key: cord-0746199-bgxz5ly1
authors: Vitiello, A.; La Porta, R.; D'Aiuto, V.; Ferrara, F.
title: Pharmacological approach for the reduction of inflammatory and prothrombotic hyperactive state in COVID-19 positive patients by acting on complement cascade
date: 2021-01-20
journal: Hum Immunol
DOI: 10.1016/j.humimm.2021.01.007
sha: 83a41dbfff30343c17020fb9ac46d84805e5964e
doc_id: 746199
cord_uid: bgxz5ly1

The novel Coronavirus SARS-CoV-2 is the viral pathogen responsible for the ongoing global pandemic, COVID-19 (Coronavirus disease 2019). To date, the data recorded indicate 1.62 Mln deaths and 72.8 Mln people infected (WHO situation report Dec 2020). On December 27, the first anti-COVID-19 vaccinations started in Europe. There are no direct antivirals against SARS-CoV-2. Understanding the pathophysiological and inflammatory/immunological processes of SARS-CoV-2 infection is essential to identify new drug therapies. In the most severe COVID-19 cases, an unregulated immunological/inflammatory system results in organ injury that can be fatal to the host in some cases. Pharmacologic approaches to normalize the unregulated inflammatory/immunologic response is an important therapeutic solution. Evidence associates a non-regulation of the “complement system” as one of the causes of generalized inflammation causing multi-organ dysfunction. Serum levels of a complement cascade mediator, factor “C5a”, have been found in high concentrations in the blood of COVID-19 patients with severe disease. In this article we discuss the correlation between complement system and COVID-19 infection and pharmacological solutions directed to regulate.

The new Coronavirus SARS-CoV-2 (COVID-19) is the cause of Severe Acute Respiratory Syndrome (SARS), a severe form of viral pneumonia. The virus spread rapidly from China to the rest of the world in a very short time and with considerable intensity and severity creating a "global emergency". To date the data recorded indicated about 1.62 million deaths and 72.8 million people infected (WHO Situation Reports Dec 2020) . SARS-CoV-2 is an RNA virus similar for about 80% of the viral genome to SARS-CoV (responsible for the 2003 outbreak) (Wu, Y., et al. 2020) . In vitro studies confirm that the virus penetrates human cells by binding to ACE-2 protein, the angiotensinconverting enzyme 2, which is part of the renin-angiotensin system (RAS) (Guoping, L., et al. 2020 ) and is considered as a possible receptor protein. Patients infected with this virus are also known to exhibit changes and variations in the concentrations of enzymatic components of the RAS during days of illness (Huang, C., et al. 2020; . SARS-CoV-2 infection can also have a totally asymptomatic or mildly symptomatic course. In a percentage of cases, the infection has a course consisting of an asymptomatic or mildly symptomatic initial phase and subsequent phases characterized by a generalized inflammatory state causing multi-organ tissue injury and respiratory distress syndrome (Wang D et al. 2020; Chen N et al. 2020) . According to the observational studies conducted, most of the patients considered as severe cases have bilateral interstitial pneumonia and a hyperactive inflammatory state that is not only localized in the lung tissue, but in all tissues of the body, causing multi-organ dysfunction and high risk of thrombosis. (Toshiaky I et al. 2020) The presence of generalized inflammation in all organs and the resulting increased risk of thrombosis requires timely anti-inflammatory/immunomodulatory and anticoagulant treatment. The generalized inflammatory state responsible for severe injury is caused by hyperactivation of components of the host inflammatory/immune system characterized by a sudden and elevated release of cytokines, an event called a "cytokine storm" that leads to severe and sometimes fatal tissue damage. A major contributor to this dysregulation of the inflammatory/immune system is attributed to an overactive and unregulated complement system. (Gao, T., et al. 2020) In fact, elevated serum levels of C5a (complement cascade component) have been reported in blood in COVID-19 patients with severe staging, suggesting an important hyperactivation of the complement system.

The pathophysiology of COVID-19 infection and the mechanisms underlying the severe tissue damage is still not fully understood and clear. In the early stages of infection, an adequate inflammatory/immunological response is of paramount importance for the host organism to fight the virus and avoid more severe stages of infection. In the more severe stages of infection, an inadequate and dysregulated inflammatory/immunological response to the infection may be responsible for elevated accumulation of immune cells and inflammatory mediators (cytokine storm) in tissues which may lead to damage of the lung architecture and multi-organ damage. In the early stages of viral infection, a proper initial inflammatory response attracts T cells to the site of infection where they can fight and eliminate virus-infected cells. In the more severe stages of infection, if the immune response has not been sufficient to completely eliminate the viral load, a hyperactive and unregulated inflammatory/immunologic response, induced by a cytokine storm, can lead to severe lung injury, generalized inflammation, and multi-organ dysfunction. (Tay, M.Z et al. 2020) . Evidence has shown that in severe cases of COVID-19, the high concentration of proinflammatory mediators (particularly IL-2, IL-6, and TNF-α) is responsible for tissue injury. A key contributor to the state of excessive inflammatory activation in severe cases is the complement system. The complement system is part of the immune system that provides an innate defense against pathogens and mediates inflammatory responses. It consists of numerous plasma proteins that, when activated, interact with various cells and mediators of the immune system. (Mastellos D. et al. 2003 ) These activations and interactions vary throughout the stages of a viral infection, the complement system is responsible for a dual action, innate immunity response, as well as in the events that occur later during the adaptive immune reaction. Thus, the complement response can lead to an acute inflammatory reaction that, by the immune system, aims to eliminate host pathogens. In some cases, however, as was demonstrated during COVID-19 infection, an overactive and unregulated complement system can be deleterious to the host itself and contribute to the state of systemic inflammation, however, a lack of an inadequate contribution of the complement system can promote viral replication and infection and a failure of the host's defenses to respond. Adequate and regulated activation of the complement system is therefore essential in combating COVID-19 infection. From a molecular perspective, complement is well known to promote immune cell activation and pro-inflammatory states; complement mediators, particularly C3a and C5a are able to activate neutrophils, mast cells, monocytes/macrophages, T cells, and B cells. (Markiewski MM et al. 2007 ). As noted above, the mediator C5a, in high concentrations in patients with moderate and severe COVID-19 along with elevated IL-6 (Cugno M, et al. 2020) , has been shown in plasma, suggesting that complement plays a key role. The SARS-CoV-2 genome encodes for spike protein (S), which is essential for entry into cells through the ACE-2 membrane. The complement system can be activated through three pathways, the classical, alternative, and lectin pathway, SARS-CoV-2 spike protein (S) is detected by mannan-binding lectin (MBL) which induces complement activation in lectin-mediated SARS-CoV infection. (Ip, W. K., et al.2020) ( Zhou, Y et al.2020) .

The classical pathway is activated by binding of IgM-or IgG-induced natural antibodies, which form immunocomplexes with viral antigens. (Fig.1) 

Schematic representation of complement activation by SARS-CoV-2. SARS-CoV-2 infection mediates complement system activation via the classical, lectin and alternative pathways. The classical pathway is activated by the binding of IgM or IgG-induced antibodies, which form immunocomplexes with viral antigens, the lectin pathway is activated by the binding of SARS-CoV-2 proteins to an MBL protein (Mannan-binding lectin). Complement activation leads to the formation of a membrane attachment complex (MAC) on the surface of the target cells. C3a and C5a mediate complement actions such as inflammation, coagulation, platelet activation, leukocyte recruitment and endothelial cell activation, which are observed in COVID-19 infection. If hyperactivated, the complement leads to an inflammatory pulmonary complement-mediated lesion.

The C5 or C3 inhibition, blocking the generation of proinflammatory mediators.

Activated components of the complement system are important effector molecules that attract, activate, and regulate innate and adaptive immune cells (Laumonnier, Y et al. 2020) . Studies have shown that there is a correlation between complement and the coagulation cascade. (Riedl M., et al. 2020; Perico L et al 2020) Several evidences show that patients with severe COVID-19 have a hypercoagulable state. (Emert R, et al. 2020) The hypothesis that dysregulated complement activation may also be responsible for hypercoagulability in severe COVID-19 patients is not fully understood. (Gavriilaki E et al. 2020) Exposure of the endothelium to C5b-9 leads to the release of VWF (Von Willembrand factor) (Bettoni S, et al. 2017; Feng S, et al. 2015) which leads to platelet activation and aggregation, contributing to a prothrombotic state with thrombus formation and arteriolar occlusion and thrombotic microangiopathy (TMA) (Ota et al. 2005 2.2 Pharmacological approach with a "terminal complement inhibitor".

On December 27, 2020, the first COVID-19 vaccinations began in Europe. Currently, drug therapy management of the COVID-19 positive patient is limited to symptomatic and palliative treatment.

To date, there are no antivirals directed against SARS-CoV-2. A potential pharmacological therapeutic approach against COVID-19 is modifying and regulating agents of the complement cascade. Clinical signs of severely ill patients with COVID-19 showed significant complement activation (Gao, T., et al.2020) suggesting that blocking the complement cascade in the most severe stages could be an effective therapeutic target to avoid severe severe complications. Evidence has shown rapid clinical improvement in patients with COVID-19 mediated by complement cascade inhibitory agents. (Mastaglio, S et al. 2020 ; Diurno, F. et al. 2020) . In this direction, a pharmacological solution to prevent the multi-organ inflammatory alteration and the risk of thrombosis is the blockade of the complement protein C5, thus inhibiting the activation of the terminal portion of the cascade with the antibody Eculizumab (Reis ES et al. 2018) or Ravulizumab.

Drug therapy with Eculizumab in particular has already been shown to be effective in thrombotic, hematologic and inflammatory diseases. The identification of new effective and safe therapeutic solutions for COVID-19 positive patients is a great challenge for the whole scientific world. Inhibiting terminal complement factor could be an important strategy to reduce the inflammatory state and the risk of thrombosis that can cause severe injury and in some cases death. Wellstructured clinical studies will provide us with the clinical evidence needed to investigate this important scientific hypothesis. Evidence shows that the acute respiratory syndrome caused by SARS-CoV-2, is closely associated with activation of the C3 component of complement (Jan C. Holter et al. 2020) . This suggests that C3 inhibition may alleviate the inflammatory pulmonary complications of SARS-CoV-2 infection. (Kulkarni H et al 2020) Furthermore, the upstream placement of C3 signaling in the innate immune cascade further supports the broader antiinflammatory potential of C3 blockade with pharmacological agents. (Fang S et al 2020) C3

inhibition could simultaneously block C3a and C5a generation, as well as intrapulmonary C3

activation and IL-6 release from alveolar macrophages, or other cells expressing C3a receptors (C3aRs) and/or C5a receptors (C5aRs), thereby ameliorating lung injury. (Kim AHJ et al 2020) Proximal complement inhibitors (which target C3 or its upstream activators) might be more effective in combating severe COVID-19 stages than C5 inhibitors, but these hypotheses have yet to be demonstrated by clinical evidence. Several clinical trials are underway to test complement system-modifying agents by different targets. (Fig. 2 ) Important information such as the right time window of administration for optimal intervention, the patient populations that could benefit from pharmacological modulation of complement have yet to be established. (Riedemann et al 2017) . In a phase 2 study (Vlaar et al.2020 ) a significant temporary increase in D-dimer was observed at the start of Anti-C5a antibody therapy, this suggests a direct or indirect profibrinolytic effect and is consistent with the lower pulmonary embolism rate observed in the treatment group versus the control group. (Magro C et al 2020 , Ackermann M et al 2020 . Activation of coagulation in COVID-19 could be initiated by direct virus-induced endothelial injury resulting in upregulation of tissue factor,suppressed fibrinolysis and production of other procoagulant proteins. (Noris M et al 2020) In particular, activation of C5a has been shown to directly induce endothelial tissue factor upregulation, ( Ikeda k et al.2020) activation of neutrophil-mediated coagulation, and to change inflammatory cells from a profibrinolytic (t-PA release) to a prothrombotic phenotype. In these advanced stages of COVID-19, C3 inhibition has the potential to largely control not only ARDS but also systemic inflammation affecting the microvascular beds of the kidney, brain, and other vital organs, which appears to be a complication in severe cases. Complement is a key player in protective immunity against pathogens, but its excessive or deregulated activation can result in collateral tissue injury.

SARS-CoV-2 can cause an abnormal inflammatory/immunologic response responsible for the most severe complications and tissue damage, in some cases fatal. Regulation of the inflammatory/immunologic response in patients with ongoing SARS-CoV-2 infection with specific pharmacologic agents may be of great clinical benefit. In particular, an essential element of the immune system, termed the "complement system," has been shown to be responsible for much of the inflammatory/immunologic dysregulation and cytokine cascade that occurs in the most severe phases of viral infection. Pharmacological modulation of the complement system (including in combination with other immunomodulants and anticoagulants) may be useful in managing the dysregulated and generalized inflammatory state, endothelial injury, and hypercoagulable state and preventing COVID-19 cases from having severe complications. Too many questions remain unanswered. First, what is the optimal time to initiate anti-complement therapy? Which protein of the complement system is the most effective target against COVID-19? Will treatment with anticomplement therapies increase the risk of complications from other infections? And finally, does complement inhibition reduce the risk of thrombotic events? Ongoing clinical trials will provide the necessary answers.

Pulmonary vascular endothelialitis, thrombosis, and angiogenesis in COVID-19

Interaction between multimeric von Willebrand factor and complement: a fresh look to the pathophysiology of microvascular thrombosis

Epidemiological and clinical characteristics of 99 cases of 2019 novel coronavirus pneumonia in Wuhan, China: a descriptive study

Complement activation in patients with COVID-19: A novel therapeutic target

Eculizumab treatment in patients with COVID19: Preliminary results from real life ASL Napoli 2 Nord experience

COVID-19 and hypercoagulability in the outpatient setting

Decreased complement C3 levels are associated with poor prognosis in patients with COVID-19: A retrospective cohort study

Afshar-Kharghan V. von Willebrand factor is a cofactor in complement regulation

Antirheumatic in SARS-CoV-2: benefit or risk?

The added value of pirfenidone to fight inflammation and fibrotic state induced by SARS-CoV-2 : Anti-inflammatory and anti-fibrotic therapy could solve the lung complications of the infection?

Are multiple sclerosis therapies safe in severe acute respiratory syndrome coronavirus 2 times?

Remdesivir and COVID-19

Potential pharmacological approach in the regulation of ACE-2 and DPP-IV in diabetic COVID-19 patient

Scientific and pharmacological rationale for the treatment of cardiac damage caused by covid 19

Highly pathogenic coronavirus N protein aggravates lung injury by MASP2-mediated complement over-activation

Assessing ACE2 expression patterns in lung tissues in the pathogenesis of COVID-19

Complement activation contributes to severe acute respiratory syndrome coronavirus pathogenesis. mBio

Severe COVID-19 infection and thrombotic microangiopathy: success does not come easily

Systemic complement activation is associated with respiratory failure in COVID-19 hospitalized patients

Clinical features of patients infected with 2019 novel coronavirus in Wuhan, China. Lancet

C5a induces tissue factor activity on endothelial cells

Mannose-binding lectin in severe acute respiratory syndrome coronavirus infection

The beneficial and pathogenic roles of complement in COVID-19

Targeting complement activation in COVID-19

Novel insights into the expression pattern of anaphylatoxin receptors in mice and men

Characterization of serious adverse drug reactions as cause of emergency department visit in children: a 5-years active pharmacovigilance study

Complement associated microvascular injury and thrombosis in the pathogenesis of severe COVID-19 infection: a report of five cases

The role of complement in inflammatory diseases from behind the scenes into the spotlight

The first case of COVID-19 treated with the complement C3 inhibitor AMY-101

Complement: structure, functions, evolution, and viral molecular mimicry

The case of complement activation in COVID-19 multiorgan impact

Fremeaux-Bacchi V. Spectrum of complement-mediated thrombotic microangiopathies: pathogenetic insights identifying novel treatment approaches

Terminal complement components mediate release of von Willebrand factor and adhesion of platelets in arteries of allografts

Immunity, endothelial injury and complement-induced coagulopathy in COVID-19

Safety profile after prolonged C3 inhibition

Controlling the anaphylatoxin C5a in diseases requires a specifically targeted inhibition

Author Correction: Complement as a target in COVID-19?

Complement Inhibition in Coronavirus Disease (COVID)-19: A Neglected Therapeutic Option

The trinity of COVID-19: immunity, inflammation and intervention

Coagulopathy in COVID-19

Cytokine storm and colchicine potential role in fighting SARS-CoV-2 pneumonia

Correlation between renin-angiotensin system and Severe Acute Respiratory Syndrome Coronavirus 2 infection: What do we know?

Remdesivir versus ritonavir/lopinavir in COVID-19 patients

Therapeutic Strategies for SARS-CoV-2 acting on ACE-2

Pharmacological agents to therapeutic treatment of cardiac injury caused by Covid-19

Anti-C5a antibody IFX-1 (vilobelimab) treatment versus best supportive care for patients with severe COVID-19 (PANAMO): an exploratory, open-label, phase 2 randomised controlled trial

Complement. First of two parts

Complement. Second of two parts

Clinical Characteristics of 138 Hospitalized Patients With 2019 Novel Coronavirus-Infected Pneumonia in Wuhan, China

Pharmacokinetics and Pharmacodynamics of Eculizumab, and Possibilities for an Individualized Approach to Eculizumab

Compensation of ACE2 Function for Possible Clinical Management of 2019-nCoV-Induced Acute Lung Injury

A single asparagine-linked glycosylation site of the severe acute respiratory syndrome coronavirus spike glycoprotein facilitates inhibition by mannose-binding lectin through multiple mechanisms

I, The undersigned, Francesco Ferrara and any other author, declare that: The manuscript was written entirely by the authors;  All authors made an equal contribution in the development of the paper;  We have no conflict of interest;  We have not received funding/source;  There are no sensitive data and no patients were recruited for this study;  The document does not conflict with ethical legislation.  The authors accept the full TRANSFER OF COPYRIGHT to the journal.