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While complement is a contributor to disease severity in severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections, all three complement pathways might be activated by the virus. Lectin pathway activation occurs through different pattern recognition molecules, including mannan binding lectin (MBL), a protein shown to interact with SARS-CoV-2 proteins. However, the exact role of lectin pathway activation and its key pattern recognition molecule MBL in COVID-19 is still not fully understood. We therefore investigated activation of the lectin pathway in two independent cohorts of SARS-CoV-2 infected patients, while also analysing MBL protein levels and potential effects of the six major single nucleotide polymorphisms (SNPs) found in the MBL2 gene on COVID-19 severity and outcome. We show that the lectin pathway is activated in acute COVID-19, indicated by the correlation between complement activation product levels of the MASP-1/C1-INH complex (p=0.0011) and C4d (p<0.0001) and COVID-19 severity. Despite this, genetic variations in MBL2 are not associated with susceptibility to SARS-CoV-2 infection or disease outcomes such as mortality and the development of Long COVID. In conclusion, activation of the MBL-LP only plays a minor role in COVID-19 pathogenesis, since no clinically meaningful, consistent associations with disease outcomes were noted.

The complement system, composed of the three activation pathways, has both protective and pathogenic roles in the development of systemic lupus erythematosus (or lupus), a prototypic autoimmune disease. The classical pathway contributes to the clearance of immune complexes (ICs) and apoptotic cells, whereas the alternative pathway (AP) exacerbates renal inflammation. The role of the lectin pathway (LP) in lupus has remained largely unknown. Mannose-binding lectin (MBL)-associated serine proteases (MASPs), which are associated with humoral pattern recognition molecules (MBL or ficolins), are the enzymatic constituents of the LP and AP. MASP-1 encoded by the gene significantly contributes to the activation of the LP. After the binding of MBL/ficolins to pathogens or self-altered cells, MASP-1 autoactivates first, then activates MASP-2, and both participate in the formation of the LP C3 convertase C4b2a, whereas, MASP-3, the splice variant of the gene, is required for the activation of the zymogen of factor D (FD), and finally participates in the formation of the AP C3 convertase C3bBb. To investigate the roles of MASP-1 and MASP-3 in lupus, we generated gene knockout lupus-prone MRL/ mice ( MRL/ mice), lacking both MASP-1 and MASP-3, and analyzed their renal disease. As expected, sera from MRL/ mice had no or markedly reduced activation of the LP and AP with zymogen forms of complement FD. Compared to their wild-type littermates, the MRL/ mice had maintained serum C3 levels, little-to-no albuminuria, as well as significantly reduced glomerular C3 deposition levels and glomerular pathological score. On the other hand, there were no significant differences in the levels of serum anti-dsDNA antibody, circulating ICs, glomerular IgG and MBL/ficolins deposition, renal interstitial pathological score, urea nitrogen, and mortality between the wild-type and MRL/ mice. Our data indicate that MASP-1/3 plays essential roles in the development of lupus-like glomerulonephritis in MRL/ mice, most likely activation of the LP and/or AP.

The objective of the present study was to elucidate the association between glomerular complement depositions belonging to the alternative (AP) and lectin (LP) pathways, and clinical findings of lupus nephritis (LN). Immunofluorescence (IF) was performed on 17 LN patients using antibodies against factor B, factor H, properdin, mannose-binding lectin (MBL) and L-ficolin. Compared with factor B/factor H negative patients (n = 9), positive patients (n = 8) showed longer duration of LN (p < 0.05) and more severe interstitial fibrosis (p < 0.05). Eleven patients had properdin deposition in glomeruli, and in three of them, with a duration of LN of less than 1 month, factor B was undetectable. Compared with properdin negative patients (n = 6), positive patients (n = 11) showed significantly higher urinary protein excretion (p < 0.01). MBL/L-ficolin positive patients (n = 11) also had significantly higher urinary protein excretion (p < 0.05) compared with negative patients (n = 6). An independent association was found between glomerular deposition of properdin and that of MBL/L-ficolin (p < 0.01) in addition to factor B/factor H. Traces of glomerular activation of AP and LP reflected the clinical status of LN. It appears that glomerular deposition of each complement component, especially properdin, may be an index of the histological activity of LN.

The lectin pathway of complement aids in removing apoptotic cells and maintenance of tissue homeostasis. However, its role in SLE pathogenesis remains unknown. This study aimed to assess the association of ficolins, mannose-binding lectin (MBL), and other pathogen recognition molecules (PRMs) of the lectin pathway and their corresponding autoantibodies with various clinical manifestations and disease activity in SLE patients from Western India. In this cross-sectional study, 282 clinically diagnosed SLE patients were included. Serum levels of ficolins, antigenic MBL, MBL-associated serine proteases (MASPs), MBL-associated protein 44 (MAp44), Collectin liver-1 (CL-L1), and their corresponding autoantibodies were quantified using ELISA. Group differences were analyzed using Mann-Whitney U tests, while associations/relationships were evaluated using chi-square tests and Spearman’s correlations. Serum levels of ficolin-2 ( p < 0.001 ), MASP-3 ( p = 0.030 ), and MAp44 ( p < 0.001 ) were significantly elevated, while antigenic MBL ( p < 0.001 ) and MASP-1 ( p < 0.001 ) were significantly reduced in SLE patients compared to healthy controls (HCs). Renal involvement was associated with elevated ficolin-1 ( p = 0.009 ), while hematological manifestations were linked to reduced MASP-1 ( p = 0.018 ), MASP-3 ( p = 0.002 ), and MAp44 ( p = 0.002 ) levels. Mucocutaneous manifestations were associated with elevated MAp44 ( p < 0.001 ) and anti-ficolin-1 ( p = 0.038 ) autoantibodies. Anti-ficolin-1 ( p = 0.001 ), anti-ficolin-2 ( p = 0.001 ), and anti-ficolin-3 ( p < 0.001 ) autoantibodies were significantly elevated in SLE patients compared to HCs. Anti-ficolin-2 autoantibodies were negatively correlated with ficolin-2 ( r =-0.153, p  = 0.015). Anti-MBL antibodies were correlated with SLEDAI ( r  = 0.169, p  = 0.007) and anti-dsDNA antibodies ( r  = 0.178, p  = 0.005). These findings indicate altered levels of lectin pathway-associated PRMs and their corresponding autoantibodies in SLE. Their association with clinical manifestations, disease activity, and complement-related parameters, suggest their potential as novel biomarkers in SLE.

Ficolins are a group of oligomeric lectins with subunits consisting of both collagen-like and fibrinogen-like domains. The majority of ficolins identified in vertebrates and invertebrates to date recognize N-acetylglucosamine (GlcNAc). X-ray crystallographic analysis of human ficolins has shown that the fibrinogen-like domain binds to the N-acetylated moiety. Ficolins in serum are associated with MBL-associated serine protease (MASP). The ficolin-MASP complex binds directly to carbohydrates present on the surface of a variety of Gram-positive and Gram-negative bacteria through ficolin. Binding of the complex initiates complement activation via the lectin pathway, leading to generation of opsonic fragments of complement components, such as C3b, and to lysis of the bacteria by the membrane attack complex. Thus, serum ficolins play an important role in innate immunity.

Activation of classical and lectin complement pathways contributes to several human diseases. Empasiprubart is a humanized recycling monoclonal antibody that inhibits both pathways by binding to the CCP2 domain of complement factor 2 (C2), an interaction that is dependent on both Ca 2+ and pH. Here, we resolve the crystal structure of empasiprubart complexed with C2, providing the molecular basis of its Ca 2+ dependency, and report a randomized, double-blind, placebo-controlled trial to assess the safety and tolerability (primary objectives) in addition to pharmacokinetics, pharmacodynamics, and immunogenicity (secondary objectives) of empasiprubart in 78 healthy participants (NCT04532125). A single intravenous (IV) dose of empasiprubart reduces circulating C2 levels by up to 99% and dose-dependently inhibits the classical and lectin pathways. Multiple IV empasiprubart doses reinforce reductions in free C2 levels, which persist until the endpoint of the study at 41 weeks. This prolonged reduction is in line with the empasiprubart elimination half-life (70–88 days). Single and multiple ascending doses of empasiprubart are generally safe and well tolerated. Overall, our results reveal in atomic detail the mechanism of empasiprubart and demonstrate that it is a first-in-class anti-C2 therapeutic antibody for use in complement-mediated diseases. Though the complement system is pivotal in the defence against infections, pathologic activation of the system contributes to disease. Here, authors show that their recently developed monoclonal antibody against complement factor 2, empasiprubart, inhibits the classical and lectin pathways in a clinical trial, and its crystal structure provides basis for its inhibitory properties, such as Ca 2+ binding.

In the evolution of the complement system, a major humoral innate immune factor, the existence of multiple isotypes of the complement components is considered as a key strategy to enhance innate immune defense. Complement C4 is also diversified in a wide range of vertebrate species including teleost fish, possibly supporting the robust activation mechanism of the complement. To better understand the functional diversity of C4 isotypes in the teleost complement system, two C4 isotypes, C4-1 and C4-2, sharing only 32% amino acid sequence identity, were examined for binding specificities towards model target molecules representing microbe antigens and towards Gram-positive and -negative bacteria. The results suggest that C4-1 and C4-2 behave similarly in binding to the tested targets, despite the predicted difference in binding specificity based on the thioester catalytic site. The participation of C4-1 in the classical and lectin pathways of complement activation was also explored using pathway-specific activating enzyme complexes, C1r/s and MBL-MASP2. As a result, C4-1 can be activated in both the classical and the lectin pathways, at higher efficiency in the classical pathway. Taken together, the present results imply that both C4-1 and C4-2 isotypes are fully functional in the complement activation cascades, probably playing comparable roles in innate immunity.

Background We aimed to investigate the involvement of the classical/lectin complement pathway in anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV) by exploring the complement activation fragment C4d in association to AAV activity. Methods Forty patients with active AAV and twenty population-based controls were included. The study included 27 (67.5%) patients with a diagnosis of GPA and 13 (32.5%) with MPA. Twenty-four patients (60%) were anti-proteinase 3 (PR3)-ANCA positive and 16 (40%) anti-myeloperoxidase (MPO)-ANCA positive. Thirty-three (82.5%) patients had kidney involvement. A follow-up sample obtained after induction therapy (median 6 months) was available for 24 of the patients, of whom 20 were in remission. Plasma C4d was analysed by ELISA detecting an epitope that arises upon complement-mediated cleavage. Plasma complement factor 4 (C4) and the soluble terminal complement complex (sTCC) were analysed by ELISA. The C4d/C4 ratio was calculated. HLA-DRB1-typing and immunohistochemistry for C4d in kidney biopsies were performed. Results Patients with active AAV had higher C4d, sTCC levels and C4d/C4 ratio than controls (p < 0.001, p = 0.004, p < 0.001). C4d, sTCC levels and C4d/C4 ratio all decreased from active disease to remission (p = 0.010, p = 0.009, p = 0.011). C4d levels in AAV patients in remission remained higher than population-based controls (p = 0.026). Active anti-PR3-ANCA patients had higher C4d levels and C4d/C4 ratio than anti-MPO-ANCA patients (p = 0.001, p = 0.007). Patients with active AAV and kidney involvement had lower C4d levels than patients without (p = 0.04). C4d levels and C4d/C4 ratio correlated positively with the percentage of normal glomeruli in kidney biopsies. The immunohistochemistry was negative for C4d in kidney biopsies. Conclusions The specific C4d assay revealed activity in the classical/lectin complement pathway in AAV, which reflected general disease activity, but was not associated specifically with kidney involvement. C4d levels differed depending on anti-PR3/MPO-ANCA subtypes suggesting differences in complement activation and underlying pathogenetic mechanisms. The findings imply that the classical/lectin complement pathway may play a more significant role in AAV pathogenesis than previously reported and that plasma C4d levels and C4d/C4 ratio may be biomarker candidates for disease activity and treatment outcome monitoring.

Complement activation is considered to contribute to the pathogenesis of severe SARS-CoV-2 infection, mainly by generating potent immune effector mechanisms including a strong inflammatory response. Involvement of the lectin complement pathway, a major actor of the innate immune anti-viral defense, has been reported previously. It is initiated by recognition of the viral surface Spike glycoprotein by mannose-binding lectin (MBL), which induces activation of the MBL-associated protease MASP-2 and triggers the proteolytic complement cascade. A role for the viral nucleoprotein (N) has also been reported, through binding to MASP-2, leading to protease overactivation and potentiation of the lectin pathway. In the present study, we reinvestigated the interactions of the SARS-CoV-2 N protein, produced either in bacteria or secreted by mammalian cells, with full-length MASP-2 or its catalytic domain, in either active or proenzyme form. We could not confirm the interaction of the N protein with the catalytic domain of MASP-2 but observed N protein binding to proenzyme MASP-2. We did not find a role of the N protein in MBL-mediated activation of the lectin pathway. Finally, we showed that incubation of the N protein with MASP-2 results in proteolysis of the viral protein, an observation that requires further investigation to understand a potential functional significance in infected patients.

The nucleocapsid (N) protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a viral structural protein that is abundant in the circulation of infected individuals. Previous published studies reported controversial data about the role of the N protein in the activation of the complement system. It was suggested that the N protein directly interacts with mannose-binding lectin-associated serine protease-2 (MASP-2) and stimulates lectin pathway overactivation/activity. In order to check these data and to reveal the mechanism of activation, we examined the effect of the N protein on lectin pathway activation. We found that the N protein does not bind to MASP-2 and MASP-1 and it does not stimulate lectin pathway activity in normal human serum. Furthermore, the N protein does not facilitate the activation of zymogen MASP-2, which is MASP-1 dependent. Moreover, the N protein does not boost the enzymatic activity of MASP-2 either on synthetic or on protein substrates. In some of our experiments, we observed that MASP-2 digests the N protein. However, it is questionable, whether this activity is biologically relevant. Although surface-bound N protein did not activate the lectin pathway, it did trigger the alternative pathway in 10% human serum. Additionally, we detected some classical pathway activation by the N protein. Nevertheless, we demonstrated that this activation was induced by the bound nucleic acid, rather than by the N protein itself.