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.

Complement Activation Induces Excessive T Cell Cytotoxicity in Severe COVID-19 (preprint)

Severe COVID-19 is linked to both dysfunctional immune response and unrestrained immunopathogenesis, and it remains unclear if T cells contribute to disease pathology. Here, we combined single-cell transcriptomics and proteomics with mechanistic studies assessing pathogenic T cell functions and inducing signals. We identified activated, CD16+ T cells with increased cytotoxic functions in severe COVID-19. CD16 expression enabled immune complex-mediated, T cell receptor-independent degranulation and cytotoxicity not found in other diseases. CD16+ T cells from COVID-19 patients promoted microvascular endothelial cell injury and release of neutrophil and monocyte chemoattractants. CD16+ T cell clones persisted beyond acute disease maintaining their cytotoxic phenotype. Age-dependent generation of C3a in severe COVID-19 induced activated CD16 + cytotoxic T cells. The proportion of activated CD16+ T cells and plasma levels of complement proteins upstream of C3a correlated with clinical outcome, supporting a pathological role of exacerbated cytotoxicity and complement activation in COVID-19.Funding: This work was supported by the German Research Foundation (DFG): SA1383/3-1 to B.S.; SFB-TR84 114933180 to L.E.S., S.B., P.G., S.H. and W.M.K. INST 37/1049-1, INST 216/981- 1, INST 257/605-1, INST 269/768-1, INST 217/988-1, INST 217/577-1, and EXC2151- 390873048 to J.L.S.; GRK 2168 – 272482170, ERA CVD (00160389 to J.L.S.; SFB 1454 – 432325352 to A.C.A. and J.L.S.; SFB TR57 and SPP1937 to J.N.; GRK2157 to A.-E.S.; and ME 3644/5-1 to H.E.M.; RTG2424 to N.B.; SFB-TRR219 322900939, BO3755/13-1 Project- ID 454024652 to P.B.; the Berlin University Alliance (BUA) (PreEP-Corona grant to L.E.S. and V.M.C.); the Berlin Institute of Health (BIH) (to L.E.S., V.M.C.,B.S. and W.M.K.); Helmholtz- Gemeinschaft Deutscher Forschungszentren, Germany (sparse2big to J.L.S.), EU projects SYSCID (733100 to J.L.S.); European Research Council Horizon 2020 (grant agreement No 101001791 to P.B.); the DZIF, Germany (TTU 04.816 and 04.817 to J.N.); the Hector Foundation (M89 to J.N.); the EU projects ONE STUDY (260687), BIO-DrIM (305147) and INsTRuCT (860003) to B.S.); German Registry of COVID-19 Autopsies through Federal Ministry of Health (ZMVI1-2520COR201 to P.B.); Federal Ministry of Education and Research (DEFEAT PANDEMICs, 01KX2021 and STOP-FSGS-01GM1901A to P.B.); the Berlin Senate to German Rheumatism Research Centre (DRFZ); the Berlin Brandenburg School for regenerative Therapies (BSRT) to C.B.; the German Federal Ministry of Education and Research (BMBF) projects RECAST (01KI20337) to B.S., V.M.C., L.E.S and M.R.; VARIPath (01KI2021) to V.M.C.; NUM COVIM (01KX2021) to L.E.S., V.M.C., F.K., J.L.S., J.N. and B.S.; RAPID to and S.H.,; SYMPATH to N.S. and W.M.K.; PROVID to S.H. and W.M.K.; ZissTrans (02NUK047E) to N.B; National Research Node ‘Mass spectrometry in Systems Medicine (MSCoresys) (031L0220A) to M.R. and N.B.; Diet–Body–Brain (DietBB) (01EA1809A) to J.L.S.; the UKRI/NIHR through the UK Coronavirus Immunology Consortium (UK-CIC), the Francis Crick Institute through the Cancer Research UK (FC001134), the UK Medical Research Council (FC001134), the Wellcome Trust (FC001134 and IA 200829/Z/16/Z) to M.R.; a Charité 3R project (to B.S., S.H., W.M.K.); and an intramural grant from the Department of Genomics & Immunoregulation at the LIMES Institute to A.C.A. We are grateful to the patients and donors volunteering to participate in this study making this research possible in the first place and wish for a speedy and full recovery.Conflict of Interest: V.M.C. is named together with Euroimmun GmbH on a patent application filed recently regarding SARS-CoV-2 diagnostics via antibody testing. A.R.S. and H.E.M. are listed asinventors on a patent application by the DRFZ Berlin in the field of mass cytometry.Ethical Approval: The study was approved by the Institutional Review board of Charité(EA2/066/20).

Complement activation induces excessive T cell cytotoxicity in severe COVID-19 (preprint)

Severe COVID-19 is linked to both dysfunctional immune response and unrestrained immunopathogenesis, and it remains unclear if T cells also contribute to disease pathology. Here, we combined single-cell transcriptomics and proteomics with mechanistic studies to assess pathogenic T cell functions and inducing signals. We identified highly activated, CD16+ T cells with increased cytotoxic functions in severe COVID-19. CD16 expression enabled immune complex-mediated, T cell receptor-independent degranulation and cytotoxicity not found in other diseases. CD16+ T cells from COVID-19 patients promoted microvascular endothelial cell injury and release of neutrophil and monocyte chemoattractants. CD16+ T cell clones persisted beyond acute disease maintaining their cytotoxic phenotype. Age-dependent generation of C3a in severe COVID-19 induced activated CD16+ cytotoxic T cells. The proportion of activated CD16+ T cells and plasma levels of complement proteins upstream of C3a correlated with clinical outcome of COVID-19, supporting a pathological role of exacerbated cytotoxicity and complement activation in COVID-19.

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.

Remdesivir-Ivermectin combination displays synergistic interaction with improved in vitro antiviral activity against SARS-CoV-2 (preprint)

A key element to the prevention and management of the COVID-19 pandemic is the development of effective therapeutics. Drug combination strategies of repurposed drugs offer a number of advantages to monotherapies including the potential to achieve greater efficacy, the potential to increase the therapeutic index of drugs and the potential to reduce the emergence of drug resistance. Combination of agents with antiviral mechanisms of action with immune-modulatory or anti-inflammatory drug is also worthy of investigation. Here, we report on the in vitro synergistic interaction between two FDA approved drugs, remdesivir (RDV) and ivermectin (IVM) resulting in enhanced antiviral activity against SARS-CoV-2, the causative pathogen of COVID-19. These findings warrant further investigations into the clinical potential of this combination, together with studies to define the underlying mechanism.

HLA-C* 04:01 is a Genetic Risk Allele for Severe Course of COVID-19 (preprint)

Background Since the beginning of the coronavirus disease 2019 (COVID-19) pandemic, there has been increasing demand to identify predictors of severe clinical course in patients infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Human leukocyte antigen alleles (HLA) have been suggested as potential genetic host factors. We sought to evaluate this hypothesis by conducting an international multicenter study using HLA sequencing with subsequent independent validation. Methods We analyzed a total of 332 samples. First, we enrolled 233 patients in Germany, Spain, and Switzerland for HLA and whole exome sequencing. Furthermore, we validated our results in a public data set (United States, n=99). Patients older than 18 years presenting with COVID-19 were included, representing the full spectrum of the disease. HLA candidate alleles were identified in the derivation cohort (n=92) and tested in two independent validation cohorts (n=240). Results We identified HLA-C* 04:01 as a novel genetic predictor for severe clinical course in COVID-19. Carriers of HLA-C* 04:01 had twice the risk of intubation when infected with SARS-CoV-2 (hazard ratio 2.1, adjusted p-value=0.0036). Importantly, these findings were successfully replicated in an independent data set. Furthermore, our findings are biologically plausible, as HLA-C* 04:01 has fewer predicted bindings sites with relevant SARS-CoV-2 peptides as compared to other HLA alleles. Exome sequencing confirmed findings from HLA analysis. Conclusions HLA-C* 04:01 carriage is associated with a twofold increased risk of intubation in patients infected with SARS-CoV-2. Testing for HLA-C* 04:01 could have clinical implications to identify high-risk patients and individualize management.

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.

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.