Optimized ACE2 decoys neutralize antibody-resistant SARS-CoV-2 variants through functional receptor mimicry and treat infection in vivo.

As severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) evolves to escape natural antibodies, it also loses sensitivity to therapeutic antibody drugs. By contrast, evolution selects for binding to ACE2, the cell-surface receptor required for SARS-CoV-2 infection. Consistent with this, we find that an ACE2 decoy neutralizes antibody-resistant variants, including Omicron, with no loss in potency. To identify design features necessary for in vivo activity, we compare several enzymatically inactive, Fc effector-silenced ACE2-Fc decoys. Inclusion of the ACE2 collectrin-like domain not only improves affinity for the S protein but also unexpectedly extends serum half-life and is necessary to reduce disease severity and viral titer in Syrian hamsters. Fc effector function is not required. The activity of ACE2 decoy receptors is due, in part, to their ability to trigger an irreversible structural change in the viral S protein. Our studies provide a new understanding of how ACE2 decoys function and support their development as therapeutics to treat ACE2-dependent coronaviruses.

Generating 3D Spheres and 2D Air-Liquid Interface Cultures of Human Induced Pluripotent Stem Cell-Derived Type 2 Alveolar Epithelial Cells.

In the lung, the alveolar epithelium is a physical barrier from environmental stimuli and plays an essential role in homeostasis and disease. Type 2 alveolar epithelial cells (AT2s) are the facultative progenitors of the distal lung epithelium. Dysfunction and injury of AT2s can result from and contribute to various lung diseases. Improved understanding of AT2 biology is, thus, critical for understanding lung biology and disease; however, primary human AT2s are generally difficult to isolate and limited in supply. To overcome these limitations, human induced pluripotent stem cell (iPSC)-derived type 2 alveolar epithelial cells (iAT2s) can be generated through a directed differentiation protocol that recapitulates in vivo lung development. iAT2s grow in feeder-free conditions, share a transcriptomic program with human adult primary AT2s, and execute key functions of AT2s such as production, packaging, and secretion of surfactant. This protocol details the methods for maintaining self-renewing iAT2s through serial passaging in three-dimensional (3D) culture or adapting iAT2s to air-liquid interface (ALI) culture. A single-cell suspension of iAT2s is generated before plating in 3D solubilized basement membrane matrix (hereafter referred to as "matrix"), where they self-assemble into monolayered epithelial spheres. iAT2s in 3D culture can be serially dissociated into single-cell suspensions to be passaged or plated in 2D ALI culture. In ALI culture, iAT2s form a polarized monolayer with the apical surface exposed to air, making this platform readily amenable to environmental exposures. Hence, this protocol generates an inexhaustible supply of iAT2s, producing upwards of 1 x 1030 cells per input cell over 15 passages while maintaining the AT2 program indicated by SFTPCtdTomato expression. The resulting cells represent a reproducible and relevant platform that can be applied to study genetic mutations, model environmental exposures, or screen drugs.

Population Genomics Approaches for Genetic Characterization of SARS-CoV-2 Lineages.

The genome of the Severe Acute Respiratory Syndrome coronavirus 2 (SARS-CoV-2), the pathogen that causes coronavirus disease 2019 (COVID-19), has been sequenced at an unprecedented scale leading to a tremendous amount of viral genome sequencing data. To assist in tracing infection pathways and design preventive strategies, a deep understanding of the viral genetic diversity landscape is needed. We present here a set of genomic surveillance tools from population genetics which can be used to better understand the evolution of this virus in humans. To illustrate the utility of this toolbox, we detail an in depth analysis of the genetic diversity of SARS-CoV-2 in first year of the COVID-19 pandemic. We analyzed 329,854 high-quality consensus sequences published in the GISAID database during the pre-vaccination phase. We demonstrate that, compared to standard phylogenetic approaches, haplotype networks can be computed efficiently on much larger datasets. This approach enables real-time lineage identification, a clear description of the relationship between variants of concern, and efficient detection of recurrent mutations. Furthermore, time series change of Tajima's D by haplotype provides a powerful metric of lineage expansion. Finally, principal component analysis (PCA) highlights key steps in variant emergence and facilitates the visualization of genomic variation in the context of SARS-CoV-2 diversity. The computational framework presented here is simple to implement and insightful for real-time genomic surveillance of SARS-CoV-2 and could be applied to any pathogen that threatens the health of populations of humans and other organisms.

Multiscale PHATE identifies multimodal signatures of COVID-19.

As the biomedical community produces datasets that are increasingly complex and high dimensional, there is a need for more sophisticated computational tools to extract biological insights. We present Multiscale PHATE, a method that sweeps through all levels of data granularity to learn abstracted biological features directly predictive of disease outcome. Built on a coarse-graining process called diffusion condensation, Multiscale PHATE learns a data topology that can be analyzed at coarse resolutions for high-level summarizations of data and at fine resolutions for detailed representations of subsets. We apply Multiscale PHATE to a coronavirus disease 2019 (COVID-19) dataset with 54 million cells from 168 hospitalized patients and find that patients who die show CD16hiCD66blo neutrophil and IFN-γ+ granzyme B+ Th17 cell responses. We also show that population groupings from Multiscale PHATE directly fed into a classifier predict disease outcome more accurately than naive featurizations of the data. Multiscale PHATE is broadly generalizable to different data types, including flow cytometry, single-cell RNA sequencing (scRNA-seq), single-cell sequencing assay for transposase-accessible chromatin (scATAC-seq), and clinical variables.

Human airway lineages derived from pluripotent stem cells reveal the epithelial responses to SARS-CoV-2 infection.

There is an urgent need to understand how SARS-CoV-2 infects the airway epithelium and in a subset of individuals leads to severe illness or death. Induced pluripotent stem cells (iPSCs) provide a near limitless supply of human cells that can be differentiated into cell types of interest, including airway epithelium, for disease modeling. We present a human iPSC-derived airway epithelial platform, composed of the major airway epithelial cell types, that is permissive to SARS-CoV-2 infection. Subsets of iPSC-airway cells express the SARS-CoV-2 entry factors angiotensin-converting enzyme 2 (ACE2), and transmembrane protease serine 2 (TMPRSS2). Multiciliated cells are the primary initial target of SARS-CoV-2 infection. On infection with SARS-CoV-2, iPSC-airway cells generate robust interferon and inflammatory responses, and treatment with remdesivir or camostat mesylate causes a decrease in viral propagation and entry, respectively. In conclusion, iPSC-derived airway cells provide a physiologically relevant in vitro model system to interrogate the pathogenesis of, and develop treatment strategies for, COVID-19 pneumonia.

SARS-CoV-2 Infection of Pluripotent Stem Cell-derived Human Lung Alveolar Type 2 Cells Elicits a Rapid Epithelial-Intrinsic Inflammatory Response

The most severe and fatal infections with SARS-CoV-2 result in the acute respiratory distress syndrome, a clinical phenotype of coronavirus disease 2019 (COVID-19) that is associated with virions targeting the epithelium of the distal lung, particularly the facultative progenitors of this tissue, alveolar epithelial type 2 cells (AT2s). Little is known about the initial responses of human lung alveoli to SARS-CoV-2 infection due in part to inability to access these cells from patients, particularly at early stages of disease. Here we present an in vitro human model that simulates the initial apical infection of the distal lung epithelium with SARS-CoV-2, using AT2s that have been adapted to air-liquid interface culture after their derivation from induced pluripotent stem cells (iAT2s). We find that SARS-CoV-2 induces a rapid global transcriptomic change in infected iAT2s characterized by a shift to an inflammatory phenotype predominated by the secretion of cytokines encoded by NF-kB target genes, delayed epithelial interferon responses, and rapid loss of the mature lung alveolar epithelial program. Over time, infected iAT2s exhibit cellular toxicity that can result in the death of these key alveolar facultative progenitors, as is observed in vivo in COVID-19 lung autopsies. Importantly, drug testing using iAT2s confirmed the efficacy of TMPRSS2 protease inhibition, validating putative mechanisms used for viral entry in human alveolar cells. Our model system reveals the cell-intrinsic responses of a key lung target cell to infection, providing a platform for further drug development and facilitating a deeper understanding of COVID-19 pathogenesis.

Morphological cell profiling of SARS-CoV-2 infection identifies drug repurposing candidates for COVID-19.

The global spread of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), and the associated disease COVID-19, requires therapeutic interventions that can be rapidly identified and translated to clinical care. Traditional drug discovery methods have a >90% failure rate and can take 10 to 15 y from target identification to clinical use. In contrast, drug repurposing can significantly accelerate translation. We developed a quantitative high-throughput screen to identify efficacious agents against SARS-CoV-2. From a library of 1,425 US Food and Drug Administration (FDA)-approved compounds and clinical candidates, we identified 17 hits that inhibited SARS-CoV-2 infection and analyzed their antiviral activity across multiple cell lines, including lymph node carcinoma of the prostate (LNCaP) cells and a physiologically relevant model of alveolar epithelial type 2 cells (iAEC2s). Additionally, we found that inhibitors of the Ras/Raf/MEK/ERK signaling pathway exacerbate SARS-CoV-2 infection in vitro. Notably, we discovered that lactoferrin, a glycoprotein found in secretory fluids including mammalian milk, inhibits SARS-CoV-2 infection in the nanomolar range in all cell models with multiple modes of action, including blockage of virus attachment to cellular heparan sulfate and enhancement of interferon responses. Given its safety profile, lactoferrin is a readily translatable therapeutic option for the management of COVID-19.

SARS-CoV-2 induces double-stranded RNA-mediated innate immune responses in respiratory epithelial-derived cells and cardiomyocytes.

Coronaviruses are adept at evading host antiviral pathways induced by viral double-stranded RNA, including interferon (IFN) signaling, oligoadenylate synthetase-ribonuclease L (OAS-RNase L), and protein kinase R (PKR). While dysregulated or inadequate IFN responses have been associated with severe coronavirus infection, the extent to which the recently emerged SARS-CoV-2 activates or antagonizes these pathways is relatively unknown. We found that SARS-CoV-2 infects patient-derived nasal epithelial cells, present at the initial site of infection; induced pluripotent stem cell-derived alveolar type 2 cells (iAT2), the major cell type infected in the lung; and cardiomyocytes (iCM), consistent with cardiovascular consequences of COVID-19 disease. Robust activation of IFN or OAS-RNase L is not observed in these cell types, whereas PKR activation is evident in iAT2 and iCM. In SARS-CoV-2-infected Calu-3 and A549ACE2 lung-derived cell lines, IFN induction remains relatively weak; however, activation of OAS-RNase L and PKR is observed. This is in contrast to Middle East respiratory syndrome (MERS)-CoV, which effectively inhibits IFN signaling and OAS-RNase L and PKR pathways, but is similar to mutant MERS-CoV lacking innate immune antagonists. Remarkably, OAS-RNase L and PKR are activated in MAVS knockout A549ACE2 cells, demonstrating that SARS-CoV-2 can induce these host antiviral pathways despite minimal IFN production. Moreover, increased replication and cytopathic effect in RNASEL knockout A549ACE2 cells implicates OAS-RNase L in restricting SARS-CoV-2. Finally, while SARS-CoV-2 fails to antagonize these host defense pathways, which contrasts with other coronaviruses, the IFN signaling response is generally weak. These host-virus interactions may contribute to the unique pathogenesis of SARS-CoV-2.

Actionable Cytopathogenic Host Responses of Human Alveolar Type 2 Cells to SARS-CoV-2.

Human transmission of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), causative pathogen of the COVID-19 pandemic, exerts a massive health and socioeconomic crisis. The virus infects alveolar epithelial type 2 cells (AT2s), leading to lung injury and impaired gas exchange, but the mechanisms driving infection and pathology are unclear. We performed a quantitative phosphoproteomic survey of induced pluripotent stem cell-derived AT2s (iAT2s) infected with SARS-CoV-2 at air-liquid interface (ALI). Time course analysis revealed rapid remodeling of diverse host systems, including signaling, RNA processing, translation, metabolism, nuclear integrity, protein trafficking, and cytoskeletal-microtubule organization, leading to cell cycle arrest, genotoxic stress, and innate immunity. Comparison to analogous data from transformed cell lines revealed respiratory-specific processes hijacked by SARS-CoV-2, highlighting potential novel therapeutic avenues that were validated by a high hit rate in a targeted small molecule screen in our iAT2 ALI system.

SARS-CoV-2 induces double-stranded RNA-mediated innate immune responses in respiratory epithelial derived cells and cardiomyocytes.

Coronaviruses are adept at evading host antiviral pathways induced by viral double-stranded RNA, including interferon (IFN) signaling, oligoadenylate synthetase-ribonuclease L (OAS-RNase L), and protein kinase R (PKR). While dysregulated or inadequate IFN responses have been associated with severe coronavirus infection, the extent to which the recently emerged SARS-CoV-2 activates or antagonizes these pathways is relatively unknown. We found that SARS-CoV-2 infects patient-derived nasal epithelial cells, present at the initial site of infection, induced pluripotent stem cell-derived alveolar type 2 cells (iAT2), the major cell type infected in the lung, and cardiomyocytes (iCM), consistent with cardiovascular consequences of COVID-19 disease. Robust activation of IFN or OAS-RNase L is not observed in these cell types, while PKR activation is evident in iAT2 and iCM. In SARS-CoV-2 infected Calu-3 and A549 ACE2 lung-derived cell lines, IFN induction remains relatively weak; however activation of OAS-RNase L and PKR is observed. This is in contrast to MERS-CoV, which effectively inhibits IFN signaling as well as OAS-RNase L and PKR pathways, but similar to mutant MERS-CoV lacking innate immune antagonists. Remarkably, both OAS-RNase L and PKR are activated in MAVS knockout A549 ACE2 cells, demonstrating that SARS-CoV-2 can induce these host antiviral pathways despite minimal IFN production. Moreover, increased replication and cytopathic effect in RNASEL knockout A549 ACE2 cells implicates OAS-RNase L in restricting SARS-CoV-2. Finally, while SARS-CoV-2 fails to antagonize these host defense pathways, which contrasts with other coronaviruses, the IFN signaling response is generally weak. These host-virus interactions may contribute to the unique pathogenesis of SARS-CoV-2. SIGNIFICANCE: SARS-CoV-2 emergence in late 2019 led to the COVID-19 pandemic that has had devastating effects on human health and the economy. Early innate immune responses are essential for protection against virus invasion. While inadequate innate immune responses are associated with severe COVID-19 diseases, understanding of the interaction of SARS-CoV-2 with host antiviral pathways is minimal. We have characterized the innate immune response to SARS-CoV-2 infections in relevant respiratory tract derived cells and cardiomyocytes and found that SARS-CoV-2 activates two antiviral pathways, oligoadenylate synthetase-ribonuclease L (OAS-RNase L), and protein kinase R (PKR), while inducing minimal levels of interferon. This in contrast to MERS-CoV which inhibits all three pathways. Activation of these pathways may contribute to the distinctive pathogenesis of SARS-CoV-2.

SARS-CoV-2 Infection of Pluripotent Stem Cell-Derived Human Lung Alveolar Type 2 Cells Elicits a Rapid Epithelial-Intrinsic Inflammatory Response.

A hallmark of severe COVID-19 pneumonia is SARS-CoV-2 infection of the facultative progenitors of lung alveoli, the alveolar epithelial type 2 cells (AT2s). However, inability to access these cells from patients, particularly at early stages of disease, limits an understanding of disease inception. Here, we present an in vitro human model that simulates the initial apical infection of alveolar epithelium with SARS-CoV-2 by using induced pluripotent stem cell-derived AT2s that have been adapted to air-liquid interface culture. We find a rapid transcriptomic change in infected cells, characterized by a shift to an inflammatory phenotype with upregulation of NF-κB signaling and loss of the mature alveolar program. Drug testing confirms the efficacy of remdesivir as well as TMPRSS2 protease inhibition, validating a putative mechanism used for viral entry in alveolar cells. Our model system reveals cell-intrinsic responses of a key lung target cell to SARS-CoV-2 infection and should facilitate drug development.

SARS-CoV-2 Infection of Pluripotent Stem Cell-derived Human Lung Alveolar Type 2 Cells Elicits a Rapid Epithelial-Intrinsic Inflammatory Response.

The most severe and fatal infections with SARS-CoV-2 result in the acute respiratory distress syndrome, a clinical phenotype of coronavirus disease 2019 (COVID-19) that is associated with virions targeting the epithelium of the distal lung, particularly the facultative progenitors of this tissue, alveolar epithelial type 2 cells (AT2s). Little is known about the initial responses of human lung alveoli to SARS-CoV-2 infection due in part to inability to access these cells from patients, particularly at early stages of disease. Here we present an in vitro human model that simulates the initial apical infection of the distal lung epithelium with SARS-CoV-2, using AT2s that have been adapted to air-liquid interface culture after their derivation from induced pluripotent stem cells (iAT2s). We find that SARS-CoV-2 induces a rapid global transcriptomic change in infected iAT2s characterized by a shift to an inflammatory phenotype predominated by the secretion of cytokines encoded by NF-kB target genes, delayed epithelial interferon responses, and rapid loss of the mature lung alveolar epithelial program. Over time, infected iAT2s exhibit cellular toxicity that can result in the death of these key alveolar facultative progenitors, as is observed in vivo in COVID-19 lung autopsies. Importantly, drug testing using iAT2s confirmed an antiviral dose-response to remdesivir and demonstrated the efficacy of TMPRSS2 protease inhibition, validating a putative mechanism used for viral entry in human alveolar cells. Our model system reveals the cell-intrinsic responses of a key lung target cell to infection, providing a physiologically relevant platform for further drug development and facilitating a deeper understanding of COVID-19 pathogenesis.