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A global vision of the biology of SARS-CoV2, COVID-19 pathology and associated therapeutic pathways

MacKenzieJun 17, 2021, 2:10:04 AM

https://www.francesoir.fr/archive-scientifique-libre/une-vision-globale-de-la-biologie-du-sars

Author (s): Jean-François Lesgards, doctor in biochemistry, and Dominique Cerdan, doctor in pharmacy, for FranceSoir

Introduction:

Infection with the new coronavirus SARS-CoV-2 (Severe acute respiratory syndrome-coronavirus-2), at the origin of the pathology COVID-19 (Coronavirus disease-19), is responsible for a global pandemic with health consequences and devastating economics. Since its appearance in China in the winter of 2019, more than 175 million cases and more than 3.77 million deaths have been recorded in just over a year. In most cases (over 85%), COVID-19 is a mild illness and a large majority of infected individuals are asymptomatic or present with a paucisymptomatic illness (with few symptoms) characterized by fever, flu syndrome, anosmia (loss of smell), and ageusia (loss of taste). In 10 to 15% of cases on the contrary, COVID-19 leads to hospitalization which can lead to hypoxemic pneumonia. The most severe form of the disease (affecting 5 to 10% of hospitalized patients) corresponds to acute respiratory distress syndrome (ARDS), sometimes associated with other organ failures and justifying the admission of patients to intensive care.

SARS-CoV2 : genetic characteristics and differences with SARS-CoV1 and MERS

The origin of severe acute respiratory syndrome coronavirus 2 (SARS-CoV2) is still controversial.

Genomic analyzes show that the virus is probably a chimera: that is, it contains genetic material from different species. A chimera is not necessarily synthetic, and RNA from viruses of different species can combine naturally over time as well.

Summarizing the work of geneticists, SARS-COV2 appears to be for the most part of its sequence closer to a bat coronavirus (CoV RaTG13), while its receptor binding domain (RBD) of human host is almost identical to that of a pangolin coronavirus.

The particular key site of furin and its importance in the penetration of the virus into human cells

There is a very special site in the protein structure of SARS-CoV2 that has become famous for all those interested in SARSCoV2. This site is called the furin cleavage site or “furin site” and it is found in the famous spike protein (peak in French) of SARS-CoV2.

We can imagine that these points are complex for non-scientists and the public in general, but it is probably one of the most important points and which can explain a major part of the toxicity of this virus in combination with another key point: the peculiarity of the virus receptor in humans, the angiotensin-2 converting enzyme (or ACE2) which is found on the surface of cells that line the human airways.

These points may explain, in particular, the strong penetrability and replication of the virus in its human host as well as the activation of certain inflammatory pathways which ensue in COVID-19.

The spike protein has two subunits with different roles (figure 1). This spike or S protein which we very often hear about and which is the target of vaccines (colored red in figure 2) is directly associated with the name of crown that we give to coronaviruses. It is responsible for anchoring the virus on its ACE2 receptor present in the respiratory tract but also expressed in many organs (see below).

If we detail a little (figure 1), this S protein is made up of 2 parts: the first, called S1, recognizes the target of the virus and the second S2 helps the virus, once anchored to the cell, to fuse with the membrane cellular. Once the virus's outer membrane fuses with that of the affected cell, the viral genome is injected into the cell, hijacking its protein-making machinery, and forcing it to generate new viruses.

The fusion process is started by the fusion peptide marked in yellow, but in order for it to engage in this action, a protein (an enzyme) must cut, literally like with scissors, the protein S in order to activate it. , at a specific location: one of the sites marked by diamonds in figure 1.

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Figure 1 : Precise diagram of the Spike protein and its different elements: in particular S1 (host receptor binding site by l) and S2 and the furin site

The virus does not have its own scissors, so it uses various enzymes from its victims capable of cutting proteins (proteases). There are several types of such proteases as can be seen with these diamonds of different colors (furin, trypsin, cathepsin…). But not all proteases are created equal, and not all cell types have the proteases the virus needs. Furin is one of the most effective and it is found not only on the surface of cells, but also inside. In addition, as seen in this figure 1, the amino acids of the furin site are very different from those of other enzymes if we compare SARS-CoV2 to SARS-CoV1 whose replication rate is much lower (Chu H and al., 2020).

As seen in figure 2, SARS-CoV2 has a group of amino acids PRRAR (where R = Arginine, a basic amino acid) which creates a basic site very attractive for the furins which will cut the protein and activate entry of the virus. This concentration of amino acids R in SARS-CoV2 is quite simply UNIQUE if we compare it to the 3000 coronaviruses known to date (in none of the close relatives of bats or pangolin for example) even if this type of repetition is present in human immunodeficiency virus (HIV), influenza, human cytoMegalo virus (herpes) and respiratory syncytial virus, yellow fever, or Zika and Ebola.

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Figure 2 : Amino acid comparison of the furin site between SARS-CoV2 and other coronaviruses believed to be closest

Furin cuts proteins in strictly defined places, namely after an RxxR sequence (i.e. Arg-XX-Arg, where X can be any amino acid). In addition, if the arginine is also in the second or third place (i.e. RRxR or RxRR), then the cleavage efficiency is greatly increased.

This is well known to biochemists who are able to fabricate these sequences in the laboratory to increase the virulence of viruses in what are called gain of function (GOF) studies.

All this is very well described in the recent article by Nicholas Wade in a real journalistic work as well as in various scientific journals (Segreto R and Deigein Y, 2020).

It is recalled here that more than 200 scientists have called for the cessation of this type of work to manufacture chimeric viruses in general supposed to prevent pandemics! The problem, they said, is that it increases the likelihood of a pandemic occurring in a lab accident.(which has happened often for decades!) and finally under the presidency of Barack Obama, on October 17, 2014, the United States government and the NIH (National Institutes of Health), the main body of the United States government responsible for biomedical and public health research announced an unprecedented suspension on funding for 21 GOF studies involving research on influenza, MERS-CoV (Middle East Respiratory Syndrome Coronavirus) and SARS (US Government Gain) virus -of-Function, 2014).

It was surprisingly at this time that funding from NIH and NIAID (National Institute of Allergies and Infectious Diseases) such as US funding No. R01AI110964 of $ 3.37 million from the NIAID of the famous Dr Fauci (director since 1984 and AIDS, Ebola, Zika etc) are referred to the Wuhan Institute of Virology (WIV) of Dr Zenghli Shi.

It is with this type of funding (NIH grants R01AI089728 (to FL) and R01AI110700) that in 2015 the Chinese researcher Zhengli Shi (the famous “Batwoman”) and her team published (Yang Y et al., 2015) in collaboration with American laboratories the manipulation carried out successfully on the genome of a coronavirus allowing it to be contagious to humans by the modification of the spike protein attaching to the human cell: "To evaluate the genetic modifications required for the bat coronavirus HKU4 infects human cells, we have reworked the “spike” protein of the HKU4 virus (from bats) in order to strengthen its capacity to allow viral entry into human cells. To this end, we have introduced two single mutations, S746R and N762A, in the HKU4 (bat coronavirus) tip. "

Conclusion of the study: “Mutations of these motifs in animal coronavirus entry proteins (spike) have demonstrated dramatic effects on viral entry into human cells. "

One of the surest ways to make a virus more lethal is to give it a furin cleavage site writes Dr. Steven Quay “At least eleven gain-of-function experiments, adding a furin site to make a virus more deadly. infectious diseases, are published in the open literature, in particular by Dr Zhengli Shi, head of coronavirus research at the Wuhan Institute of Virology ”.

This has been known for years as this patent shows in one of its claims (US Patent 7,223,390 B2, 2004): “Viruses according to claim 8, in which 1000 times more infectious viruses are produced from host cells which do not have no furin expression compared to host cells which express furin. "

Dr Ralph S Baric (University of North Carolina), global coronavirus specialist and mentor to Dr Z Shi, Dr Fauci (director of NIAID, close to NIH directors, Bill Gates and member of its scientific board, the Democratic Party and financial support of the US-China / Wuhan projects) and Dr Peter Daszac (director of the EcoHealth Alliance) through whom millions of dollars have passed between the NIAID and Wuhan (WIV) are the scientists most aware of this work and in particularly of what has been done in Wuhan for many years, with Dr Shi of course.

It should also be noted that Peter Daszac was the quickest to proclaim loud and clear that the virus could only be of natural origin and that the Chinese researchers of the WIV had to be protected and did not hesitate to force the hand (literally since a series of public emails prove it, including researchers like RS Baric) :, so that researchers sign (very early: in March 2020) an article in the Lancet on this subject (Calisher C et al., 2020) “We stand in solidarity in strongly condemning conspiracy theories suggesting that COVID-19 is not of natural origin. »All without ANY scientific argument! While we have already heard him declare in an interview that coronaviruses are viruses very easy to handleand that "some of them can cause SARS disease in humanized mouse models." They are not treatable with therapeutic monoclonal antibodies and you cannot vaccinate against them with a vaccine ”. The funding of this association which hires mercenary researchers around the world saw its funding cut in 2020 by the Trump administration and then resumed normally recently (see article Nicholas Wade also). In addition, Dr Peter Daszac is part of the "independent commission" mandated to determine whether the origin of the virus is natural or artificial ...

Dr Baric (also present in the first tests of remdesivir and in the first publication of the Moderna vaccine) has worked for many years with Dr Fauci, Daszac and Shi and says in another interview that we can totally manipulate the genome of coronaviruses without leaving any visible trace! He can be considered the world's best coronavirus researcher.

Regarding Dr Fauci, many observers, Americans in particular, such as Colonel Lawrence Sellin , former research director in the P4 laboratory at Fort Detrick recently said that this collaboration with China and the Chinese government was continuing on different viruses and pathogens ( which can be likened to high treason):

“ Anthony Fauci still appears to be funding the functional research gain that has taken place over the past two years. And that the Chinese military is involved in the planning and execution of this research. "

Coming back to this spike protein (the one whose messenger RNA is injected by vaccines so that our cells produce it), and "which does everything" comments Daszac, also determines which animals the virus can or cannot infect, because the ACE2 receptors (or other targets for other viruses) may differ in structure depending on the species. It should also be noted that this SARS-CoV2 supposed to come from bats is much more suited to the human receptor than to the receptors of bats.

This mutation of the furin site present in SARS-CoV2 and unique in coronaviruses also increases its possibility of being cleaved (therefore activated) by other enzymes! Like plasmin, trypsin etc which significantly facilitates the entry of the virus into the bronchial epithelial cells (Figure 3). These enzymes can be found in greater amounts in patients with pre-existing hypertension, diabetes, coronary artery disease, heart disease, cerebrovascular disease, chronic obstructive pulmonary disease (COPD) and renal dysfunction, in short in people with co-morbidities in the lungs. COVID19 (Ji HL et al., 2020).

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Figure 3 : Plasmin (ogen) increases the pathogenicity of COVID-19

Plasmin cleaves SARS-CoV-2 S protein extracellularly, increasing its ability to bind to angiotensin converting enzyme 2 (ACE2) receptors in host cells, and possibly facilitating entry and exit. virus fusion. Plasmin breaks down excess fibrin to elevate D-dimer and other fibrin degradation products which lowers platelets and causes bleeding

According to (Ji HL et al., 2020)

It should also be noted that other potential co-receptors / attachment factors such as neuropilins, heparan sulfate, sialic acid, CD147 are also being studied as avenues for entry of the virus into the body (Zamorano Cuervo N et al., 2020) (Cantuti-Castelvetri L et al, 2020).

Another specificity of the virus: blocking the activity of interferon

Normally, the response to type I interferon (IFN-I) is essential to provide effective protection against viral infections. The production of IFN-I is rapidly triggered by the recognition by host sensors of pathogen-associated molecular models (PAMPs), such as the nucleic acids of viral RNAs. This normally then converges into a response that rapidly induces the expression of hundreds of genes called interferon-stimulated genes (ISGs). This antiviral signaling cascade occurs in virtually all types of cells exposed to IFN-I. ISGs, as well as other downstream molecules controlled by IFN-I (including pro-inflammatory cytokines), have a variety of functions, ranging from direct inhibition of viral replication upon recruitment and activation of various immune cells. A robust, punctual and localized IFN-I response is a first line of defense against viral infection because it promotes virus elimination, induces tissue repair and elicits a prolonged adaptive immune response against viruses.

It is clear that SARS-Cov-2 is a poor inducer of the IFN-I response in vitro and in animal models as well as in humans since the levels of IFN-I in the serum of infected patients are below detection levels of commonly used tests (Sa Ribero M et al., 2020) (Hadjadj J et al., 2020) (Lee JS and Shin EC, 2020) (Figure 4).

SARS-CoV2 induces even less IFN-I than SARS-CoV and these coronaviruses contain several proteins allowing to escape the production or responses induced by interferon (Spiegel M et al., 2005) (Hu Y et al., 2017) (Sa Ribero M et al., 2020).

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Figure 4: Modeling of the failure of interferon (IFN-I) to control SARS-CoV-2 infection, leading to COVID-19

While IFN-I can control viral infection (upper panel), IFN-I deficiency is believed to play a key role in the pathogenesis of SARS-CoV-2 (lower panel). As indicated for related coronaviruses, delayed IFN-I signaling is associated with viral replication and severe complications, i.e., strong inflammation or "cytokine storm", especially via accumulation of monocytes. resulting in pulmonary immunopathology, vascular leakage and suboptimal T cell response

IFNAR, alpha and beta interferon receptor; IFN-I, type I interferon; ISG, gene stimulated by IFN; pDC, plasmacytoid dendritic cell; SARS-CoV-2, severe respiratory syndrome coronavirus-2.

According to (Sa Ribero M et al., 2020)

Consequence of the virus binding with the ACE2 receptor and the destabilization of the ACE2 / ACE balance

The second aspect, besides the very particular genetics of SARS-Cov2, is the target of this virus, namely the angiotensin-2 converting enzyme (or ACE2).

The ACE2 protein, the entry receptor for the SARS-CoV2 virus, plays a beneficial role in our body: vasodilator, antioxidant, anti-inflammatory, while its counterpart, the ACE protein, is on the contrary vasoconstrictor, pro-oxidant, and pro-inflammatory. .

In almost all pathological conditions, in particular those of the cardiovascular system but also neurodegenerative (Alzheimer's), there is an increase in the ACE / ACE2 ratio within organs and systems (Bernardi S et al., 2012) (Lavrentyev EN et al., 2009) (Mizuiri S et al., 2008) (Yuan YM et al., 2015) (Kehoe PG et al., 2016).

A growing body of evidence suggests that the host immune response strongly contributes to severe forms of MERS-CoV, SARS-CoV and SARS-CoV-2 infections (Huang C et al., 2020) (Wong C et al., 2004) (Mahallawi WH et al., 2018).

Organs affected and link to COVID long

Scientific literature allows to observe that SARS-CoV2 has an impact on the human body far beyond the lungs and shows a complex interaction with the human host which is not always correlated with the expression levels of the receptor. input (ACE2). Numerous studies have identified viral components (RNA, proteins) of SARS-CoV-2 in several organs (pharynx, trachea, lungs, blood, heart, vessels, intestines, brain, male genitals and kidneys) and body fluids (mucus , saliva, urine, cerebrospinal fluid, semen and breast milk) (Figure 5) (Trypsteen W et al., 2020). However, apart from the lungs, researchers were only able to detect infectious viruses in stool and urine in a limited number of patients with SARS-CoV2.

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Figure 5: Overview of the expression level of ACE2 receptors
The color gradient (orange) indicates the low or high level of expression of ACE2 in tissues or biological fluids. The highest levels were detected in the oral cavity, the gastrointestinal tract and the male reproductive system. From (Trypsteen W et al., 2020)

This vision helps lay the foundation for better diagnosis and treatment of COVID-19 patients, including long-term COVID. Indeed, the majority of patients with COVID-19 present with various other symptoms in addition to respiratory disorders, including neurological, cardiovascular, intestinal and renal dysfunctions (Argenziano MG et al., 2020) (Huang Cet al., 2020) (Lin L et al., 2020) (Chu KH et al., 2005) (Mao L et al., 2020).

Interestingly, the presence of SARS-CoV-2 in different tissues does not always correlate exactly with the levels of ACE2. For example, high viral loads are extracted from the lungs which have medium amounts or ACE2. In the gastrointestinal tract, viral loads peak in the colon, while ACE2 expression is highest in the small intestine (Trypsteen W et al., 2020). A plausible explanation for this apparent mismatch could be the fact that few ACE2 molecules might be enough to cause effective infection with SARS-CoV2. Indeed, several comorbidities associated with the severity of the COVID-19 disease such as smoking, diabetes, COPD (chronic obstructive pulmonary disease), obesity and

Surprisingly at first glance, the abundance of ACE2 receptors in plasma is high in children, who often present minor symptoms during SARS-CoV2 infection, while it decreases in the elderly, who are more at risk of serious disease (Bénéteau-Burnat B et al., 190) (Chen J et al., 2020).

This apparent paradox could be explained by the difference between ACE2 molecules bound to the membrane and those which are soluble. While the membrane-bound form acts as a host cell receptor for SARS-CoV2, soluble ACE2 could neutralize free virions (Ciaglia E et al., 2020).

With regard to the brain and damage to the nervous system, some authors also note that the addition of the furin site allows the mutant virus to infect nerve cells (Cheng J et al., 2019).

Movements of the COVID-19 virus to the brain via the cribriform plaque near the olfactory bulb (see figure 6) may be an additional pathway that could allow the virus to reach and affect the brain (Baig AM et al., 2020) ( Cheng J et al., 2019). Additionally, symptoms such as an altered sense of smell (anosmia) in the early stages of COVID19 should be further investigated as well as the consequences on the central nervous system (CNS) (Baig AM et al., 2020).

In fact ACE2 receptors are expressed in the human CNS, particularly in the spinal cord, spinal ganglion, black substance of the brainstem, choroid plexuses, hypothalamus, hippocampus, middle temporal gyrus and posterior cingulate cortex. (Chen R et al., 2020) (Shiers S et al. 2020) (Baig AM et al., 2020).

Previous studies have shown the ability of SARSCoV to cause neuronal death in mice by invading the brain via the nose near the olfactory lining (Netland, J et all, 2008). Recently, a study reported manifestations of COVID-19 that involved 214 patients, of which 78 (36.4%) patients had neurological manifestations (Mao, L et al., 2020).

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Figure 6: Human olfactory system. 1: Odor bulb 2: Mitral cells 3: Bone (Cribriforme plate) 4: Nasal epithelium 5: Glomerulus 6: Odor receptor cells

Viral spread in the mouse brain has also been studied with a focus on the olfactory bulb and regions of the hippocampus, as these regions have been shown to be primarily infected with the reference viral strain ( Le Coupanec A et al., 2010).

This virus is therefore not only a respiratory virus and this is particularly relevant in the treatment of long COVIDs.

How the SARS-CoV2 virus induces COVID-19 disease: induced inflammatory processes ("cytokines storm") and associated biochemical pathways

The main hallmark of severe cases of COVID-19 is pneumonia, with potential acute respiratory distress syndrome (ARDS) that requires patients to be treated in the intensive care unit.

After the replication phase of the virus a strong inflammation occurs. Fifteen percent of patients develop dysfunctional and harmful procoagulant and inflammatory disease, which results in life-threatening respiratory distress.

The famous "cytokine storm" is an uncontrolled release of cytokines (inflammation modulating molecules) that has been observed in some infectious and non-infectious diseases. But this term is often used, including by many physicians and researchers, without sufficiently specifying the biochemical pathways it involves and the potential therapies that may result from it. The spike protein therefore allows great dissemination of the virus through the epithelia and then through the bloodstream to the various organs mentioned above.

The down-regulation of the ACE2 protein described above and the imbalance of the ACE2 / ACE balance. The consequences of this imbalance may contribute to the hyper-inflammation observed in severe cases of COVID-19 passing through 4 major biochemical pathways (Figure 7):

1 / Deregulation of the renin-angiotensin-aldosterone system (ACE / angiotensin II / AT1R axis)

2 / The attenuation of the Mas receiver (ACE2 / MasR axis)

3 / The increased activation of [des-Arg9] -bradykinin (ACE2 / bradykinin B1R / DABK axis)

4 / Activation of the complement system including components C5a and C5b-9

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Figure 7: The downregulation of ACE2 driven by SARS-CoV-2 leads to a set of complex and intertwined molecular interactions via at least four axes consisting of deregulation of the ACE2 / angiotensin II / AT1R axis, a attenuation of the ACE2 / MasR axis, increased activation of ACE2 / bradykinin B1R / DABK axis and activation of the complement cascade resulting in a tornado of inflammatory cytokine responses. According to (Mahmudpour M et al., 2020)

1 / The renin-angiotensin-aldosterone system (RAAS or RAAS for English speakers) is, in mammals, one of the most important regulatory systems for autonomic, cardiovascular and pulmonary functions. It is a hormonal system organized around the kidney , which allows in particular to preserve homeostasis hydrosodium (the balance between Na + ions and water) and which in particular regulates blood pressure and also plays a role in inflammation (Pacurari M et al., 2014). As mentioned previously, the ACE2 protein, the entry receptor for the SARSCoV2 virus, plays a beneficial role: vasodilator, antioxidant, anti-inflammatory, while its counterpart, the ACE protein, is, on the contrary, vasoconstrictor, pro-oxidant, and pro-inflammatory. ACE2 plays this role by controlling the amount of an essential peptide, angiotensin II, produced by ACE from another small protein or peptide, angiotensin I. Angiotensin I is produced by kidney thanks to the enzyme renin from the angiotensinogenic peptide which is produced in the liver and released into the plasma) (figure 8).

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Figure 8: Renin angiotensin system and role of the ACE2 / ACE balance

As ACE2 is in deficit because of the presence and multiplication of the virus, the balance tilts in favor of the inflammation produced by angiotensin II which is no longer degraded through the many mediators that result from it. , especially in the lungs. This inflammation occurs through the interaction between angiotensin II and the AT1 receptor ( Angiotensin II type 1 receptor, AT1R) in the kidney and the vascular system (Crowley SD and Rudemiller NP, 2017) (Jia H, 2016).

A significant production of cytokines results from this, such as the growth factor TFG-β (Transforming Growth Factor Beta) stimulated during the activation of RAAS. Angiotensin II can in particular activate the pivotal pathway of nuclear factor kappa B (NF-κB) (Okamoto H et al., 2021) (Jamaluddin M et al. 2000) (Ruiz-Ortega M et al., 2001), a major component of the inflammatory response which in turn leads to an increase in other cytokines important in COVID-19: IL-6, TNFα, IL-1B, IL-10, MCP1 (monocyte chemoattractant protein 1), AT1, le platelet-derived growth factor beta (PDGF-B).

2 / The ACE2 / MasR receptors, Ang- (1–7) and Ang- (1–7) are the constituents of

the other arm of the RAAS system which counteracts and attenuates the effects of the first axis ACE-Ang II-AT1R described in 1 / (figure 9).

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Figure 9: Schematic overview of RAAS and its biological functions Angiotensinogen is secreted by the liver and is converted to angiotensin I (AngI) via renin, a protease produced in the kidneys. AngI is then converted to AngII by the catalytic action of angiotensin converting enzyme (ACE) and binds to angiotensin II type 1 (AT1) and type 2 (AT2) receptors. AngII binds to the angiotensin type 1 receptor (AT1R) to promote actions such as vasoconstriction, cell proliferation, fibrosis, and inflammation.
ACE2 converts Ang-I and Ang-II to angiotensin (1–7). Ang (1–7) binds to the MAS receptor (MASR) to promote vasodilation, vascular protection, anti-fibrosis, anti-proliferation, anti-inflammation and anti-angiogenesis actions . According to (Medina-Enríquez MM et al., 2020)

This is an axis of protection against inflammation which is weakened because of the monopolization of the ACE2 receptor by the virus. The beneficial effect of ACE2 is through the cleavage of angiotensin II into the Ang- (1–7) peptide. It is this last molecule which has a vasodilator effect, antiproliferative (anti-cancer), anti-thrombotic and anti-inflammatory properties and which is therefore found in deficit. Ang- (1–7) reduces the expression of inflammatory factors such as NF-κB, IL-6, TNFα and IL-8 (Peiró C et al., 2020) (Verdecchia P et al., 2020). In addition to its antiviral activity, ivermectin inhibits the production of NF-κB, IL1 and IL-6 in vitro and in vivo (Zhang X et al., 2008).

3 / Infection with SARS-CoV2 by depleting ACE2 also increases the levels of des-Arg9-bradykinin (DABK) which is a known pulmonary inflammatory (peptide) factor. DABK is a bioactive metabolite of bradykinin which is associated with lung damage and inflammation, primarily through BR1 receptors in endothelial cells (lining the blood vessels) of the lungs. The kinin-kallikrein system includes kininogen, the enzyme kallikrein, bradykinin (BK-1–9 or BK) and des-Arg-9-BK or DABK (Figure 10).

ACE2 participates in the degradation of des-Arg9-bradykinin, a process ultimately inhibited by the downregulation of ACE2 induced by the virus and thus leading to a loss of control of this inflammatory pathway (Sodhi CP et al., 2018).

Crosstalk between angiotensin, the kinin-kallikrein system, as well as the inflammatory and coagulation systems play a critical role in COVID-19. Cardiac complications and coagulopathies involve crosses between these systems and should be a more tested route of study to prevent or control ARDS in patients with severe COVID-19 (Tolouian T, et al., 2020) (van de Veerdonk FL et al., 2020).

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Figure 10: Action of ACE and ACE2 enzymes in the KKK (kininogen-kinin-kallikrein) system
A. ACE degrades bradykinin, a vasodilator peptide acting primarily through BR2 receptors. Bradykinin can also be converted by kininase I to des-Arg9-bradykinin (DABK), which promotes vasoconstriction and pro-inflammatory effects upon interaction with BR1 receptors.
B. SARS-CoV-2 mediated imbalance in the KKK system with predominant pro-inflammatory effect of des-Arg9-bradykinin. According to De (Goldin CJ et al., 2020)

Indeed, a review of the literature suggests that the control of the bradykinin pathway may be a good option in the management of COVID-19 in patients with vascular pathologies and that a supportive treatment of respiratory complications and cardiology is necessary in COVID-19 patients, in particular by C1-inhibitors (C1-INH) (Haybar H et al., 2021) (Mansour E et al., 2021) (van de Veerdonk FL et al., 2020). This inflammatory pathway appears to be much more important in COVID-19 than the first two.

4 / The complement system.

The complement system works as an immune surveillance system that responds to infection. It contributes to the innate immune response which includes the cells and the mechanisms allowing the body's defense against infectious agents immediately (in the absence of antibodies ) because it does not require cell division, conversely of the adaptive immune system which confers a later but more durable protection and which requires a cell division (lymphocyte B and T). This system includes many proteins (about 30) which play a key functional role in the defense against microorganisms, including viruses. Its excessive activation during COVID-19 participates in cytokine storm, endothelial inflammation (endothelitis) and thromboses that accompany the disease.

There are three biochemical pathways that activate the complement system: the classical complement pathway, the alternate complement pathway and the lectins pathway (proteins that bind to sugars) binding mannose and N-acetylglucosamine residues , of the sugars present. in bacterial and viral membranes. The lectin pathway is a priority in COVID-19.

Viral inactivation by the complement cascade involves absorption and clearance (or elimination) of the virus by phagocytosis (ingestion of the virus by immune cells), which prevents attachment to their receptors, lysis of the virus by formation of pores and destruction of its membrane by formation of a membrane attack complex (C5b-9) (MAC) (Spear Get al., 2001). The increase in Ang II production and the activation of AT1R (cf 1 /) are accompanied by a pro-inflammatory response via the activation of the complement cascade comprising C5a, C5b-9. Complementary factor 5a (C5a) is the most powerful inflammatory peptide in the complement cascade which induces the release of numerous pro-inflammatory cytokines including TNF-α, IL-1, IL-6, NF-κB, activator Protein-1 (AP-1) (Viedt C et al., 2000). In addition, increased production of C3a leads to the production of pro-inflammatory cytokines such as IL-1, IL-6 and TNF-α.

The involvement of the complement system in the pathogenesis of infection by syncytial virus, MERS-CoV and SARS-CoV is already known from several studies (Gao T et al., 2020) (Gralinski LE et al. , 2018) (Jiang Yet al., 2018). Hyperactivation of complement components includes C5a in serum and C5b-9 in lungs was observed in hDPP4 transgenic mice infected with MERS-CoV. Lung and spleen induced damage and inflammatory responses were attenuated by blocking the C5a - C5aR axis in these transgenic mice (Jiang Yet al., 2018).

If the S protein (spike) by binding to the ACE2 receptor can trigger these inflammatory pathways, another protein much less talked about is perhaps responsible, by a much more direct immuno-inflammatory reaction of the most serious effects observed in patients. patients with severe forms who die from COVID-19.

N protein: more inflammatory than S protein?

In COVID19, an essential aspect of triggering complement-induced hyperinflammation, part of the innate immune response, appears to be effectively linked to the N protein of the virus (Bumiller-Bini V et al., 2021) (Ma L et al., 2021) (Dobó Jet al., 2018).

The N protein is linked to the RNA of the virus that it surrounds (helical nucleocapsid) and constitutes more than 50% of the proteins of the virus (see figure 11). They are highly immunogenic: the N proteins of SARS-CoV, MERS-CoV and SARS-CoV-2 bind to the MASP-2 protein a protease (serine protease) which is key in the pathway for the activation of complement via lectins, resulting in aberrant activation of complement and worsening inflammatory lung damage. By blocking the N protein: MASP-2 interaction or suppressing complement activation, the N protein-induced effects on complement hyperactivation and lung damage in vitro and in vivo can be significantly reduced (Gao T et al. ., 2020).

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Figure 11: N protein attached to viral RNA

Important observation: in the lung tissues recovered during autopsies of subjects who died of Covid, different molecules of the biochemical pathway of lectins were found: MBL (mannan-binding lectin), MASP-2, C4alpha, C3 or C5b- antibody. 9 (Gao T et al., 2020).

Figure 12 below describes the different activation pathways of the complement cascade, the priority pathway in the immune response to the N protein is the lectin pathway.

Also, lectin-type receptors (CLRs), in particular DC-SIGN (but also L-SIGN, MR, and MGL) on the surface of cells of the innate immune system (such as macrophages and dendritic cells) can direct immune responses of the host against the virus (S proteins in particular) by identifying specific sugars (glycans: Oligomannose N-glycans) associated with virus proteins (Gao C et al., 2020).

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Figure 12: Pathways of complement activation by SARS-CoV-2
Activation of the classical pathway is initiated by binding of the C1 complex to immunoglobulins or to endogenous ligands. This complex will be able to cleave C4 and C2 to form the classic C3 convertase (C4b2a).
The lectin pathway is activated by the binding of an MBL-MASP-2 complex to the surface of a pathogen. This complex, linked to the pathogen, then cleaves C4 and C2. These cleavages thus form a C3 convertase (C4b2a).
The alternate pathway acts as a monitoring system by maintaining a low level of activation of the complement system through a process known as “tick-over” which allows the formation of alternate C3 convertase (C3bBb). According to (Chouaki Benmansour N et al., 2021)

The three pathways lead to the formation of a C3 convertase making it possible to cleave the C3 into C3a, an anaphylatoxin, and into C3b, at the origin of the formation of the C5 convertase (C4b2a3b or C3bBb3b). The C5 is then cleaved into C5a and C5b, which will initiate the final stages of the complement cascade. This leads to the formation of the membrane attack complex (C5b-9, MAC) and allows pathogens to be lysed (figure 13).

jfl13.png

Figure 13: Formation of MAC (membrane attack complex) and virus lysis

The anaphylatoxins will trigger the cytokine storm, the degranulation of mast cells which release histamine, the activation of leukocytes and their infiltration into the pulmonary alveoli. Regarding mast cells and the release of histamine induced by this activation of complement, this probably explains the positive and curative effect of antihistamines proposed and observed in numerous studies and in the practice of numerous physicians. The complement system is highly regulated by circulating proteins in the blood (C1inh, FI, C4BP, FH, clusterin, vitronectin) and membranes (CR1, MCP, DAF, CD59).

Blocking the complement by specific therapies represents a therapeutic hope in the most severe forms of the disease. The inhibition of downstream complement pathways, such as C3 and C5 fractions and their receptors, thus constitutes a possible approach to contain the pathogenesis of ARDS according to (Ram Kumar Pandian S et al., 2020) (Maglakelidze Net al. ., 2020).

But it would seem even more efficient upstream administration of C1-esterase inhibitors which considerably reduced the mortality rate and pulmonary inflammation dependent on MASP-2 in mice infected with adenovirus expressing the N protein of SARS. -Co V. The C1-esterase inhibitor adequately suppresses classical and complement lectin pathways and may effectively reduce lung damage in patients with severe COVID-19, particularly at the microvascular level (Jodele S et al. Köhl J, 2020).

The portion of viral N protein responsible for the interaction with MASP-2 has recently been identified and corresponds to residues 115-123 in SARS-CoV-2 (GTGPEAGLP) (Figure 14). While the amino acid sequence of this portion is highly conserved in SARS-CoV, MERS-CoV, and SARS-CoV-2, it is very different from other coronaviruses associated with mild illness, such as coronaviruses. humans 229E (and others like OC43, NL63 and HKU1) (Flude BM et al., 2021).

jfl14.png

Figure 14: Sequence alignment (C-terminal domain) of the reactive part of the N protein of human coronaviruses with MASP-2. According to (Flude BM et al., 2021)

Remarkably, the N proteins of different bat coronaviruses not yet known to infect humans are characterized by the presence of this conserved sequence (figure 15), suggesting the potential risk of emergence of new coronavirus infections. zoonotic in the future, with the same severe consequences as SARS-CoV, MERS-CoV and SARS-CoV2.

jfl15.png

Figure 15: Sequence alignment (C-terminal domain) of the reactive part of the N protein of coronavirus with MASP-2 (humans vs bats). According to (Flude BM et al., 2021)

Majority activation of innate immunity versus acquired immunity

The evoked particular importance of the bradykinin and complement pathways correlates well with what studies measuring biomarkers of COVID-19 show in many subjects, namely that the severity of SARS-CoV2 infection is linked to uncontrolled innate immune responses and an alteration of adaptive immune responses mainly due to depletion of T lymphocytes and lymphopenia (lower than normal lymphocyte count, less than 1500 per mm³) (Jeannet R et al. , 2020) (Diao B et al., 2020) (Lucas C et al., 2020).

The Th1 helper lymphocyte pathway is largely altered, which may explain the poor T cell responses and persistent SARSCoV2 viral load in the blood. There is also a decrease in CD4 + and CD8 + T lymphocytes which express the HLA-DR and CD38 activation markers in many patients who show little or no lymphocyte activation response (Gutiérrez-Bautista JF et al., 2020).

A weak activation of the Th2 helper lymphocytes is observed not and on the contrary hypereosinophilia: eosinophils (family of granulocytes or polynuclear cells) also belong to the system of innate immunity (Xie G et al., 2021).

Activation of the complement cascade causing thrombosis

If the virus has not been cleared by the immune system at the epithelial barrier, it will then progress to the wall of the vessels (the vascular endothelium). The endothelial cells that make up this epithelium express the ACE2 receptor and viral penetration into these cells will be the cause of endothelial inflammation, called endothelitis or endothelialitis (Ackermann M et al ;, 2020). Endothelitis is associated with the thrombotic phenomena observed in COVID-19, such as microthrombosis (Magro C et al., 2020) and macrovascular thrombosis, as well as pulmonary embolism, in particular (Llitjos JFet al;, 2020 ).

The complement has been associated with the endothelitis lesions seen in severe forms of COVID-19. Histological deposits of MASP-2, C4 and MAC, as well as macrophages overexpressing the C5a receptor, C5aR1, have in fact been found in endothelitis lesions and microthrombi (Eriksson O et al., 2020). Complement and coagulation (contact phase, or intrinsic pathway of coagulation3) are closely linked (Kenawy HI et al., 2020). Indeed, MASP-1 and MASP-2 have a catalytic role for prothrombin and fibrinogen, leading to thrombotic complications (Berlin et al., 2020; Jodele and Köhl, 2020) (Figure 14).

MASP 1 and / or 2 have increased expression in patients with risk factors for COVID-19: diabetes, high blood pressure and cardiovascular disease, chronic kidney disease, chronic obstructive pulmonary disease and cerebrovascular disease (Bumiller-Bini V et al., 2021).

Studies and reviews of the literature highlight the role of MASP binding to SARS-COV-2, activation of the lectin pathway and blood coagulation, as well as their associations with COVID-19 comorbidities . It is therefore probably a pathway or the key pathway making the link between inflammation and the phenomena of coagulation in the final phase of severe forms of COVID-19.

This has major and underestimated implications for the care of ICU and intubated patients. Heparin and antithrombin have also shown a major impact on the outcome of treatment and prevention of disseminated intravascular coagulation associated with COVID-19 reducing mortality (Tang et al., 2020). In the presence of heparin, antithrombin and C1-inhibitors (C1-INH) are effective inhibitors of MASP-1 (Bumiller-Bini V et al., 2021) blocking both the al lectin pathway and the cascade coagulation (Figure 16). C1-INHs also block MASP-2 (Kajdácsi et al., 2020). Interestingly, C1- INH deficiency is suggested as a direct result of SARS-CoV-2 infection, resulting in loss of physiological pathway control of lectins and coagulation (Thomson et al., 2020). Narsoplimab,

Finally, for some authors, the activation of coagulation in severe forms, comparable to a picture of disseminated intravascular coagulation (DIC) with thrombocytopenia (platelet deficit), consumption of coagulation proteins and elevation of D-dimers could be linked to the production and accumulation of platelet-activating immune complexes (Nazy I et al., 2021). The detoxification of these immune complexes normally carried out by the complement system could be compromised due to the consumption of complement factors throughout the immuno-inflammatory phase described above.

jfl16.png

Figure 16: MASP in the coagulation cascade during COVID-19

The participation of MASP in coagulation contributes significantly to the formation of clots. On the right side of the figure, MASP-1 activates endothelial cells through protease activated receptors (PAR). This activation has a characteristic response profile, and different from that stimulated by thrombin: activated endothelial cells begin to secrete pro-inflammatory cytokines and interleukins 6 and 8 (IL-6 and IL-8). Platelet aggregation also occurs. IL-6 and IL-8 contribute to the chemotaxis of neutrophils and macrophages.

On the left, the MASP and MBL / MASP complexes participate in the coagulation cascade at different levels. Like factor Xa (FXa) in the coagulation cascade, MASP-2 cleaves prothrombin to thrombin l. MASP-1 participates in the cleavage of prothrombin to thrombin and the cleavage of fibrinogen to fibrin. These complexes cleave fibrinogen into fibrin, contributing to the formation of clots in the coagulation cascade.

According to (Bumiller-Bini V et al., 2021)

Mechanisms of action of antiviral and anti-inflammatory molecules of interest

Ivermectin

Ivermectin has antiviral activity against SARS-CoV2. Indeed, studies show that it effectively reduces the viral load in patients with COVID-19 (Kory P et al., 2021). Caly and Druce reported that ivermectin inhibits SARS-CoV-2 in vitro within 48 h at 5 μM (IC50 = 2 μM or 1750 ng / ml) (Caly L et al., 2020). Other authors have found even more effective values: EC50 of 0.14 μM (Bernigaud C et al., 2020) which confirms the effectiveness observed in vivo if we take into account that ivermectin is concentrated in the lungs (Lifschitz, A et al., 2000) and given other physicochemical and pharmacological parameters (high pKa and log P, large dilution volume) (Boer F, 2003) (González Canga A et al., 2008).

A plausible and well characterized antiviral mechanism of ivermectin is the inhibition of transfer of viral proteins to the cell nucleus by proteins called importins (Sen Gupta PS et al., 2020). Ivermectin could also interfere between the virus (RBD site) and its ACE2 receptor (Lehrer S et al., 2020). But antivirals alone must be taken very early and often are not enough to prevent symptoms and the deterioration of the condition. However, the clinical results of ivermectin are spectacular with a reduction in mortality of around 75% in the average of meta-analyzes carried out by independent researchers (Lawrie T, 2021) (Kory P et al., 2021). This is explained by the potent anti-inflammatory activity of ivermectin which inhibits the production of key inflammatory actors involved in COVID-19, in particular NF-κB, IL1 and IL-6 in vitro and in vivo (Zhang X et al., 2008) (Ci X et al., 2009). Ivermectin is also capable of reducing the production of inflammatory mediators such as NO, prostaglandin E2 (PGE2) or cyclooxygenase-2 (COX2) (Zhang X et al., 2009). Ivermectin could in particular inhibit the formation of NF-κB induced by the activation of lectin-type receptors (CLRs) activated by the N protein of the virus (Kingeter LM et al., 2012). Ivermectin is also capable of reducing the production of inflammatory mediators such as NO, prostaglandin E2 (PGE2) or cyclooxygenase-2 (COX2) (Zhang X et al., 2009). Ivermectin could in particular inhibit the formation of NF-κB induced by the activation of lectin-type receptors (CLRs) activated by the N protein of the virus (Kingeter LM et al., 2012). Ivermectin is also capable of reducing the production of inflammatory mediators such as NO, prostaglandin E2 (PGE2) or cyclooxygenase-2 (COX2) (Zhang X et al., 2009). Ivermectin could in particular inhibit the formation of NF-κB induced by the activation of lectin-type receptors (CLRs) activated by the N protein of the virus (Kingeter LM et al., 2012).

Hydroxychloroquine and Azithromycin

Hydroxychloroquine has an excellent known activity against coronaviruses in vitro and in particular against SARS-CoV2 (Wang Met al., 2020).

Despite the controversies, hydroxychloroquine in the presence of azithromycin reduces viral load, hospital stay and mortality, which is clear from independent studies and meta-analyzes: https://hcqmeta.com/ (Lounnas V et al., 2021)

Regarding the antiviral mechanisms of hydroxychloroquine, there are potentially many (Devaux C et al, 2020):

interferes with the glycosylation of ACE2 receptors, thus preventing the binding of SARS-CoV-2 to target cells
can modulate the acidification of endosomes, thereby inhibiting the movement of viruses within the cell
inhibit the replication of the virus thanks to the decrease in the activation of the cellular mitogenic protein (MAP) kinase (set of protein kinases necessary for the induction of mitosis or cell division)
alter the maturation of M proteins and interfere with the assembly and budding of virions

Like ivermectin, hydroxychloroquine also has anti-inflammatory activity which is in addition to antiviral activity. Hydroxychloroquine has an anti-inflammatory effect on Th17-related cytokines (IL-6, IL-17 and IL-22) in healthy individuals, and patients with systemic lupus erythematosus and rheumatoid arthritis (Silva JCd et al., 2013).

One study reported that hydroxychloroquine did not cause higher negative conversion rates, but reduced clinical symptoms through anti-inflammatory properties and improved lymphopenia (Tang W et al., 2020). However, there are many inflammatory pathways in the body and the anti-inflammatory activity of ivermectin seems more suited to the inflammatory pathways associated with COVID-19, in particular to counter the effects of the RAAS system dysregulation associated with the disorder of the ACE2 / ACE balance as well as activation linked to complement and CRLs.

Azithromycin, in addition to its well-established antibacterial properties, has shown antiviral activity against SARSCov2 in vitro as well as in clinical studies in improving symptoms, alone or in synergy with hydroxychloroquine (Damle B et al., 2020) (Lepere P et al., 2021).

In addition, azithromycin increases interferon in the reaction to different viruses and this mechanism is also proposed for SARS-Cov2 (Bleyzac N et al., 2020).

C1-inhibitor

In fact, a global review of the literature on immunoinflammation suggests that controlling the bradykinin pathway may be a good option in the management of COVID-19 in patients with vascular pathologies and that a Supportive treatment of respiratory and cardiological complications is necessary in COVID-19 patients, in particular with C1-inhibitors (C1-INH) (Haybar H et al., 2021) (Mansour E et al., 2021) (van de Veerdonk FL et al., 2020).

In addition, this C1-inhibitor (C1-INH) or C1 esterase inhibitor is a serine protease inhibitor ( serpin ), the main function of which is to inhibit the complement system, but it seems that this is one of the pathways or the main route of inflammation and also leading to coagulation phenomena in severe forms. C1-INH adequately and upstream inhibits classical and complement lectin pathways and may effectively reduce lung damage in patients with severe COVID-19, particularly at the microvascular level (Jodele S and Köhl J , 2020). This would most likely be more effective than blocking this complement pathway downstream (Ram Kumar Pandian S et al., 2020) (Maglakelidze Net al., 2020).

This has major and underestimated implications for the care of ICU and intubated patients.

Heparin and antithrombin have also shown a major impact on the outcome of treatment and prevention of disseminated intravascular coagulation associated with COVID-19 reducing mortality (Tang et al., 2020). In the presence of heparin, antithrombin and C1-inhibitors (C1-INH) are effective inhibitors of MASP-1 (Bumiller-Bini V et al., 2021) blocking both the lectin pathway and the cascade coagulation. C1-INHs also block MASP-2 (Kajdácsi et al., 2020). Interestingly, C1-INH deficiency is proposed as a direct consequence of SARS-CoV-2 infection, resulting in loss of physiological control of the lectin pathway and of coagulation (Thomson et al., 2020). The MASP-2 inhibitor narsoplimab has similar effects (Rambaldi et al., 2020).

Corticosteroids (dexamethasone)

Corticosteroids have different modes of action depending on the dose. At moderate doses dexamethasone is used as an adjunct treatment for viral pneumonia. Dexamethasone binds to the glucocorticoid receptor on the cell membrane, and the formation of this complex leads to the translocation of the corticosteroid into the cell, where it travels to the nucleus. It acts on a wide variety of genes and can inhibit the production of pro-inflammatory cytokines such as interleukin IL-1, IL-2, IL-6, IL-8, TNF, IFN-gamma, VEGF and prostaglandins. Five of these factors are linked to severe forms of COVID-19 (Ahmed MH and Hassan A, 2020) (Zhong J, et al., 2020).

Finally, and this is perhaps a key point of their effectiveness, corticosteroids can also inhibit the complement pathway but, again, less upstream or less directly than C1-INH (Das A, Rana S, 2021 ) (Lappin DF, Whaley K, 1991).

Antihistamines

The activation of complement by lectins also triggers the degranulation of mast cells which release histamine, the activation of leukocytes and their infiltration into the pulmonary alveoli. Regarding mast cells and the release of histamine induced, this probably explains the positive and curative effect of antihistamines proposed and observed in numerous studies and in the practice of numerous physicians (Reznikov LR et al., 2021) (Malone RWet al. , 2020) (Motta Junior JDS et al., 2020) (Eldanasory OA et al., 2020). Antiviral activity by interaction with the ACE2 receptor is also proposed.

Conclusion:

The molecular clarification of these axes of inflammation in the context of COVID-19 will elucidate a panoply of therapeutic strategies to face the "cytokine storm" in order to prevent and treat the acute respiratory distress syndrome associated with this pathology.

We could write "should have allowed" instead of "will allow" but unfortunately we can regret since the beginning of this crisis that the link between researchers in biochemistry with clinical research and field physicians including in intensive care is not optimal and to the great disadvantage of patients. This great waste of scientific knowledge which would make it possible to best treat patients affected by COVID is a chronic problem that existed before the crisis and which concerns many diseases but never this had been so visible! In particular in favoritism towards certain profitable molecules and therapies for the pharmaceutical industry to the detriment of known molecules, safer, much more effective but much less profitable.

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Author (s): Jean-François Lesgards, doctor in biochemistry, and Dominique Cerdan, doctor in pharmacy, for FranceSoir

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