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

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.

Next generation plasma proteome profiling of COVID-19 patients with mild to moderate symptoms

COVID-19 has caused millions of deaths globally, yet the cellular mechanisms underlying the various effects of the disease remain poorly understood. Recently, a new analytical platform for comprehensive analysis of plasma protein profiles using proximity extension assays combined with next generation sequencing has been developed. Here, we describe the analysis of the plasma profiles of COVID-19 patients with mild and moderate symptoms by comparing the protein levels in newly diagnosed patients with the plasma profiles in the same individuals after recovery 14 days later. The study has identified more than 200 proteins that are significantly elevated at infection and many of these are related to cytokine response and other immune-related functions. In addition, several other proteins are shown to be elevated, including SCARB2, a host cell receptor protein involved in virus entry. A comparison with the plasma protein response in patients with severe symptoms shows a highly similar pattern, but with some interesting differences. In conclusion, the results will facilitate further studies to understand the molecular mechanism of the immune-related response of the SARS-CoV-2 virus.

Rapid and Sensitive Detection of SARS-CoV-2 Infection Using Quantitative Peptide Enrichment LC-MS/MS Analysis

Reliable, robust, large-scale molecular testing for SARS-CoV-2 is essential for monitoring the ongoing Covid-19 pandemic. We have developed a scalable analytical approach to detect viral proteins based on peptide immunoaffinity enrichment combined with liquid chromatography - mass spectrometry (LC-MS). This is a multiplexed strategy, based on targeted proteomics analysis and read-out by LC-MS, capable of precisely quantifying and confirming the presence of SARS-CoV-2 in PBS swab media from combined throat/nasopharynx/saliva samples. The results reveal that the levels of SARS-CoV-2 measured by LC-MS correlate well with their corresponding RT-PCR readout (r=0.79). The analytical workflow shows similar turnaround times as regular RT-PCR instrumentation with a quantitative readout of viral proteins corresponding to cycle thresholds (Ct) equivalents ranging from 21 to 34. Using RT-PCR as a reference, we demonstrate that the LC-MS-based method has 100% negative percent agreement (estimated specificity) and 95% positive percent agreement (estimated sensitivity) when analyzing clinical samples collected from asymptomatic individuals with a Ct within the limit of detection of the mass spectrometer (Ct [≤]30). These results suggest that a scalable analytical method based on LC-MS has a place in future pandemic preparedness centers to complement current virus detection technologies.

Screening for high amounts of SARS-CoV-2 identifies pre-symptomatic subjects among healthy healthcare workers

BackgroundPre-symptomatic subjects are spreaders of SARS-CoV-2 infection, and strategies that could identify these subjects, particularly in hospital settings, are needed. MethodsWe tested a cohort of 9449 employees at work at the Karolinska University Hospital, Stockholm, Sweden for SARS-CoV-2 RNA and antibodies, linked the screening results to sick leave records and examined the association between screening results and past or future sick leave using multinomial logistic regression. ResultsWe found that healthcare workers with high amounts of SARS-CoV-2 virus, as indicated by the Cycle threshold (Ct) value in the PCR, had the highest risk for sick leave in the two weeks after testing (OR 11{middle dot}97 (CI 95% 6{middle dot}29-22{middle dot}80)) whereas subjects with low amounts of virus had the highest risk for sick leave in the past three weeks before testing (OR 6{middle dot}31 (4{middle dot}38-9{middle dot}08)). Only 2{middle dot}5% of employees were SARS-CoV-2 positive while 10{middle dot}5% were positive by serology and 1{middle dot}2% were positive in both tests. Serology-positive subjects were not at excess risk for future sick leave (OR 1{middle dot}06 (95% CI, 0{middle dot}71-1{middle dot}57)), but virus-positive subjects had a 7{middle dot}23 fold (95% CI, 4{middle dot}52-11{middle dot}57)) increased risk for sick leave within two weeks post testing. ConclusionsScreening of asymptomatic healthcare workers for high amounts of SARS-CoV-2 virus using Ct values will identify pre-symptomatic subjects who will develop disease in the next few weeks. Identification of potentially contagious, pre-symptomatic subjects is likely critical for protecting patients and healthcare workers. Main pointHealthy healthcare workers with low amounts of SARS-CoV-2 nucleic acids will previously have had the disease. Presence of a high amount of SARS-CoV-2 nucleic acids predicts future symptomatic disease.

Combined metabolic cofactor supplementation accelerates recovery in mild-to-moderate COVID-19

There is a need to treat COVID-19 patients suffering from respiratory problems, resulting in decreased oxygen levels and thus leading to mitochondrial dysfunction and metabolic abnormalities. Here, we investigated if a high oral dose of a mixture of Combined Metabolic Activators (CMA) can restore metabolic function and thus aid the recovery of COVID-19 patients. We conducted a placebo-controlled, open-label phase 2 study and a double-blinded phase 3 clinical trials to investigate the time of symptom-free recovery on ambulatory patients using a mixture of CMA consisting of NAD+ and glutathione precursors. The results of both studies showed that the time to complete recovery was significantly shorter in the CMA group (6.6 vs 9.3 days) in phase 2 and (5.7 vs 9.2 days) in phase 3 trials. A comprehensive analysis of the blood metabolome and proteome showed that the plasma levels of proteins and metabolites associated with inflammation and antioxidant metabolism are significantly improved in patients treated with the metabolic activators as compared to placebo. The results show that treating patients infected with COVID-19 with a high dose of CMAs leads to a more rapid symptom-free recovery, suggesting a role for such a therapeutic regime in the treatment of infections leading to respiratory problems.

The protein expression profile of ACE2 in human tissues

The novel SARS-coronavirus 2 (SARS-CoV-2) poses a global challenge on healthcare and society. For understanding the susceptibility for SARS-CoV-2 infection, the cell type-specific expression of the host cell surface receptor is necessary. The key protein suggested to be involved in host cell entry is Angiotensin I converting enzyme 2 (ACE2). Here, we report the expression pattern of ACE2 across >150 different cell types corresponding to all major human tissues and organs based on stringent immunohistochemical analysis. The results were compared with several datasets both on the mRNA and protein level. ACE2 expression was mainly observed in enterocytes, renal tubules, gallbladder, cardiomyocytes, male reproductive cells, placental trophoblasts, ductal cells, eye and vasculature. In the respiratory system, the expression was limited, with no or only low expression in a subset of cells in a few individuals, observed by one antibody only. Our data constitutes an important resource for further studies on SARS-CoV-2 host cell entry, in order to understand the biology of the disease and to aid in the development of effective treatments to the viral infection.