Elevated plasma Complement Factor H Regulating Protein 5 is associated with venous thromboembolism and COVID-19 severity (preprint)

Venous thromboembolism (VTE), comprising both deep vein thrombosis (DVT) and pulmonary embolism (PE) is a common, multi-causal disease with potentially serious short- and long-term complications. In clinical practice, there is a need for improved plasma biomarker-based tools for VTE diagnosis and risk prediction. We used multiplex proteomics profiling to screen plasma from patients with suspected acute VTE, and a case-control study of patients followed up after ending anticoagulant treatment for a first VTE. With replication in 5 independent studies, together totalling 1137 patients and 1272 controls, we identify Complement Factor H Related Protein (CFHR5), a regulator of the alternative pathway of complement activation, as a novel VTE associated plasma biomarker. Using GWAS analysis of 2967 individuals we identified a genome-wide significant pQTL signal on chr1q31.3 associated with CFHR5 levels. We showed that higher CFHR5 levels are associated with increased thrombin generation in patient plasma and that recombinant CFHR5 enhances platelet activation in vitro. Thrombotic complications are a frequent feature of COVID-19; in hospitalised patients we found CFHR5 levels at baseline were associated with short-time prognosis of disease severity, defined as maximum level of respiratory support needed during hospital stay. Our results indicate a clinically important role for regulation of the alternative pathway of complement activation in the pathogenesis of VTE and pulmonary complications in acute COVID-19. Thus, CFHR5 is a potential diagnostic and/or risk predictive plasma biomarker reflecting underlying pathology in VTE and acute COVID-19.

Replication of SARS-CoV-2 in Adipose Tissue Determines Organ and Systemic Lipid Metabolism in Hamsters and Humans (preprint)

Obesity increases the risk for poor outcome in patients with coronavirus disease-19 (COVID-19). However, the role of adipose tissue for viral propagation and potential metabolic implications are not understood. We detected SARS-CoV-2 in adipose tissue of overweight but not lean male COVID-19 patients. SARS-CoV-2 replicates to high titres in cultured mature adipocytes, a process depending on lipid accumulation and mobilization. After intranasal inoculation, we observed high viral replication in fat depots of Golden Syrian hamsters, demonstrating dissemination from the respiratory tract and subsequent propagation in adipose tissue. Following induction of pro-inflammatory responses, expression of de novo lipogenesis enzymes was suppressed in adipose tissue. This specific down-regulation was reflected by lipidomic alterations in plasma of SARS-CoV-2 infected hamsters as well as in hospitalized COVID-19 patients. Overall, our study highlights that adipose tissue is an important site of SARS-CoV-2 replication, contributing to dysregulation of systemic lipid metabolism.Funding: This study was supported by a rapid response grant from the Federal Ministry of Health (BMG; ZMV I 1-2520COR501 to GG), by DFG grants SCHE522/4-1 (LS) and SFB1328, project- ID:335447727 (JH). As part of the National Network University Medicine (NUM) funded by the Federal Ministry of Education and Research (BMBF, Germany), this work was funded within the research consortium DEFEAT PANDEMIcs, grant number 01KX2021 (FH, PL, KP, BO).Declaration of Interests: The authors declare no competing interests.Ethics Approval Statement: The Ethics Committee of the Hamburg Chamber of Physicians reviewed and approved the studies (PV7311, 2020-10353-BO-ff, WF-051/20, WF-053/20). For the preparation of primary human white adipocytes, biopsies of subcutaneous and visceral adipose tissues were taken during bariatric surgery at the Department of General, Visceral and Thoracic Surgery, University Medical Center Hamburg-Eppendorf. All participants signed an informed consent and the study was approved by the Ethics Committee of the Hamburg Chamber of Physicians (PV4889).

Digital PCR to quantify ChAdOx1 nCoV-19 copies in blood and tissues (preprint)

Vaccination with the adenoviral-vector based Astra Zeneca ChAdOx1 nCov-19 vaccine is efficient and safe. However, in rare cases vaccinated individuals developed life-threatening thrombotic complications, including thrombosis in cerebral sinus and splanchnic veins. Monitoring of the applied vector in vivo represents an important precondition to study the molecular mechanisms underlying vaccine-driven adverse effects now referred to as vaccine-induced immune thrombotic thrombocytopenia (VITT). We previously have shown that digital PCR is an excellent tool to quantify transgene copies in vivo . Here we present a highly sensitive digital PCR for in-situ quantification of ChAdOx1 nCoV-19 copies. Using this method, we quantified vector copies in human serum 24, 72 and 168 hours post vaccination, and in a variety of murine tissues in an experimental vaccination model 30 minutes post injection. We describe a method for high-sensitivity quantitative detection of ChAdOx1 nCoV-19 with possible implications to elucidate the mechanisms of severe ChAdOx1 nCov-19 vaccine complications.

Sex hormone dysregulations are associated with disease severity in critically ill male COVID-19 patients - a retrospective analysis (preprint)

BACKGROUNDMale sex was repeatedly identified as a risk factor for death and intensive care admission. However, it is yet unclear whether sex hormones are associated with disease severity in COVID-19 patients. We sought to characterize sex differences in hormone levels and cytokine responses in critically ill COVID-19 patients.METHODSWe performed a retrospective cohort study of critically ill COVID-19 patients. Males and females were compared. Multivariate regression was performed to assess the association between sex hormones, cytokine responses and the requirement for extracorporeal membrane oxygenation (ECMO) treatment.RESULTSWe analyzed sex hormone levels (estradiol and testosterone) of n=181 male and female individuals. These consisted of n=50 critically ill COVID-19 patients (n=39 males, n=11 females), n=42 critically ill non-COVID-19 patients (n=27 males, n=15 females), n=39 non-COVID-19 patients with coronary heart diseases (CHD) (n=25 males, n=14 females) and n=50 healthy individuals (n=30 males, n=20 females). We detected highest estradiol levels in critically ill male COVID-19 patients compared to non-COVID-19 patients (p=0.0123), patients with CHD (p=0.0002) or healthy individuals (p=0.0007). Lowest testosterone levels were detected in critically ill male COVID-19 patients compared to non-COVID-19 patients (p=0.0094), patients with CHD (p=0.0068) or healthy individuals (p<0.0001). No statistically significant differences in sex hormone levels were detected in critically ill female COVID-19 patients, albeit similar trends in estradiol levels were observed. In critically ill male COVID-19 patients, cytokine and chemokine responses (IFN-γ, p=0.0301; IL-1RA, p=0.0160; IL-6, p=0.0145; MCP-1, p=0.0052; MIP-1α, p=0.0134) were significantly elevated in those with higher Sequential Organ Failure Assessment (SOFA) scores (8-11). Linear regression analysis revealed that herein IFN-γ levels correlate with estradiol levels in male and female COVID-19 patients (R2=0.216, =0.0009). Male COVID-19 patients with elevated estradiol levels were more likely to receive ECMO treatment in the course of their ICU stay (p=0.0009). CONCLUSIONS We identified high estradiol and low testosterone levels as a hallmark of critically ill male COVID-19 patients. Elevated estradiol levels in critically ill male COVID-19 patients were positively associated with IFN-γ levels and increased risk for ECMO requirement. 

Towards Understanding ChAdOx1 nCov-19 Vaccine-induced Immune Thrombotic Thrombocytopenia (VITT) (preprint)

BackgroundSARS-CoV-2 vaccine ChAdOx1 nCov-19 rarely causes vaccine-induced immune thrombotic thrombocytopenia (VITT) that—like autoimmune heparin-induced thrombocytopenia—is mediated by platelet-activating anti-platelet factor 4 (PF4) antibodies.MethodsWe investigated vaccine, PF4, and VITT patient-derived anti-PF4 antibody interactions using dynamic light scattering, 3D-super-resolution microscopy, and electron microscopy. Mass spectrometry was used to analyze vaccine composition. We investigated the mechanism for early post-vaccine inflammatory reactions as potential co-stimulant for anti-PF4 immune response. Finally, we evaluated VITT antibodies for inducing release of procoagulant DNA-containing neutrophil extracellular traps (NETs), and measured DNase activity in VITT patient serum.ResultsBiophysical analyses showed formation of complexes between PF4 and vaccine constituents, including virus proteins that were recognized by VITT antibodies. EDTA, a vaccine constituent, increased microvascular leakage in mice allowing for circulation of virus- and virus-producing cell culture-derived proteins. Antibodies in normal sera cross-reacted with human proteins in the vaccine and likely contribute to commonly observed acute ChAdOx1 nCov-19 post-vaccination inflammatory reactions. Polyphosphates and DNA enhanced PF4-dependent platelet activation by VITT antibodies. In the presence of platelets, PF4 enhanced VITT antibody-driven procoagulant NETs formation, while DNase activity was reduced in VITT sera, with granulocyte-rich cerebral vein thrombosis observed in a VITT patient.ConclusionsChAdOx1 nCoV-19 vaccine constituents (i) form antigenic complexes with PF4, (ii) EDTA increases microvascular permeability, and (iii) vaccine components cause acute inflammatory reactions. Antigen formation in a proinflammatory milieu offers an explanation for anti-PF4 antibody production. High-titer anti-PF4 antibodies activate platelets and induce neutrophil activation and NETs formation, fueling the VITT prothrombotic response.

Design of Specific Primer Set for Detection of B.1.1.7 SARS-CoV-2 Variant using Deep Learning (preprint)

The SARS-CoV-2 variant B.1.1.7 lineage, also known as clade GR from Global Initiative on Sharing All Influenza Data (GISAID), Nextstrain clade 20B, or Variant Under Investigation in December 2020 (VUI - 202012/01), appears to have an increased transmissability in comparison to other variants. Thus, to contain and study this variant of the SARS-CoV-2 virus, it is necessary to develop a specific molecular test to uniquely identify it. Using a completely automated pipeline involving deep learning techniques, we designed a primer set which is specific to SARS-CoV-2 variant B.1.1.7 with >99% accuracy, starting from 8,923 sequences from GISAID. The resulting primer set is in the region of the synonymous mutation C16176T in the ORF1ab gene, using the canonical sequence of the variant B.1.1.7 as a reference. Further in-silico testing shows that the primer set's sequences do not appear in different viruses, using 20,571 virus samples from the National Center for Biotechnology Information (NCBI), nor in other coronaviruses, using 487 samples from National Genomics Data Center (NGDC). In conclusion, the presented primer set can be exploited as part of a multiplexed approach in the initial diagnosis of Covid-19 patients, or used as a second step of diagnosis in cases already positive to Covid-19, to identify individuals carrying the B.1.1.7 variant.

Defective NET clearance contributes to sustained FXII activation in COVID-19-associated pulmonary thrombo-inflammation (preprint)

Background Coagulopathy and inflammation are hallmarks of Coronavirus disease 2019 (COVID-19) and are associated with increased mortality. Clinical and experimental data have revealed a role for neutrophil extracellular traps (NETs) in COVID-19 disease. The mechanisms that drive thrombo-inflammation in COVID-19 are poorly understood. Methods We performed proteomic analysis and immunostaining of postmortem lung tissues from COVID-19 patients and patients with other lung pathologies. We further compared coagulation factor XII (FXII) and DNase activities in plasma samples from COVID-19 patients and healthy control donors and determined NET-induced Factor XIII (FXII) activation using a chromogenic substrate assay. Findings FXII expression and activity were increased in the lung parenchyma, within the pulmonary vasculature and in fibrin-rich alveolar spaces of postmortem lung tissues from COVID-19 patients. In agreement with this, plasma FXII activation (FXIIa) was increased in samples from COVID-19 patients. Furthermore, FXIIa colocalized with NETs in COVID-19 lung tissue indicating that NETs accumulation leads to FXII contact activation in COVID-19. We further showed that an accumulation of NETs is partially due to impaired NET clearance by extracellular DNases as DNase substitution improved NET dissolution and reduced FXII activation in vitro . Interpretation Collectively, our study supports that the NETs/FXII axis contributes to the pathogenic chain of procoagulant and proinflammatory responses in COVID-19. Targeting both, NETs and FXIIa, could provide a strategy to mitigate COVID-19-induced thrombo-inflammation. Funding This study was supported by the European Union (840189), the Werner Otto Medical Foundation Hamburg (8/95) and the German Research Foundation (FR4239/1-1, A11/SFB877, B08/SFB841 and P06/KFO306).

Structural basis for broad coronavirus neutralization (preprint)

Three highly pathogenic {beta}-coronaviruses crossed the animal-to-human species barrier in the past two decades: SARS-CoV, MERS-CoV and SARS-CoV-2. SARSCoV-2 has infected more than 64 million people worldwide, claimed over 1.4 million lives and is responsible for the ongoing COVID-19 pandemic. We isolated a monoclonal antibody, termed B6, cross-reacting with eight {beta}-coronavirus spike glycoproteins, including all five human-infecting {beta}-coronaviruses, and broadly inhibiting entry of pseudotyped viruses from two coronavirus lineages. Cryoelectron microscopy and X-ray crystallography characterization reveal that B6 binds to a conserved cryptic epitope located in the fusion machinery and indicate that antibody binding sterically interferes with spike conformational changes leading to membrane fusion. Our data provide a structural framework explaining B6 cross-reactivity with {beta}-coronaviruses from three lineages along with proof-of-concept for antibody-mediated broad coronavirus neutralization elicited through vaccination. This study unveils an unexpected target for next-generation structure-guided design of a pan-coronavirus vaccine.

Non-covalent TMPRSS2 inhibitors identified from virtual screening (preprint)

The SARS-CoV-2 pandemic has prompted researchers to pivot their efforts to finding anti-viral compounds and vaccines. In this study, we focused on the human host cell transmembrane protease serine 2 (TMPRSS2), which plays an important role in the viral life cycle by cleaving the spike protein to initiate membrane fusion. TMPRSS2 is an attractive target and has received significant attention for the development of drugs against SARS and MERS. Starting with comparative structural modeling and binding model analysis, we developed an efficient pharmacophore-based approach and applied in a large-scale in silico database screening for small molecule inhibitors against TMPRSS2. A number of novel inhibitors were identified, providing starting points for further development of drug candidates for the treatment of COVID-19.

Environmental Dependence of the Structure of the C-terminal Domain of the SARS-CoV-2 Envelope Protein (preprint)

The SARS-CoV-2 envelope protein (E) is involved in a broad spectrum of functions in the cycle of the virus, including assembly, budding, envelope formation, and pathogenesis. To enable these activities, E is likely to be capable of changing its conformation depending on environmental cues. To investigate this issue, here we characterised the structural properties of the C-terminal domain of E (E-CTD), which has been reported to interact with host cell membranes. We first studied the conformation of the E-CTD in solution, finding characteristic features of a disordered protein. By contrast, in the presence of large unilamellar vesicles and micelles, which mimic cell membranes, the E-CTD was observed to become structured. The E-CTD was also found to display conformational changes with osmolytes. Furthermore, prolonged incubation of the E-CTD under physiological conditions resulted in amyloid-like fibril formation. Taken together, these findings indicate that the E-CTD can change its conformation depending on its environment, ranging from a disordered state, to a membrane-bound folded state, and an amyloid state. Our results thus provide insight into the structural basis of the role of E in the viral infection process.

The majority of male patients with COVID-19 present low testosterone levels on admission to Intensive Care in Hamburg, Germany: a retrospective cohort study. (preprint)

Background. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continues to spread worldwide and pose a major public health burden. There is increasing evidence that men are more likely to die from SARS-CoV-2 infection than women. However, underlying factors that mediate the observed sex bias in coronavirus disease 2019 (COVID-19) remain unknown. Methods. In this retrospective cohort, we included COVID-19 patients who were admitted to an Intensive Care Unit at the University Hospital Hamburg-Eppendorf, Germany. We obtained demographic data of all patients who were discharged or had died by 29th April 2020. We systematically analyzed sex hormones as well as cytokine and chemokine responses in male and female patients with laboratory-confirmed SARS-CoV-2 infections upon hospital admission. We used uni- and multivariable linear regression methods to identify potential risk factors for disease severity in males and females. Findings. All enrolled patients (n=45; n=35 males and n=10 females) presented comorbidities with hypertension being the most common (45.7% in males; 40% in females), followed by cancer (35% in males; 40% in females), obesity (34.3% in males and 30% in females), type II diabetes (25.7% in males and 20% in females) and chronic heart diseases (8.6% in males and 0% in females). We detected that the vast majority of male COVID-19 patients present low testosterone (68.6%) and low dihydrotestosterone (48.6%) levels. In contrast, most female COVID-19 patients have elevated testosterone levels (60%) without alterations in dihydrotestosterone levels. Both, female and male COVID-19 patients may present elevated estradiol levels (45.7% in males and 40% in females). Disease severity defined by SOFA score correlates with elevated cytokine responses (e.g. IL-6) in males and IL-2 in females. In male COVID-19 patients, testosterone levels negatively correlate with inflammatory IL-2 and IFN-{gamma}, whereas estradiol levels positively correlate with the inflammatory cytokine IL-6. Vice versa, in female COVID-19 patients, testosterone levels positively correlate with inflammatory cytokines (e.g. IL-6). Interpretation. We here show that critically ill male COVID-19 patients suffer from severe testosterone and dihydrotestosterone deficiencies. Both androgens are required to mount antiviral immune responses to combat infection in males.