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Human lung organoids as a model for respiratory virus replication and countermeasure performance in human hosts

  • Caitlin E. Edwards
    Affiliations
    Department of Epidemiology, The University of North Carolina at Chapel Hill, Chapel Hill, North Carolina
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  • Aleksandra Tata
    Affiliations
    Department of Cell Biology, Duke University School of Medicine, Durham, North Carolina
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  • Ralph S. Baric
    Correspondence
    Reprint requests: Ralph S. Baric, Department of Epidemiology, The University of North Carolina at Chapel Hill, Michael Hooker Research Building, CB #7435, Chapel Hill, NC 27599, USA.
    Affiliations
    Department of Epidemiology, The University of North Carolina at Chapel Hill, Chapel Hill, North Carolina

    Department of Microbiology and Immunology, The University of North Carolina at Chapel Hill, Chapel Hill, North Carolina
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Open AccessPublished:July 15, 2022DOI:https://doi.org/10.1016/j.trsl.2022.07.002
      Human respiratory viruses induce a wide breadth of disease phenotypes and outcomes of varying severity. Innovative models that recapitulate the human respiratory tract are needed to study such viruses, understand the virus-host interactions underlying replication and pathogenesis, and to develop effective countermeasures for prevention and treatment. Human organoid models provide a platform to study virus-host interactions in the proximal to distal lung in the absence of a human in vivo model. These cultures fill the niche of a suitable ex vivo model that represents the in vivo lung environment and encapsulates the structure and function of the native human lung.

      Abbreviations:

      ARDS (Acute respiratory distress syndrome), AT1 (Alveolar epithelial type 1 cells), AT2 (Alveolar epithelial type 2 cells), ALI (Air-liquid interface), COPD (Chronic obstructive pulmonary disease), HPIV (Parainfluenza), hCoV (Human coronavirus), HA (Hemagglutinin), hPSC (Human pluripotent stem cells), hDLO (Human distal lung organoids), NA (Neuraminidase), RSV (Respiratory Syncytial Virus)

      Human respiratory viruses

      Human respiratory virus infections cause a wide spectrum of asymptomatic to severe disease phenotypes each year, resulting in high morbidity and mortality outcomes globally. Common respiratory viruses, such as human influenza, respiratory syncytial virus (RSV), parainfluenza (HPIV), rhinoviruses and common cold coronaviruses (CoV), represent diverse families of viruses characterized by different genome organizations, unique transmission patterns and pathogenic outcomes in the upper and lower respiratory tract of human populations. Contemporary human respiratory viruses oftentimes cause seasonal epidemics, such as influenza, RSV, and HPIV, or have widespread endemic transmission across the calendar year, such as the common cold CoV and rhinovirus strains. While infections usually cause mild upper respiratory tract disease, patients may progress to more serious lower respiratory tract infections like bronchiolitis and croup, or to life threatening pneumonias that can progress to acute lung injury and/or organizing chronic pneumonias with fibrosis. Moreover, due to waning immune responses in upper respiratory mucosal compartments, coupled with antigenic changes in response to host immune memory responses, many contemporary respiratory viruses have the potential to cause dramatic spikes in disease prevalence, hospitalization, and mortality, associated with epidemic or pandemic spread.
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      Recent advances in technologies, like single-cell RNA sequencing, GeoMx, and CODEX Multiplexed Tissue Staining and Image Acquisition and related technologies allow for detailed characterization of human host innate and acquired immunologic responses at single cell resolution. Although biopsies and bronchial alveolar lavage fluids provide for infrequent but targeted longitudinal sampling opportunities, however, most of these genomic analyses focus on samples derived from end stage lung samples associated with lethal outcomes, thereby missing many of the critical time-ordered events associated with infection, clearance, disease progression and/or repair of the lung. Consequently, the virologic, host and immunologic factors that contribute to altered disease outcomes in humans remains a critical question in viral pathogenesis, the development of treatments and vaccines. Moreover, direct acting antivirals have limited opportunities for reversing disease progression in human patients unless given early in infection. Especially evident after influenza, RSV, and emerging coronavirus infections, disease severity is associated with complex immune pathologic and host response patterns, which can progress to end stage lung diseases. Therefore, later stage pathologic features require treatment modalities that focus on mitigating disease enhancing host response networks and/or immunopathologic signatures, dictating the need for host and immune based interventions that do not directly target live virus replication. Variation in disease severity can be attributed to microvariation in virus strain and sequence variants, changes in virus antigenicity, cell tropism, mutations that enhance virus replication/gene expression and the presence of viral genes that antagonize host innate immune responses. Moreover, natural genetic variation in outbred human populations, which present either as strong monogenic or polygenic traits, can regulate virus disease severity across individuals and promote pathogenic or protective immune/host responses that promote lethal disease.
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      Human primary cell and organoid model systems of infection

      Human organoid models provide a reliable platform to study virus-host interactions in the proximal to distal lung in the absence of a human in vivo model. Alternatively, these cultures provide an ex vivo model that presents the in vivo environment and recapitulates the structure and function of the native human lung. While commonly used 2D human airway epithelial cultures provide a resource for understanding viral fitness and potential efficacy of clinical interventions, they fail to represent the 3D lung structure and interactions between cells that is conferred by organoid models. Additionally, such ex vivo models can be adapted to represent differing compartments of the respiratory tract, providing novel opportunities to study virus tropism, virus tissue specific host response patterns, lung function, disease pathology, and provide a suitable platform to target therapeutics of various viruses by focusing on distinct cell types or areas of the lung.
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      Fig 1
      Fig 1A, Human primary lung cells can be harvested from the proximal to distal lung to represent various lung compartments including large airway, small airway, and alveoli. Harvested primary cells represent a heterogeneous population of key lung cells important in virus-host interactions, as well as natural human lung function B, Each of these cell types express either ACE2, TMPRSS2, or both, indicating a strong model for studying human epidemic coronaviruses such as SARS-CoV and SARS-CoV-2. C, SARS-CoV-2 has been studied in these lung models and depicts a gradients of replication efficiency from the proximal to distal lung.
      3D models, such as human organoids, involve arranged complex structures of multiple cells that can self-organize to replicate in vivo tissue structure and morphology while mimicking cellular interactions and the dynamic regulation of signaling pathways in an ex vivo culture. Organoids derived from human pluripotent stem cells (hPSC) are the most common and well-known type of human organoids that can be derived from peripheral blood and differentiated towards airway and alveolar epithelial cells.
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      These cells are then co-cultured with mesenchymal and endothelial cells to stimulate the generation and maturation of a 3D organoid structure that represents a section of the human lung. Once matured, hPSC derived organoids can possess both upper and lower respiratory-like epithelium containing basal cells, ciliated cells or alveolar-like structures, respectively. However, hPSC-derived organoids may not completely recapitulate the functionality and gene expression profiles compare to mature cells of adult human lungs. Despite this limitation, hPSC remain a reliable tool for studying both viral-host interactions, pathogenesis, and clinical interventions as they better replicate the human airway than previously developed models of the human respiratory tract.
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      This advancement allows for a dynamic environment that more closely recapitulates the alveolar space of the human lung and fills a critical niche for understanding viral pathogenesis and host interaction.
      More recently, distal lung organoids, derived from AT2 cells or basal appear to form intact lumens lined with differentiated ciliated and club cells, providing new insights into complex virus-host interactions with respiratory cell epitheliums.
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      After differentiation into mature cultures, cultures can be evaluated as either an apical in or apical out polarity (“flipped” organoids), the latter orientation presents critical host receptors, such as ACE2 or DPP4, on exposed organoid surfaces that promote efficient infection by respiratory viruses.
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      Receptor expression for various respiratory viruses on the surface of human organoids is critical to modeling of viral replication in these cultures and understanding the virus-host interactions. However, the expression of these receptors in the human respiratory tract is cell and organoid dependent. For example, while α2,3 and α2,6 linked sialic acids, the receptors for avian and human influenza viruses, respectively, are abundant on most airway cells, ACE2 and DPP4, the receptors for epidemic human coronaviruses, are more localized.
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      ACE2 expression in organotypic human airway epithelial cultures and airway biopsies.
      However, ACE2 is also expressed in AT2 cells, indicating that AT2 derived organoids serve as a strong ex vivo model to study these viruses and potential interventions. Lastly, expression of DPP4, the receptor for epidemic hCoV MERS-CoV, follows a more specific pattern and is rarely found on the surface epithelium of the proximal airway with a slight increase in expression in the distal airway.
      • Meyerholz DK
      • Lambertz AM
      • McCray PB.
      Dipeptidyl peptidase 4 distribution in the human respiratory tract: implications for the middle east respiratory syndrome.
      Additionally, DPP4 is predominantly found on AT1 and AT2 cells of the alveoli, as well as macrophages and fibroblasts, indicating a unique role of such primary cells to study MERS-CoV replication, pathogenesis, and repair in human organoids of various derivation.
      • Meyerholz DK
      • Lambertz AM
      • McCray PB.
      Dipeptidyl peptidase 4 distribution in the human respiratory tract: implications for the middle east respiratory syndrome.
      ,
      • Zhang T
      • Tong X
      • Zhang S
      • et al.
      The roles of dipeptidyl peptidase 4 (DPP4) and DPP4 inhibitors in different lung diseases: new evidence.
      As indicated, systems of human lung organoids provide reproducible models for applications in viral replication, and pathogenesis, including understanding virus-host interactions, exploring viral induced tissue damage and innate immune pathways and subsequent tissue regeneration responses, and the testing of novel clinical interventions such as prophylactic and therapeutic drugs that target viral genes or host pathways essential for efficient virus growth (Fig 2). Human lung organoids that recapitulate the human respiratory environment provide a model to explore the function of lung-related genes and signaling pathways, as well as immune responses following viral infection. With this tool, modeling of lung diseases becomes a multi-factorial process that allows for the further understanding of age-dependent viruses and outcomes, such as the predominance of RSV in infant lungs or age-exacerbated adverse events following coronavirus infection, by modifying the age at which organoids are used following stem cell proliferation. Further, these interactions between virus infection and respiratory epithelial tissue can be teased out to understand the underlying mechanisms that drive viral induced tissue damage. Additionally, as organoids derived from stem cells and progenitor cells retain their programming, by capturing the regenerative process of in vivo lung processes, researchers can study, and mimic processes of lung repair suggested from an in vivo study or autopsy of afflicted human lung tissue. Lastly, the multiple types of human organoid models provide a platform for screening drugs to prevent or alleviate respiratory disease by understanding disease pathology in ex vivo tissues and applying these findings to effective clinical interventions.
      Fig 2
      Fig 2Human pluripotent (hPSC) or embryonic (hESC) stem cells can be cultured and differentiated into primary airway cells that can generate 2D and 3D cultures. Additionally, human primary cells fr om the large airway, small airway, and alveoli can be used to develop models of human lung tissue including ALI models, organoid models, or both. Each of these models has unique analytical and clinical testing applications that fill a critical niche in studying human respiratory viruses.

      Drug testing and development

      Prior to clinical trial testing of potential clinical interventions, drug development and testing in vitro must be completed and demonstrate potential efficacy. Historically, pre-clinical testing of these drugs has been completed in 2D human primary cells, such as large bronchial cultures. These cultures have provided a pathway to in vivo small animal models and ultimately human clinical trials of many antivirals, such as those used against the pandemic virus SARS-CoV-2, including remdesivir, molnupiravir, and pegylated IFNλ, which have proven effective in clinical trials.
      • Sheahan TP
      • Sims AC
      • Leist SR
      • et al.
      Comparative therapeutic efficacy of remdesivir and combination lopinavir, ritonavir, and interferon beta against MERS-CoV.
      ,
      • Sheahan TP
      • Sims AC
      • Graham RL
      • et al.
      Broad-spectrum antiviral GS-5734 inhibits both epidemic and zoonotic coronaviruses.
      ,
      • Fischer WA
      • Eron JJ
      • Holman W
      • et al.
      A phase 2a clinical trial of molnupiravir in patients with COVID-19 shows accelerated SARS-CoV-2 RNA clearance and elimination of infectious virus.
      • Dinnon KH
      • Leist SR
      • Schäfer A
      • et al.
      A mouse-adapted model of SARS-CoV-2 to test COVID-19 countermeasures.
      • Beigel JH
      • Tomashek KM
      • Dodd LE
      • et al.
      Remdesivir for the treatment of covid-19 — final report.
      However, in future studies involving the development of antivirals, human organoid systems could provide a more robust environment for pre-clinical testing, which provides detailed insights into drug delivery, especially in the gas exchange region of the lung that is oftentimes associated with life threatening disease.
      Remdesivir, an approved nucleoside analog inhibitor effective at blocking SARS-CoV-2 infection in human patients, was first tested in primary lung cell cultures, such as large airway cultures, followed by robust mouse models of human disease.
      • Pruijssers AJ
      • George AS
      • Schäfer A
      • et al.
      Remdesivir inhibits SARS-CoV-2 in human lung cells and chimeric SARS-CoV expressing the SARS-CoV-2 RNA polymerase in mice.
      A strength of these early studies were applications to show that broad antiviral activity exists against an array of unique coronaviruses that could replicate efficiently in primary cells, supporting its application as a potentially effective antiviral against a newly emerged coronavirus. In animal models, the drug was effective, although efficacy waned in concert with reducing virus titers later in infection. Similar findings became evident in human trials. Upon successful development and approval of remdesivir to inhibit infection, it became widely used as a control for testing novel prophylactic and therapeutic treatments.
      • Pruijssers AJ
      • George AS
      • Schäfer A
      • et al.
      Remdesivir inhibits SARS-CoV-2 in human lung cells and chimeric SARS-CoV expressing the SARS-CoV-2 RNA polymerase in mice.
      Interestingly, this drug was also utilized as confirmation of the development of human tonsillar epithelial organoids as an ex vivo model to study viral infections, such as SARS-CoV-2.
      • Kim HK
      • Kim H
      • Lee MK
      • et al.
      Generation of human tonsil epithelial organoids as an ex vivo model for SARS-CoV-2 infection.
      It was found that remdesivir could successfully decrease viral RNA in these organoid cultures in a dose-dependent manner, indicating that these novel type organoids could be used as a preclinical and translational research platform for testing of future clinical interventions.
      Similarly, molnupiravir is a second anti-viral authorized by the FDA for treatment against SARS-CoV-2 and causes error catastrophe following treatment of virally infected cells.
      • Sheahan TP
      • Sims AC
      • Zhou S
      • et al.
      An orally bioavailable broad-spectrum antiviral inhibits SARS-CoV-2 in human airway epithelial cell cultures and multiple coronaviruses in mice.
      This orally available antiviral effectively inhibited SARS-CoV-2 replication in human airway epithelial cultures, as was seen with remdesivir.
      • Sheahan TP
      • Sims AC
      • Zhou S
      • et al.
      An orally bioavailable broad-spectrum antiviral inhibits SARS-CoV-2 in human airway epithelial cell cultures and multiple coronaviruses in mice.
      The broad-spectrum activity of this ribonucleoside analog successfully inhibited all three emergent epidemic coronaviruses, SARS-CoV, MERS-CoV, and SARS-CoV-2, when administered both prophylactically and therapeutically.
      • Sheahan TP
      • Sims AC
      • Zhou S
      • et al.
      An orally bioavailable broad-spectrum antiviral inhibits SARS-CoV-2 in human airway epithelial cell cultures and multiple coronaviruses in mice.
      Moreover, molnupiravir was also tested in human organoid models, in conjunction with an in vivo mouse model to demonstrate the efficacy in complex human lung tissue, as well as robust SARS-CoV, SARS-CoV-2, and MERS-CoV mouse models of human disease.
      • Wahl A
      • Gralinski LE
      • Johnson CE
      • et al.
      SARS-CoV-2 infection is effectively treated and prevented by EIDD-2801.
      Later, immunodeficient mice were implanted with human lung tissue in the form of organoids and again infected with all three emergent coronaviruses, as well as two SARS-like bat coronaviruses that have yet to infect human hosts but remain poised for potential zoonosis. These human lung organoids demonstrated robust infection of AT2 cells and ciliated airway cells but effectively blocked or reduced infection when treated prophylactically or therapeutically with molnupiravir, respectively.
      • Wahl A
      • Gralinski LE
      • Johnson CE
      • et al.
      SARS-CoV-2 infection is effectively treated and prevented by EIDD-2801.
      Molnupiravir is a prime example of the utilization of human respiratory organoids to further demonstrate the efficacy of clinical interventions in human lung tissue, following 2D models, and the ability to use this data to propagate potential interventions into human clinical trials. It has been approved for use in some human patients.
      Lastly, pegylated type III interferon, or peg-IFN-λ1, has recently completed final phase III clinical trials for the inhibition of SARS-CoV-2 in human patients.
      Eiger's single-dose peginterferon lambda for COVID-19 reduced risk of hospitalization or ER visits by 50% in a predominantly vaccinated population in phase 3 TOGETHER study.
      Similarly, it has been suggested that peg-IFN-λ1 can potently block replication in human airway cells, both prophylactically and therapeutically, as also demonstrated in the molnupiravir studies. Studies with peg-IFN-λ1 would largely benefit from testing in human organoid models as the type III interferon receptors are largely found in epithelial cells, including the cells that compromise the lung epithelium prominent in lung organoids models.
      • Dinnon KH
      • Leist SR
      • Schäfer A
      • et al.
      A mouse-adapted model of SARS-CoV-2 to test COVID-19 countermeasures.
      As seen with the previous pre-clinical and clinical candidates, in vitro testing of peg-IFN-λ1a was confirmed by successful inhibition of infection in vivo in small animal models.
      • Dinnon KH
      • Leist SR
      • Schäfer A
      • et al.
      A mouse-adapted model of SARS-CoV-2 to test COVID-19 countermeasures.
      Additionally, peg-IFN-λ1a was demonstrated to block SARS-CoV replication in human primary cells, a finding that could be further studied in human lung organoids and have important potential to successfully block future emergent or zoonotic coronaviruses. Importantly, all of these antiviral drugs work well if delivered early in infection during peak rises in virus replication and spread. After acute virus infection (eg, day 7), direct acting antivirals and therapeutic antibodies provide little benefit as pathogenic host responses and immunopathologic phases of disease do not depend on significant levels of virus replication and spread.

      Future directions

      In recent years, much progress has been made in developing in vitro primary airway and alveolar culture models. Organoids established using epithelial cells isolated from different regions of the respiratory tract provide a valuable platform for unraveling SARS-CoV-2 virus infection, replication kinetics, and tropism. Although several studies demonstrated many applications of human organoids, the current models lack the complexity and do not fully represent the adult human lung. There is an urgent need to develop human-relevant complex organoid models, incorporating cellular components including endothelial, immune, and mesenchymal cells that closely recapitulate the niche of infected epithelial cells found in human COVID-19 lungs. This approach will provide revolutionary new perspectives for better understanding of SARS-CoV-2 viral infection and cellular responses.
      Present studies primarily focused on studying viral entry, replication, and host cell responses. More studies are needed to fully understand the molecular mechanisms and critical factors that control highly pathogenic respiratory tract infections, including SARS-CoV-2 infection and the long-term chronic disease outcomes associated with COVID-19 infection in the upper and lower respiratory tract
      • Dinnon KH
      • Leist SR
      • Okuda K
      • et al.
      A model of persistent post SARS-CoV-2 induced lung disease for target identification and testing of therapeutic strategies.
      . Future studies will also need to address how various host factors such as age, sex, and genetic variation influences the potential of stem cells in the airway and alveoli following SARS-CoV-2 infection. Organoid models coupled with genetic perturbations to simulate genetic variants identified in GWAS will be valuable in determining genetic factors that modulate infection efficiency, viral propagation, and immune invasion.
      Finally, more research is needed to explore further development and validation of effective antiviral therapeutics for COVID-19 treatment. Recent studies in COVID-19 infected patients and organoid models have shown that, following viral infection, alveolar AT2 cells activate aberrant pathways including interferon signaling and undergo apoptosis. Studies have also shown that AT2s acquire a transitional cell state previously implicated in idiopathic pulmonary fibrosis was also observed in lung specimens collected from COVID-19 autopsies.
      • Dinnon KH
      • Leist SR
      • Okuda K
      • et al.
      A model of persistent post SARS-CoV-2 induced lung disease for target identification and testing of therapeutic strategies.
      • Lechowicz K
      • Drożdżal S
      • Machaj F
      • et al.
      COVID-19: the potential treatment of pulmonary fibrosis associated with SARS-CoV-2 infection.
      • Ravaglia C
      • Doglioni C
      • Chilosi M
      • et al.
      Clinical, radiological, and pathological findings in patients with persistent lung disease following SARS-CoV-2 infection.
      Therefore, there is a need to identify novel therapeutic targets that preserve AT2 cells function and enable them to fully restore the alveolar structure without undergoing transitional state. Organoid platforms that phenocopy the chronic disease states often initiated by viral infections offer novel opportunities for new drug discovery and disease control, especially after virus clearance. With the advent of scalable organoid culture models now it is possible to perform high-throughput genetic and pharmacological screens to identify new therapeutics against COVID-19 and restore lung structure and function.

      Acknowledgements

      Conflicts of Interest: All authors have read the journal's policy on disclosure of potential conflicts of interest and have none to declare.
      Supported in part by National Institute of Health U19 grant awards AI116484 and AI108197 to RSB.
      All authors have read the journal's authorship agreement and the manuscript has been reviewed and approved by all named authors. This manuscript conforms to relevant ethical guidelines for human and animal research.

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