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The emergence of the coronavirus disease 2019 (Covid-19) pandemic, caused by a newly discovered coronavirus called the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), has necessitated a fast and thorough understanding of its pathophysiology.1, 2 Presently, Covid-19 management is limited to palliative and symptomatic treatment. Measures should be taken to develop more effective therapies.3
The complement system___which consists of more than 30 types of soluble plasma proteins or membrane proteins___is sensitive to cellular damage and pathogens.4, 5 It can play important roles, for instance, in protecting the host against pathogens or clearing the body of apoptotic and necrotic cells, as well as in destroying immune complexes.6 In viral infections, the complement system can exert different antiviral roles, including directly neutralising virus particles through opsonisation, membrane attack complex (MAC) formation on virions or virus-infected cells, targeting viral components inside the cell for proteasomal degradation. It also plays a role in regulating and promoting other immune responses, the effectiveness of which depends on the infectious agent and genetic diversity of individuals.7 The complement system is conventionally activated by three main independent, but overlapping, pathways, including classical, alternative, and lectin, and it has been reported that SARS-CoV-2 activates these pathways, either directly or indirectly.8-10 While the classical pathway is activated following the formation of antibody-antigen complexes or acute phase proteins such as C-reactive protein (CRP), the alternative cascade is stimulated by non-self-proteins, lipids, and carbohydrates such as bacterial endotoxin or yeast wall. Mannose-binding lectin (MBL), multiple ficolins, and collectins are the pattern recognition molecules in the lectin pathway and can trigger this pathway through interactions that they are involved in. In all three pathways, MAC or C5b6789 (C5b-9) is ultimately formed, which leads to cell lysis by forming pores in the membranes of pathogens like enveloped viruses or virus-infected host cells. Active components such as C3a, C3b, C4a, and C5a are also produced in these pathways. The C5a fragment is a potent anaphylatoxin that causes neutrophil and macrophage recruitment and activation. The C3b fragment stimulates the phagocytic activity of neutrophils and macrophages. Also, C3a, C4a, and C5a fragments stimulate basophils and mast cells, resulting in the release of inflammatory factors such as histamine and serotonin.4, 5, 11-19
Many infectious agents such as viruses and bacteria can exploit the complement system to facilitate tissue invasion.20 This system also mediates the inflammatory response to infection, which can be tissue-destructive and results in multi-organ failure (MOF) and the clinical syndrome of sepsis.21, 22 Therefore, it functions as a double edge sword since its harmful effects in the response to infection may outweigh its beneficial effects. This might be the case in Covid-19 infection, where emerging evidence of complement activation is reported, and in which the highest mortality is reported among patients with MOF, severe pneumonitis, and systemic sepsis.13, 23-25 The aim of the present review is to summarise current knowledge about the interaction of SARS-CoV-2 with the complement system and to critically appraise complement inhibition as a new target for Covid-19 treatment.
2 VIROLOGY AND LIFE CYCLE OF SARS-CoV-2Coronaviruses (Co-Vs) that can cause disease in humans and animals, belong to the order Nidovirales, the family Coronaviridae and the subfamily Orthocoronavirinae, which is subdivided into four genera: alpha-coronavirus, beta-coronavirus, gamma-coronavirus, and delta-coronavirus.26, 27 SARS-CoV-2, which belongs to beta-coronaviruses, is an enveloped virus having a non-segmented, single-stranded, and positive-sense RNA genome (∼30 kb). Noteworthy, the genomic RNA (gRNA) of this virus possesses a 5′cap structure and a 3′poly-A tail and can thus function as a messenger RNA (mRNA) to be immediately translated into the viral polyproteins.28
SARS-CoV-2 has four major structural proteins, including envelope protein (E), spike glycoprotein (S), membrane protein (M), and nucleocapsid protein (N), all of which are located near the 3′ end of the viral genome. The E protein is a small protein that acts as a viroporin. It participates in virus assembly and plays an important role in viral morphogenesis.29 The M protein can also be involved, for example, in assembling and shaping the envelope of the virus.30 The N protein, which is a phosphoprotein that can bind to RNA, is therefore involved in processes such as virus transcription and replication.29 SARS-CoV-2 must be able to bind to a suitable receptor at the cell surface to begin its life cycle. Superficial S glycoproteins of the virus are in the form of homotrimer, containing 2 domains, namely, S1 and S2. In the S1 domain, there is a region called the receptor-binding domain (RBD) through which viral spike binds to the host cell surface receptor, namely, angiotensin-converting enzyme-2 (ACE2). Moreover, the S2 domain participates in the fusion of the viral envelope with the host cell membrane.31, 32 It is worth noting that SARS-CoV-2, similar to SARS-CoV, uses human ACE2 as an entry receptor, while Middle East respiratory syndrome coronavirus (MERS-CoV) binds specifically to transmembrane dipeptidyl peptidase 4 (DPP4) receptor. After the virus attachment to ACE2, the slicing of S glycoprotein, as well as its activation, occurs by the host's transmembrane serine protease 2 (TMPRSS2). In addition to ACE2 and TMPRSS2, some other receptors such as CD147, heparan sulphate proteoglycans (HSPGs), a wide range of the host membrane proteases (i.e. TMPRSS4 and furin), extracellular proteases (i.e. elastin, plasmin, trypsin, and factor Xa protease), and cathepsins may also facilitate the SARS-CoV-2 entry.28, 33, 34 After binding to the receptor, the virus enters host cells mainly via clathrin-mediated endocytosis.35
In addition to the structural proteins, the SARS-CoV-2 genome is also composed of 14 open reading frames (ORFs), that ORF1a and ORF1ab translate to replicase polyprotein 1a (PP1a) and polyprotein 1ab (PP1ab), respectively.28 Proteinases cleave these polyproteins to produce 16 nonstructural proteins (Nsp1-16), which eventually form the viral replicase complex replication and transcription complex (RTC).35 Replication initiates in the double-membrane vesicles (DMVs), and a viral enzyme named RNA-dependent RNA polymerase (RdRp) is responsible for viral replication.35, 36 The positive-strand genome is then used as a template for constructing full-length negative-strand RNA and producing subgenomic (sg)RNA.36 Translation of sgRNAs results in the production of accessory proteins as well as major structural proteins (N, S, M, and E) that enter the endoplasmic reticulum Golgi intermediate compartment (ERGIC), where the assembly and budding of new virus particles of SARS-CoV-2 are done.37 Eventually, after transporting the newly assembled viral particles in vesicles (i.e. exosomes) to the cell surface and subsequently their fusion with the part of the host plasma membrane, new viruses are released through exocytosis into the extracellular space.38
3 AN OVERVIEW OF THE IMMUNE PATHOGENESIS OF COVID-19Generally, invading pathogens such as viruses are recognized by pattern recognition receptors (PRRs) expressed in/on immune cells.39 Viruses induce multiple main immune responses, including augmenting in interferons type I (IFNs) synthesis, elevation in inflammatory factors secretion, and dendritic cells (DCs) maturation.39 SARS-CoV-2 causes activation of both innate and acquired immune responses. Helper T lymphocytes (Th; also known as T CD4+ cells) activate B cells for the production of virus-specific antibodies, and T CD8+ cells directly kill the cells which are infected by the virus. Besides, Th cells generate mediators and pro-inflammatory cytokines to assist other immune cells. Recent studies have demonstrated that SARS-CoV-2 can stimulate apoptosis of T cells and suppress their functions and therefore contribute to inhibiting the immune defence of the host. In addition, antibodies and complement factors for example C3a and C5a are vital in fighting against viral infections.40-44 Therefore, Covid-19 pathogenesis can be a result of the immune system's overreaction or its abnormal response with unclear aetiology in some cases. This leads to the local generation of inflammatory chemokines, cytokines, and free radicals in very high amounts which contribute to intense damage to organs, including the lungs. This, in the worst-case state, leads to failure in multiple organs and finally death.45, 46 Indeed, the majority of patients who become critically ill following SARS-CoV-2 infection beget acute respiratory distress syndrome (ARDS), which is considered the major cause of death in Covid-19.47 But it is unclear that what is the exact reason that this immuno-pathological event is common in SARS-CoV-2 infections.48
A principal mechanism explained for ARDS is known to be cytokine storm, which is a fatal systemic uncontrolled inflammatory response within SARS-CoV infection and is caused by secretion of high levels of pro-inflammatory chemokines (CCL5, CCL3, CCL2, CXCL10, CXCL9, CXCL8 and, etc.) and cytokines (IL-1β, IL-12, IL-33, IL-6, IL-18, IFN-γ, IFN-α, TGF-β, TNF-α and, etc.) by effector cells of the immune system.46, 47, 49, 50 Individuals infected severely by SARS-CoV or MERS-CoV indicated higher amounts of IFN-α, IL-6, CXCL-10, CXCL-8, and CCL5 in serum in comparison with mild to moderate infected individuals.51 The mentioned cytokine storm causes a severe attack of the immune system to body organs and results in MOF, ARDS, and eventually death in severe SARS-CoV-2 infected cases, just similar to MERS-CoV and SARS-CoV infections.48, 52 Furthermore, considerably higher serum concentrations of pro-inflammatory cytokines, including IL-1β, IL-6, IL-2, TNF-α, IL-17, GM-CSF, G-CSF, and the chemokines CXCL8, CCL7, CCL3, CCL2, and CXCL10, were reported in most patients who critically suffer from the severe form of Covid-19.53, 54 These chemokines attract monocytes and neutrophils to inflamed tissues and reinforce tissue damage. Notably, overproduction of CXCL10 and CCL7 is connected with the severity and lethality of the disease. IL-6 levels are also dramatically elevated in these cases and are often derived from neutrophils and macrophages accumulated in the lung.55-58 Production of high levels of IL-6, combined with delayed lower IFN, can change Th1 response to Th17 response and therefore declines the efficiency of antiviral activity and increases tissue damage via accumulation of macrophages and neutrophils.59 Neutrophils and macrophages, through a positive loop, lead to a hyper-inflammatory state and cytokine storm.58, 60
As mentioned above, there are other expressed receptors on the T cell surface like CD147, which mediates the virus entering.61 Besides, SARS-CoV-2 might enter the cells through other receptors or other ways of cell entry including antibody-dependent enhancement (ADE). ADE might take place when immune complexes consisting of virus–antibody attach to complement receptors or Fc receptors or as an alternative by changing the conformation of envelope glycoproteins, which are necessary for the fusion of the virus into host cells.62 In addition, lymphopenia seen in Covid-19 suffering patients can be explained by increased expression levels of programed cell death protein 1 (PD-1) on T CD8+ cells, which causes exhaustion of T cells.25, 61 Altogether, it can be said that several major events, including cytokine storm, lymphopenia, increased neutrophil-to-lymphocyte ratio (NLR), ADE phenomenon, exhaustion and dysfunction of lymphocytes, as well as granulocyte and monocyte abnormalities, potentially contribute to the dysregulation of immune response and the exacerbation of Covid-19 illness.34, 63 For better understanding, the detailed information on Covid-19 immunopathogenesis is presented in Figure 1.
An overview of the immune pathogenesis of Covid-19. The virus degradation is occurred by the antigen-presenting cells (APCs) such as dendritic cells (DCs) and subsequently, the virus antigens are presented by APCs-derived major histocompatibility complexes (MHCs) to the T cells. After T CD8+ cells clonal expansion through T cell receptor (TCR) and MHC-I interaction, infected cells are directly attacked via cytotoxic proteins, like perforin and granzymes released from effector T cells (cytotoxic T cells or CTLs). Moreover, T CD8+ cells result in great inflammation by producing various inflammatory cytokines. Besides, the exhausted T CD8+ cell phenotype is induced by the cytokine storm created via different immune cells. On the issue of the CD4+ T cells contribution, they can activate SARS-CoV-2-specific B cells to differentiate into plasma cells (PCs) and secrete antibodies to neutralise the virus. As a result of the antibodies elevation especially IgG, a mechanism entitled antibody-dependent enhancement (ADE) happens by which the pathogenesis of Covid-19 is boosted via enhanced viral replication. Also, Th polarization is done in favour of Th17 than Th1 due to the cytokine storm which, in turn, results in delayed expression of IFN-γ. These huge amounts of cytokines coupled with reactive oxygen species (ROS) generated from immune cells lead to multi-organ failure (MOF) and ARDS
4 THE ROLE OF THE COMPLEMENT SYSTEM IN SARS-CoV AND ARDSAs mentioned earlier, ARDS is known as an immune-driven condition that in addition to SARS-CoV-2, is also seen in patients who severely suffer from SARS-CoV.64 Since at least three decades ago, the activation of the complement system in ARDS has been well identified.65-67 But, the relationship between complement activation and ARDS pathogenesis has not been well-investigated___even though the contributing role of C5b-9 in the pathogenesis of ARDS was questioned,67 and it has been demonstrated that C5a could play a key role in viral infections, especially in acute lung injury (ALI).68
Previously, it has been shown that mannose-binding lectin (MBL) can bind to the SARS-CoV spike glycoprotein in vitro.69, 70 One retrospective analysis displayed that people who had deficient serum MBL levels were more probable to become infected with SARS-CoV infection than those with high MBL levels.71 In another study, the effect of the complement system on SARS-CoV infection in mice lacking C3 (C3−/−) with C57BL/6J control mice was investigated. The results of this study showed that in SARS-CoV-infected C3−/− mice compared to infected C57BL/6J mice, the infiltration of neutrophils and inflammatory monocytes was lower, the levels of cytokines and chemokines in the lungs and sera were decreased, and also less weight loss and respiratory distress were seen in these mice. This mouse experiment indicated that activation of complement component C3 intensifies illness in SARS-CoV-associated ARDS. It was moreover found that the complement system does not affect the control of virus replication.64 A recent study in hDPP4-transgenic (hDPP4-Tg) mice examined the role of complement in lung damage caused by MERS-CoV infection. In this study, an increase in C5a and C5b-9 concentrations in sera and lung tissues were observed respectively. Generally, when the C5aR receptor is targeted, C5a production is inhibited, inflammatory responses diminish, and spleen and lung tissue damage is reduced. Also, inhibition of C5a–C5aR interaction can lead to the regulation of the host's immune response.72 Additionally, Gralinski et al. showed that the complement system has a vital role in SARS-CoV pathogenesis and also demonstrated that inhibition of the complement pathway may be considered as an effective therapeutic way for coronavirus-related disorders.64 To provide a better overview, the role of the complement system in ARDS was represented in Figure 2.
The role of the complement system in ARDS. By the over-activation of the complement system in Covid-19 patients, the amounts of C3a, C4a, and C5a anaphylatoxins are considerably increased which results in enormous inflammation. Moreover, C5a induces inflammation and damages tissue by recruiting activated neutrophils into the lung and also by the increase in the quantity of different inflammatory cytokines which all together lead to lung tissue damage and ARDS
5 THE COMPLEMENT SYSTEM IN COVID-19The fragment crystallisable (Fc) of antibodies created against viral antigens can activate the complement classical pathway, cellular cytotoxicity, and phagocytosis by interacting with complement soluble proteins (i.e. C1q) and Fc receptors expressed on the cell surface of leucocytes, thereby making an important contribution to clear the body of several viral infections.73 It has been observed that convalescent plasma injection in people with Covid-19 can have therapeutic effects.74 Besides, according to a recent study by Natarajan et al., convalescent plasma therapy not only neutralises the virus using plasma antibodies but also has extra-neutralising effects, namely, antibody-dependent cell-mediated phagocytosis (ADCP), antibody-dependent cellular cytotoxicity (ADCC), and antibody-dependent complement deposition (ADCD) that are all Fc-dependent activities.75 Nevertheless, over-activation of the complement system can act as one of the pathophysiological factors in Covid-19. In some patients with severe Covid-19 infection, systemic activation of the complement system and deposition of terminal complement proteins (C5b-9), as well as C4d and MASP-2 in the lungs and skin were observed. Viral S glycoprotein was also observed at the site of these deposits, indicating that the virus-infected endothelium was one of the target sites of the complement system.76 A recently published meta-analysis revealed that lower serum levels of complement C3 and C4 components, indicative of complement over-activation and product consumption, are meaningfully associated with the increased disease severity and mortality of patients suffering Covid-19. Thus, investigating C3 and C4 might be helpful in predicting adverse clinical outcomes in these patients.77 Based on a single-centre case series from Italy, enhanced plasma levels of C5a and soluble C5b-9 (sC5b-9) were reported in moderate Covid-19 patients requiring continuous positive airway pressure and also severe Covid-19 cases requiring mechanical ventilation.78 In the early stages of Covid-19, in those who are infected with SARS-CoV-2 for the first time, the body lacks memory cells or immunoglobulin G (IgG) against viral spike glycoproteins and may use IgA to fight the virus on mucosal surfaces. Indeed, IgA complexes, as well as SARS-CoV–S-N-glycan (N-330), can bind to MBL and activate the lectin pathway in the mucosal surfaces.69
There is evidence indicating the potent immunohistochemical staining for MBL, MASP-2, C3, C4, and C5b-9 in the lung of Covid-19 patients. Type I and type II alveolar epithelial cells are proposed to be the principal target sites for complement deposition. Likewise, the existence of MBL and MASP-2 in the lung indicates a probable function of the lectin pathway in complement deposition.79 Indeed, type II alveolar cells are the principal target cells for SARS-CoV-2 and can release several complement factors such as ficolin-1 (FCN-1) and possibly collectin-11 (CL-11). C3a, C3b, C5a, and C5b-9 are also formed as a result of complement activation arising from the presence of the mentioned ligand-recognition molecules and other substantial factors of the lectin pathway.25
Noteworthy, increased production of C3a and C5a fragments leads to the infiltration of neutrophils and monocytes into the alveolar sac that causes damage to the lungs and is itself an aggravating factor of ARDS following Covid-19 (Figure 2).80, 81 Activated neutrophils generate web-like neutrophil extracellular traps (NETs) in a process termed NETosis, which is a type of programed cell death that contributes to the host defence against pathogens. Both activated neutrophils and NETs contain components like properdin, C3, and factor B, which are required for the formation and stabilising of the bimolecular C3 convertase of the alternative complement pathway (C3bBb), thereby causing inflammatory cascade amplification. Complement activation is associated with NETs because it induces pro-inflammatory cytokines production and also accelerates C3a and C5a formation which in turn activates monocytes, eosinophils, and neutrophils. Therefore, while NETs are effective in host defence, their sustained formation that occurs in Covid-19, may induce an inflammatory reactions cascade and a hypercoagulable state that damage the adjacent tissues.82
It has also been indicated that C5a can stimulate adaptive immune cells such as T and B cells, consequently causing the release of several pro-inflammatory cytokines such as TNF-α, IL-1β, IL-6, and IL-8 from these cells.83 IL-6 and the inflammatory cytokine storm have been shown to be associated with the severity of disease in Covid-19 patients. In this regard, sarilumab, as a wholly human IgG1 monoclonal antibody (mAb) that can bind to both the membrane-bound and soluble forms of IL-6 receptors (IL-6R) with high affinity, has been considered in treating Covid-19.84 Inhibition of IL-6 by another anti-IL-6R mAb such as tocilizumab has also been reported to be effective for mitigating the cytokine storm and Covid-19 mortality.83, 85 C5a fragments are also one of the strongest chemotactic factors that mediate the migration and accumulation of inflammatory cells, induce their degranulation, and provoke the release of the enzymes and free radicals leading to an excessive inflammation.86 These events cause damage to the epithelium and increase vascular permeability, resulting in the accumulation of inflammatory infiltrates in the pulmonary alveoli and pleura which lead to dyspnoea and pulmonary insufficiency.68 The effects of Covid-19 lung illness on other organs can be explicated by the impact of toxic concentrations of circulating inflammatory factors or via blood-borne infection.25
It has been reported that the crosstalk between complement components and coagulation may also lead to thrombotic microangiopathy (TMA) and hypercoagulable state in Covid-19. Indeed, C3a and C5a fragments induce mast cell degranulation and activate the endothelial cell, which, in turn, trigger prothrombotic events mainly through stimulating the expression of tissue factor (TF) and secretion of von-Willebrand factor (vWF), respectively (Figure 3).87 Other components of the complement system like MASP-1 and MASP-2 proteins could also accelerate coagulopathy by augmenting clot formation through converting fibrinogen to fibrin and prothrombin to thrombin.87 Given that the C3a fragment can stimulate platelet, it is reasonable to assume that the activation of the complement system in SARS-CoV-2-infected patients may increase the risk of thrombosis.88 Taken together, these data suggest that SARS-CoV-2-induced over-activation of the complement system may exacerbate Covid-19 illness either through the accumulation of inflammatory infiltrates in the pulmonary alveoli or the induction of a hypercoagulable state leading to fatal TMA. To provide a well-conceptualised overview, Figure 3 presents the plausible role of the complement activation in Covid-19. Also, the concentration of complement components in different studies conducted on Covid-19 patients in comparison with the healthy controls is summarised in Table 1. On the basis of this Table, the plasma concentrations of C3a, C5a, and sC5b-9 are increased in most patients suffering Covid-19, indicating excessive complement activation.
The complement activation in Covid-19. Through the binding of SARS-CoV-2 to the angiotensin-converting enzyme 2 (ACE2) receptor, the virus enters the endothelial cells and activates different complement pathways. C3b takes part in the creation of the trimolecular C5 convertases converting the C5 into C5a and C5b fragments. In following, by joining C6, C7, C8, and C9 one after another to the C5b, the membrane attack complex (MAC) is ultimately formed. The components that are produced during complement activation stimulate inflammation via several procedures. The C5a causes an excessive influx of activated neutrophils (Neu) and macrophages (MQ). Moreover, the anaphylatoxins C3a and C5a stimulate basophils and mast cells to release histamine and serotonin resulting in considerable inflammation. Recruiting leucocytes cause the release of cytokines and reactive oxygen species (ROS) that assist in inflammation. The C3a and C5a fragments also induce mast cell degranulation and activate the endothelial cells, which consequently initiate prothrombotic events mainly through stimulating the expression of tissue factor (TF) and secretion of von-Willebrand factor (vWF), respectively, which prompt the coagulation cascade. Besides, MASP1/2 converts prothrombin to thrombin which, in turn, leads to the formation of fibrin from fibrinogen, and this process also ends up with coagulopathy
TABLE 1. The concentration of complement components in Covid-19 patients compared to healthy controls Sample Studied subjects Concentration changes Ref. C1q Plasma Patients: Mild (N = 32) and severe (N = 39) Decreased in severe relative to mild 89 Serum/plasma Patients (N = 102): Hospitalised patients, with and without oxygen supplementation (N = 36 and 31, respectively), ICU patients (N = 35); outpatients (N = 26) No difference was found between the groups 90 For all patients, were within the normal range MBL Plasma Patients with (N = 23) and without respiratory failure (N = 16) No difference was found between the two groups 13 Plasma Critically ill patients (N = 65); controls (N = 72) Increased in patients relative to controls 91 C3 Serum Patients: Severe (N = 30) and non-severe (N = 30); controls (N = 30) Increased in non-severe relative to controls, decreased in severe relative to non-severe 92 Serum/plasma Patients (N = 102): Hospitalised patients, with and without oxygen supplementation (N = 36 and 31, respectively), ICU patients (N = 35); outpatients (N = 26) Decreased in ICU patients relative to other groups 90 Serum Patients (N = 30) were divided into two groups: Diarrhoea (N = 15) and non-diarrhoea (N = 15) Some decrement in patients (47%) than the normal range 93 No difference was found between the two groups Serum Patients: Survivors (N = 169) and non-survivors (N = 67) Reduced in non-survivors relative to survivors 94 Serum Covid-19 patients with type 2 diabetes (T2D; N = 37) and without T2D (NMD: N = 33) No difference was found between the two groups 95 Some decrement (9.7%) in NMD group than the normal range Some decrement (19.4%) or increment (2.8%) in T2D group than the normal range Serum Patients (N = 182) Some increment (12.4%) or reduction (18.6%) than the normal range 96 Serum Patients (N = 72); controls (N = 20) Reduced in patients relative to controls 97 Serum Patients: Survivors (N = 414) and non-survivors (N = 125) Decreased in non-survivors relative to survivors 98 C3a Plasma Severe patients receiving maintenance haemodialysis (N = 19); haemodialysis controls (N = 10) Increased in patients relative to controls 99 Plasma Patients (N = 102): Hospitalised patients, with and without oxygen supplementation (N = 36 and 31, respectively), ICU patients (N = 35); outpatients (N = 26) Increased in patients relative to outpatients 90 Increased with disease severity Plasma Patients (N = 122); controls (N = 10) Increased in patients (particularly ICU patients during the entire illness course) relative to controls 100 C3c Plasma Patients (N = 122); controls (N = 10) Inc
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