Lectin Pathway Mediates Complement Activation by SARS-CoV-2 Proteins

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.

Keywords: COVID-19; SARS-CoV-2; complement system; innate immunity; lectin pathway.

Figure 1

Binding of LP recognition molecules and C1q to SARS-CoV-2 proteins. A microtitre ELISA plate was coated with either S, N or control ligands (mannan for MBL, N-acetyl BSA for FCN2, zymosan for CL-11 or immune complexes for C1q). Following incubation with blocking buffer and washing steps, serial dilutions of NHS, starting at 5% were added to detect binding of LP recognition molecules and C1q from NHS to SARS-CoV-2 proteins. Human MBL (A), FCN2 (B), Cl-11 (C) and C1q (D) were assayed by ELISA. A significant binding of LP recognition molecules to S and N proteins was clearly observed. No C1q binding to any of the viral proteins was detected. Results are means of duplicates ± SD.

Figure 2

Detection of C3b and C4b deposition on SARS-CoV-2 proteins. An ELISA plate was coated with SARS-CoV-2 proteins (S or N) or zymosan and incubated with serial dilutions of NHS (starting at 5%) for 1 h at 37°C. C3b or C4b deposition were detected using antibodies against human C3c or C4c. High levels of C3b (A) and C4b (B) deposition were observed on surface immobilised SARS-CoV-2 proteins.

Figure 3

HG4 inhibits lectin pathway mediated C4b deposition. A microtiter ELISA plate was coated with mannan and blocked with BSA. Different concentrations of HG4 antibody were mixed with 2% NHS and incubated on the plate. Bound C4b was detected using anti C4c antibodies.

Figure 4

MASP-2 binds directly to SARS-Cov-2 N-protein and mediates complement C4 activation. (A) Microtiter plates were coated with 2.5μg/well N-protein or BSA as a control. Residual binding sites were blocked using 1% BSA. Serial dilutions of rMASP-2 were added, and binding was detected using an anti-MASP-2 mAb. MASP-2 bound to the NP protein in a concentration-dependent and saturable manner (A). (B) In a parallel experiment, 1μg of rMASP-2 in barbital buffered saline (BBS) was added to wells coated with NP or BSA. After 1 hr at 37°C, wells were washed and 1μg of purified human C4 was added to each well. After 1 hr incubation at 37°C, the supernatant was collected and separated on SDS-PAGE. The results showed that rMASP-2 binds directly to NP and cleaves C4. Addition of HG4 (a mAb that inhibits MASP-2) inhibited MASP-2 mediated C4 cleavage. Purified C4 was run on the gel as a control.

Figure 5

Transfection of HEK 293T promotes high levels of expression of SARS-CoV-2 surface proteins. The expression levels of S protein on the surface of HEK 293 T cells were measured using serum from convalescent SARS-CoV-2 PCR positive patients or NHS (PCR negative) followed by anti-human IgG Alexa fluor 647 antibodies. Significant high levels of IgG binds to S were observed on the surface of HEK T293 cells.

Figure 6

Inhibition of LP impairs complement C3b deposition on SARS-CoV-2 surface proteins. HEK 293T cells expressing SARS-CoV-2 S protein were used. Cells were incubated with 2.5% NHS with 100nM HG4 or an isotype control antibody at 37C for 30 min. Cells were washed and C3b deposition was detected using rabbit anti human C3b followed by goat anti rabbit FITC labelled antibodies. A significant C3b deposition was detected on HEK 293T cells expressing S protein. Inhibition of MASP-2 using HG4 significantly impairs complement C3b deposition on the surface of HEK 293T cells expressing S protein.