Underlying mechanisms contributing to the severity of SARS-CoVs' complications
In this deterioration series, I elaborate on the mechanisms that contribute to the severity of SARS-CoV-2 (and, likewise, SARS-CoV-1 (2003)) cases. Previously, I discussed the role of the Renin-Angiotensin-Aldosterone and Kallikrein-Kinin System, hypercoagulation and thromboinflammation and SARS-CoV-2 involvement of the Central Nervous System (either by direct invasion or through systemic diseases following COVID). These mechanisms are strongly intertwined.
As I will discuss in this message, the Complement System is a major contributor to hyperinflammation and tissue damage following coronavirus infection. The Complement interacts, by default, with blood platelets and endothelial cells and contributes to hypercoagulation- and the other way around (Paragraph 2). As can be gathered from biopsy and autopsy reports following COVID-19, depositions found in the microvasculature of COVID patients indicate Lectin Pathway-mediated damage (Paragraph 3). Lastly, I will discuss therapeutical options to target the Complement cascade in COVID-19 (Paragraph 4). In order to write this message, I analyzed research papers from a body of knowledge spanning the last 3 decades. With regards to the Complement cascade, thromboinflammation, hypercoagulation and neurological involvement, it is clear that SARS-CoV-2 is similar to SARS-CoV-1 (2003). It cannot be stressed enough that therapeutic options for treatment of 2003-SARS complications and hematological complications in general should be revisited.
1 The complement system: mediator between innate and adaptive immunity;
1.1 Classical Pathway;
1.2 Alternative Pathway;
1.3 Lectin Pathway;
2 Contribution to SARS-CoV-2 severity;
2.1 Complement-induced Acute Lung Injury (ALI) and ARDS;
2.2 Complement anaphylatoxin C5a and IL-8 induction of Reactive Oxygen Species (ROS);
2.3 Interaction of the Complement System and NETs;
2.4 Complement System and coagulation;
2.5 Complement en endothelial dysfunction;
2.6 Do SARS-Coronaviruses specifically target the Lectin Pathway?
3 Cases of SARS-CoV-2 extensive complement deposition damage of the microvasculature (non-Diffuse Alveolar Damage/DAD);
4 Pharmaceutical intervention targeting the Complement cascade in COVID-19
1 The complement system: mediator between innate and adaptive immunity
The complement system refers to a system mediating between the innate and adaptive host immune response against invasion by pathogens, such as viruses. The adaptive immune response is what is needed to ward off bespoke invader. One function is pathogen lysis by the C56-9 (C5 to 9 = C5bC6C7 to C8 to C9) Membrane Attack Complex (MAC). By doing so, the pathogen, for example a virus, is cut and subsequently stripped from its contents. Opsonization is the process in which antibodies bind to the pathogen. Complement C1 binds to IgG, in order to initiate a cascade, which results in the binding of C3b to the pathogen. C3b subsequently adheres to the C3b receptor found on phagocytes. This induces phagocytosis, a process in which phagocytes (protective cells) ingest pathogens inside their membrane in order to form an isolating particle containing the pathogens (the phagosome). Activation of the complement cascade to initiate an immunity response or inflammation is a normal response to invasion of the body, but turns out to be detrimental when overactivation of the complement system occurs. The functions of the complement pathways are: (1) opsonization in order to constitute phagocytosis; (2) initiating inflammation through recruitment of neutrophils and monocytes by anaphylatoxins; (3) lysis of the pathogen by the MAC (Microbiology Principles and Explorations 10th edition, J.G. Black, December 2017).
There are three complement pathways: the classical (CP), alternative (AP) and lectin (LP) complement pathway. Upon programmed cell death (apoptosis), the complement pathways are activated on the cell surface. C3 is the key complement protein. Any of these pathways that produces C3b contributes to amplification of C3 convertase, resulting in a self-amplification loop (Complement System I: molecular mechanisms of activation and regulation, Frontiers in Immunology, June 2015, Volume 6 Article 262).
The relevance of the complement pathways with regards to SARS-CoV-2 is that complement anaphylatoxins C3a and C5a are hypothesized to contribute to cytokine storms in severe COVID cases. C5a attracts neutrophils and monocytes and is involved in the release of Reactive Oxygen Species (ROS), mast cell degranulation and vascular permeability (Inhibiting the C5-C5a receptor axis, Molecular Immunology, Volume 48, Issue 14, August 2011, p. 1631-1642; The case of complement activation in COVID-19 multiorgan impact, Kidney International (2020), 98, 314-322).
1.1 Classical pathway
The classical pathway is initiated by IgM or IgG antigen-antibody complexes binding to complement protein C1q (a protein that is well able to recognize pathogenic patterns in viruses and bacteria and which is capable of recognizing lipopolysaccharide (LPS)), in order to activate C1r which cleaves C1q. C1s cleaves C4 into C4a and C4b. C2 binds to C4b and is cleaved by C1s, to release C2a. C1qrs cleaves C4 and C2, resulting in the C3 convertase C4b2a. C3 is then cleaved into C3a and C3b. C3a is an anaphylatoxin, an inflammation mediator.
C3b is an opsonin, a "tagger", that binds to C4b2a to form C5 convertase C4b2a3b. C5 convertase cleaves C5 into C5a and C5b. Like C3a, C5a is an anaphylatoxin that mediates the inflammatory response by activating neutrophils. C5b forms with C6 (C5bC6) and binds C7, C8 and 12 molecules of C9 to form the TCC C5b-9 MAC that lyses the virus.
Thus: the classical pathway initiates inflammation via C3a and C5a (anaphylatoxins) or MAC (C5b) and opsonization and phagocytosis via C3b (opsonin). IgG strongly interacts with complement protein C1q. The presence of IgG determines the strength of the classical pathway activation of the complement system.
1.2 Alternative pathway
The alternative pathway (also known as the properdin pathway) is permanently active at a lower level to detect pathogens invading the body. Tick-over is a spontaneous hydrolysis of C3 into a fluid C3(H20), which binds to factor B (fB) to be cleaved by factor D (fD) to form the C3 convertase C3(H20) to amplify more C3a anaphylatoxins and C3b. C3b contributes to the alternative pathway amplification convertase, resulting in a C3 self-amplification loop. Factor D cleaves factor B into fragments Ba and Bb. When C3b attaches to fragment Bb, Properdin (Factor P) stabilizes the C3 convertase C3bBb. C3 is then cleaved into C3a and C3b.
In addition, C3b opsonizes the pathogen and binds a C3 convertase to generate C5 convertase C3bBb3b. C5 is then cleaved into the anaphylatoxin C5a that recruits neutrophils and monocytes, while C5b is involved in initiating the MAC. Factor H is a regulator of the AP and the amplification loop. Factor H inhibits the C3 convertase and concurs with factor B for binding C3b. Factor H and factor I (fI) and membrane-bound CD46, CD55 and Membrane Cofactor Proteine (MCP) serve as inactivators of C3b.
In contrast to the classical pathway and lectin pathway (CP and LP), the alternative pathway lacks C1q to serve as its "memory" in the recognition of viruses. This is where properdin and P-selectin come to aid by recruiting the C3 convertase C3(H20) to the surface of cells (Overview of Complement Activation, Seminars in Nephrology, 2013 Nov; 33(6): 479-492; see also Functional Characterization of Alternative and Classical Pathway C3/C5 Convertase Activity and Inhibition Using Purified Models, Frontiers in Immunology, 23 July 2018).
1.3 Lectin pathway
The lectin pathway (LP) uses Mannose-Binding Lectins (MBLs) and ficolins to recognize carbohydrate ligands on the surface of pathogens (sugary patterns). Mannose-Associated Serine Proteases (MASP) are substitute for C1 proteases C1r and C1s which are key in the recognition of viruses and bacteria. MBL forms complexes with MASP-1 and MASP-2, leading to cleavage of C4 and C2 and formation of the C3 convertase C4b2a. The binding of C3b to C3 convertase C4b2a form the C5 convertases that cleave C5 into C5a and C5b. While anaphylatoxin C5a is known for its contribution to inflammation, C5b forms the C5-9 MAC complex.
Each of these factors are involved in cytokine hyperactivation, cell injury, endothelial damage, hypercoagulation and thrombotic events, with the MAC, anaphylatoxins and overactivation of the lectin pathway as key factors.
2 Contribution to SARS-CoV-2 severity
2.1 Complement induced Acute Lung Injury (ALI) and ARDS
As discussed briefly, amplification of anaphylatoxins C3a and C5a, the formation of the amplification loop and the formation of the MAC via C3 and C5 convertases contributes to immune-mediated injury. C5a constitutes inflammation through recruitment of neutrophils, monocytes, eosinophils, phagocytic cells, granule-based enzymes and T-lymphocytes (The role of C5a in acute lung injury induced by highly pathogenic viral infections, Emerging Microbes and Infections (2015) 4, e28, 27 April 2015). What is typical for SARS-Coronaviruses, is a rapid progression towards ARDS. As is the case with avian influenzaviruses, pulmonary fibro proliferative changes are observed at 11 days after symptom onset, with progression towards Acute Lung Injury (ALI). Increased levels of C5a are found in the bronchoalveolar lavage fluid (BALF), but this is not the case with seasonal Influenza A (IVA). Not only are SARS-CoV-1 and SARS-CoV-2 entirely different from influenzaviruses, as they do not share an ancestor, Acute Lung Injury associated with excess C5a complement activation is a characteristic for SARS-CoVs, avian influenzaviruses and MERS-CoVs.
2.2 Complement anaphylatoxin C5a and IL-8 induction of Reactive Oxygen Species (ROS)
C5a as well as interleukin IL-8 is synthesized by pulmonary epithelial cells, macrophages, endothelial cells and neutrophils, among other pulmonary cells. C5a is reported to be able to amplify IL-8 to increase neutrophil counts and further pulmonary dysfunction. After activation of neutrophils and monocytes by C5a, an oxidative burst is generated, followed by the release of Reactive Oxygen Species (ROS) (The case of complement activation in COVID-19 Multiorgan Impact, Kidney International (2020) 98, 314-222). Higher amounts of ROS are observed in fibrotic lung cells.
2.3 Interaction of the Complement System and NETs
C5a in association with granulocyte-macrophage colony-stimulating factor (GM CSF) is reported to be able to induce the release of NETs (neutrophil extracellular traps) and to activate macrophages and endothelial cells to promote vascular leakage and the release of NETs. NETs increase permeability of the pulmonary capillary barrier and induces inflammatory cytokine release (The role of C5a in acute lung injury induced by highly pathogenic viral infections, Emerging Microbes and Infections (2015) 4, e28, 27 April 2015). NETs and hypoxia further contribute to endotheliopathy. While component opsonization induces NET formation, blockade of complement receptors CR1 and CR3 inhibits NETosis (programmed cell death). Moreover, neutrophils and NETs contain C3, Factor B and Factor P (Properdin), which are key to the generation and stabilization of C3 convertase and complement cascade in the Alternative Pathway (AP). Priming neutrophils with lipopolysaccharides ('sugary' LPS) or TNF-α stimulates the release of Properdin, after which C3b is found on these neutrophils (NETosis, complement and coagulation: a triangular relationship, Cellular & Molecular Immunology, 2019 January;16(1):19-27).
When Interferon-gamma (IFN-γ) is used, C5a stimulates NETosis. C5a upregulates Toll-like receptors (TLR) to induce the NET response. The NETosis response is significantly enhanced when neutrophils are primed with Tumor Necrosis Factor-alpha (TNF-α). Factor H acts as a cofactor with Factor I in the inhibition of the Alternative Pathway by degrading C3b. Factor H is recruited to NETs when C3b is deposited on NETs. It is hypothesized that H is recruited to prevent deviant complement activation and MAC formation to protect neutrophil membranes from lysis. In their turn, pathogens are able to recruit Factor H on their surface to prevent C3b opsonization. In addition to induction of the complement system, NETs activate the coagulation pathway.
2.4 Complement system and coagulation
C5a is able to increase tissue factor activity in circulating form and on endothelial cells. Inhibition of C3 and C5 is shown to lead to reduced expression of tissue factor (The key roles of complement and tissue factor in E.coli-induced coagulation in human whole blood, Clinical & Experimental Immunology, October 2015, Vol. 182 Issue 1, p. 81-89). On mast cells, the complement system is able to enhance tissue factor and create a prothrombotic state. MASP-1 and MASP-2, the mannose proteases that initiate the Lectin Pathway, cleave prothrombin to an active thrombin and these proteases activate fibrinogen and factor XII, which stabilizes fibrin. Factor XII in its turn, cleaves C1 to activate the Classical Pathway. Complement inhibitor C1-INH inhibits factor XII and thrombin, while C4b-binding protein C4BP prevents coagulation inhibition by inhibiting protein S.
MAC formation (C5b-9) on platelets stimulates the release of prothrombotic Factor V. Platelets have C3a receptors, indicating that C3 and MAC are platelet activators. In addition, C5a and MAC induce the release of P-selectin from platelet alpha-granules and Ultra-Large Von Willebrand Factor Multimers (ULVWF) that promote platelet adhesion and shedding of thrombomodulin (TM). The MAC induces the release of procoagulant microparticles (PMP). VWF multimers can be bound by C3b to initiate the Alternative Pathway.
Thrombin acts as a C5-convertase in C3 depleted mice models. C3 -/- (knock-out) models have normal C5a levels, despite being deprived of the usual C3b formation. Thrombin is shown to convert C3 and C5 into C3a and C5a. Moreover, thrombin cleaves C5 into a C5b fragment that forms a MAC with significantly higher lysic potential than regularly generates C5b. Hence, thrombin activates C5 in a C3-independent matter (Thrombin generates previously unidentified C5 products that support the terminal complement activation pathway, Thrombosis and Hemostasis, August 23, 2012, Blood Vol. 120, number 8). Beside thrombin, plasmin, FX and FXI generate C3a and C5a, furthering inflammation.
To prevent thrombosis, mast cells express enzymatically active tissue plasminogen activator (t-PA), which induces fibrinolysis, the breakdown of fibrin clots. Anaphylatoxin C5a induces mast cells to express PA inhibitor PAI-I. As a result, the thrombotic state prevails (New Aspects in Thrombotic Research: Complement induced switch in Mast Cells from a profibrinolytic to a prothrombotic phenotype, Pathophysiology of Haemostasis and Thrombosis, 2003/2004; 33: 438-441). MASP-1 is able to activate Thrombin-Activated Fibrinolytic Inhibitor (TAFI). In its turn, fibrinolytic activator plasmin activates C3 and C5, C3-convertase independently inducing the release of C5a and the formation of a C3-convertase independent MAC (Complement Activation in Arterial and Venous Thrombosis is Mediated by Plasmin, EBioMedicine, March 01. 2016, Volume 5, p. 175-182).
2.5 Complement and endothelial dysfunction
C5a interacts directly with the C5aR receptor on endothelial cells. The MAC induces endothelial activation. The dysruption of the endothelium by the MAC induces endotheliitis, prompting the release of IL-6 and IL-1β. Both C5a and the MAC stimulate the endothelium to release IL-8 and monocyte chemoattractant protein MCP-1 and to express adhesion molecules ICAM-1, E-selectin and VCAM. The MAC and C5a induce the release of P-selectin and VWF multimers (ULVWF) in order to promote platelet adhesion and the release of Thrombomodulin (TM) from the endothelium.
These mechanisms increase inflammation, coagulation and vascular permeability. Endothelial cells contain heparan on their surface to moderate inflammation and inhibit coagulation. Damage of endothelial cells by the MAC formation and anaphylatoxin C5a leads to loss of heparan. The shedding of heparan by damaged endothelial cells impairs anti-coagulation, furthering the thrombotic state (The role of C5a and antibody in the release of heparan sulfate from endothelial cells, European Journal of Immunology, November 1991, Vol. 21, Issue 11, p. 2887-2890).
2.6 Do SARS-Coronaviruses specifically target the Lectin Pathway?
As of 2010, it has been hypothesized and reported that Mannose Binding Lectin (MBL) is able to bind to the Spike glycoprotein of SARS-CoV-1, dependent on an N-glycosylation site of SARS-Coronavirus (A Single Asparagine-Linked Glycosylation Site of the Severe Acute Respiratory Syndrome Coronavirus Spike Glycoprotein Facilitates Inhibition by Mannose-Binding Lectin through Multiple Mechanisms, Journal of Virology, September 2010, Vol. 84, No. 17). According to a preprint on SARS-CoV-2, lung tissue of a patient revealed strong staining for MBL, C4, C3, MAC in alveolar epithelial cells, inflammatory cells and pneumocytes in alveolar spaces (Highly pathogenic coronavirus N protein aggravates lung injury by MASP-2-mediated complement over-activation, posted June 18, 2020). Although caution be taken with any preprint, reports of biopsies and autopsies following coronavirus infection support the hypothesis that overactivation of the Lectin Pathway is involved.
3 Cases of SARS-CoV-2 extensive complement deposition damage of the microvasculature (non-DAD)
A report of five cases confirms the activation of both the Alternative Pathway and Lectin Pathway. Depositions of MAC (C5b-9), C4d and MASP-2 were observed in the microvasculatory in SARS-CoV-2 patients. Patient 1 showed severe hemorrhagic pneumonitis with significant fibrin deposition within the septal capillary, endothelial cell necrosis and thrombotic nectrotizing capillary injury syndrome. C3d was similarly deposited in the septal capillary. Extensive C4d and MAC deposition was observed in the alveolar septal capillary. MAC deposition was also seen in normal appearing dermal capillaries. Patient 2 showed a similar pattern of septal capillary injury, hemorrhagic pneumonitis, fibrin deposition and dominant deposition of MAC formations in the microvasculature. Noteworthy, MAC depositions were found in normal appearing lung tissues, while C4d was solely observed in the injured microvasculature. MASP-2 demonstrated granular staining in the interalveolar septa. Patient 1 as well as patient 2 did not show Diffuse Alveolar Damage (DAD); no hyaline membranes were observed.
Patient 3 showed purpuric buttocks, thrombogenic vasculopathy with necrosis, interstitial and perivascular neutrophilia, breakdown of white blood cells and extensive MAC deposits in the microvasculature. In patient 4, purpuric patches were seen on foot soles and hand palms. Occlusive thrombi were found in the artery, including extensive deposits of MAC formations, C3d and C4d. Brain infarctions and a complete infarction in the area supplied by the left middle cerebral artery were revealed on CT. Like patient 3 and 4, patient 5 showed purpuric patching. Perivascular lymphocytic infiltrates and thrombi were accompanied by MAC and C4d (Complement associated microvascular injury and thrombosis in the pathogenesis of severe COVID-19: A report of five cases, Translational Research, June 2020, Vol. 220). A report of 31 COVID patients investigated the plasma levels of soluble C5b-9 (MAC) and C5a. While both the MAC and C5a were significantly elevated, C5a remained within normal range in some patients (Complement activation in patients with COVID-19: A novel therapeutic target, Journal of Allergy and Clinical Immunology, July 2020, Vol. 146, number 1).
4 Pharmaceutical intervention targeting the Complement cascade in COVID-19
C3 knock-out mice models were shown to be protected from lung inflammation. Viral titers are similar in the C3 wild type models and C3 knock-outs. C3 knock-outs did not show respiratory failure. While MCP-1 is expressed highly in both C3 models and C3 knock-outs, Granulocyte colony-stimulating factor (G-CSF), IL-6, Tumor Necrosis Factor-alpha and IL-1a comprised highly produced cytokines and chemokines in C3 models. In models treated with a C3a receptor antagonist or antibodies against C5a, lung inflammation and injury are significantly reduced following MERS-Coronavirus (Complement Activation Contributes to SARS Coronavirus Pathogenesis, Host-Microbe Biology, 9 October 2018, Vol. 9, Issue 5, e01753-18).
Consecutive with the previously mentioned reports of COVID patients, see paragraph 3, blockade of the Mannan-Binding Lectin-Associated Serine Protease-2 (MASP-2) by Narsoplimab or Eculizumab might be considered to prevent Lectin Pathway-mediated hyperinflammation.
AMY-101 is a C3 inhibitor that prevents cleavage of C3, formation of C3 and C5, subsequently preventing formation of anaphylatoxins C3a and C5a and the MAC formation. IFX is a monoclonal antibody that specifically targets C5a, thereby preventing the release of inflammatory cytokines induced by C5a.
Therapeutic options for pharmaceutical inhibition of the complement cascade are:
Narsoplimab- human antibody targeting MASP-2;
Avdoralimab- monoclonal antibody prevents the binding of C5a to its receptor C5aR
Eculizumab- anti-C5 monoclonal antibody preventing cleavage of C5 into C5a
Ravulizumab- antibody against C5
C1-INH- C1 classical pathway inhibitor, approved for treating hereditary angioedema
IFX- monoclonal antibody targeting C5a
AMY-101- C3 inhibitor, preventing cleavage of C3, formation of C3 and C5 and subsequently preventing formation of C3a, C5a and MAC
(Complement Inhibition in COVID-19: A neglected therapeutic option, Frontiers in Immunology, July 2020, Volume 11, Article 1661; see also Complement System I: molecular mechanisms of activation and regulation, Frontiers in Immunology, June 2015, Volume 6 Article 262).
In this deterioration series, I elaborate on the mechanisms that contribute to the severity of SARS-CoV-2 (and, likewise, SARS-CoV-1 (2003)) cases. Previously, I discussed the role of the Renin-Angiotensin-Aldosterone and Kallikrein-Kinin System, hypercoagulation and thromboinflammation and SARS-CoV-2 involvement of the Central Nervous System (either by direct invasion or through systemic diseases following COVID). These mechanisms are strongly intertwined.
As I will discuss in this message, the Complement System is a major contributor to hyperinflammation and tissue damage following coronavirus infection. The Complement interacts, by default, with blood platelets and endothelial cells and contributes to hypercoagulation- and the other way around (Paragraph 2). As can be gathered from biopsy and autopsy reports following COVID-19, depositions found in the microvasculature of COVID patients indicate Lectin Pathway-mediated damage (Paragraph 3). Lastly, I will discuss therapeutical options to target the Complement cascade in COVID-19 (Paragraph 4). In order to write this message, I analyzed research papers from a body of knowledge spanning the last 3 decades. With regards to the Complement cascade, thromboinflammation, hypercoagulation and neurological involvement, it is clear that SARS-CoV-2 is similar to SARS-CoV-1 (2003). It cannot be stressed enough that therapeutic options for treatment of 2003-SARS complications and hematological complications in general should be revisited.
1 The complement system: mediator between innate and adaptive immunity;
1.1 Classical Pathway;
1.2 Alternative Pathway;
1.3 Lectin Pathway;
2 Contribution to SARS-CoV-2 severity;
2.1 Complement-induced Acute Lung Injury (ALI) and ARDS;
2.2 Complement anaphylatoxin C5a and IL-8 induction of Reactive Oxygen Species (ROS);
2.3 Interaction of the Complement System and NETs;
2.4 Complement System and coagulation;
2.5 Complement en endothelial dysfunction;
2.6 Do SARS-Coronaviruses specifically target the Lectin Pathway?
3 Cases of SARS-CoV-2 extensive complement deposition damage of the microvasculature (non-Diffuse Alveolar Damage/DAD);
4 Pharmaceutical intervention targeting the Complement cascade in COVID-19
1 The complement system: mediator between innate and adaptive immunity
The complement system refers to a system mediating between the innate and adaptive host immune response against invasion by pathogens, such as viruses. The adaptive immune response is what is needed to ward off bespoke invader. One function is pathogen lysis by the C56-9 (C5 to 9 = C5bC6C7 to C8 to C9) Membrane Attack Complex (MAC). By doing so, the pathogen, for example a virus, is cut and subsequently stripped from its contents. Opsonization is the process in which antibodies bind to the pathogen. Complement C1 binds to IgG, in order to initiate a cascade, which results in the binding of C3b to the pathogen. C3b subsequently adheres to the C3b receptor found on phagocytes. This induces phagocytosis, a process in which phagocytes (protective cells) ingest pathogens inside their membrane in order to form an isolating particle containing the pathogens (the phagosome). Activation of the complement cascade to initiate an immunity response or inflammation is a normal response to invasion of the body, but turns out to be detrimental when overactivation of the complement system occurs. The functions of the complement pathways are: (1) opsonization in order to constitute phagocytosis; (2) initiating inflammation through recruitment of neutrophils and monocytes by anaphylatoxins; (3) lysis of the pathogen by the MAC (Microbiology Principles and Explorations 10th edition, J.G. Black, December 2017).
There are three complement pathways: the classical (CP), alternative (AP) and lectin (LP) complement pathway. Upon programmed cell death (apoptosis), the complement pathways are activated on the cell surface. C3 is the key complement protein. Any of these pathways that produces C3b contributes to amplification of C3 convertase, resulting in a self-amplification loop (Complement System I: molecular mechanisms of activation and regulation, Frontiers in Immunology, June 2015, Volume 6 Article 262).
The relevance of the complement pathways with regards to SARS-CoV-2 is that complement anaphylatoxins C3a and C5a are hypothesized to contribute to cytokine storms in severe COVID cases. C5a attracts neutrophils and monocytes and is involved in the release of Reactive Oxygen Species (ROS), mast cell degranulation and vascular permeability (Inhibiting the C5-C5a receptor axis, Molecular Immunology, Volume 48, Issue 14, August 2011, p. 1631-1642; The case of complement activation in COVID-19 multiorgan impact, Kidney International (2020), 98, 314-322).
1.1 Classical pathway
The classical pathway is initiated by IgM or IgG antigen-antibody complexes binding to complement protein C1q (a protein that is well able to recognize pathogenic patterns in viruses and bacteria and which is capable of recognizing lipopolysaccharide (LPS)), in order to activate C1r which cleaves C1q. C1s cleaves C4 into C4a and C4b. C2 binds to C4b and is cleaved by C1s, to release C2a. C1qrs cleaves C4 and C2, resulting in the C3 convertase C4b2a. C3 is then cleaved into C3a and C3b. C3a is an anaphylatoxin, an inflammation mediator.
C3b is an opsonin, a "tagger", that binds to C4b2a to form C5 convertase C4b2a3b. C5 convertase cleaves C5 into C5a and C5b. Like C3a, C5a is an anaphylatoxin that mediates the inflammatory response by activating neutrophils. C5b forms with C6 (C5bC6) and binds C7, C8 and 12 molecules of C9 to form the TCC C5b-9 MAC that lyses the virus.
Thus: the classical pathway initiates inflammation via C3a and C5a (anaphylatoxins) or MAC (C5b) and opsonization and phagocytosis via C3b (opsonin). IgG strongly interacts with complement protein C1q. The presence of IgG determines the strength of the classical pathway activation of the complement system.
1.2 Alternative pathway
The alternative pathway (also known as the properdin pathway) is permanently active at a lower level to detect pathogens invading the body. Tick-over is a spontaneous hydrolysis of C3 into a fluid C3(H20), which binds to factor B (fB) to be cleaved by factor D (fD) to form the C3 convertase C3(H20) to amplify more C3a anaphylatoxins and C3b. C3b contributes to the alternative pathway amplification convertase, resulting in a C3 self-amplification loop. Factor D cleaves factor B into fragments Ba and Bb. When C3b attaches to fragment Bb, Properdin (Factor P) stabilizes the C3 convertase C3bBb. C3 is then cleaved into C3a and C3b.
In addition, C3b opsonizes the pathogen and binds a C3 convertase to generate C5 convertase C3bBb3b. C5 is then cleaved into the anaphylatoxin C5a that recruits neutrophils and monocytes, while C5b is involved in initiating the MAC. Factor H is a regulator of the AP and the amplification loop. Factor H inhibits the C3 convertase and concurs with factor B for binding C3b. Factor H and factor I (fI) and membrane-bound CD46, CD55 and Membrane Cofactor Proteine (MCP) serve as inactivators of C3b.
In contrast to the classical pathway and lectin pathway (CP and LP), the alternative pathway lacks C1q to serve as its "memory" in the recognition of viruses. This is where properdin and P-selectin come to aid by recruiting the C3 convertase C3(H20) to the surface of cells (Overview of Complement Activation, Seminars in Nephrology, 2013 Nov; 33(6): 479-492; see also Functional Characterization of Alternative and Classical Pathway C3/C5 Convertase Activity and Inhibition Using Purified Models, Frontiers in Immunology, 23 July 2018).
1.3 Lectin pathway
The lectin pathway (LP) uses Mannose-Binding Lectins (MBLs) and ficolins to recognize carbohydrate ligands on the surface of pathogens (sugary patterns). Mannose-Associated Serine Proteases (MASP) are substitute for C1 proteases C1r and C1s which are key in the recognition of viruses and bacteria. MBL forms complexes with MASP-1 and MASP-2, leading to cleavage of C4 and C2 and formation of the C3 convertase C4b2a. The binding of C3b to C3 convertase C4b2a form the C5 convertases that cleave C5 into C5a and C5b. While anaphylatoxin C5a is known for its contribution to inflammation, C5b forms the C5-9 MAC complex.
Each of these factors are involved in cytokine hyperactivation, cell injury, endothelial damage, hypercoagulation and thrombotic events, with the MAC, anaphylatoxins and overactivation of the lectin pathway as key factors.
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| The complement system: Classic Pathway, Alternative Pathway and Lectin Pathway |
2 Contribution to SARS-CoV-2 severity
2.1 Complement induced Acute Lung Injury (ALI) and ARDS
As discussed briefly, amplification of anaphylatoxins C3a and C5a, the formation of the amplification loop and the formation of the MAC via C3 and C5 convertases contributes to immune-mediated injury. C5a constitutes inflammation through recruitment of neutrophils, monocytes, eosinophils, phagocytic cells, granule-based enzymes and T-lymphocytes (The role of C5a in acute lung injury induced by highly pathogenic viral infections, Emerging Microbes and Infections (2015) 4, e28, 27 April 2015). What is typical for SARS-Coronaviruses, is a rapid progression towards ARDS. As is the case with avian influenzaviruses, pulmonary fibro proliferative changes are observed at 11 days after symptom onset, with progression towards Acute Lung Injury (ALI). Increased levels of C5a are found in the bronchoalveolar lavage fluid (BALF), but this is not the case with seasonal Influenza A (IVA). Not only are SARS-CoV-1 and SARS-CoV-2 entirely different from influenzaviruses, as they do not share an ancestor, Acute Lung Injury associated with excess C5a complement activation is a characteristic for SARS-CoVs, avian influenzaviruses and MERS-CoVs.
2.2 Complement anaphylatoxin C5a and IL-8 induction of Reactive Oxygen Species (ROS)
C5a as well as interleukin IL-8 is synthesized by pulmonary epithelial cells, macrophages, endothelial cells and neutrophils, among other pulmonary cells. C5a is reported to be able to amplify IL-8 to increase neutrophil counts and further pulmonary dysfunction. After activation of neutrophils and monocytes by C5a, an oxidative burst is generated, followed by the release of Reactive Oxygen Species (ROS) (The case of complement activation in COVID-19 Multiorgan Impact, Kidney International (2020) 98, 314-222). Higher amounts of ROS are observed in fibrotic lung cells.
2.3 Interaction of the Complement System and NETs
C5a in association with granulocyte-macrophage colony-stimulating factor (GM CSF) is reported to be able to induce the release of NETs (neutrophil extracellular traps) and to activate macrophages and endothelial cells to promote vascular leakage and the release of NETs. NETs increase permeability of the pulmonary capillary barrier and induces inflammatory cytokine release (The role of C5a in acute lung injury induced by highly pathogenic viral infections, Emerging Microbes and Infections (2015) 4, e28, 27 April 2015). NETs and hypoxia further contribute to endotheliopathy. While component opsonization induces NET formation, blockade of complement receptors CR1 and CR3 inhibits NETosis (programmed cell death). Moreover, neutrophils and NETs contain C3, Factor B and Factor P (Properdin), which are key to the generation and stabilization of C3 convertase and complement cascade in the Alternative Pathway (AP). Priming neutrophils with lipopolysaccharides ('sugary' LPS) or TNF-α stimulates the release of Properdin, after which C3b is found on these neutrophils (NETosis, complement and coagulation: a triangular relationship, Cellular & Molecular Immunology, 2019 January;16(1):19-27).
When Interferon-gamma (IFN-γ) is used, C5a stimulates NETosis. C5a upregulates Toll-like receptors (TLR) to induce the NET response. The NETosis response is significantly enhanced when neutrophils are primed with Tumor Necrosis Factor-alpha (TNF-α). Factor H acts as a cofactor with Factor I in the inhibition of the Alternative Pathway by degrading C3b. Factor H is recruited to NETs when C3b is deposited on NETs. It is hypothesized that H is recruited to prevent deviant complement activation and MAC formation to protect neutrophil membranes from lysis. In their turn, pathogens are able to recruit Factor H on their surface to prevent C3b opsonization. In addition to induction of the complement system, NETs activate the coagulation pathway.
2.4 Complement system and coagulation
C5a is able to increase tissue factor activity in circulating form and on endothelial cells. Inhibition of C3 and C5 is shown to lead to reduced expression of tissue factor (The key roles of complement and tissue factor in E.coli-induced coagulation in human whole blood, Clinical & Experimental Immunology, October 2015, Vol. 182 Issue 1, p. 81-89). On mast cells, the complement system is able to enhance tissue factor and create a prothrombotic state. MASP-1 and MASP-2, the mannose proteases that initiate the Lectin Pathway, cleave prothrombin to an active thrombin and these proteases activate fibrinogen and factor XII, which stabilizes fibrin. Factor XII in its turn, cleaves C1 to activate the Classical Pathway. Complement inhibitor C1-INH inhibits factor XII and thrombin, while C4b-binding protein C4BP prevents coagulation inhibition by inhibiting protein S.
MAC formation (C5b-9) on platelets stimulates the release of prothrombotic Factor V. Platelets have C3a receptors, indicating that C3 and MAC are platelet activators. In addition, C5a and MAC induce the release of P-selectin from platelet alpha-granules and Ultra-Large Von Willebrand Factor Multimers (ULVWF) that promote platelet adhesion and shedding of thrombomodulin (TM). The MAC induces the release of procoagulant microparticles (PMP). VWF multimers can be bound by C3b to initiate the Alternative Pathway.
Thrombin acts as a C5-convertase in C3 depleted mice models. C3 -/- (knock-out) models have normal C5a levels, despite being deprived of the usual C3b formation. Thrombin is shown to convert C3 and C5 into C3a and C5a. Moreover, thrombin cleaves C5 into a C5b fragment that forms a MAC with significantly higher lysic potential than regularly generates C5b. Hence, thrombin activates C5 in a C3-independent matter (Thrombin generates previously unidentified C5 products that support the terminal complement activation pathway, Thrombosis and Hemostasis, August 23, 2012, Blood Vol. 120, number 8). Beside thrombin, plasmin, FX and FXI generate C3a and C5a, furthering inflammation.
To prevent thrombosis, mast cells express enzymatically active tissue plasminogen activator (t-PA), which induces fibrinolysis, the breakdown of fibrin clots. Anaphylatoxin C5a induces mast cells to express PA inhibitor PAI-I. As a result, the thrombotic state prevails (New Aspects in Thrombotic Research: Complement induced switch in Mast Cells from a profibrinolytic to a prothrombotic phenotype, Pathophysiology of Haemostasis and Thrombosis, 2003/2004; 33: 438-441). MASP-1 is able to activate Thrombin-Activated Fibrinolytic Inhibitor (TAFI). In its turn, fibrinolytic activator plasmin activates C3 and C5, C3-convertase independently inducing the release of C5a and the formation of a C3-convertase independent MAC (Complement Activation in Arterial and Venous Thrombosis is Mediated by Plasmin, EBioMedicine, March 01. 2016, Volume 5, p. 175-182).
2.5 Complement and endothelial dysfunction
C5a interacts directly with the C5aR receptor on endothelial cells. The MAC induces endothelial activation. The dysruption of the endothelium by the MAC induces endotheliitis, prompting the release of IL-6 and IL-1β. Both C5a and the MAC stimulate the endothelium to release IL-8 and monocyte chemoattractant protein MCP-1 and to express adhesion molecules ICAM-1, E-selectin and VCAM. The MAC and C5a induce the release of P-selectin and VWF multimers (ULVWF) in order to promote platelet adhesion and the release of Thrombomodulin (TM) from the endothelium.
These mechanisms increase inflammation, coagulation and vascular permeability. Endothelial cells contain heparan on their surface to moderate inflammation and inhibit coagulation. Damage of endothelial cells by the MAC formation and anaphylatoxin C5a leads to loss of heparan. The shedding of heparan by damaged endothelial cells impairs anti-coagulation, furthering the thrombotic state (The role of C5a and antibody in the release of heparan sulfate from endothelial cells, European Journal of Immunology, November 1991, Vol. 21, Issue 11, p. 2887-2890).
2.6 Do SARS-Coronaviruses specifically target the Lectin Pathway?
As of 2010, it has been hypothesized and reported that Mannose Binding Lectin (MBL) is able to bind to the Spike glycoprotein of SARS-CoV-1, dependent on an N-glycosylation site of SARS-Coronavirus (A Single Asparagine-Linked Glycosylation Site of the Severe Acute Respiratory Syndrome Coronavirus Spike Glycoprotein Facilitates Inhibition by Mannose-Binding Lectin through Multiple Mechanisms, Journal of Virology, September 2010, Vol. 84, No. 17). According to a preprint on SARS-CoV-2, lung tissue of a patient revealed strong staining for MBL, C4, C3, MAC in alveolar epithelial cells, inflammatory cells and pneumocytes in alveolar spaces (Highly pathogenic coronavirus N protein aggravates lung injury by MASP-2-mediated complement over-activation, posted June 18, 2020). Although caution be taken with any preprint, reports of biopsies and autopsies following coronavirus infection support the hypothesis that overactivation of the Lectin Pathway is involved.
3 Cases of SARS-CoV-2 extensive complement deposition damage of the microvasculature (non-DAD)
A report of five cases confirms the activation of both the Alternative Pathway and Lectin Pathway. Depositions of MAC (C5b-9), C4d and MASP-2 were observed in the microvasculatory in SARS-CoV-2 patients. Patient 1 showed severe hemorrhagic pneumonitis with significant fibrin deposition within the septal capillary, endothelial cell necrosis and thrombotic nectrotizing capillary injury syndrome. C3d was similarly deposited in the septal capillary. Extensive C4d and MAC deposition was observed in the alveolar septal capillary. MAC deposition was also seen in normal appearing dermal capillaries. Patient 2 showed a similar pattern of septal capillary injury, hemorrhagic pneumonitis, fibrin deposition and dominant deposition of MAC formations in the microvasculature. Noteworthy, MAC depositions were found in normal appearing lung tissues, while C4d was solely observed in the injured microvasculature. MASP-2 demonstrated granular staining in the interalveolar septa. Patient 1 as well as patient 2 did not show Diffuse Alveolar Damage (DAD); no hyaline membranes were observed.
Patient 3 showed purpuric buttocks, thrombogenic vasculopathy with necrosis, interstitial and perivascular neutrophilia, breakdown of white blood cells and extensive MAC deposits in the microvasculature. In patient 4, purpuric patches were seen on foot soles and hand palms. Occlusive thrombi were found in the artery, including extensive deposits of MAC formations, C3d and C4d. Brain infarctions and a complete infarction in the area supplied by the left middle cerebral artery were revealed on CT. Like patient 3 and 4, patient 5 showed purpuric patching. Perivascular lymphocytic infiltrates and thrombi were accompanied by MAC and C4d (Complement associated microvascular injury and thrombosis in the pathogenesis of severe COVID-19: A report of five cases, Translational Research, June 2020, Vol. 220). A report of 31 COVID patients investigated the plasma levels of soluble C5b-9 (MAC) and C5a. While both the MAC and C5a were significantly elevated, C5a remained within normal range in some patients (Complement activation in patients with COVID-19: A novel therapeutic target, Journal of Allergy and Clinical Immunology, July 2020, Vol. 146, number 1).
4 Pharmaceutical intervention targeting the Complement cascade in COVID-19
C3 knock-out mice models were shown to be protected from lung inflammation. Viral titers are similar in the C3 wild type models and C3 knock-outs. C3 knock-outs did not show respiratory failure. While MCP-1 is expressed highly in both C3 models and C3 knock-outs, Granulocyte colony-stimulating factor (G-CSF), IL-6, Tumor Necrosis Factor-alpha and IL-1a comprised highly produced cytokines and chemokines in C3 models. In models treated with a C3a receptor antagonist or antibodies against C5a, lung inflammation and injury are significantly reduced following MERS-Coronavirus (Complement Activation Contributes to SARS Coronavirus Pathogenesis, Host-Microbe Biology, 9 October 2018, Vol. 9, Issue 5, e01753-18).
Consecutive with the previously mentioned reports of COVID patients, see paragraph 3, blockade of the Mannan-Binding Lectin-Associated Serine Protease-2 (MASP-2) by Narsoplimab or Eculizumab might be considered to prevent Lectin Pathway-mediated hyperinflammation.
AMY-101 is a C3 inhibitor that prevents cleavage of C3, formation of C3 and C5, subsequently preventing formation of anaphylatoxins C3a and C5a and the MAC formation. IFX is a monoclonal antibody that specifically targets C5a, thereby preventing the release of inflammatory cytokines induced by C5a.
Therapeutic options for pharmaceutical inhibition of the complement cascade are:
Narsoplimab- human antibody targeting MASP-2;
Avdoralimab- monoclonal antibody prevents the binding of C5a to its receptor C5aR
Eculizumab- anti-C5 monoclonal antibody preventing cleavage of C5 into C5a
Ravulizumab- antibody against C5
C1-INH- C1 classical pathway inhibitor, approved for treating hereditary angioedema
IFX- monoclonal antibody targeting C5a
AMY-101- C3 inhibitor, preventing cleavage of C3, formation of C3 and C5 and subsequently preventing formation of C3a, C5a and MAC
(Complement Inhibition in COVID-19: A neglected therapeutic option, Frontiers in Immunology, July 2020, Volume 11, Article 1661; see also Complement System I: molecular mechanisms of activation and regulation, Frontiers in Immunology, June 2015, Volume 6 Article 262).
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| Targeting the complement cascade through pharmaceutical inhibitors |
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| Therapeutic options targeting the complement cascade in SARS-CoV-2/COVID-19 |
