections (RTI) are a major cause of morbidity and mortality in humans. A large number of RTIs is caused by viruses, often resulting in more severe disease in infants, elderly and the immunocompromised. Upon viral infection, most individuals experience common cold-like symptoms associated with an upper RTI. However, in some cases a severe and sometimes life-threatening lower RTI may develop. Reproducible and scalable in vitro culture models that accurately reflect the human respiratory tract are needed to study interactions between respiratory viruses and the host, and to test novel therapeutic interventions. Multiple in vitro respiratory cell culture systems have been described, but the majority of these are based on immortalized cell lines. Although useful for studying certain aspects of viral infections, such monomorphic, unicellular systems fall short in creating an understanding of the processes that occur at an integrated tissue level. Novel in vitro models involving primary human airway epithelial cells and, more recently, human airway organoids, are now in use. In this review, we describe the evolution of in vitro cell culture systems and their characteristics in the context of viral RTIs, starting from advances after immortalized cell cultures to more recently developed organoid systems. Furthermore, we describe how these models are used in studying virus-host interactions, e.g. tropism and receptor studies as well as interactions with the innate immune system. Finally, we provide an outlook for future developments in this field, including co-factors that mimic the microenvironment in the respiratory tract.

/pdf/in-vitro-modelling-of-respiratory-virus-infections-in-human-3ffhiht87g.pdf

In Vitro Modelling of Respiratory Virus Infections in Human Airway Epithelial Cells - A Systematic Review.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the etiological agent of Coronavirus Disease 2019 (COVID-19). There is a dire need for novel effective antivirals to treat COVID-19, as the only approved direct-acting antiviral to date is remdesivir, targeting the viral polymerase complex. A potential alternate target in the viral life cycle is the main SARS-CoV-2 protease 3CLpro (Mpro). The drug candidate PF-00835231 is the active compound of the first anti-3CLpro regimen in clinical trials. Here, we perform a comparative analysis of PF-00835231, the pre-clinical 3CLpro inhibitor GC-376, and the polymerase inhibitor remdesivir, in alveolar basal epithelial cells modified to express ACE2 (A549+ACE2 cells). We find PF-00835231 with at least similar or higher potency than remdesivir or GC-376. A time-of-drug-addition approach delineates the timing of early SARS-CoV-2 life cycle steps in A549+ACE2 cells and validates PF-00835231’s early time of action. In a model of the human polarized airway epithelium, both PF-00835231 and remdesivir potently inhibit SARS-CoV-2 at low micromolar concentrations. Finally, we show that the efflux transporter P-glycoprotein, which was previously suggested to diminish PF-00835231’s efficacy based on experiments in monkey kidney Vero E6 cells, does not negatively impact PF-00835231 efficacy in either A549+ACE2 cells or human polarized airway epithelial cultures. Thus, our study provides in vitro evidence for the potential of PF-00835231 as an effective SARS-CoV-2 antiviral and addresses concerns that emerged based on prior studies in non-human in vitro models. Importance The arsenal of SARS-CoV-2 specific antiviral drugs is extremely limited. Only one direct-acting antiviral drug is currently approved, the viral polymerase inhibitor remdesivir, and it has limited efficacy. Thus, there is a substantial need to develop additional antiviral compounds with minimal side effects and alternate viral targets. One such alternate target is its main protease, 3CLpro (Mpro), an essential component of the SARS-CoV-2 life cycle processing the viral polyprotein into the components of the viral polymerase complex. In this study, we characterize a novel antiviral drug, PF-00835231, which is the active component of the first-in-class 3CLpro-targeting regimen in clinical trials. Using 3D in vitro models of the human airway epithelium, we demonstrate the antiviral potential of PF-00835231 for inhibition of SARS-CoV-2.

/pdf/a-comparative-analysis-of-sars-cov-2-antivirals-in-human-300agu78cl.pdf

A comparative analysis of SARS-CoV-2 antivirals in human airway models characterizes 3CLpro inhibitor PF-00835231 as a potential new treatment for COVID-19.

The interplay of type-2 inflammation and antiviral immunity underpins asthma exacerbation pathogenesis. Virus infection induces type-2 inflammation–promoting chemokines CCL17 and CCL22 in asthma; h...

Rhinovirus-induced CCL17 and CCL22 in Asthma Exacerbations and Differential Regulation by STAT6

Respiratory viral infections, particularly those caused by rhinovirus, exacerbate chronic respiratory inflammatory diseases, such as asthma and chronic obstructive pulmonary disease (COPD). Airway epithelial cells are the primary site of rhinovirus replication and responsible of initiating the host immune response to infection. Numerous studies have reported that the anti-viral innate immune response (including type I and type III interferon) in asthma is less effective or deficient leading to the conclusion that epithelial innate immunity is a key determinant of disease severity during a rhinovirus induced exacerbation. However, deficient rhinovirus-induced epithelial interferon production in asthma has not always been observed. We hypothesized that disparate in vitro airway epithelial infection models using high multiplicity of infection (MOI) and lacking genome-wide, time course analyses have obscured the role of epithelial innate anti-viral immunity in asthma and COPD. To address this, we developed a low MOI rhinovirus model of differentiated primary epithelial cells obtained from healthy, asthma and COPD donors. Using genome-wide gene expression following infection, we demonstrated that gene expression patterns are similar across patient groups, but that the kinetics of induction are delayed in cells obtained from asthma and COPD donors. Rhinovirus-induced innate immune responses were defined by interferons (type-I, II, and III), interferon response factors (IRF1, IRF3, and IRF7), TLR signaling and NF-κB and STAT1 activation. Induced gene expression was evident at 24 h and peaked at 48 h post-infection in cells from healthy subjects. In contrast, in cells from donors with asthma or COPD induction was maximal at or beyond 72-96 h post-infection. Thus, we propose that propensity for viral exacerbations of asthma and COPD relate to delayed (rather than deficient) expression of epithelial cell innate anti-viral immune genes which in turns leads to a delayed and ultimately more inflammatory host immune response.

/pdf/airway-epithelial-cell-immunity-is-delayed-during-rhinovirus-3e55pia6jo.pdf

Airway Epithelial Cell Immunity Is Delayed During Rhinovirus Infection in Asthma and COPD.

The airway epithelium remains the first line of defense against insult, since it lies at the interface between the external environment and the internal milieu. Although thought to play a simple barrier role in this capacity, it has now been recognized to respond to the external environment and infectious stimuli via mediator release that direct signals with immune and mesenchymal cells. Specifically, in response to viral has developed a multifaceted approach to trapping and eliminating virus. A compromised epithelial barrier in diseases such as asthma, chronic obstructive pulmonary disease, or cystic fibrosis dramatically increases the risk of pathogenic infection. During infection, airway epithelial cells become the primary site of replication for respiratory viruses such as rhinovirus (RV) and initiate the hosts’ immune responses. This chapter will discuss the structure of the airway highlighting the now broad number of cell types that comprise it. It will discuss these in the context of health and inflammatory respiratory disease as well as their role in facilitating viral infection. In addition, this chapter will summarize the primary roles of the airway, including its mucociliary, barrier, and reparative functions in these settings. We will examine the interaction between the airway epithelium and RV, which promotes viral attachment, subsequent entry, and replication, and elaborate on the innate response of the airway to viral infection. Finally, we review the literature on interventional studies conducted to combat RV infection.

Ground zero—the airway epithelium

Club cells are found in human small airways where they play an important role in immune defense, xenobiotic metabolism, and repair after injury. Over the past few years, data from single-cell RNA sequencing (scRNA-seq) studies has generated new insights into club cell heterogeneity and function. In this review we integrate findings from scRNA-seq experiments with earlier in vitro, in vivo, and microscopy studies and highlight the many ways club cells contribute to airway homeostasis. We then discuss evidence for loss of club cells or club cell products in the airways of patients with chronic obstructive pulmonary disease (COPD) and discuss potential mechanisms through which this might occur.

Ngan Fung Li

Papers

Airway Epithelial Cell Immunity Is Delayed During Rhinovirus Infection in Asthma and COPD.

Ground zero—the airway epithelium

An Update in Club Cell Biology and Its Potential Relevance to Chronic Obstructive Pulmonary Disease.