Single-cell-resolved interspecies comparison shows a shared inflammatory axis and a dominant neutrophil-endothelial program in severe COVID-19.

A key issue for research on COVID-19 pathogenesis is the lack of biopsies from patients and of samples at the onset of infection. To overcome these hurdles, hamsters were shown to be useful models for studying this disease. Here, we further leverage the model to molecularly survey the disease progression from time-resolved single-cell RNA sequencing data collected from healthy and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-infected Syrian and Roborovski hamster lungs. We compare our data to human COVID-19 studies, including bronchoalveolar lavage, nasal swab, and postmortem lung tissue, and identify a shared axis of inflammation dominated by macrophages, neutrophils, and endothelial cells, which we show to be transient in Syrian and terminal in Roborovski hamsters. Our data suggest that, following SARS-CoV-2 infection, commitment to a type 1- or type 3-biased immunity determines moderate versus severe COVID-19 outcomes, respectively.

TMPRSS2-mediated SARS-CoV-2 uptake boosts innate immune activation, enhances cytopathology, and drives convergent virus evolution.

The accessory protease transmembrane protease serine 2 (TMPRSS2) enhances severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) uptake into ACE2-expressing cells, although how increased entry impacts downstream viral and host processes remains unclear. To investigate this in more detail, we performed infection assays in engineered cells promoting ACE2-mediated entry with and without TMPRSS2 coexpression. Electron microscopy and inhibitor experiments indicated TMPRSS2-mediated cell entry was associated with increased virion internalization into endosomes, and partially dependent upon clathrin-mediated endocytosis. TMPRSS2 increased panvariant uptake efficiency and enhanced early rates of virus replication, transcription, and secretion, with variant-specific profiles observed. On the host side, transcriptional profiling confirmed the magnitude of infection-induced antiviral and proinflammatory responses were linked to uptake efficiency, with TMPRSS2-assisted entry boosting early antiviral responses. In addition, TMPRSS2-enhanced infections increased rates of cytopathology, apoptosis, and necrosis and modulated virus secretion kinetics in a variant-specific manner. On the virus side, convergent signatures of cell-uptake-dependent innate immune induction were recorded in viral genomes, manifesting as switches in dominant coupled Nsp3 residues whose frequencies were correlated to the magnitude of the cellular response to infection. Experimentally, we demonstrated that selected Nsp3 mutations conferred enhanced interferon antagonism. More broadly, we show that TMPRSS2 orthologues from evolutionarily diverse mammals facilitate panvariant enhancement of cell uptake. In summary, our study uncovers previously unreported associations, linking cell entry efficiency to innate immune activation kinetics, cell death rates, virus secretion dynamics, and convergent selection of viral mutations. These data expand our understanding of TMPRSS2's role in the SARS-CoV-2 life cycle and confirm its broader significance in zoonotic reservoirs and animal models.

Pharmacological inhibition of bromodomain and extra-terminal proteins induces an NRF-2-mediated antiviral state that is subverted by SARS-CoV-2 infection.

Inhibitors of bromodomain and extra-terminal proteins (iBETs), including JQ-1, have been suggested as potential prophylactics against SARS-CoV-2 infection. However, molecular mechanisms underlying JQ-1-mediated antiviral activity and its susceptibility to viral subversion remain incompletely understood. Pretreatment of cells with iBETs inhibited infection by SARS-CoV-2 variants and SARS-CoV, but not MERS-CoV. The antiviral activity manifested itself by reduced reporter expression of recombinant viruses, and reduced viral RNA quantities and infectious titers in the culture supernatant. While we confirmed JQ-1-mediated downregulation of expression of angiotensin-converting enzyme 2 (ACE2) and interferon-stimulated genes (ISGs), multi-omics analysis addressing the chromatin accessibility, transcriptome and proteome uncovered induction of an antiviral nuclear factor erythroid 2-related factor 2 (NRF-2)-mediated cytoprotective response as an additional mechanism through which JQ-1 inhibits SARS-CoV-2 replication. Pharmacological inhibition of NRF-2, and knockdown of NRF-2 and its target genes reduced JQ-1-mediated inhibition of SARS-CoV-2 replication. Serial passaging of SARS-CoV-2 in the presence of JQ-1 resulted in predominance of ORF6-deficient variant, which exhibited resistance to JQ-1 and increased sensitivity to exogenously administered type I interferon (IFN-I), suggesting a minimised need for SARS-CoV-2 ORF6-mediated repression of IFN signalling in the presence of JQ-1. Importantly, JQ-1 exhibited a transient antiviral activity when administered prophylactically in human airway bronchial epithelial cells (hBAECs), which was gradually subverted by SARS-CoV-2, and no antiviral activity when administered therapeutically following an established infection. We propose that JQ-1 exerts pleiotropic effects that collectively induce an antiviral state in the host, which is ultimately nullified by SARS-CoV-2 infection, raising questions about the clinical suitability of the iBETs in the context of COVID-19.

90K/LGALS3BP expression is upregulated in COVID-19 but may not restrict SARS-CoV-2 infection.

Glycoprotein 90K, encoded by the interferon-stimulated gene LGALS3BP, displays broad antiviral activity. It reduces HIV-1 infectivity by interfering with Env maturation and virion incorporation, and increases survival of Influenza A virus-infected mice via antiviral innate immune signaling. Its antiviral potential in SARS-CoV-2 infection remains largely unknown. Here, we analyzed the expression of 90K/LGALS3BP in 44 hospitalized COVID-19 patients at multiple levels. We quantified 90K protein concentrations in serum and PBMCs as well as LGALS3BP mRNA levels. Complementary, we analyzed two single cell RNA-sequencing datasets for expression of LGALS3BP in respiratory specimens and PBMCs from COVID-19 patients. Finally, we analyzed the potential of 90K to interfere with SARS-CoV-2 infection of HEK293T/ACE2, Calu-3 and Caco-2 cells using authentic virus. 90K protein serum concentrations were significantly elevated in COVID-19 patients compared to uninfected sex- and age-matched controls. Furthermore, PBMC-associated concentrations of 90K protein were overall reduced by SARS-CoV-2 infection in vivo, suggesting enhanced secretion into the extracellular space. Mining of published PBMC scRNA-seq datasets uncovered monocyte-specific induction of LGALS3BP mRNA expression in COVID-19 patients. In functional assays, neither 90K overexpression in susceptible cell lines nor exogenous addition of purified 90K consistently inhibited SARS-CoV-2 infection. Our data suggests that 90K/LGALS3BP contributes to the global type I IFN response during SARS-CoV-2 infection in vivo without displaying detectable antiviral properties in vitro.

Nonproductive exposure of PBMCs to SARS-CoV-2 induces cell-intrinsic innate immune responses.

Cell-intrinsic responses mounted in PBMCs during mild and severe COVID-19 differ quantitatively and qualitatively. Whether they are triggered by signals emitted by productively infected cells of the respiratory tract or result from physical interaction with virus particles remains unclear. Here, we analyzed susceptibility and expression profiles of PBMCs from healthy donors upon ex vivo exposure to SARS-CoV and SARS-CoV-2. In line with the absence of detectable ACE2 receptor expression, human PBMCs were refractory to productive infection. RT-PCR experiments and single-cell RNA sequencing revealed JAK/STAT-dependent induction of interferon-stimulated genes (ISGs) but not proinflammatory cytokines. This SARS-CoV-2-specific response was most pronounced in monocytes. SARS-CoV-2-RNA-positive monocytes displayed a lower ISG signature as compared to bystander cells of the identical culture. This suggests a preferential invasion of cells with a low ISG baseline profile or delivery of a SARS-CoV-2-specific sensing antagonist upon efficient particle internalization. Together, nonproductive physical interaction of PBMCs with SARS-CoV-2- and, to a much lesser extent, SARS-CoV particles stimulate JAK/STAT-dependent, monocyte-accentuated innate immune responses that resemble those detected in vivo in patients with mild COVID-19.

Vaccine-associated enhanced respiratory pathology in COVID-19 hamsters after TH2-biased immunization.

Vaccine-associated enhanced respiratory disease (VAERD) is a severe complication for some respiratory infections. To investigate the potential for VAERD induction in coronavirus disease 2019 (COVID-19), we evaluate two vaccine leads utilizing a severe hamster infection model: a T helper type 1 (TH1)-biased measles vaccine-derived candidate and a TH2-biased alum-adjuvanted, non-stabilized spike protein. The measles virus (MeV)-derived vaccine protects the animals, but the protein lead induces VAERD, which can be alleviated by dexamethasone treatment. Bulk transcriptomic analysis reveals that our protein vaccine prepares enhanced host gene dysregulation in the lung, exclusively up-regulating mRNAs encoding the eosinophil attractant CCL-11, TH2-driving interleukin (IL)-19, or TH2 cytokines IL-4, IL-5, and IL-13. Single-cell RNA sequencing (scRNA-seq) identifies lung macrophages or lymphoid cells as sources, respectively. Our findings imply that VAERD is caused by the concerted action of hyperstimulated macrophages and TH2 cytokine-secreting lymphoid cells and potentially links VAERD to antibody-dependent enhancement (ADE). In summary, we identify the cytokine drivers and cellular contributors that mediate VAERD after TH2-biased vaccination.

Key benefits of dexamethasone and antibody treatment in COVID-19 hamster models revealed by single-cell transcriptomics.

For coronavirus disease 2019 (COVID-19), effective and well-understood treatment options are still scarce. Since vaccine efficacy is challenged by novel variants, short-lasting immunity, and vaccine hesitancy, understanding and optimizing therapeutic options remains essential. We aimed at better understanding the effects of two standard-of-care drugs, dexamethasone and anti-severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) antibodies, on infection and host responses. By using two COVID-19 hamster models, pulmonary immune responses were analyzed to characterize effects of single or combinatorial treatments. Pulmonary viral burden was reduced by anti-SARS-CoV-2 antibody treatment and unaltered or increased by dexamethasone alone. Dexamethasone exhibited strong anti-inflammatory effects and prevented fulminant disease in a severe disease model. Combination therapy showed additive benefits with both anti-viral and anti-inflammatory potency. Bulk and single-cell transcriptomic analyses confirmed dampened inflammatory cell recruitment into lungs upon dexamethasone treatment and identified a specifically responsive subpopulation of neutrophils, thereby indicating a potential mechanism of action. Our analyses confirm the anti-inflammatory properties of dexamethasone and suggest possible mechanisms, validate anti-viral effects of anti-SARS-CoV-2 antibody treatment, and reveal synergistic effects of a combination therapy, thus informing more effective COVID-19 therapies.

Single-cell analysis of arthritogenic alphavirus-infected human synovial fibroblasts links low abundance of viral RNA to induction of innate immunity and arthralgia-associated gene expression.

Infection by (re-)emerging RNA arboviruses including Chikungunya virus (CHIKV) and Mayaro virus primarily cause acute febrile disease and transient polyarthralgia. However, in a significant subset of infected individuals, debilitating arthralgia persists for weeks over months up to years. The underlying immunopathogenesis of chronification of arthralgia upon primary RNA-viral infection remains unclear. Here, we analysed cell-intrinsic responses to ex vivo arthritogenic alphaviral infection of primary human synovial fibroblasts isolated from knee joints, one the most affected joint types during acute and chronic CHIKV disease. Synovial fibroblasts were susceptible and permissive to alphaviral infection. Base-line and exogenously added type I interferon (IFN) partially and potently restricted infection, respectively. RNA-seq revealed a CHIKV infection-induced transcriptional profile that comprised upregulation of expression of several hundred IFN-stimulated and arthralgia-mediating genes. Single-cell virus-inclusive RNA-seq uncovered a fine-tuned switch from induction to repression of cell-intrinsic immune responses depending on the abundance of viral RNA in an individual cell. Specifically, responses were most pronounced in cells displaying low-to-intermediate amounts of viral RNA and absence of virus-encoded, fluorescent reporter protein expression, arguing for efficient counteraction of innate immunity in cells expressing viral antagonists at sufficient quantities. In summary, cell-intrinsic sensing of viral RNA that potentially persists or replicates at low levels in synovial fibroblasts and other target cell types in vivo may contribute to the chronic arthralgia induced by alphaviral infections. Our findings might advance our understanding of the immunopathophysiology of long-term pathogenesis of RNA-viral infections.

Temporal omics analysis in Syrian hamsters unravel cellular effector responses to moderate COVID-19.

In COVID-19, immune responses are key in determining disease severity. However, cellular mechanisms at the onset of inflammatory lung injury in SARS-CoV-2 infection, particularly involving endothelial cells, remain ill-defined. Using Syrian hamsters as a model for moderate COVID-19, we conduct a detailed longitudinal analysis of systemic and pulmonary cellular responses, and corroborate it with datasets from COVID-19 patients. Monocyte-derived macrophages in lungs exert the earliest and strongest transcriptional response to infection, including induction of pro-inflammatory genes, while epithelial cells show weak alterations. Without evidence for productive infection, endothelial cells react, depending on cell subtypes, by strong and early expression of anti-viral, pro-inflammatory, and T cell recruiting genes. Recruitment of cytotoxic T cells as well as emergence of IgM antibodies precede viral clearance at day 5 post infection. Investigating SARS-CoV-2 infected Syrian hamsters thus identifies cell type-specific effector functions, providing detailed insights into pathomechanisms of COVID-19 and informing therapeutic strategies.