Abstract
Early and persistent activation of complement is considered to play a key role in the pathogenesis of COVID-19. Complement activation products orchestrate a proinflammatory environment that might be critical for the induction and maintenance of a severe inflammatory response to SARS-CoV-2 by recruiting cells of the cellular immune system to the sites of infection and shifting their state of activation towards an inflammatory phenotype. It precedes pathophysiological milestone events like the cytokine storm, progressive endothelial injury triggering microangiopathy, and further complement activation, and causes an acute respiratory distress syndrome (ARDS). To date, the application of antiviral drugs and corticosteroids have shown efficacy in the early stages of SARS-CoV-2 infection, but failed to ameliorate disease severity in patients who progressed to severe COVID-19 pathology. This report demonstrates that lectin pathway (LP) recognition molecules of the complement system, such as MBL, FCN-2 and CL-11, bind to SARS-CoV-2 S- and N-proteins, with subsequent activation of LP-mediated C3b and C4b deposition. In addition, our results confirm and underline that the N-protein of SARS-CoV-2 binds directly to the LP- effector enzyme MASP-2 and activates complement. Inhibition of the LP using an inhibitory monoclonal antibody against MASP-2 effectively blocks LP-mediated complement activation. FACS analyses using transfected HEK-293 cells expressing SARS-CoV-2 S protein confirm a robust LP-dependent C3b deposition on the cell surface which is inhibited by the MASP-2 inhibitory antibody. In light of our present results, and the encouraging performance of our clinical candidate MASP-2 inhibitor Narsoplimab in recently published clinical trials, we suggest that the targeting of MASP-2 provides an unsurpassed window of therapeutic efficacy for the treatment of severe COVID-19.
Highlights
Coronaviruses (CoVs) are single-stranded RNA viruses causing life threatening respiratory infection in humans and other species
This work demonstrates that the lectin pathway (LP) recognition molecules MBL, FCN-2 and CL-11 bind to S
N proteins of SARS-CoV-2 with subsequent activation of LPmediated C3b and C4b deposition. These findings clearly show the activation of the LP on SARS-CoV-2 surface proteins and N protein, confirming the central role of LP activation in the immunopathogenesis of COVID-19
Summary
Coronaviruses (CoVs) are single-stranded RNA viruses causing life threatening respiratory infection in humans and other species. The C3 activation product C3b can bind to zymogen complement factor B (FB), forming a complex that can in turn convert more C3 into C3a and C3b if C3b-bound FB is cleaved by a serine protease called FD [10]. Nunc MaxiSorp microtiter ELISA plates were coated with 10 mg/ mL of purified recombinant SARS-CoV-2 proteins S and N in coating buffer (10 mM Tris-HCl, 140 mM NaCl, pH 7.4). To measure C3 and C4 activation, Nunc MaxiSorp microtiter plates were coated with 100 mL of 10 mg/mL mannan (Promega), or 100 mL of 10 mg/mL SARS-CoV-2 proteins in coating buffer. After 1 hour, the plate was washed and MASP-2 binding to SARS-CoV-2 proteins was detected using monoclonal antibodies against MASP-2 followed by HRP-conjugated rabbit anti-human IgG and the chromogenic substrate ELISA Colorimetric TMB Reagent (Sigma). C4 cleavage mediated via MASP-2 was detected using SDS-PAGE and Coomassie bule staining under reducing conditions
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