Neural Network-Assisted Humanization of COVID-19 Hamster scRNAseq Data Reveals Matching Severity States in Human Disease (preprint)

Translating findings from animal models to human disease is essential for dissecting disease mechanisms, developing and testing precise therapeutic strategies. The coronavirus disease 2019 (COVID-19) pandemic has highlighted this need, particularly for models showing disease severity-dependent immune responses. Single-cell transcriptomics (scRNAseq) is well poised to reveal similarities and differences between species at the molecular and cellular level with unprecedented resolution. However, computational methods enabling detailed matching are still scarce. Here, we provide a structured scRNAseq-based approach that we applied to scRNAseq from blood leukocytes originating from humans and hamsters affected with moderate or severe COVID-19. Integration of COVID-19 patient data with two hamster models that develop moderate (Syrian hamster, Mesocricetus auratus) or severe (Roborovski hamster, Phodopus roborovskii) disease revealed that most cellular states are shared across species. A neural network-based analysis using variational autoencoders quantified the overall transcriptomic similarity across species and severity levels, showing highest similarity between neutrophils of Roborovski hamsters and severe COVID-19 patients, while Syrian hamsters better matched patients with moderate disease, particularly in classical monocytes. We further used transcriptome-wide differential expression analysis to identify which disease stages and cell types display strongest transcriptional changes. Consistently, hamsters response to COVID-19 was most similar to humans in monocytes and neutrophils. Disease-linked pathways found in all species specifically related to interferon response or inhibition of viral replication. Analysis of candidate genes and signatures supported the results. Our structured neural network-supported workflow could be applied to other diseases, allowing better identification of suitable animal models with similar pathomechanisms across species. Key PointsO_LINeural networks can successfully match disease states between animal models and humans using single-cell data as shown for COVID-19 C_LIO_LIModerately diseased patients best matched Syrian hamster cells; severely diseased patients best matched Roborovski hamster neutrophils C_LI

Single-cell-resolved interspecies comparison identifies a shared inflammatory axis and a dominant neutrophil-endothelial program in severe COVID-19 (preprint)

Key issues for research of COVID-19 pathogenesis are 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 leveraged the model to molecularly survey the disease progression from time-resolved single-cell RNA-sequencing data collected from healthy and SARS-CoV-2-infected Syrian and Roborovski hamster lungs. We compared our data to human COVID-19 studies, including BALF, nasal swab, and post-mortem lung tissue, and identified 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.

Pharmacological inhibition of bromodomain and extra-terminal proteins induces NRF-2-mediated inhibition of SARS-CoV-2 replication and is subject to viral antagonism (preprint)

Inhibitors of bromodomain and extra-terminal proteins (iBETs), including JQ-1, have been suggested as potential therapeutics against SARS-CoV-2 infection. However, molecular mechanisms underlying JQ-1-induced antiviral activity and its susceptibility to viral antagonism remain incompletely understood. iBET treatment transiently inhibited infection by SARS-CoV-2 variants and SARS-CoV, but not MERS-CoV. Our functional assays confirmed JQ-1-mediated downregulation of ACE2 expression and multi-omics analysis uncovered induction of an antiviral NRF-2-mediated cytoprotective response as an additional antiviral component of JQ-1 treatment. Serial passaging of SARS-CoV-2 in the presence of JQ-1 resulted in predominance of ORF6-deficient variants. JQ-1 antiviral activity was transient in human bronchial airway epithelial cells (hBAECs) treated prior to infection and absent when administered therapeutically. We propose that JQ-1 exerts pleiotropic effects that collectively induce a transient antiviral state that is ultimately nullified by an established SARS-CoV-2 infection, raising questions on their clinical suitability in the context of COVID-19.

SARS-CoV-2 entry route impacts a range of downstream viral and cellular processes (preprint)

SARS-CoV-2 entry is promoted by both cell-surface TMPRSS2 and endolysosomal cathepsins. To investigate the impact of differentially routed virions on host and viral processes, lung epithelial cells expressing distinct combinations of entry factors were infected with authentic viruses. Entry route determined early rates of viral replication and transcription, egress and inhibitor sensitivity, with differences observed between virus strains. Transcriptional profiling revealed that induction of innate immunity was correlated to viral genome and transcript abundance in infected cells. Surface entry triggered early activation of antiviral responses, reducing cumulative virion production, while endolysosomal entry delayed antiviral responses and prolonged virus shedding due to extended cell viability. The likely molecular footprints of escape from antiviral effector targeting were also recorded in viral genomes and correlated with entry route-dependent immune status of cells. TMPRSS2 orthologues from diverse mammals, but not zebra fish, facilitated infection enhancement, which was more pronounced for ancestral strains. Leveraging RNA-seq and scRNA-seq datasets from SARS-CoV-2 infected hamsters, we validate aspects of our model in vivo. In summary, we demonstrate that distinct cellular and viral processes are linked to viral entry route, collectively modulating virus shedding, cell-death rates and viral genome evolution.

90K/LGALS3BP Expression is Upregulated in COVID-19 but Does Not Restrict SARS-CoV-2 Infection (preprint)

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. Here, we analyzed the expression of 90K/LGALS3BP in 44 hospitalized COVID-19 patients. 90K protein serum levels 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.

A live attenuated vaccine confers superior mucosal and systemic immunity to SARS-CoV-2 variants (preprint)

Vaccines are a cornerstone in COVID-19 pandemic management. Here, we compare immune responses to and preclinical efficacy of the mRNA vaccine BNT162b2, an adenovirus-vectored spike vaccine, and the live-attenuated-virus vaccine candidate sCPD9 after single and double vaccination in Syrian hamsters. All regimens containing sCPD9 showed superior efficacy. The robust immunity elicited by sCPD9 was evident in a wide range of immune parameters after challenge with heterologous SARS-CoV-2 including rapid viral clearance, reduced tissue damage, fast differentiation of pre-plasmablasts, strong systemic and mucosal humoral responses, and rapid recall of memory T cells from lung tissue. Our results demonstrate that use of live-attenuated vaccines may offer advantages over available COVID-19 vaccines, specifically when applied as booster, and may provide a solution for containment of the COVID-19 pandemic.

Non-productive exposure of PBMCs to SARS-CoV-2 induces cell-intrinsic innate immunity responses (preprint)

Cell-intrinsic responses mounted in vivo 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 are, at least partially, resulting 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. Bulk and single cell RNA-sequencing revealed JAK/STAT-dependent induction of interferon-stimulated genes, but not pro-inflammatory 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 base-line profile or delivery of a SARS-CoV-2-specific sensing antagonist upon efficient particle internalization. Together, non-productive physical interaction of PBMCs with SARS-CoV-2- but not SARS-CoV particles stimulates JAK/STAT-dependent, monocyte-accentuated innate immune responses that resemble those detected in vivo in patients with mild COVID-19.

In contrast to TH2-biased approaches, TH1 COVID-19 vaccines protect Syrian hamsters from severe disease in the absence of dexamethasone-treatable vaccine-associated enhanced respiratory pathology (preprint)

Since December 2019, the novel human coronavirus SARS-CoV-2 has spread globally, causing millions of deaths. Unprecedented efforts have enabled development and authorization of a range of vaccines, which reduce transmission rates and confer protection against the associated disease COVID-19. These vaccines are conceptually diverse, including e.g. classical adjuvanted whole-inactivated virus, viral vectors, and mRNA vaccines. We have analysed two prototypic model vaccines, the strongly TH1-biased measles vaccine-derived candidate MeVvac2-SARS2-S(H) and a TH2-biased Alum-adjuvanted, non-stabilized Spike (S) protein side-by-side, for their ability to protect Syrian hamsters upon challenge with a low-passage SARS-CoV-2 patient isolate. As expected, the MeVvac2-SARS2-S(H) vaccine protected the hamsters safely from severe disease. In contrast, the protein vaccine induced vaccine-associated enhanced respiratory disease (VAERD) with massive infiltration of eosinophils into the lungs. Global RNA-Seq analysis of hamster lungs revealed reduced viral RNA and less host dysregulation in MeVvac2-SARS2-S(H) vaccinated animals, while S protein vaccination triggered enhanced host gene dysregulation compared to unvaccinated control animals. Of note, mRNAs encoding the major eosinophil attractant CCL-11, the TH2 response-driving cytokine IL-19, as well as TH2-cytokines IL-4, IL-5, and IL-13 were exclusively up-regulated in the lungs of S protein vaccinated animals, consistent with previously described VAERD induced by RSV vaccine candidates. IL-4, IL-5, and IL-13 were also up-regulated in S-specific splenocytes after protein vaccination. Using scRNA-Seq, T cells and innate lymphoid cells were identified as the source of these cytokines, while Ccl11 and Il19 mRNAs were expressed in lung macrophages displaying an activated phenotype. Interestingly, the amount of viral reads in this macrophage population correlated with the abundance of Fc-receptor reads. These findings suggest that VAERD is triggered by induction of TH2-type helper cells secreting IL-4, IL-5, and IL-13, together with stimulation of macrophage subsets dependent on non-neutralizing antibodies. Via this mechanism, uncontrolled eosinophil recruitment to the infected tissue occurs, a hallmark of VAERD immunopathogenesis. These effects could effectively be treated using dexamethasone and were not observed in animals vaccinated with MeVvac2-SARS2-S(H). Taken together, our data validate the potential of TH2-biased COVID-19 vaccines and identify the transcriptional mediators that underlie VAERD, but confirm safety of TH1-biased vaccine concepts such as vector-based or mRNA vaccines. Dexamethasone, which is already in use for treatment of severe COVID-19, may alleviate such VAERD, but in-depth scrutiny of any next-generation protein-based vaccine candidates is required, prior and after their regulatory approval.

Insights into standards of care: dexamethasone and antibodies against COVID-19 in hamster models (preprint)

Rationale: In face of the ongoing SARS-CoV-2 pandemic, effective and well-understood treatment options are still scarce. While vaccines have proven instrumental in fighting SARS-CoV-2, their efficacy is challenged by vaccine hesitancy, novel variants and short-lasting immunity. Therefore, understanding and optimization of therapeutic options remains essential. Objectives: We aimed at generating a deeper understanding on how currently used drugs, specifically dexamethasone and anti-SARS-CoV-2 antibodies, affect SARS-CoV-2 infection and host responses. Possible synergistic effects of both substances are investigated to evaluate combinatorial treatments. Methods: By using two COVID-19 hamster models, pulmonary immune responses were analyzed to characterize effects of treatment with either dexamethasone, anti-SARS-CoV-2 spike monoclonal antibody or a combination of both. scRNA sequencing was employed to reveal transcriptional response to treatment on a single cell level. Measurements and main results: Dexamethasone treatment resulted in similar or increased viral loads compared to controls. Anti-SARS-CoV-2 antibody treatment alone or combined with dexamethasone successfully reduced pulmonary viral burden. Dexamethasone exhibited strong anti-inflammatory effects and prevented fulminant disease in a severe COVID-19-like 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 candidate subpopulation of neutrophils specifically responsive to dexamethasone. Conclusions: Our analyses i) confirm the anti-inflammatory properties and indicate possible modes of action for dexamethasone, ii) validate anti-viral effects of anti-SARS-CoV-2 antibody treatment, and iii) reveal synergistic effects of a combination therapy and can thus inform more effective COVID-19 therapies.

Early and rapid identification of COVID-19 patients with neutralizing type I-interferon auto-antibodies by an easily implementable algorithm (preprint)

Purpose Six-19% of critically ill COVID-19 patients display circulating auto-antibodies against type I interferons (IFN-AABs). Here, we establish a clinically applicable strategy for early identification of IFN-AAB-positive patients for potential subsequent clinical interventions. Methods We analysed sera of 430 COVID-19 patients with severe and critical disease from four hospitals for presence of IFN-AABs by ELISA. Binding specificity and neutralizing activity were evaluated via competition assay and virus-infection-based neutralization assay. We defined clinical parameters associated with IFN-AAB positivity. In a subgroup of critically ill patients, we analyzed effects of therapeutic plasma exchange (TPE) on the levels of IFN-AABs, SARS-CoV-2 antibodies and clinical outcome. Results The prevalence of neutralizing AABs to IFN- and IFN-{omega} in COVID-19 patients was 4.2% (18/430), while being undetectable in an uninfected control cohort. Neutralizing IFN-AABs were detectable exclusively in critically affected, predominantly male (83%) patients (7.6% IFN- and 4.6% IFN-{omega} in 207 patients with critical COVID-19). IFN-AABs were present early post-symptom onset and at the peak of disease. Fever and oxygen requirement at hospital admission co-presented with neutralizing IFN-AAB positivity. IFN-AABs were associated with higher mortality (92.3% versus 19.1 % in patients without IFN-AABs). TPE reduced levels of IFN-AABs in three of five patients and may increase survival of IFN-AAB-positive patients compared to those not undergoing TPE. Conclusion IFN-AABs may serve as early biomarker for development of severe COVID-19. We propose to implement routine screening of hospitalized COVID-19 patients according to our algorithm for rapid identification of patients with IFN-AABs who most likely benefit from specific therapies.

Post-entry, spike-dependent replication advantage of B.1.1.7 and B.1.617.2 over B.1 SARS-CoV-2 in an ACE2-deficient human lung cell line (preprint)

Epidemiological data demonstrate that B.1.1.7, and even more, B.1.617.2 SARS-CoV-2 are more transmissible and infections are associated with a higher mortality than B.1 virus infection. Intrinsic properties underlying their enhanced spread in the human population remain unknown. B.1.1.7 virus isolates displayed inferior or equivalent spread in most cell lines and primary cells compared to B.1 SARS-CoV-2, and were outcompeted by the latter. Lower infectivity and delayed entry kinetics of B.1.1.7 viruses were accompanied by inefficient proteolytic processing of spike. B.1.1.7 viruses failed to escape from neutralizing antibodies, but slightly dampened induction of innate immunity. The lung cell line NCI-H1299 supported 24- and 595-fold increased growth of B.1.1.7 and B.1.617.2 viruses, respectively, in the absence of detectable ACE2 expression and in a spike-determined fashion. Superior spread in ACE2-deficient NCI-H1299 cells suggests that variants of concern employ a distinct set of cellular cofactors that may be unavailable in standard cell culture lines.

A realistic touch-transfer method reveals low risk of transmission for SARS-CoV-2 by contaminated coins and bank notes (preprint)

The current severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic has created a significant threat to global health. While respiratory aerosols or droplets are considered as the main route of human-to-human transmission, secretions expelled by infected individuals can also contaminate surfaces and objects, potentially creating the risk of fomite-based transmission. Consequently, frequently touched objects such as paper currency and coins have been suspected as a potential transmission vehicle. To assess the risk of SARS-CoV-2 transmission by banknotes and coins, we examined the stability of SARS-CoV-2 and bovine coronavirus (BCoV), as surrogate with lower biosafety restrictions, on these different means of payment and developed a touch transfer method to examine transfer efficiency from contaminated surfaces to skin. Although we observed prolonged virus stability, our results, including a novel touch transfer method, indicate that the transmission of SARS-CoV-2 via contaminated coins and banknotes is unlikely and requires high viral loads and a timely order of specific events.

Longitudinal omics in Syrian hamsters integrated with human data unravel cellular effector responses to moderate COVID-19 (preprint)

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 model for moderate COVID-19, we conducted a detailed longitudinal analysis of systemic and pulmonary cellular responses, and corroborated it with datasets from COVID-19 patients. Monocyte-derived macrophages in lungs exerted the earliest and strongest transcriptional response to infection, including induction of pro-inflammatory genes, while epithelial cells showed weak activation. Without evidence for productive infection, endothelial cells reacted, 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 preceded viral clearance at day 5 post infection. Investigating SARS-CoV-2 infected Syrian hamsters can thus identify cell type-specific effector functions, provide detailed insights into pathomechanisms of COVID-19, and inform therapeutic strategies.

Longitudinal omics in Syrian hamsters integrated with human data unravel complexity of moderate immune responses to SARS-CoV-2 (preprint)

In COVID-19, the immune response largely determines disease severity and is key to therapeutic strategies. Cellular mechanisms contributing to inflammatory lung injury and tissue repair in SARS-CoV-2 infection, particularly endothelial cell involvement, remain ill-defined. We performed detailed spatiotemporal analyses of cellular and molecular processes in SARS-CoV-2 infected Syrian hamsters. Comparison of hamster single-cell sequencing and proteomics with data sets from COVID-19 patients demonstrated inter-species concordance of cellular and molecular host-pathogen interactions. In depth vascular and pulmonary compartment analyses (i) supported the hypothesis that monocyte-derived macrophages dominate inflammation, (ii) revealed endothelial inflammation status and T-cell attraction, and (iii) showed that CD4+ and CD8+ cytotoxic T-cell responses precede viral elimination. Using the Syrian hamster model of self-limited moderate COVID-19, we defined the specific roles of endothelial and epithelial cells, among other myeloid and non-myeloid lung cell subtypes, for determining the disease course.

A comprehensive library of fluorescent constructs of SARS-CoV-2 proteins and their initial characterization in different cell types (preprint)

Comprehensive libraries of plasmids for SARS-CoV-2 proteins with various tags (e.g. Strep, HA, Turbo) are now available. They enable the identification of numerous potential protein-protein interactions between the SARS-CoV-2 virus and host proteins. To facilitate further cellular investigations, notably by imaging techniques, we present here a large library of SARS CoV-2 protein constructs fused with green and red fluorescent proteins and their initial characterization in various human cell lines including lung epithelial cell models (A549, BEAS-2B), as well as in budding yeast. The localization of a few SARS-CoV-2 proteins matches their proposed interactions with host proteins. These include the localization of Nsp13 to the centrosome, Orf3a to late endosomes, and Orf9b to mitochondria.

Genetic Conservation of SARS-CoV-2 RNA Replication Complex in Globally Circulating Isolates from Humans and Minks Predicts Minimal Pre-Existing Resistance to Remdesivir (preprint)

Remdesivir (RDV) exhibits potent antiviral activity against SARS-CoV-2 and is currently the only drug approved for the treatment of COVID-19. However, little is currently known about the potential for pre-existing resistance to RDV and the possibility of SARS-CoV-2 genetic diversification that might impact RDV efficacy as the virus continue to spread globally. In this study, > 90,000 SARS-CoV-2 sequences from globally circulating clinical isolates and >300 from mink isolates collected through early September 2020 were analyzed for genetic diversity in the RNA replication complex (nsp7, nsp8, nsp10, nsp12, nsp13, and nsp14) with a focus on the RNA-dependent RNA polymerase (nsp12), the molecular target of RDV. Overall, low genetic variation was observed with only 12 amino acid substitutions present in the entire RNA replication complex in [≥]0.5% of analyzed sequences with the highest overall frequency (82.2%) observed for nsp12 P323L that consistently increased over time. Low sequence variation in the RNA replication complex was also observed among the mink isolates. Importantly, the coronavirus Nsp12 mutations previously selected in vitro in the presence of RDV were identified in only 2 isolates (0.002%) within all the analyzed sequences. In addition, among the sequence variants observed in [≥]0.5% clinical isolates, including P323L, none were located near the established polymerase active site or sites critical for the RDV mechanism of inhibition. In summary, the low diversity and high genetic stability of the RNA replication complex observed over time predicts a minimal global risk of pre-existing SARS-CoV-2 resistance to RDV.

IFITM proteins promote SARS-CoV-2 infection and are targets for virus inhibition (preprint)

Interferon-induced transmembrane proteins (IFITMs 1, 2 and 3) are thought to restrict numerous viral pathogens including severe acute respiratory syndrome coronaviruses (SARS-CoVs). However, most evidence comes from single-round pseudovirus infection studies of cells that overexpress IFITMs. Here, we verified that artificial overexpression of IFITMs blocks SARS-CoV-2 infection. Strikingly, however, endogenous IFITM expression was essential for efficient infection of genuine SARS-CoV-2 in human lung cells. Our results indicate that the SARS-CoV-2 Spike protein interacts with IFITMs and hijacks them for efficient viral entry. IFITM proteins were expressed and further induced by interferons in human lung, gut, heart and brain cells. Intriguingly, IFITM-derived peptides and targeting antibodies inhibited SARS-CoV-2 entry and replication in human lung cells, cardiomyocytes and gut organoids. Our results show that IFITM proteins are important cofactors for SARS-CoV-2 infection of human cell types representing in vivo targets for viral transmission, dissemination and pathogenesis and suitable targets for therapeutic approaches.

Human Lungs Show Limited Permissiveness for SARS-CoV-2 Due to Scarce ACE2 Levels But Strong Virus-Induced Immune Activation in Alveolar Macrophages (preprint)

SARS-CoV-2 utilizes the ACE2 transmembrane peptidase as essential cellular entry receptor. Several studies have suggested abundant ACE2 expression in the human lung, inferring strong permissiveness to SARS-CoV-2 infection with resultant alveolar damage and lung injury. Against this expectation, we provide evidence that ACE2 expression must be considered scarce, thereby limiting SARS-CoV-2 propagation in the human alveolus. Instead, spectral imaging of ex vivo infected human lungs and COVID-19 autopsy samples depicted that alveolar macrophages were frequently positive for SARS-CoV-2, indicating viral phagocytosis. Single-cell transcriptomics of SARS-CoV-2 infected human lung tissue further revealed strong inflammatory and anti-viral activation responses in macrophages and monocytes, comparable to those induced by MERS-CoV, but with virus-specific gene expression profiles. Collectively, our findings indicate that severe lung injury in COVID-19 likely results from an overwhelming immune activation rather than direct viral damage of the alveolar compartment.Funding: ACH, LES, SH were supported by Berlin University Alliance GC2 Global Health (Corona Virus Pre-Exploration Project). ACH, SH, TW and CD were supported by BMBF (RAPID) and ACH, SH by BMBF (alvBarriereCOVID-19). KH, LB, SL, SH, CD, TW, ACH were funded by BMBF (NFN-COVID 19, Organo-Strat). KH, NS, LES, MW, SH, ADG, CD, TW and ACH were supported by DFG (SFB-TR 84). ACH was supported by BIH, Charite 3R, and Charité-Zeiss MultiDim. KH was supported by BMBF (Camo-COVID-19). MW, NS and SH was supported by BMBF (PROVID). MW and NS was supported by BIH and BMBF (SYMPATH, CAPSyS, NAPKON). BO and DB were funded through the BIH Clinical Single Cell Bioinformatics Pipeline. LB was supported by the BMBF (CoIMMUNE), the DFG (KFO 342) and the IZKF of the Medical Faculty of the WWU. Conflict of Interest: The authors declare no competing interests.Ethical Approval: The study was approved by the ethics committee at the Charité clinic (projects EA2/079/13) and Ärztekammer Westfalen-Lippe and of the Westfälischen Wilhelms-Universität (AZ: 2016-265-f-S). Written informed consent was obtained from all patients.

Delayed viral clearance and exacerbated airway hyperinflammation in hypertensive COVID-19 patients (preprint)

In COVID-19, hypertension and cardiovascular diseases have emerged as major risk factors for critical disease progression. Concurrently, the impact of the main anti-hypertensive therapies, angiotensin-converting enzyme inhibitors (ACEi) and angiotensin receptor blockers (ARB), on COVID-19 severity is controversially discussed. By combining clinical data, single-cell sequencing data of airway samples and in vitro experiments, we assessed the cellular and pathophysiological changes in COVID-19 driven by cardiovascular disease and its treatment options. Anti-hypertensive ACEi or ARB therapy, was not associated with an altered expression of SARS-CoV-2 entry receptor ACE2 in nasopharyngeal epithelial cells and thus presumably does not change susceptibility for SARS-CoV-2 infection. However, we observed a more critical progress in COVID-19 patients with hypertension associated with a distinct inflammatory predisposition of immune cells. While ACEi treatment was associated with dampened COVID-19-related hyperinflammation and intrinsic anti-viral responses, under ARB treatment enhanced epithelial-immune cell interactions were observed. Macrophages and neutrophils of COVID-19 patients with hypertension and cardiovascular comorbidities, in particular under ARB treatment, exhibited higher expression of CCL3, CCL4, and its receptor CCR1, which associated with critical COVID-19 progression. Overall, these results provide a potential explanation for the adverse COVID-19 course in patients with cardiovascular disease, i.e. an augmented immune response in critical cells for the disease course, and might suggest a beneficial effect of clinical ACEi treatment in hypertensive COVID-19 patients.

IFITM proteins promote SARS-CoV-2 infection in human lung cells (preprint)

Interferon-induced transmembrane proteins (IFITMs 1, 2 and 3) restrict numerous viral pathogens and are thought to prevent infection by severe acute respiratory syndrome coronaviruses (SARS-CoVs). However, most evidence comes from single-round pseudoparticle infection of cells artificially overexpressing IFITMs. Here, we confirmed that overexpression of IFITMs blocks pseudoparticle infections mediated by the Spike proteins of {beta}-coronaviruses including pandemic SARS-CoV-2. In striking contrast, however, endogenous IFITM expression promoted genuine SARS-CoV-2 infection in human lung cells both in the presence and absence of interferon. IFITM2 was most critical for efficient entry of SARS-CoV-2 and enhanced virus production from Calu-3 cells by several orders of magnitude. IFITMs are expressed and further induced by interferons in the lung representing the primary site of SARS-CoV-2 infection as well as in other relevant tissues. Our finding that IFITMs enhance SARS-CoV-2 infection under conditions approximating the in vivo situation shows that they may promote viral invasion during COVID-19. HIGHLIGHTSO_LIOverexpression of IFITM1, 2 and 3 restricts SARS-CoV-2 infection C_LIO_LIEndogenous IFITM1, 2 and 3 boost SARS-CoV-2 infection of human lung cells C_LIO_LIIFITM2 is critical for efficient entry of SARS-CoV-2 in Calu-3 cells C_LI