Extensive thrombosis after COVID-19 vaccine: cause or coincidence

Authors: Luís Lourenço Graça ,1 Maria João Amaral ,2 Marco Serôdio,3 Beatriz Costa2

SUMMARY
A 62-year-old Caucasian female patient presented with abdominal pain, vomiting and fever 1 day after administration of COVID-19 vaccine. Bloodwork revealed anaemia and thrombocytosis. Abdominal CT angiography showed a mural thrombus at the emergence of the coeliac trunk, hepatic and splenic arteries, and extensive thrombosis of the superior and inferior mesenteric veins, splenic and portal veins, and the inferior vena cava, extending to the
left common iliac vein. The spleen displayed extensive areas of infarction. Etiological investigation included assessment of congenital coagulation disorders and acquired causes with no relevant findings. Administration of COVID-19 vaccine was considered a possible cause of the extensive multifocal thrombosis. After reviewing relevant literature, it was considered
that other causes of this event should be further investigated. Thrombosis associated with COVID-19vaccine is rare and an etiological relationship should only be considered in the appropriate context and after investigation of other, more frequent, causes.

BACKGROUND
During the COVID-19 pandemic, the pharmaceutical industry is under immense pressure to develop effective and safe vaccines, and as such clinical trials have been expedited in order to make them available to help fight this health crisis. In this context, timely communication between healthcare institutions and regulatory entities is especially important. Reports of thrombosis due to administration of these vaccines have been causing an important
discussion in the scientific community as well as social alarm. However, it is important to note that this is a rare complication and more frequent causes of extensive arterial and venous thrombosis should be considered and investigated.1

CASE PRESENTATION
A 62-year-old Caucasian female patient, with personal history of obesity (body mass index of
30kg/m2), asthma and rhinitis, presented to the emergency department with abdominal pain,
nausea, vomiting and fever (38°C) 1day after administration of the first dose of COVID-19 vaccine(from AstraZeneca). On physical examination, she presented epigastric and left iliac fossa tenderness as the only abnormal finding. The patient denied recent epistaxis and gastrointestinal or genitourinary blood loss.

INVESTIGATIONS
Blood tests revealed microcytic hypochromic anemia (hemoglobin 7g/L), thrombocytosis (780×109/L),increased levels of inflammatory parameters (leucocytes 13×109/L; C reactive protein 31.07mg/dL) and slightly increased levels of liver enzymes and function (AST 36, ALP 126U/L, GGT 72U/L, LDH 441U/L, total bilirubin 1.3mg/dL, direct bilirubin 0.5mg/dL). The patient was tested for COVID-19 with nasopharyngeal PCR tests at admission and on the fifth day of hospitalization. Both tests were negative. Abdominal CT angiography (CTA) showed a mural thrombus at the emergence of the coeliac trunk, with total occlusion (figure 1), as well as at the hepatic and splenic arteries. There was also extensive thrombosis of the superior and inferior mesenteric veins and its tributaries, splenic and portal veins, including the splenoportal confluent (figure 2). There was a filiform thrombus at the distal portion of the inferior vena cava, extending to the left common iliac vein, non-occlusive (figure 3). Spleen presented extensive areas of infarction (figure 1). Coeliac trunk occlusion due to paradoxical embolism was excluded by transthoracic echocardiogram. No interatrial communication was detected. Re-evaluation CTA 5days after the diagnosis was identical. Etiological investigation included assessment of congenital coagulation disorders and acquired causes. Regarding congenital disorders, personal and family history of important thrombotic events, thrombosis in unusual sites and abortions were assessed with no relevant findings. Molecular testing for factor V Leiden mutation and prothrombin gene20210 G/A mutation were both negative. Acquired causes of a coagulation disorder, such as neoplastic, infectious and autoimmune disorders, like antiphospholipid syndrome (APS), were also investigated. Thorax, abdomen, pelvic and brain CT did not detect any suspicious lesions. Tumor biomarkers—carcinoembryonic antigen, alpha fetoprotein, carbohydrate antigen 19-9, cancer antigen 125, cancer antigen 15-3, neuron-specific enolase and chromogranin A—were negative. The patient refused to undergo upper digestive endoscopy and colonoscopy. Despite increased levels of inflammatory parameters at admission (leukocytosis and C reactive protein), these values decreased during the hospitalization period. Blood and urine cultures were also negative. Anticardiolipin IgG and IgM and antibeta-2-glycoprotein IgG and IgM were negative, excluding APS.

DIFFERENTIAL DIAGNOSIS
In the presence of venous and arterial thrombosis, the etiological investigation should include

assessment of congenital and acquired coagulation disorders, as well as the presence of interatrial communication that could explain the coeliac trunk occlusion due to paradoxical embolism. As previously stated, these etiological factors were assessed with no specific findings, with the exception of digestive endoscopic study, which was refused by the patient. In this context, and given the fact that the presentation took place 1day after administration of the first dose of COVID-19 vaccine, we hypothesize that the vaccine might be the cause of the extensive arterial and venous thrombosis. This case was immediately reported to INFARMED, the Portuguese authority for drugs and health products. Vaccine-induced thrombotic thrombocytopenia (VITT) was also considered a differential diagnosis. However, the patient did
not present with thrombocytopenia, which is a key criteria for VITT, and therefore the presence of this syndrome was unlikely.COVID-19 tests at admission and on the fifth day of hospitalization were negative; however, she was not tested prior to the onset of the event and therefore it was not possible to exclude

recent COVID-19 infection, which may predispose to thrombosis, even during the convalescent phase.
TREATMENT
At presentation, there were no signs of organ ischemia that required revascularization procedure or intestinal resection. Considering the anemia, the patient was not a candidate for
fibrinolysis. The treatment was empiric endovenous antibiotherapy and transfusion of two units of red blood cells. Anticoagulation with low molecular weight heparin (LMWH) 1mg/kg
two times per day was initiated and maintained during hospitalisation, with monitoring of anti-Xa levels. After hospitalization,in an outpatient setting, the patient was initiated on edoxaban.

OUTCOME AND FOLLOW-UP
Re-evaluation CTA 28 days after presentation revealed a portal vein with a filiform caliber, with a cavernomatous transformation. There was only permeability of the left branch of the portal
vein, with venous collateralization in the hepatic hilum. Coeliac trunk was still occluded, with permeability of the gastroduodenal artery and the right hepatic artery, and apparent occlusion at the emergence of the left hepatic artery, although with distal repermeabilisation. Partial thrombus persisted in the lumen of the left common iliac vein and inferior infrarenal vena cava. At the follow-up consultation, 1month after discharge, the patient was clinically asymptomatic.

DISCUSSION
Venous and arterial thrombotic disorders have long been considered separate pathophysiological entities due to their anatomical differences and distinct clinical presentations. In particular, arterial thrombosis is seen largely as a phenomenon of platelet
activation, whereas venous thrombosis is mostly a matter of activation of the clotting system.2
There is increasing evidence regarding a link between venous and arterial thromboses. These two vascular complications share several risk factors, such as age, obesity, diabetes mellitus, blood Figure 1 CT angiography arterial phase, axial image: a mural thrombus is observed at the coeliac trunk emergence, with total occlusion. Splenic parenchyma without enhancement after contrast administration can also be observed, translating to extensive infarct areas.
Figure 2 CT angiography portal phase, coronal image: portal vein thrombosis (A) extending to the splenoportal confluent (B) can be observed. Figure 3 CT angiography portal phase, coronal image: a non-occlusive filiform thrombus at the distal portion of the inferior vena cava can be observed, extending to the left common iliac vein. on April 13, 2022 by guest. Protected by copyright. http://casereports.bmj.com/ BMJ Case Rep: first published as 10.1136/bcr-2021-244878 on 16 August 2021. Downloaded from Graça LL, et al. BMJ Case Rep 2021;14:e244878. doi:10.1136/bcr-2021-244878 3

Case report hypertension, hypertriglyceridaemia and metabolic syndrome.3 Moreover, there are many examples of conditions accounting for both venous and arterial thromboses, such as APS, hyperhomocysteinaemia, malignancies, infections and use of hormonal treatment.3 In this case, in accordance with the literature, the patient is 62 years old and obese, with no other findings. Hyperhomocysteinaemia and digestive tract malignancies were not excluded. Recent studies have shown that patients with venous thromboembolism are at a higher risk of arterial thrombotic complications than matched control individuals. Therefore, it is speculated that
the two vascular complications may be simultaneously triggered by biological stimuli responsible for activating coagulation and inflammatory pathways in both the arterial and the venous system.3 The modified adenovirus vector COVID-19 vaccines (ChAdOx1nCoV-19 by Oxford/AstraZeneca and Ad26.COV2.S by Johnson & Johnson/Janssen) and mRNA-based COVID-19 vaccines(BNT162b2 mRNA by Pfizer/BioNTech and mRNA-1273 by Moderna) have shown both safety and efficacy against COVID-19 in phase III clinical trials and are now being used in global vaccination programmes.4Rare cases of postvaccine-associated cerebral venous thrombosis(CVT) from use of COVID-19 vaccines which use a viral vector, including the mechanism of VITT, have emerged in real-worldvaccination.4 On the other hand, the incidence and pathogenesis of CVT after mRNA COVID-19 vaccines remain unknown. However Fan et al4
presented three cases and Dias et al5reported two cases of CVT in patients who took an mRNA vaccine (BNT162b2 mRNA by Pfizer/BioNTech). In both cases, causality has not been proven.
In a recent editorial, three independent descriptions of persons with a newly described syndrome, VITT, were highlighted, characterized by thrombosis and thrombocytopenia that developed 5–24 days after initial vaccination with ChAdOx1 nCoV-19 (AstraZeneca), a recombinant adenoviral vector encoding the spike protein of SARS-CoV-2.6VITT is also characterized by the presence of CVT, thrombosis in the portal, splanchnic and hepatic veins, as well as acute arterial thromboses, platelet counts of 20–30×109 /L, high levels of D-dimers and low levels of fibrinogen, suggesting systemic activation of coagulation.6 In our case, similarities were found with VITT regarding thrombosis in the portal, splanchnic and hepatic veins, as well as acute arterial thromboses and high levels of D-dimers. On the other hand, timing of the event (1day after vaccination), high levels of fibrinogen and absence of thrombocytopenia, which is a key criteria for VITT, point to a different direction. Moreover, the
presence of thrombocytosis allowed for a safe use of LMWH for anticoagulation, with monitoring of anti-Xa levels. Most of the cases reported so far of venous and arterial thrombosis as a complication of AstraZeneca’s COVID-19 vaccine have occurred in women under the age of 60 years, associated with thrombocytopenia, within 2weeks of receiving their first dose of the vaccine.7As for the mechanism, it is thought that the vaccine may trigger an immune response leading to an atypical heparin-induced thrombocytopenia-like disorder. In contrast with the literature, our patient presented with thrombocytosis, not thrombocytopaenia.7 Smadja et al8reported that between 13 December 2020 and
16 March 2021 (94 days), 361734967 people in the international COVID-19 vaccination data set received vaccination and795 venous and 1374 arterial thrombotic events were reported in
Vigibase on 16 March 2021. Spontaneous reports of thrombotic events are shared in 1197 for Pfizer/BioNtech’s COVID-19 vaccine,325 for Moderna’s COVID-19 vaccine and 639 for AstraZeneca’sCOVID-19 vaccine.7 The reporting rate for cases of venous (VTE) and arterial (ATE) thrombotic events during this time period among the total number of people vaccinated was 0.21 cases of thrombotic events per 1million person vaccinated-days.7For VTE and ATE, the rates were 0.075 and 0.13 cases per 1million persons vaccinated, respectively, and the timeframe between vaccinationand ATE is the same for the three vaccines (median of 2days),
although a significant difference in terms of VTE was identified between AstraZeneca’s COVID-19 vaccine (median of 6days) and both mRNA vaccines (median of 4days).8 The first paper addressing this issue was published in the New England Journal of Medicine and described 11 patients, 9 of themwomen.9 Nine patients had cerebral venous thrombosis, three had
splanchnic vein thrombosis, three had pulmonary embolism and four had other thromboses. All 11 patients, as well as another 17 for whom the researchers had blood samples, tested positive for antibodies against platelet factor 4 (PF4). These antibodies are also observed in people who develop heparin-induced thrombocytopenia. However, none of the patients had received heparin before their symptoms started.9Our patient did not present thrombocytopenia, so anti-PF4 antibodies were not tested. Thus, considering the anemia, thrombocytosis and thrombosis diagnosed 1day after the first dose ofCOVID-19 vaccine, it seems prudent to continue investigation for other causes of this event, such as hematological malignancies or others.

REFERENCES
1 Burch J, Enofe I. Acute mesenteric ischaemia secondary to portal, splenic and superior
mesenteric vein thrombosis. BMJ Case Rep 2019;12:e230145.
2 Singer DE, Albers GW, Dalen JE, et al. Antithrombotic therapy in atrial fibrillation:
American College of chest physicians evidence-based clinical practice guidelines (8th
edition). Chest 2008;133:546S–92.
3 Ageno W, Becattini C, Brighton T, et al. Cardiovascular risk factors and venous
thromboembolism: a meta-analysis. Circulation 2008;117:93–102.
4 Fan BE, Shen JY, Lim XR, et al. Cerebral venous thrombosis post BNT162b2 mRNA
SARS-CoV-2 vaccination: a black Swan event. Am J Hematol 2021. doi:10.1002/
ajh.26272. [Epub ahead of print: 16 Jun 2021].
5 Dias L, Soares-Dos-Reis R, Meira J, et al. Cerebral venous thrombosis after BNT162b2
mRNA SARS-CoV-2 vaccine. J Stroke Cerebrovasc Dis 2021;30:105906.
6 Cines DB, Bussel JB. SARS-CoV-2 vaccine-induced immune thrombotic
thrombocytopenia. N Engl J Med 2021;384:2254–6.
7 AstraZeneca’s COVID-19 vaccine: EMA finds possible link to very rare cases of unusual
blood clots with low blood platelets. Available: https://www.ema.europa.eu/en/news/
astrazenecas-covid-19-vaccine-ema-finds-possible-link-very-rare-cases-unusual-bloodclots-low-blood [Accessed Apr 2021].
8 Smadja DM, Yue Q-Y, Chocron R, et al. Vaccination against COVID-19: insight from
arterial and venous thrombosis occurrence using data from VigiBase. Eur Respir J
2021;58:2100956.
9 Wise J. Covid-19: rare immune response may cause clots after AstraZeneca vaccine, say
researchers. BMJ 2021;373:n954.

Spike protein in mRNA COVID vaccines: One of the most bioactive substances known to mankind

Source: https://www.planet-today.com/2022/03/spike-protein-in-mrna-covid-vaccines.html

The spike protein present in Wuhan coronavirus (COVID-19) vaccines is one of the most bioactive and potentially damaging substances known to mankind. It penetrates the blood-brain barrier, cell nucleus and even affects DNA replication. The spike protein appears to reprogram the immune system in a strange way. The BNT162b2 mRNA vaccine against the COVID-19 virus has been shown to reprogram both adaptive and innate immune responses. When it penetrates the cell nuclei, the free-floating spike protein inhibits DNA repair. There had been immune system problems in the vaccinated, and it is becoming apparent that they do not actually develop broad natural immunity. Instead, they produce more S antibodies against the spike protein that they were originally vaccinated with. A recent surveillance report from the U.K. Health Security Agency showed that N antibody levels appear to be lower in individuals who acquire infection following two doses of the vaccine. This means that the vaccines interfere with the immune system’s ability to produce antibodies against the virus following infection. In the case of the N antibody, this is shown to be against the nucleocapsid protein, which serves as the shell of the virus and is an important part of the immune system response of the unvaccinated population. (Related: After you are vaccine damaged, if you complain about symptoms you will be REQUIRED to take psychiatric medications until your “disorder” is cured.) If any mutations to the spike protein of the COVID virus occur in the future, the vaccinated will be more vulnerable and may possibly be unprotected due to their inability to produce the N antibody. Meanwhile, the unvaccinated would have much better immunity to any mutations due to their ability to produce both S and N antibodies after infection. America’s Front Line Doctors also warned that vaccines are turning people’s bodies into walking spike protein factories, which causes the body to create antibodies to them. “First, these vaccines ‘mis-train’ the immune system to recognize only a small part of the virus [the spike protein]. Variants that differ, even slightly, in this protein are able to escape the narrow spectrum of antibodies created by the vaccines,” AFLDS explained. “Second, the vaccines create ‘vaccine addicts,’ meaning persons become dependent upon regular booster shots because they have been ‘vaccinated’ only against a tiny portion of a mutating virus.” The group also cited Australian Health Minister Dr. Kerry Chant, who said that COVID will become endemic and people will have to get used to taking endless vaccines. Finally, there is the simple fact that the vaccines do not, in any way, prevent infection in the nose and upper airways, which is where fully vaccinated people tend to show the highest viral loads. Immune problems and other vaccine infections Vaccinated individuals have also encountered immune problems and reinfections. These conditions, dubbed VAIDS (or Vaccine Acquired Immune Deficiency Syndrome), have been very concerning as they could be damaging to individuals. While not an official scientific term, it is important to bring attention to VAIDS, especially for those who are concerned about the immune health of their vaccinated loved ones. In late January, an anti-mandate rally in Italy reiterated the claim that COVID-19 vaccines were toxic and that they could cause a variety of medical catastrophes down the line. Professor Luc Montagnier, a Nobel Prize winner for medicine for his discovery of the human immunodeficiency virus (HIV) said himself that those who received the third dose of COVID vaccines should go to the laboratory and take AIDS tests, then sue their governments. If Montagnier and other dissident experts are correct about “the great die-off,” then around one to two billion deaths are to be expected in the near future. If the estimation seems alarming, then people should be more aware of the rising number of adverse effects, including cancers and cardiac problems, that developed worldwide. Even Pfizer itself has a long list of possible adverse events from its vaccines, with nine pages of illnesses barely scratching the surface.

OPINION

Here’s how to detox from the COVID spike protein – from the jab or the virus


Spike proteins can circulate in your body after infection or injection, causing damage to cells, tissues and organs, but the World Council for Health has compiled a list of medications to prevent this.

Thu Dec 23, 2021 – 10:38 am EST

Note: This article is an opinion and the treatments that are recommended in it have not been proven as an effective means to eliminate the spike protein from COVID or mRNA vaccines. Damage to endothelial linings of vessels and organs by the COVID-19 spike protein and how to reverse it requires new research and randomized clinical trials to determine if any treatment can detox the body of the spike protein that causes Long-haul diseases.

STORY AT-A-GLANCE

  • If you had COVID-19 or received a COVID-19 injection, you may have dangerous spike proteins circulating in your body
  • Spike proteins can circulate in your body after infection or injection, causing damage to cells, tissues and organs
  • The World Council for Health has released a spike protein detox guide, which provides straightforward steps you can take to potentially lessen the effects of toxic spike protein in your body
  • Spike protein inhibitors and neutralizers include pine needles, ivermectin, neem, N-acetylcysteine (NAC) and glutathione
  • The top 10 spike protein detox essentials include vitamin D, vitamin C, nigella seed, quercetin, zinc, curcumin, milk thistle extract, NAC, ivermectin and magnesium

(Mercola) – Have you had COVID-19 or received a COVID-19 injection? Then you likely have dangerous spike proteins circulating in your body. While a spike protein is naturally found in SARS-CoV-2, no matter the variant, it’s also produced in your body when you receive a COVID-19 shot. In its native form in SARS-CoV-2, the spike protein is responsible for the pathologies of the viral infection.

In its wild form it’s known to open the blood-brain barrier, cause cell damage (cytotoxicity) and, as Dr. Robert Malone – the inventor of the mRNA and DNA vaccine core platform technology – said in a commentary on News Voice, the protein “is active in manipulating the biology of the cells that coat the inside of your blood vessels — vascular endothelial cells, in part through its interaction with ACE2, which controls contraction in the blood vessels, blood pressure and other things.”

It’s also been revealed that the spike protein on its own is enough to cause inflammation and damage to the vascular system, even independent of a virus.

Now, the World Council for Health (WCH) – a worldwide coalition of health-focused organizations and civil society groups that seek to broaden public health knowledge – has released a spike protein detox guide, which provides straightforward steps you can take to potentially lessen the effects of toxic spike protein. You can view their full guide of natural remedies, including dosages, at the end of this article.

Why should you consider a spike protein detox?

Spike proteins can circulate in your body after infection or injection, causing damage to cells, tissues and organs. “Spike protein is a deadly protein,” Dr. Peter McCullough, an internist, cardiologist and trained epidemiologist, says in a video. It may cause inflammation and clotting in any tissue in which it accumulates.

For instance, Pfizer’s biodistribution study, which was used to determine where the injected substances end up in the body, showed the COVID spike protein from the shots accumulated in “quite high concentrations” in the ovaries.

Further, a Japanese biodistribution study for Pfizer’s jab found that vaccine particles move from the injection site to the blood, after which circulating spike proteins are free to travel throughout the body, including to the ovaries, liver, neurological tissues and other organs. WCH noted:

“The virus spike protein has been linked to adverse effects, such as: blood clots, brain fog, organizing pneumonia, and myocarditis. It is probably responsible for many of the Covid-19 [injection] side effects … Even if you have not had any symptoms, tested positive for Covid-19, or experienced adverse side effects after a jab, there may still be lingering spike proteins inside your body.

In order to clear these after the jab or an infection, doctors and holistic practitioners are suggesting a few simple actions. It is thought that cleansing the body of spike protein … as soon as possible after an infection or jab may protect against damage from remaining or circulating spike proteins.”

Spike protein inhibitors and neutralizers

A group of international doctors and holistic practitioners who have experience helping people recover from COVID-19 and post-injection illness compiled natural options for helping to reduce your body’s spike protein load. The following are spike protein inhibitors, which means they inhibit the binding of the spike protein to human cells:

Prunella vulgarisPine needles
EmodinNeem
Dandelion leaf extractIvermectin

Ivermectin, for example, docks to the SARS-CoV-2 spike receptor-bending domain attached to ACE2, which may interfere with its ability to attach to the human cell membrane. They also compiled a list of spike protein neutralizers, which render it unable to cause further damage to cells. This includes:

N-acetylcysteine (NAC)Glutathione
Fennel teaStar anise tea
Pine needle teaSt. John’s wort
Comfrey leafVitamin C

The plant compounds in the table above contain shikimic acid, which may counteract blood clot formation and reduce some of the spike protein’s toxic effects. Nattokinase, a form of fermented soy, may also help to reduce the occurrence of blood clots.

How to protect your ACE2 receptors and detox IL-6

Spike protein attaches to your cells’ ACE2 receptors, impairing the receptors’ normal functioning. This blockage may alter tissue functioning and could be responsible for triggering autoimmune disease or causing abnormal bleeding or clotting, including vaccine-induced thrombotic thrombocytopenia.

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Ivermectin, hydroxychloroquine (with zinc), quercetin (with zinc) and fisetin (a flavonoid) are examples of substances that may naturally protect your ACE2 receptors. Ivermectin works in this regard by binding to ACE2 receptors, preventing the spike protein from doing so.

Interleukin 6 (IL-6) is a proinflammatory cytokine that is expressed post-injection, and its levels increase in people with COVID-19. It’s for this reason that the World Health Organization recommends IL-6 inhibitors for people who are severely ill with COVID-19. Many natural IL-6 inhibitors, or anti-inflammatories, exist and may be useful for those seeking to detox from COVID-19 or COVID-19 injections:

Boswellia serrata (frankincense)Dandelion leaf extract
Black cumin (Nigella sativa)Curcumin
Krill oil and other fatty acidsCinnamon
FisetinApigenin
QuercetinResveratrol
LuteolinVitamin D3 (with vitamin K)
ZincMagnesium
Jasmine teaSpices
Bay leavesBlack pepper
NutmegSage

How to detox from Furin and Serine Protease

To gain entry into your cells, SARS-CoV-2 must first bind to an ACE2 or CD147 receptor on the cell. Next, the spike protein subunit must be proteolytically cleaved (cut). Without this protein cleavage, the virus would simply attach to the receptor and not get any further.

“The furin site is why the virus is so transmissible, and why it invades the heart, the brain and the blood vessels,” Dr. Steven Quay, a physician and scientist, explained at a GOP House Oversight and Reform Subcommittee on Select Coronavirus Crisis hearing.

The existence of a novel furin cleavage site on SARS-CoV-2, while other coronaviruses do not contain a single example of a furin cleavage site, is a significant reason why many believe SARS-CoV-2 was created through gain-of-function (GOF) research in a laboratory. Natural furin inhibitors, which prevent cleavage of the spike protein, can help you detox from furin and include:

  • Rutin
  • Limonene
  • Baicalein
  • Hesperidin

Serine protease is another enzyme that’s “responsible for the proteolytic cleavage of the SARS-CoV-2 spike protein, enabling host cell fusion of the virus.” Inhibiting serine protease may therefore prevent spike protein activation and viral entry into cells. WCH compiled several natural serine protease inhibitors, which include:

Green teaPotato tubers
Blue green algaeSoybeans
N-acetyl cysteine (NAC)Boswellia

Time-restricted eating and healthy diet for all

In addition to the targeted substances mentioned above, WCH was wise to note that a healthy diet is the first step to a healthy immune system. Reducing your consumption of processed foods and other proinflammatory foods, including vegetable (seed) oils, is essential for an optimal immune response.

Time-restricted eating, which means condensing your meals into a six- to eight-hour window, is also beneficial. This will improve your health in a variety of ways, primarily by improving your mitochondrial health and metabolic flexibility. It can also increase autophagy, which helps your body clear out damaged cells. As noted by WCH:

“This method … is used to induce autophagy, which is essentially a recycling process that takes place in human cells, where cells degrade and recycle components. Autophagy is used by the body to eliminate damaged cell proteins and can destroy harmful viruses and bacteria post-infection.”

Another strategy to boost your health and longevity, and possibly to help detox spike protein, is regular sauna usage. As your body is subjected to reasonable amounts of heat stress, it gradually becomes acclimated to the heat, prompting a number of beneficial changes to occur in your body.

These adaptations include increased plasma volume and blood flow to your heart and muscles (which increase athletic endurance) along with increased muscle mass due to greater levels of heat-shock proteins and growth hormone. It’s a powerful detoxification method due to the sweating it promotes.

Top 10 spike protein detox essentials and the full guide

Below you can find WCH’s full guide of useful substances to detox from toxic spike proteins, including recommended doses, which you can confirm with your holistic health care practitioner. If you’re not sure where to start, the following 10 compounds are the “essentials” when it comes to spike protein detox. This is a good place to begin as you work out a more comprehensive health strategy:

Vitamin DVitamin C
NACIvermectin
Nigella seedQuercetin
ZincMagnesium
CurcuminMilk thistle extract

World Council for Health’s spike protein detox guide

SubstanceNatural Source(s)Where to GetRecommended Dose
IvermectinSoil bacteria (avermectin)On prescription0.4 mg/kg weekly for 4 weeks, then monthly
*Check package instructions to determine if there are contraindications prior to use
HydroxychloroquineOn prescription200 mg weekly for 4 weeks
*Check package instructions to determine if there are contraindications prior to use
Vitamin CCitrus fruits (e.g. oranges) and vegetables (broccoli, cauliflower, brussels sprouts)Supplement: health food stores, pharmacies, dietary supplement stores, online6-12 g daily (divided evenly between sodium ascorbate (several grams), liposomal vitamin C (3-6 g) & ascorbyl palmitate (1–3 g)
Prunella Vulgaris (commonly known as self-heal)Self-heal plantSupplement: health food stores, pharmacies, dietary supplement stores, online7 ounces (207 ml) daily
Pine NeedlesPine treeSupplement: health food stores, pharmacies, dietary supplement stores, onlineConsume tea 3 x daily (consume oil/resin that accumulates in the tea also)
NeemNeem treeSupplement: health food stores, pharmacies, dietary supplement stores, onlineAs per your practitioner’s or preparation instructions
Dandelion Leaf ExtractDandelion plantSupplement (dandelion tea, dandelion coffee, leaf tincture): natural food stores, pharmacies, dietary supplement stores, onlineTincture as per your practitioner’s or preparation instructions
N-Acetyl Cysteine (NAC)High-protein foods (beans, lentils, spinach, bananas, salmon, tuna)Supplement: health food stores, pharmacies, dietary supplement stores, onlineUp to 1,200 mg daily (in divided doses)
Fennel TeaFennel plantSupplement: health food stores, pharmacies, dietary supplement stores, onlineNo upper limit. Start with 1 cup and monitor body’s reaction
Star Anise TeaChinese evergreen tree (Illicium verum)Supplement: health food stores, pharmacies, dietary supplement stores, onlineNo upper limit. Start with 1 cup and monitor body’s reaction
St John’s WortSt John’s wort plantSupplement: health food stores, pharmacies, dietary supplement stores, onlineAs directed on supplement
Comfrey LeafSymphytum plant genusSupplement: health food stores, pharmacies, dietary supplement stores, onlineAs directed on supplement
Lumbrokinase
Serrapeptidase
Or Nattokinase
Natto (Japanese soybean dish)Supplement: health food stores, pharmacies, dietary supplement stores, online2-6 capsules 3-4 times a day on empty stomach one hour before or two hours after a meal
Boswellia serrataBoswellia serrata treeSupplement: health food stores, pharmacies, dietary supplement stores, onlineAs directed on supplement
Black Cumin (Nigella Sativa)Buttercup plant familyGrocery stores, health food stores
CurcuminTurmericGrocery stores, health food stores
Fish OilFatty/oily fishGrocery stores, health food storesUp to 2,000 mg daily
CinnamonCinnamomum tree genusGrocery store
Fisetin (Flavonoid)Fruits: strawberries, apples, mangoes Vegetables: onions, nuts, wineSupplement: health food stores, pharmacies, dietary supplement stores, onlineUp to 100 mg daily Consume with fats
ApigeninFruits, veg & herbs parsley, chamomile, vine-spinach, celery, artichokes, oreganoSupplement: health food stores, pharmacies, dietary supplement stores, online50 mg daily
Quercetin (Flavonoid)Citrus fruits, onions, parsley, red wineSupplement: health food stores, pharmacies, dietary supplement stores, onlineUp to 500 mg twice daily, Consume with zinc
ResveratrolPeanuts, grapes, wine, blueberries, cocoaSupplement: health food stores, pharmacies, dietary supplement stores, onlineUp to 1,500 mg daily for up to 3 months
LuteolinVegetables: celery, parsley, onion leaves
Fruits: apple skins, chrysanthemum flowers
Supplement: health food stores, pharmacies, dietary supplement stores, online100-300 mg daily (Typical manufacturer recommendations)
Vitamin D3Fatty fish, fish liver oilsSupplement: health food stores, pharmacies, dietary supplement stores, online5,000–10,000 IU daily or whatever it takes to get to 60-80 ng/ml as tested in your blood
Vitamin KGreen leafy vegetablesSupplement: health food stores, pharmacies, dietary supplement stores, online90-120 mg daily (90 for women, 120 for men)
ZincRed meat, poultry, oysters, whole grains, milk productsSupplement: health food stores, pharmacies, dietary supplement stores, online11-40 mg daily
MagnesiumGreens, whole grains, nutsSupplement: health food stores, pharmacies, dietary supplement stores, onlineUp to 350 mg daily
Jasmine TeaLeaves of common jasmine or Sampaguita plantsGrocery store, health food storesUp to 8 cups per day
SpicesGrocery store
Bay LeavesBay leaf plantsGrocery store
Black PepperPiper nigrum plantGrocery store
NutmegMyristica fragrans tree seedGrocery store
SageSage plantGrocery store
RutinBuckwheat, asparagus, apricots, cherries, black tea, green tea, elderflower teaSupplement: health food stores, pharmacies, dietary supplement stores, online500-4,000 mg daily (consult health care provider before taking higher-end doses)
LimoneneRind of citrus fruits such as lemons, oranges, and limesSupplement: health food stores, pharmacies, dietary supplement stores, onlineUp to 2,000 mg daily
BaicaleinScutellaria plant genusSupplement: health food stores, pharmacies, dietary supplement stores, online100-2,800 mg
HesperidinCitrus fruitSupplement: health food stores, pharmacies, dietary supplement stores, onlineUp to 150 mg twice daily
Green TeaCamellia sinensis plant leavesGrocery storeUp to 8 cups of tea a day or as directed on supplement
Potatoes tubersPotatoesGrocery store
Blue Green AlgaeCyanobacteriaSupplement: health food stores, pharmacies, dietary supplement stores, online1-10 grams daily
Andrographis PaniculataGreen chiretta plantSupplement: health food stores, pharmacies, dietary supplement stores, online400 mg x 2 daily
*Check for contraindications
Milk Thistle ExtractSilymarinSupplement; Health food stores, pharmacies, dietary supplement stores, online200 mg x 3 daily
Soybeans (organic)SoybeansGrocery store, health food stores

Reprinted with permission from Mercola

The spike protein of SARS-CoV-2 induces endothelial inflammation through integrin α5β1 and NF-κB signaling

Authors: Juan Pablo Robles 1Magdalena Zamora 1Elva Adan-CastroLourdes Siqueiros-MarquezGonzalo Martinez de la EscaleraCarmen Clapp

Open AccessDOI:https://doi.org/10.1016/j.jbc.2022.101695

Vascular endothelial cells (ECs) form a critical interface between blood and tissues that maintains whole-body homeostasis. In COVID-19, disruption of the EC barrier results in edema, vascular inflammation, and coagulation, hallmarks of this severe disease. However, the mechanisms by which ECs are dysregulated in COVID-19 are unclear. Here, we show that the spike protein of SARS-CoV-2 alone activates the EC inflammatory phenotype in a manner dependent on integrin ⍺5β1 signaling. Incubation of human umbilical vein ECs with whole spike protein, its receptor-binding domain, or the integrin-binding tripeptide RGD induced the nuclear translocation of NF-κB and subsequent expression of leukocyte adhesion molecules (VCAM1 and ICAM1), coagulation factors (TF and FVIII), proinflammatory cytokines (TNF⍺, IL-1β, and IL-6), and ACE2, as well as the adhesion of peripheral blood leukocytes and hyperpermeability of the EC monolayer. In addition, inhibitors of integrin ⍺5β1 activation prevented these effects. Furthermore, these vascular effects occur in vivo, as revealed by the intravenous administration of spike, which increased expression of ICAM1, VCAM1, CD45, TNFα, IL-1β, and IL-6 in the lung, liver, kidney, and eye, and the intravitreal injection of spike, which disrupted the barrier function of retinal capillaries. We suggest that the spike protein, through its RGD motif in the receptor-binding domain, binds to integrin ⍺5β1 in ECs to activate the NF-κB target gene expression programs responsible for vascular leakage and leukocyte adhesion. These findings uncover a new direct action of SARS-CoV-2 on EC dysfunction and introduce integrin ⍺5β1 as a promising target for treating vascular inflammation in COVID-19.

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https://www.jbc.org/action/showPdf?pii=S0021-9258%2822%2900135-1

Elevated Expression of Serum Endothelial Cell Adhesion Molecules in COVID-19 Patients

Authos: Ming Tong,1Yu Jiang,2Da Xia,3Ying Xiong,3Qing Zheng,4Fang Chen,2Lianhong Zou,2Wen Xiao,2 and Yimin Zhu2

J Infect Dis. 2020 Sep 15; 222(6): 894–898.Published online 2020 Jun 24. doi: 10.1093/infdis/jiaa349 PMCID: PMC7337874PMID: 32582936

Abstract

In a retrospective study of 39 COVID-19 patients and 32 control participants in China, we collected clinical data and examined the expression of endothelial cell adhesion molecules by enzyme-linked immunosorbent assays. Serum levels of fractalkine, vascular cell adhesion molecule-1 (VCAM-1), intercellular adhesion molecule 1 (ICAM-1), and vascular adhesion protein-1 (VAP-1) were elevated in patients with mild disease, dramatically elevated in severe cases, and decreased in the convalescence phase. We conclude the increased expression of endothelial cell adhesion molecules is related to COVID-19 disease severity and may contribute to coagulation dysfunction.Keywords: COVID-19, fractalkine, endothelial cell adhesion molecules, D-dimer, coagulopathy

In December 2019, a severe public health event, manifested mainly with fever and respiratory tract symptoms, broke out in Wuhan, China, and quickly spread throughout the country and the world [1], which was named coronavirus disease 2019 (COVID-19) by the World Health Organization. As of 1 May 2020, more than 3 million cases have been confirmed, while more than 200 000 patients have died, and the number is continuing to increase.

COVID-19 causes a systemic inflammatory response, involving dysregulation and misexpression of many inflammatory cytokines [1]. The recruitment and activation of inflammatory cells depend on the expression of many classes of inflammatory mediators, such as cytokines (interleukin [IL]-1, IL-6, and IL-18), chemokines (fractalkine [FKN]), and adhesion molecules (intercellular adhesion molecule 1 [ICAM-1)] and vascular cell adhesion molecule-1 [VCAM-1]) [2]. Pathological evidence of venous thromboembolism, direct viral infection of the endothelial cells, and diffuse endothelial inflammations have been reported in recent studies [23]. Therefore, it is of significance to investigate the expression of endothelial cell adhesion molecules in COVID-19.

Here, we collected clinical data and blood samples from confirmed COVID-19 patients in the Fourth People’s Hospital of Yiyang in Hunan, China, and performed enzyme-linked immunosorbent assays (ELISAs) to study the expression of inflammatory mediators and endothelial cell adhesion molecules in COVID-19 patients.Go to:

METHODS

Study Participants

A retrospective study was conducted. From 1 February to 10 March 2020, 39 COVID-19 patients were recruited at the Infectious Disease Ward in the Fourth People’s Hospital of Yiyang, Hunan, China, and 32 uninfected participants were recruited from the physical examination center of Hunan Provincial People’s Hospital. All patients tested positive for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and were hospitalized. Nine patients were diagnosed with severe pneumonia, while 30 had mild disease. Mild pneumonia was defined as positivity in quantitative reverse transcription polymerase chain reaction (qRT-PCR) tests, with typical chest tomography imaging features of viral pneumonia [4], while severe pneumonia was defined as mild pneumonia plus 1 of the following criteria: (1) respiratory distress with a respiratory rate ≥ 30 times per minute;

(2) oxygen saturation ≤ 93% at rest; (3) oxygenation index ≤ 300 mmHg (1 mmHg = 0.133 kPa); (4) respiratory failure requiring ventilation; (5) refractory shock; and

(6) admission to the intensive care unit for other organ failure. All patients were given interferon-α2b (5 million units twice daily, atomization inhalation) and lopinavir plus ritonavir (500 mg twice daily, orally) as antiviral therapy. All patients with severe disease received preventive anticoagulant treatment with low-molecular-weight heparin (LMWH) 5000 IU/day by subcutaneous injection for 7 days. No patients died during the observation period.

The criteria for discharge were: (1) absence of fever for at least 3 days; (2) significant improvement in both lungs on chest computed tomography (CT); (3) clinical remission of respiratory symptoms; and (4) repeated negativity in RT-PCR tests of throat swab samples at least 24 hours apart.

Clinical data were measured at enrolment. The study was approved by the Medical Ethics Review Board of Hunan Provincial People’s Hospital (No. 2020-10). All study participants provided written informed consent.

Sample Collection

Blood samples were collected at admission from each patient in a fasting state and repeated during the convalescence period for severe cases. Serum lipids, glucose, C-reaction protein (CRP), and D-dimer were determined by conventional laboratory methods. Blood samples of control subjects were also collected and tested. The obtained blood samples were placed in tubes containing EDTA and immediately centrifuged at 1500g and stored at −80°C.

Enzyme-Linked Immunosorbent Assay

Quantitative determination of IL-18, tumor necrosis factor-α (TNF-α), interferon-γ (IFN-γ), FKN, VCAM-1, ICAM-1, and vascular adhesion protein-1 (VAP-1) was performed using commercially available ELISA kits (BOSTER).

Statistical Analysis

Categorical variables were reported as number and percentages, and significance was detected by χ2 or Fisher exact test. The continuous variables were compared using independent group t tests and described using mean and standard deviation if normally distributed, or compared using the Mann-Whitney U test and Kruskal-Wallis H test and described using median and interquartile range (IQR) value if not. Paired comparisons of the severe group were analyzed with the Nemenyi test. Statistical analysis was performed by SPSS version 19.0. Two-sided P values < .05 were considered statistically significant.

Patient and Public Involvement

In this retrospective study, no patients were directly involved in the study design, question proposal, or the outcome measurements. No patients were asked for input concerning interpretation or recording of the results.

RESULTS

Patient Characteristics

Demographic information is shown in Table 1. Briefly, 20 patients were male, 19 patients were female, 16 controls were male, and 16 were female, and the median ages in the control, mild, and severe groups were 52, 49, and 54 years, respectively. Nine patients had severe disease, while 30 cases had mild disease. No significant differences were found between patients with mild disease and control participants in age, smoking, cardiovascular disease (CVD), autoimmune disease, low-density lipoprotein cholesterol (LDL-C), triglycerides, total cholesterol (CHO), glucose, and D-dimer. Significant differences in D-dimer were observed between the severe disease and control participants (median, 4.49 vs 0.34, respectively; P < .05), while no significant differences in age, smoking, CVD, autoimmune disease, and the levels of triglycerides, LDL-C, CHO, and glucose were observed. Significant differences in age (median, 54 vs 49; P < .05), triglycerides (median, 0.93 vs 1.29; P < .05), D-dimer (median, 4.49 vs 0.35; P < .05), and length of stay (mean, 16.6 vs 10.6; P < .05) were observed between patients with severe and mild disease, respectively, while no significant differences in smoking, CVD, autoimmune disease, and the levels of LDL-C, CHO, and glucose were observed.

Table 1.

Characteristics of Study Participants

CharacteristicOverallControlMild DiseaseSevere Disease
Sex, male/female, n/n36/3516/1616/144/5
Age, y, median (25, 75 percentile)a50 (42, 57)52 (44, 60)49 (25, 55)54 (47, 75)*
Current smoker, n (%)10 (14)6 (192 (7)2 (22)
Cardiovascular disease, n (%)8 (11)5 (16)1 (3)2 (22)
Autoimmune disease, n (%)0 (0)0 (0)0 (0)0 (0)
LDL-C, mmol/L, median (25, 75 percentile)a1.81 (1.53, 2.17)1.81 (1.45, 2.03)1.81 (1.52, 2.34)2.11 (1.58, 2.41)
Triglycerides, mmol/L, median (25, 75 percentile)a1.17 (0.93, 1.54)1.24 (0.93, 1.54)1.29 (0.96, 1.64)0.93 (0.71, 1.03)*
Total cholesterol, mmol/L, mean ± SD3.91 ± 1.143.96 ± 1.483.83 ± 0.914.00 ± 0.98
Glucose, mmol/L, median (25, 75 percentile)a5.5 (4.3, 6.7)5.2 (4.3, 6.7)5.5 (4.4, 6.7)6.2 (4.7, 7.8)
D-dimer, mg/L, median (25, 75 percentile)a0.37 (0.25, 0.58)0.34 (0.25, 0.46)0.35 (0.15, 0.52)4.49 (1.29, 7.00)b,,*
Length of stay, d, mean ± SD12.0 ± 4.310.6 ± 3.516.6 ± 3.5*

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Abbreviation: LDL-C, low-density lipoprotein cholesterol.

aCompared with control group.

b P value <.05 compared with mild disease group.

*< .05.

Expression of Inflammatory Mediators and Endothelial Cell Adhesion Molecules in COVID-19 Patients and Uninfected Participants

The serum levels of the following were higher in patients with mild disease than in control participants: FKN (median, 880.1 vs 684.6 pg/mL; P < .01); VCAM-1 (median, 3742.3 vs 891.4 pg/mL; P < .01); ICAM-1 (median, 2866.1 vs 1287.4 pg/mL; P < .01); VAP-1 (median, 16.81 vs 16.68 pg/mL; P = .41) (Figure 1A–D); CRP (median, 10.75 vs 1.59 mg/L; P < .01); IL-18 (median, 415.4 vs 276.5 pg/mL; P = .09); TNF-α (median, 257.1 vs 242.9 pg/mL; P < .01); and IFN-γ (median, 46.00 vs 42.51 pg/mL; P = .50) (Supplementary Figure 1A–D). Of these, CRP, TNF-α, FKN, VCAM-1, and ICAM-1 were significantly elevated.

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Figure 1.

Expression of endothelial cell adhesion molecules in COVID-19 patients and uninfected participants, the horizontal lines represent median with interquartile range: (A) fractalkine; (B) vascular cell adhesion molecule-1 (VCAM-1); (C) intercellular adhesion molecule 1 (ICAM-1); and (D) vascular adhesion protein-1 (VAP-1). * P < .05; ** P < .01.

The serum levels of the following were significantly higher in patients with severe disease than in control participants: FKN (median, 1457.5 vs 684.6 pg/mL; P < .01); VCAM-1 (median, 4991.3 vs 891.4 pg/mL; P < .01); ICAM-1 (median, 4498.2 vs 1287.4 pg/mL; P < .01); VAP-1 (median, 28.80 vs 16.68 pg/mL; P < .01) (Figure 1A–D); CRP (median, 43.64 vs 1.59 mg/L; P < .01); IL-18 (median, 670.7 vs 276.5 pg/mL; P < .01); TNF-α (median, 274.2 vs 242.9 pg/mL; P < .01); and IFN-γ (median, 76.50 vs 42.51 pg/mL; P < .01) (Supplementary Figure 1A–D).

The serum levels of the following were significantly higher in patients with severe disease than in patients with mild disease: FKN (median, 1457.5 vs 880.1 pg/mL; P < .01); VCAM-1 (median, 4991.3 vs 3742.3 pg/mL; P < .05); ICAM-1 (median, 4498.2 vs 2866.1 pg/mL; P < .05); VAP-1 (median, 28.80 vs 16.81 pg/mL; P < .01) (Figure 1A–D); CRP (median, 43.64 vs 10.75 mg/L; P < .01); IL-18 (median, 670.7 vs 415.4 pg/mL; P < .01); TNF-α (median, 274.2 vs 257.1 pg/mL; P < .05); and IFN-γ (median, 76.50 vs 46.00 pg/mL; P < .01) (Supplementary Figure 1A–D).

For severe cases, the serum levels of the following were lower in the convalescence phase than during the acute phase: FKN (median, 1028.2 vs 1457.5 pg/mL; P < .05); VCAM-1 (median, 3420.9 vs 4991.3 pg/mL; P < .01); ICAM-1 (median, 3046.9 vs 4498.2 pg/mL; P < .01); VAP-1 (median, 23.90 vs 28.80 pg/mL; P = .17) (Figure 1A–D); CRP (median, 10.20 vs 43.64 mg/L; P < .01); IL-18 (median, 514.6 vs 670.7 pg/mL; P < .01); TNF-α (median, 265.1 vs 274.2 pg/mL; P < .01); IFN-γ (median, 66.30 vs 76.50 pg/mL; P = .05) (Supplementary Figure 1A–D); and D-dimer (median, 0.45 vs 4.49; P < .01) (Supplementary Figure 2). Of these, IL-18, TNF-α, FKN, VCAM-1, ICAM-1, and D-dimer were significantly lower.Go to:

DISCUSSION

Three novel findings were identified in our study. First, the endothelial cell adhesion markers FKN, VCAM-1, and ICAM-1 were elevated in COVID-19 patients. Second, the severity of COVID-19 was associated with the serum levels of CRP, IL-18, TNF-α, IFN-γ, FKN, VCAM-1, ICAM-1, and VAP-1. Third, recovery from severe COVID-19 was associated with reductions in serum CRP, IL-18, TNF-α, FKN, VCAM-1, ICAM-1, and D-dimer levels.

Endothelial activation is related to severe COVID-19, and antiphospholipid antibodies, von Willebrand factor, and factor VIII may play a role in coagulopathy [5]. Endothelial cells express angiotensin-converting enzyme 2 (ACE2), the receptor for SARS-CoV-2 [6], and the interaction of SARS-CoV-2 and ACE2 possibly mediates endothelial activation. Endothelial cells are an essential component of the coagulation system and their integrity and functionality are critical to maintaining hemostasis, whereas endothelial cell activation or injury may result in platelet activation, thrombosis, and inflammation [7]. Dysfunctional endothelial cells activated by proinflammatory cytokines may contribute to the pathogenesis of thrombosis by altering the expression of pro- and antithrombotic factors [89].

In this cohort of COVID-19 patients, although apparent thrombosis formation was excluded by Doppler ultrasound in deep veins in the lower extremities and repeated chest CT scans, we found an interesting phenomenon in patients with severe disease, that is serum D-dimer levels were elevated during the acute phase and decreased significantly during the convalescence phase. As an indirect marker of coagulation activation, elevated D-dimer has been reported in several studies and confirmed to correlate with an increased likelihood of death in COVID-19 patients [10]. We consider that the relationship between prethrombosis levels of D-dimer and thrombotic disease is likely partly attributable to subclinical clot formation.

Severe COVID-19 is commonly complicated by coagulopathy, while disseminated intravascular coagulation may contribute to most deaths [11]. Anticoagulant treatment may decrease mortality due to coagulopathy [12]. In patients with severe disease, serum FKN, ICAM-1, VCAM-1, and D-dimer levels declined significantly after antiviral and anticoagulant treatment. In addition to stimulating the immune system to suppress viral replication and clear pathogens, interferon-α also inhibits the inflammatory immune response that leads to histological damage [13]. Hence, we speculate that the dynamic changes in these molecules resulted from the alleviation of endothelial cell injuries, the anti-inflammatory effect of medications, or recovery from COVID-19.

Limitations should be noted when interpreting the results of this study. First, the number of patients with severe disease was low, which may lead to statistical deviation. Second, due to tissue sample inaccessibility, the expression of endothelial activation molecules was not measured in tissues. Third, because we did not measure the direct biomarkers in the coagulation system, the specific disturbed pathways and mechanisms are still unknown. Fourth, due to the anti-inflammatory effect of interferon-α, the relationship between the anticoagulant effect of LMWH and the decreased expression of endothelial cell adhesion molecules in COVID-19 is still uncertain, and requires further study.

In conclusion, based on the results of this study, increased expression of endothelial cell adhesion molecules is related to COVID-19 and disease severity, and may contribute to coagulation dysfunction.

Supplementary Data

Supplementary materials are available at The Journal of Infectious Diseases online. Consisting of data provided by the authors to benefit the reader, the posted materials are not copyedited and are the sole responsibility of the authors, so questions or comments should be addressed to the corresponding author.

jiaa349_suppl_Supplementary_Figure_1

Click here for additional data file.(328K, png)

jiaa349_suppl_Supplementary_Figure_2

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Notes

Acknowledgments. We sincerely thank clinicians at the Forth People’s Hospital of Yiyang, Hunan, China.

Financial support. This work was supported by the Key Research and Development Program of Hunan Province (grant number 2020SK3011).

Potential conflicts of interest. All authors: No reported conflicts of interests. All authors have submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest. Conflicts that the editors consider relevant to the content of the manuscript have been disclosed.Go to:

References

1. Huang C, Wang Y, Li X, et al. . Clinical features of patients infected with 2019 novel coronavirus in Wuhan, China. Lancet 2020; 395:497–506. [PMC free article] [PubMed] [Google Scholar]2. Varga Z, Flammer AJ, Steiger P, et al. . Endothelial cell infection and endotheliitis in COVID-19. Lancet 2020; 395:1417–8. [PMC free article] [PubMed] [Google Scholar]3. Wichmann D, Sperhake JP, Lütgehetmann M, et al. . Autopsy findings and venous thromboembolism in patients with COVID-19 [published online ahead of print 6 May 2020]. Ann Intern Med doi: 10.7326/M20-2003. [PMC free article] [PubMed] [CrossRef] [Google Scholar]4. Shi H, Han X, Jiang N, et al. . Radiological findings from 81 patients with COVID-19 pneumonia in Wuhan, China: a descriptive study. Lancet Infect Dis 2020; 20:425–34. [PMC free article] [PubMed] [Google Scholar]5. Escher R, Breakey N, Lämmle B. Severe COVID-19 infection associated with endothelial activation. Thromb Res 2020; 190:62. [PMC free article] [PubMed] [Google Scholar]6. Turner AJ, Hiscox JA, Hooper NM. ACE2: from vasopeptidase to SARS virus receptor. Trends Pharmacol Sci 2004; 25:291–4. [PMC free article] [PubMed] [Google Scholar]7. Krüger-Genge A, Blocki A, Franke RP, Jung F. Vascular endothelial cell biology: an update. Int J Mol Sci 2019; 20:4411. [PMC free article] [PubMed] [Google Scholar]8. Wong R, Lénárt N, Hill L, et al. . Interleukin-1 mediates ischaemic brain injury via distinct actions on endothelial cells and cholinergic neurons. Brain Behav Immun 2019; 76:126–38. [PMC free article] [PubMed] [Google Scholar]9. Fong LY, Ng CT, Zakaria ZA, et al. . Asiaticoside inhibits TNF-α-induced endothelial hyperpermeability of human aortic endothelial cells. Phytother Res 2015; 29:1501–8. [PubMed] [Google Scholar]10. Zhou F, Yu T, Du R, et al. . Clinical course and risk factors for mortality of adult inpatients with COVID-19 in Wuhan, China: a retrospective cohort study. Lancet 2020; 395:1054–62. [PMC free article] [PubMed] [Google Scholar]11. Chen N, Zhou M, Dong X, et al. . Epidemiological and clinical characteristics of 99 cases of 2019 novel coronavirus pneumonia in Wuhan, China: a descriptive study. Lancet 2020; 395:507–13. [PMC free article] [PubMed] [Google Scholar]12. Tang N, Bai H, Chen X, Gong J, Li D, Sun Z. Anticoagulant treatment is associated with decreased mortality in severe coronavirus disease 2019 patients with coagulopathy. J Thromb Haemost 2020; 18:1094–9. [PubMed] [Google Scholar]13. Lee AJ, Ashkar AA. The dual nature of type I and type II interferons. Front Immunol 2018; 9:2061. [PMC free article] [PubMed] [Google Scholar]

What Role does Endothelial Infection Play in SARS-CoV-2 Infection?

Authors: By Dr. Liji Thomas, MD Reviewed by Emily Henderson, B.Sc.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) can cause inflammatory lung disease, including clot formation and hyper-permeability of the lung vessels, resulting in edema and bleeding into the lung. Inflammation also affects other organs, mediated by the cytokine storm.

This inflammation is characterized by endothelial cell dysfunction in multiple organs. The cause of this endothelialopathy is unknown. It could be due to the direct infection of endothelial cells or an indirect effect of the cytokines.

SARS-CoV-2 Virus

Image Credit: Kateryna Kon/Shutterstock.com

Integrin binding by SARS-CoV-2

Unlike earlier coronaviruses pathogenic to humans, SARS-CoV-2 has a spike protein that is linked to host recognition and viral attachment via the angiotensin-converting enzyme 2 (ACE2) receptor. A unique three-residue RGD motif outside the ACE2 recognition site may allow the spike protein to bind to endothelial proteins called integrins, that bind the RGD group.

In fact, the major integrin on endothelial cells, called αVβ3, is able to bind to multiple RGD-binding ligands. It also engages multiple extracellular matrix proteins, such as fibrinogen, fibronectin, and vitronectin, via its binding pocket. These matrix proteins regulate cell adhesion, migration, and proliferation, as well as angiogenesis.

This mutation could thus enhance SARS-CoV-2 binding to the host cell and may be responsible for the high transmissibility of this virus compared to the earlier ones, while also allowing for multiple routes of entry for the virus and promoting its dissemination within the host by two receptors.

SARS-CoV-2 thus produces marked dysregulation of the endothelial barrier, causing it to lose its integrity and producing a hyper-permeable state. This leads to shock and the rapid spread of the virus to major organs.

Endothelial infection in COVID-19

Endothelial cells are key to several physiological processes including activation of immune cells, platelet aggregation and adhesion, leukocyte adhesion, and transmigration. They are also the target of many viruses, leading to multi-organ dysfunction.

Some studies have failed to show the growth of the virus within endothelial cells, which has been attributed to the lack of expression of the angiotensin-converting enzyme 2 (ACE2) receptor on these cells.

However, it may be argued that this is due to the intrinsic differences between the endothelial monolayer grown in vitro, vs the endothelial lining of the blood vessels that handle blood flowing under shear stress; the activation of the endothelial cells by the high volume of cytokines; and the tight contact with the epithelial cells of the lung capillaries.What is a Cytokine Storm?

Other researchers have reported that SARS-CoV-2 is found in association with the endothelial cell marker CD31 within the lungs, in infected mice and non-human primates (NHPs). Even more significantly, this finding has been identified in the lung tissue of people who died of severe COVID-19.

The viral proteins were also found in endothelial cells. Moreover, infected mice showed upregulated KRAS signaling pathways in lung tissue, known to mediate cellular activation and dysfunction. Experimental evidence shows that mouse endothelial cells are infected by SARS-CoV-2.

Though all endothelial cells express ACE2, all are not the targets of the virus. Instead, it requires the co-expression of other host proteases such as the transmembrane serine protease TMPRSS2, or cathepsins, that cleave the spike protein to its fusion conformation, allowing viral entry into the host cell via endocytosis.

Endothelial cell injury

Following the viral entry into the endothelial cell, it begins to translate its proteins, replicate itself, and may directly induce cell injury and apoptosis. Along with this, endothelial cells activate T cells, though less than other antigen-presenting cells do. In fact, endothelial cells activate only antigen-specific memory or effector T cells, not naïve lymphocytes.

In so doing, endothelial cells may promote the destruction of infected cells by presenting viral proteins to CD8 T cells. Moreover, endothelial cells in the microvasculature may cause memory or effector CD4 T cells to migrate through the endothelium. Antiviral cytokines including gamma-interferon (IFN-γ) may induce class I or II major histocompatibility complex (MHC) molecules, costimulatory molecules that are typically required for T cell activation to occur.

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This means that the endothelial dysfunction caused by COVID-19 blocks lymphocyte activation via endothelial cells, causing an imbalance in the adaptive immune response.

Cytokine storm

The cytokine storm leads to a kind of overreach, causing further endothelial dysfunction. These cytokines include interleukin-6 (IL-6) that stimulates endothelial cell secretion of pro-inflammatory mediators and complement activation, thus further enhancing endothelial barrier breakdown.

Lymphocyte depletion often seen in COVID-19 could also be the result of the excessive inflammation induced by the endothelial cell injury. The reduced number of CD4 lymphocytes may cause an impaired response to the infection while also stimulating further inflammation. Thus, the hyper-inflammatory response in severe and critical COVID-19 could be due to endothelial cell infection and dysfunction.

Loss of endothelial barrier integrity

SARS-CoV-2 infection causes immune dysfunction as well as extensive endothelial injury, in addition to clotting defects and systemic microangiopathy. The poor disease outcome is mediated largely through the increased vascular permeability secondary to infection-related inflammation.

This hyper-permeability is associated with the leakage of both cellular and non-cellular components of the blood in the small blood vessels of the lung, causing the alveoli to become congested with liquid. The patient drowns in the fluid from the leaky blood vessels, which can endanger life by causing asphyxiation.

Hypercoagulability

Simultaneously, the clotting cascades are dysregulated, causing microthrombi to form throughout the circulation, along with leukocyte infiltration. The endothelial cell dysfunction may cause further inflammation and leukocyte recruitment and adhesion.

Since endothelial cells express glycosaminoglycans and thrombomodulin on their cell surface, they inhibit the clotting cascade component, thrombin, as well as a protein inhibitor of tissue factor. Many relaxing factors such as nitric oxide (NO) and prostacyclin (PGI2) are also produced by these cells, thus blocking leukocyte and platelet adhesion and migration, smooth muscle proliferation, and exerting an anti-inflammatory, anti-apoptotic effect.

When the endothelial cells are injured by the viral invasion, they cease to exert their anticoagulant effect, leading to a thrombotic tendency that manifests as extensive microthrombi, hyaline membrane formation in the small arterioles of the lung, and diffuse alveolar injury.

Elevated D-dimer levels occur with this hypercoagulable state, causing poor outcomes and higher mortality with COVID-19. Multiple procoagulant mechanisms are at work, from the exposure of the tissue factor to clotting factors in the blood to the loss of endothelial integrity and thus activation of the intrinsic clotting pathway by the exposed matrix under the endothelial cell layer, to the devastating release of van Willebrand factor (vWF), due to endothelial dysfunction. This molecule acts to bridge platelets for aggregation and clot formation.

Infection of the endothelial cells could be associated with viral invasion of the adjacent tissues, that is, of the smooth muscle cells of the arteries and the cardiac myocytes.

Therapeutic implications

Thus, SARS-CoV-2 infection of endothelial cells could be an underlying cause for the cardiovascular complications of COVID-19, including the end-stage multi-organ dysfunction. It is plausible that the endothelial cell apoptosis was seen in patients who have died of COVID-19, as well as the microthrombi scattered throughout the lung vascular bed along with right ventricular dysfunction, are associated with direct infection of the endothelial cells.

The binding of the spike protein to αVβ3 can be inhibited by the specific αVβ3-antagonist Cilengitide, an RGD tripeptide, that has a high affinity to this integrin and suppresses virus-endothelium binding at very low doses.

Other therapeutic strategies include serine protease inhibitors, renin-angiotensin-aldosterone system inhibitors, statins, heparin, corticosteroids, and IL-6 inhibitors, all of which act at least in part via stabilization and protection of endothelial integrity.

References:

Further Reading

The novel coronavirus’ spike protein plays additional key role in illness

Salk researchers and collaborators show how the protein damages cells, confirming COVID-19 as a primarily vascular disease

April 30, 2021

LA JOLLA—Scientists have known for a while that SARS-CoV-2’s distinctive “spike” proteins help the virus infect its host by latching on to healthy cells. Now, a major new study shows that the virus spike proteins (which behave very differently than those safely encoded by vaccines) also play a key role in the disease itself.

The paper, published on April 30, 2021, in Circulation Research, also shows conclusively that COVID-19 is a vascular disease, demonstrating exactly how the SARS-CoV-2 virus damages and attacks the vascular system on a cellular level. The findings help explain COVID-19’s wide variety of seemingly unconnected complications, and could open the door for new research into more effective therapies.

Representative images of vascular endothelial control cells (left) and cells treated with the SARS-CoV-2 Spike protein (right) show that the spike protein causes increased mitochondrial fragmentation in vascular cells.
Click here for a high-resolution image.
Credit: Salk Institute

“A lot of people think of it as a respiratory disease, but it’s really a vascular disease,” says Assistant Research Professor Uri Manor, who is co-senior author of the study. “That could explain why some people have strokes, and why some people have issues in other parts of the body. The commonality between them is that they all have vascular underpinnings.”

Salk researchers collaborated with scientists at the University of California San Diego on the paper, including co-first author Jiao Zhang and co-senior author John Shyy, among others.

While the findings themselves aren’t entirely a surprise, the paper provides clear confirmation and a detailed explanation of the mechanism through which the protein damages vascular cells for the first time. There’s been a growing consensus that SARS-CoV-2 affects the vascular system, but exactly how it did so was not understood. Similarly, scientists studying other coronaviruses have long suspected that the spike protein contributed to damaging vascular endothelial cells, but this is the first time the process has been documented.

In the new study, the researchers created a “pseudovirus” that was surrounded by SARS-CoV-2 classic crown of spike proteins, but did not contain any actual virus. Exposure to this pseudovirus resulted in damage to the lungs and arteries of an animal model—proving that the spike protein alone was enough to cause disease. Tissue samples showed inflammation in endothelial cells lining the pulmonary artery walls.

The team then replicated this process in the lab, exposing healthy endothelial cells (which line arteries) to the spike protein. They showed that the spike protein damaged the cells by binding ACE2. This binding disrupted ACE2’s molecular signaling to mitochondria (organelles that generate energy for cells), causing the mitochondria to become damaged and fragmented.

Previous studies have shown a similar effect when cells were exposed to the SARS-CoV-2 virus, but this is the first study to show that the damage occurs when cells are exposed to the spike protein on its own.

“If you remove the replicating capabilities of the virus, it still has a major damaging effect on the vascular cells, simply by virtue of its ability to bind to this ACE2 receptor, the S protein receptor, now famous thanks to COVID,” Manor explains. “Further studies with mutant spike proteins will also provide new insight towards the infectivity and severity of mutant SARS CoV-2 viruses.”

The researchers next hope to take a closer look at the mechanism by which the disrupted ACE2 protein damages mitochondria and causes them to change shape.

Other authors on the study are Yuyang Lei and Zu-Yi Yuan of Jiaotong University in Xi’an, China; Cara R. Schiavon, Leonardo Andrade, and Gerald S. Shadel of Salk; Ming He, Hui Shen, Yichi Zhang, Yoshitake Cho, Mark Hepokoski, Jason X.-J. Yuan, Atul Malhotra, Jin Zhang of the University of California San Diego; Lili Chen, Qian Yin, Ting Lei, Hongliang Wang and Shengpeng Wang of Xi’an Jiatong University Health Science Center in Xi’an, China.

The research was supported by the National Institutes of Health, the National Natural Science Foundation of China, the Shaanxi Natural Science Fund, the National Key Research and Development Program, the First Affiliated Hospital of Xi’an Jiaotong University; and Xi’an Jiaotong University.

DOI: 10.1161/CIRCRESAHA.121.318902

Domains and Functions of Spike Protein in SARS-Cov-2 in the Context of Vaccine Design

Authors: Xuhua Xia1,2Kenneth Lundstrom, Academic Editor and Alaa. A. A. Aljabali, Academic Editor

Abstract

The spike protein in SARS-CoV-2 (SARS-2-S) interacts with the human ACE2 receptor to gain entry into a cell to initiate infection. Both Pfizer/BioNTech’s BNT162b2 and Moderna’s mRNA-1273 vaccine candidates are based on stabilized mRNA encoding prefusion SARS-2-S that can be produced after the mRNA is delivered into the human cell and translated. SARS-2-S is cleaved into S1 and S2 subunits, with S1 serving the function of receptor-binding and S2 serving the function of membrane fusion. Here, I dissect in detail the various domains of SARS-2-S and their functions discovered through a variety of different experimental and theoretical approaches to build a foundation for a comprehensive mechanistic understanding of how SARS-2-S works to achieve its function of mediating cell entry and subsequent cell-to-cell transmission. The integration of structure and function of SARS-2-S in this review should enhance our understanding of the dynamic processes involving receptor binding, multiple cleavage events, membrane fusion, viral entry, as well as the emergence of new viral variants. I highlighted the relevance of structural domains and dynamics to vaccine development, and discussed reasons for the spike protein to be frequently featured in the conspiracy theory claiming that SARS-CoV-2 is artificially created.Keywords: COVID-19, spike protein, S-2P, SARS-CoV-2, cleavage, vaccine, protein structure, hydrophobicity, isoelectric pointGo to:

1. Introduction

SARS-CoV-2 uses its trimeric spike protein for binding to host angiotensin-converting enzyme 2 (ACE2) and for fusing with cell membrane to gain cell entry [1,2,3,4]. This is a multi-step process involving three separate S protein cleavage events to prime the SARS-2-S for interaction with ACE2 [2,3], and subsequent membrane fusion and cell entry. These processes involve different domains of the S protein interacting with host cell and other intracellular and extracellular components. Efficiency in each step could contribute to virulence and infectivity. Disrupting any of these steps could lead to medical cure.

The domain structure is very similar between SARS-S (UniProtKB: P59594) and SARS-2-S (UniprotKB: P0DTC2). Both are cleaved to generate S1 and S2 subunits at specific cleavage sites (Figure 1A). S1 serves the function of receptor-binding and contains a signal peptide (SP) at the N terminus, an N-terminal domain (NTD), and receptor-binding domain (RBD). S2 (Figure 1A) functions in membrane fusion to facilitate cell entry, and it contains a fusion peptide (FP) domain, internal fusion peptide (IFP), two heptad-repeat domains (HR1 and HR2), transmembrane domain, and a C-terminal domain [2,3,5,6,7,8]. However, there are also significant differences between SARS-S and SARS-2-S. For example, the contact amino acid sites between SARS-S and human ACE2 (hACE2) [5,7,9,10] differ from those between SARS-2-S and hACE2 [11,12,13,14]. This may explain why some antibodies that are effective against SARS-S are not effective against SARS-2-S [4], especially those developed to target the ACE2 binding site of SARS-S [15]. In this article, numerous experiments on SARS-S are considered to facilitate comparisons and to highlight differences between the two.

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Figure 1

Domain structure of SARS-S and SARS-2-S. (A) Key domains in SARS-S and SARS-2-S. SP, signal peptide; NTD, N-terminal domain; RBD, receptor-binding domain; FP, fusion peptide; IFP, internal fusion peptide; HR, heptad repeats; TM, transmembrane domain; CT, cytoplasmic tail. The top and bottom numbers in each domain pertain to SARS-S and SARS-2-S, respectively. The red arrows indicate cleavage sites, and their numbers pertain to SARS-2-S; (B) Alignment of SP between SARS-S (top) and SARS-2-S (bottom); (C,D) Alignment of two inter-domain segments; (E) HR1 in SARS-S and SARS-2-S, together with the top view of a helix showing hydrophobic positions a and d on the same side; (F) Hydrophobicity plot generated from DAMBE [16].Go to:

2. General Features of SARS-S and SARS-2S

SARS-2-S is 1273 aa long, in contrast to 1255 aa in SARS-S. Individual protein domains in the S protein tend to fold independently and are associated with specific functions. The numbers (Figure 1A) that indicate the start/end of individual domains in SARS-S and SARS-2-S may mislead readers to think that the boundary is based on some clearly recognizable physiochemical landmarks. In fact, these numbers are for rough reference only. For example, the boundaries of RBD in SARS-S mainly result from experiments with different RNA clones containing different parts of RBD [17,18,19]. The 5′ side is delimited by the site where upstream mutations/deletions do not affect receptor binding, but downstream mutations/deletions do affect receptor binding. Similarly, the 3′ side is where upstream mutations/deletions affect receptor binding, but downstream mutations/deletions do not have an effect. Boundaries of some domains are substantiated by protein structure, for example, the boundaries of RBD [11,12,13,14,20], but some are not substantiated by protein structure.

Some inter-domain segments (Figure 1C,D) could be much more conserved than neighboring domains. For example, C822, D830, L831, and C833 in SARS-S (corresponding to C840, D848, L849, and C851 in SARS-2-S) are located between FP and IFP but are highly conserved and critically important for membrane fusion [21]. Similarly, V601 in SARS-S (corresponding to V615 in SARS-2-S) does not belong to any recognized domain (Figure 1A) but is highly conserved. Replacing it by G contributes to viral escape from neutralizing antibodies [22]. Experimental mutations at sites 1111–1130 in SARS-S, upstream of HR2 (Figure 1A), are also associated with viral escape from neutralizing antibodies [23], suggesting that mutations at those sites affect protein structure. This segment is highly conserved in SARS-2-S and related viruses, and antibodies targeting this region provide broad protection against heterogeneous viral strains [23]. In short, inter-domain segments may not be functionally less important than those recognized domains, and the sequences in these inter-domain regions are no less conserved than those within domains. More studies will reveal their functions leading to more detailed structure-function maps.

Experiments with a truncated SARS-S excluding the C-terminus indicates that it is synthesized in the endoplasmic reticulum (ER), modified in the Golgi apparatus, glycosylated, and eventually exported to the membrane [24]. Spike protein synthesis following SARS-CoV infection can cause an unfolded protein response (UPR) [25], suggesting its association with the ER. The UPR restores ER homeostasis by upregulating chaperone proteins to increase the protein-folding capacity in the ER and by reducing translation and increasing protein degradation to reduce the folding load (review in [26]). When prolonged UPR fails to restore ER homeostasis, it often triggers apoptosis. Adenovirus-mediated overexpression of S2 induces apoptosis [27] and may have implications for viral pathogenicity and secondary bacterial infection.

Coronavirus S proteins are heavily glycosylated with 21–35 N-glycosylation sites [17]. Replacing these N-glycosylation sites in SARS-S alters protein folding and expression [18]. Glycosylation events have been identified mainly in two ways. The first way has been to compare the expected molecular weight of an expressed segment of S protein containing a putative N-glycosylation site against the actual molecular weight [18]. An increase in the actual molecular weight is assumed to be due to N-glycosylation. The second way has been by high resolution mass spectrometry [28]. O-glycosylation was also found in SARS-2-S [28]. Glycosylation is not required for receptor-binding in SARS-S [18] or MHV (murine hepatitis virus) [17].Go to:

3. Cleavage Sites

The S protein undergoes two crucial cleavage events, with the first splitting S1 and S2 and the second splitting S2 into FP and S2′ (Figure 1A). The most pronounced difference between SARS-S and SARS-2-S is an additional furin cleavage site (site 1, Figure 2A) resulting from an insertion of 12 nt at the boundary between S1 and S2 [8,11,29]. This additional furin cleavage site is shared among all sequenced SARS-CoV-2 genomes, but absent in all their closest known relatives such as bat RaTG13 and those isolated from pangolin [29]. The seemingly sudden appearance of this additional polybasic furin cleavage site 1 has been a lasting source of conspiracy theory that SARS-CoV-2 is man-made, which is discussed later.

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Figure 2

Cleavage sites at the S1/S2 boundary. (A) An insertion of 12 nt in SARS-CoV-2 results in a new polybasic furin cleavage site, resulting in two cleavage sites indicated by the red downward arrows. “*” indicates sites that are identical among the six viral strains. Numbers follow (B) Schematic domain structure of S protein, with the same abbreviation as in Figure 1A; (C) Tissue-specific mRNA distribution of human trypsin-like protease TMPRESS11D and FURIN, derived from [30]; (D) Cleavage site for splitting S2 into FP and S2′ domains.

The furin cleavage site was predicted in February 2020 [8] and, in May 2020, its functional importance was confirmed, i.e., that the cleavage was essential for efficient viral entry into human lung cells, especially in cell-cell fusion to form syncytium to facilitate viral spread from one cell to another [2]. This exemplifies the rapidity in the progress of SARS-2-S research.

The cleavage of the S protein into S1 and S2 is an essential step in viral entry into a host cell, and needs to occur before viral fusion with the host cell membrane [6]. Different cleavage sites targeted by different proteases are often associated with drastically different virulence and host cell tropism in various RNA viruses. For example, the low-pathogenicity forms of the H1N1 influenza virus has a cleavage site by trypsin-like proteases [31] in contrast to the high-pathogenicity forms with a furin cleavage site cleaved by furin-like proteases [32]. Trypsin-like proteases typically have a narrow tissue distribution in humans. For example, trypsin-like transmembrane serine protease 11D (gene name TMPRSS11D) is expressed only in the esophagus (Figure 2C). Another member of the trypsin family, PRSS1, is expressed mainly in the pancreas [30]. In contrast, furin-like proteases are ubiquitous (Figure 2C). Thus, if a coronavirus needs to be cleaved TMPRSS11D or PRSS1, then its cellular entry is limited to the esophagus where TMPRSS11D is expressed (Figure 2C) or the pancreas where PRSS1 is expressed. However, if the virus gains a furin cleavage site, then this restriction is removed because FURIN is ubiquitous in human tissues (Figure 2C), resulting in dramatic broadening of host cell tropism. In this context, the S protein contributes to host specificity [6], and also to tissue specificity through its differential requirement of tissue-specific proteases. For this reason, viruses with different cell tropism may accumulate tissue-specific genomic signatures [33].

Because the C-terminus of the spike protein is anchored inside the viral membrane, one might expect the distal S1 to be lost after cleavage at site 1. However, the distal S1 subunit remains non-covalently bound to the S2 unit in the prefusion conformation after cleavage at site 1 [10,11,34]. In order to stabilize the prefusion conformation to facilitate vaccine design [10,35] or structural determination [11,12], the furin site is often mutated so that it is not cleaved. For example, the cleavage site RRAR was changed to GSAS in obtaining protein structure 6VSB [12], and to SGAG in obtaining protein structures 6VXX and 6VYB [11].

The cleavage site 2 (Figure 2A) is highly conserved in all sequenced SARS-CoV-2, as well as in all its close relatives including SARS-CoV. This site is likely cleaved by cathepsin L in endosome in both SARS-S [34,36,37,38] and SARS-2-S [4]. Cathepsin L requires an aromatic residue at P2 and a hydrophobic residue at P3 [39]. Cleavage site 2 has Y at P2 and A at P3 to satisfy this requirement (Figure 2A). The low pH in endosomes is optimal for cathepsin L activity. Inhibitors of cathepsin L block SARS-CoV infection [36].

While cleavage site 1 (Figure 2A) is known to be cleaved during SARS-CoV-2 assembly, most likely by furin in the Golgi apparatus [2,11,24,40], it is less clear how cleavage site 2 (Figure 2A) is used in SARS-2-S priming. One could hypothesize if cleavage site 1 is efficient [2], then cleavage site 2 would seem redundant and may accumulate mutations in the SARS‑2‑S gene without a negative impact on the fitness of the virus. However, the amino acid sites near site 2 (VASQSIIAYT|MSLGAEN, where the vertical bar indicates the scissile bond, Figure 2A) was perfectly conserved among all SARS-2-S sequenced by 8 May 2020. In contrast, each site of the 4-AA insertion (PRRA, Figure 2A) has experienced at least one amino acid replacement. Thus, in spite of the additional furin cleavage site 1, cleavage site 2 (Figure 2A) may still be functionally important for it to be so evolutionarily conserved.

In addition to site 1 and site 2 (Figure 2A) that cleave SARS-2-S into the S1 and S2 domains, a third cleavage site also exists for cleaving S2 into FP and S2′ domains (Figure 2B,D). This site, often referred to as the S2′ site, is likely cleaved by TMPRSS2 [41,42,43,44], consistent with the finding that TMPRSS2 is needed for SARS-CoV-2 infection [3]. In particular, TMPRSS2 needs to be expressed in the target cell for it to be infected [41]. Because TMPRSS2 is active mainly in the membrane or extracellular space, the third cleavage site is not cleaved during SARS-CoV assembly [24,41]. This site can also be cleaved by trypsin. Exogenous trypsin can enhance membrane fusion and SARS-CoV infection [45,46]. Trypsin cleaves SARS-S at R797 (Figure 2D), consistent with the finding that an R797N mutation abolishes this trypsin-induced membrane fusion [34].

The temporal sequence of cleavage events is not clear, although the following order is likely: For SARS-2-S, furin cleaves at cleavage site 1 during viral assembly [2]. Then, the third cleavage site is cleaved by TMPRSS2 to yield FP and S2′ (Figure 2D) to trigger membrane fusion, syncytium formation, and viral entry into a target cell [3,11,34]. For SARS-S, cleavage site 1 does not seem to be used efficiently. The transmembrane TMPRSS2, if expressed, cleaves the third cleavage site to yield FP and S2′ and to trigger cell fusion and viral entry [3]. This may be termed the membrane-TMPRSS2 pathway of viral entry. If SARS-S is not cleaved by TMPRSS2 into FP and S2′, then the virus can enter the cell through endocytosis with cleavage site 2 cleaved by cathepsin L. This is the endosome-cathepsin pathway of viral entry [41,46].Go to:

4. The S1 Domain

4.1. The Signal Peptide

The spike protein requires a signal peptide (SP) to guide its transportation to its membrane destination. The SP consists of the first 13 amino acids with helix-forming high-hydrophobicity residues (Figure 1F), as is typical of almost all signal peptides. The only other SARS-S segment of high hydrophobicity is the transmembrane domain (TM, Figure 1F). These two hydrophobic regions at the two extremes of S are shared among diverse betacoronavirus lineages. The SP from different coronaviruses are only weakly homologous at the nucleotide or amino acid level (Figure 2B), but they share helix structure and high hydrophobicity in common.

4.2. The N-Terminal Domain (NTD)

The function of N-terminal and C-terminal domains of S1 differs among different betacoronavirus lineages. The receptor for S protein in MHV is carcinoembryonic antigen cell-adhesion molecules (CEACAMs), and the receptor-binding domain is near the N-terminal [47,48]. As receptor binding is clearly a vital function for any coronavirus, MHV’s NTD is conserved with no indels in aligned MHV S protein sequences, whereas its C-terminal domain homologous to RBD in SARS-S and SASRS-2-S is littered with many indels. For most betacoronaviruses, RBD is near the C-terminus of S1 (Figure 1A), and this RBD domain tends to be more conserved at the nucleotide and amino acid level, and also in the sliding-window hydrophobicity plot (Figure 3A) than in the NTD.

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Figure 3

Hydrophobicity (A) and protein isoelectric point (B) plots of spike protein from SARS-CoV-2 and its close relatives over sliding windows. For window-specific calculation of isoelectric point (pI), the N-terminus amino group is added to the first window and the C-terminus carboxyl added to the last window. Generated from DAMBE [49].

4.3. The Receptor-Binding Domain (RBD)

The RBD domain has a core subdomain and a receptor-binding motif that directly interact with the host ACE2 [4,5,50]. It has been used extensively as a drug target for anti-SARS-CoV drug and vaccine development [51,52,53,54,55,56,57]. A good vaccine should be safe but highly immunogenic and should not become obsolete as soon as there are viral mutations. RBD-based vaccines have been found to be highly immunogenic [58,59], even when they are expressed in yeast [60], which suggests that they fold independently of other parts of the spike protein and that the folding is robust in different folding environments. However, it is more difficult to establish the safety and long-term effect of the vaccines.

In spite of much effort to develop drugs and vaccines based on RBD, there is an inherent problem with this approach because RBD is highly variable at the sequence level [17]. The sequence variability in S1 relative to S2 is also highly visible in a sliding-window isoelectric point (pI) plot (Figure 3B). Because of high variability in S1 among different viral species, RBD-based antibodies or vaccines developed against SARS-CoV [54,55,56] typically do not offer heterologous protection against other coronaviruses such as MERS-CoV [61]. In fact, some antibodies against SARS-CoV strains in the first viral outbreak were no longer effective against SARS-CoV in the second outbreak [62], cautioning against drug development targeting variable domains. In contrast, human monoclonal antibodies against the more conserved S2 are expected to be more broadly neutralizing, which is true as demonstrated with antibodies against highly conserved HR1 and HR2 domains of SARS-S [63]. Thus, given that a virus can escape neutralizing antibodies by just a single amino acid replacement [22,64], one should develop anti-viral drugs or vaccines by targeting only highly conserved regions.Go to:

5. The S2 Domain

While the S1 domain mainly functions in receptor binding, the S2 domain functions mainly in membrane fusion. They represent two distinct steps in SARS-CoV infection [20,36] and SARS-CoV-2 infection [2,3,8]. This S2 function of membrane fusion was inferred early because many antibodies targeting S2 of coronavirus S proteins were almost always associated with disrupted membrane fusion [17]. Vaccine targeting segments 884–891 and 1116–1123 in S2 were highly effective in inducing humoral and cell-mediated immune responses [65]. These segments belong to the central helix between HR1 and HR2. However, some antibodies targeting S2 have been shown to be cytotoxic [66].

Membrane fusion requires two anchors, one at the virion side and the other at the host cell side [67,68]. In the case of SARS-S and SARS-2-S (Figure 1A), the C-terminus is anchored inside the virion, and the FP domain of S2 (or IFP domain of S2′ when FP is cleaved off) penetrates the target cell membrane to install the anchor inside the target cell [67,69].

Membrane fusion appears to have two distinct types associated with different pathways of cell entry. The first type involves a virus in a non-cellular environment (e.g., in the airway of human respiratory system) finding its way inside an epithelial cell, and the second type involves a virus in an infected cell finding its way to a neighboring cell. The first type would require fusion of the viral membrane and the target cell membrane, and the second type would be facilitated by the formation of syncytium through the cell-cell fusion [2,34].

5.1. Fusion Peptides

Many viral fusion proteins exist [67,70]. All known viral fusion peptides form trimers [67], but they often exhibit little sequence homology among different viral species, suggesting evolutionary convergence in trimer formation. The S protein needs a trigger to induce conformational change for membrane fusion [67], and the trigger is typically a cleavage event that occurs either at the cell surface at neutral pH or within an endosome at a reduced pH. These correspond to the two viral entry pathways in SARS-S and SARS-2-S, i.e., the membrane-TMPRSS2 pathway and the endosome-cathepsin L pathway [41,42,43,44,46].

The FP (or IFP when FP is cleaved off) in SARS-S and SARS-2-S (Figure 1A) serves to penetrate the target cell membrane and install an anchor inside. The TM and CT domains (Figure 1A) form an anchor inside the virion. The S2 (or S2′) between the two anchors will undergo conformational change to bring the two membranes together for fusion. The conformational change needs to be triggered by a signal that should reliably indicate the proximity between a virus and a target cell or between an infected cell and a target cell. The triggering signal most likely is TMPRSS2 expressed on the surface of a target cell [41,42,43,44,46]. Thus, the cleavage of S2 at 797R|S798 in SARS-S (where|indicates the scissile bond) or 815R|S816 in SARS-2-S (Figure 2D) by TMPRSS2, exposing IFP at the N-terminal of S2′, appears to be a reliable signal to the virus that a good target cell is within reach. This is consistent with the finding that a target cell needs to express TMPRSS2 to be infected, but altering expression of TMPRSS2 in the infected cell does not affect the efficiency of infection [41]. If no TMPRSS2 cleaves S2, then viral entry may go through the endosome-cathepsin L pathway in which endocytosis occurs resulting in S2 cleaved into FP and S2′ in endosome to trigger membrane fusion. Further research is needed to substantiate and validate the details.

5.2. The Heptad-Repeat Domains: HR-1 and HR-2

Heptad repeats (HR, Figure 1E) are characterized by repeated 7mers represented as (abcdefg)n with amino acids at positions a and d being hydrophobic. In leucine zipper transcription factors such as GCN4 in yeast [71] and XBP1 in humans [72], the d positions are occupied exclusively by leucine [73]. HRs are relatively poor in glycine (which would permit too much bending flexibility). They form helices, contain no helix-breaking prolines and no clustered charged residues, and are typically located next to hydrophobic fusion peptides in RNA viruses [74]. Hydrophobic residues, at positions a and d, are on the same side of the helix (Figure 1E) and form a hydrophobic interface with other helices. Because SARS-S and SARS-2S are homotrimers, there are three HR1 and three HR2 forming a six-helix bundle [6,68,75]. The six-helix bundle is also observed in SARS-2-S [76]. It has been inferred that helices formed from HRs are perpendicular to the viral membrane [74], which has been substantiated in both SARS-S and MERS-S [6].

Given that a viral HR typically follows an N-terminal hydrophobic region in diverse viral lineages [74], one may infer that such a configuration is favored by natural selection to serve the function of membrane fusion. In this context, the configuration of (FP + IFP + HR1) may not be as favorable as that of (IFP + HR1), the latter resulting from cleavage at the third cleavage site (Figure 2E) to split S2 to FP and S2′. This may explain why the cleavage at this site dramatically enhances membrane fusion and viral entry [3,37,41].

HR1 and HR2 are strongly conserved among SARS-S, SARS-2-S and their relatives (Figure 1E). The isoelectric point along a sliding-window is essentially identical among the six viral strains in regions from HR1 to CT (Figure 3B), in contrast to that for S1 where much scatter is observed. Structural comparisons have revealed conservation of HR1 in multiple coronaviruses [77]. Partly for this reason, antibodies have been developed that target these regions [23,63,78]. Such antibodies typically provide broad protection against multiple viruses [23,63], because sequences in this region are highly conserved. A previously developed pan-coronavirus fusion inhibitor (EK1) against HR1 in SARS-S to inhibit membrane fusion was also found to inhibit membrane fusion during infection by SARS-CoV-2 and MERS-CoV [76]. Thus, drug repurposing of anti-SARS-S drugs for fighting against SARS-CoV-2 should focus on drugs or vaccines targeting highly conserved regions of SARS-S.

Individual helix-forming segments in HR1 and HR2 can bind to each other, which creates an opportunity to use such HR1 and HR2 segments as drugs to disrupt membrane fusion [68,75]. HR2 peptides have been used to inhibit infection by MHV, but this inhibition is less effective against SARS-CoV [68].

The segment between HR1 and HR2 (Figure 3) is the central helix. There is a transitional bend between HR1 and the central helix which, when fixed with two consecutive proline residues, prevents structural transitions from prefusion to postfusion, and consequently contributes to the stabilization of the spike protein at the prefusion state which is important for vaccine development [10,35,79]. Spike proteins with these two proline replacements are known as S-2P. This is discussed further in the section on vaccine development.

5.3. The Transmembrane Domain and Cytoplasmic Tail Domain

The transmembrane (TM) domain of the S protein (Figure 1A) is known to be highly conserved in SARS-CoV-2 and its close relatives [69]. This conservation is also reflected in the hydrophobicity profile and pI profile among SARS-CoV-2 and its close relatives (Figure 3). The TM domain consists of the following three parts [69,80]: a juxtamembrane aromatic part, a central hydrophobic part, and a cysteine-rich part (Figure 4). It is followed by a highly hydrophilic cytoplasmic tail (CT) which anchors the spike inside the viral membrane.

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Figure 4

Transmembrane (TM) domain with its tripartite structure (juxtamembrane aromatic part in blue, central hydrophobic part in pink, and cysteine-rich part in purple) and the cytoplasmic tail that anchors inside the viral membrane.

The tryptophan residues in the aromatic part are strongly conserved among SARS-CoV-2 and related coronaviruses, suggesting their functional importance. Replacing them even by another aromatic residue such as phenylalanine will severely impact the efficiency of viral infection [80]. However, this finding was not supported in another study [81] in which replacing tryptophan by phenylalanine was tolerated.

The central hydrophobic part forms a helix. Because S proteins form a homotrimer, there are three transmembrane helices interacting with each other. The TM and the C-terminus contribute to the stabilization of the trimeric structure [19,24,69] which is important for membrane fusion. Destabilization of the trimeric structure is associated with reduced fusogenicity and infectivity [69]. Replacing hydrophobic residues in the central part by hydrophilic ones such as lysine decreases the efficiency of an infection [80]. Cysteine residues immediately proximal to the membrane (near the central hydrophobic part in Figure 4) are palmitoylated; replacing them by other amino acids (e.g., alanine) inhibits membrane fusion [82]. In contrast, replacing cysteine residues in the last half of the cysteine-rich part or even deleting them does not inhibit membrane fusion [82,83].

During the cell-to-cell infection stage, the membrane-proximal cysteine-rich part, and the cytoplasmic tail anchor the C-terminus of S inside the infected cell, and the N-terminal of S2 (or S2′) penetrates the membrane of a target cell and anchor the N-terminus inside, which is typical of viral fusion proteins [67]. The conformational changes of S2 (or S2′), including the tripartite TM, help to bring the membranes of infected and target cells close together to facilitate cell-cell membrane fusion and viral entry [2,34,80,84]. The anchor provided by the cysteine-rich part and CT is enhanced by the membrane-actin linker ezrin [84] which, upon phosphorylation, links specific transmembrane proteins such as S homotrimer to actin to reinforce the anchor inside the cell.Go to:

6. The Spike Protein in Vaccine Development

Almost all vaccine candidates against SARS-CoV-2 are based on the spike protein, including the FDA-approved Pfizer/BioNTech and Moderna vaccines that use mRNA encoding a modified spike protein stabilized in its prefusion conformation. It is important for the immune system to respond to the virus at the prefusion stage, because it would probably be too late for the immune system to intervene at the postfusion stage when the virus is gaining entry into an uninfected cell. Therefore, the rationale of vaccine development is to produce a spike protein stabilized in the prefusion conformation as a target to train the immune system to act against it.

Two structural studies on spike proteins, one on Betacoronavirus HKU1 [10] and the other on MERS-CoV [79], have demonstrated that replacing two consecutive amino acids by proline near the transition from HR1 to the central helix (Figure 3) would strongly contribute to the stabilization of the resulting spike protein at the prefusion conformation. These amino acid sites correspond to sites 986 and 987 in SARS-2-S (Figure 5), located at the transitional bend between HR1 and the central helix (Figure 3). Amino acids at two sites are not conserved, being NL in CoV-HKU1, VL in MERS-CoV, and KV in SARS [79], suggesting that they are probably not functionally important. However, the two amino acid replacements (K986P, V987P), shown in Figure 5, stabilize the resulting spike protein in the prefusion state and contribute to vaccine efficiency. The mutant SARS-2-S spike protein with these proline replacements is referred to as S-2P [85,86], which is encoded in the mRNA vaccine from both Pfizer/BioNTech (BNT162b2) and Moderna (mRNA-1273). A new spike protein variant (HexaPro) that includes four additional amino acid replacements by proline (F817P, A892P, A899P, and A942P) is even more stable and expressed more than the original S-2P [35].

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Figure 5

Two amino acid replacements that stabilize the spike protein at the prefusion state. (A) Amino acids KY in the native state of SARS-2-S is replaced by PP spike variant S-2P used in the FDA-approved Pfizer/BioNTech and Moderna vaccine; (B) Partial structure from 6VSB showing the two proline residues stabilizing the structural bend.Go to:

7. Structural Insights into the Emergence of New Viral Variants

Here, one example is described to illustrate how structural biology can shed light on the emergence of new viral variants. In an experiment that used neutralizing monoclonal antibodies to select neutralization-escaping SARS-CoV variants [22], one of the four variants was V601G within SARS-S at 594VAVLYQDVNCTDV606 where V601 was highlighted in bold. The identification of this infection-enhancing V601G variant is puzzling because one does not expect that such a V→G replacement would have much phenotypic effect on the S protein. First, site 601 is not involved in receptor binding. Second, both V and G are small and nonpolar. Therefore the replacement is conservative and should not cause a significant structural perturbation of the S protein. Does a replacement of a small nonpolar V by a smaller nonpolar G really matter? One cannot answer the question without structural evidence. It can only be inferred that site 601 is functionally important, and that the smallest amino acid at site 601 (or its vicinity) is beneficial to SARS-CoV.

A V601G mutation requires a transversion (i.e., from codon GUN to GGN). Because of proofreading in coronavirus genome replication [87,88,89], transversional mutations are much rarer than transitions. For this reason, V→G at site 601 is expected to occur much more frequently than D→G at site 600, because the latter requires a transition (from codon GAY to GGY) instead of a transversion. Therefore, a small G can be gained by a D600G mutation instead of a V601G mutation. The segment of 594VAVLYQDVNCTDV606 in SARS-S corresponds to 608VAVLYQDVNCTEV620 in SARS-2-S, therefore, a D600G mutation in SARS-S is equivalent to D614G in SARS-2-S. In this context, it is not surprising that a D614G variant of SARS-CoV-2 quickly increased in frequency [90], indicating a strong selective advantage.

Now, there are two alternative hypotheses concerning the selective advantage of the D614G mutation as follows: (1) the benefit is due to G being the smallest amino acid, or (2) the benefit is due to the loss of a negative charge altering electrostatic interactions. The second hypothesis may be dismissed on the following empirical grounds: Codons encoding D (GAY) could also mutate to AAY encoding N through a single transition. Such a mutation would lose the negative charge carried by D. If it is the loss of a negative charge that is beneficial, we would expect AAY and GGY to be roughly equally represented at this site. However, AAY is entirely missing in sequenced SARS-2-S, which goes against the second hypothesis. Unfortunately, exclusion of the second hypothesis neither implies confirmation of the first (because there are other alternatives), nor helps us understand why the D614G mutation enhances viral fitness. Only through structural studies [91] can we hope to gain a mechanistic understanding of the effect of the D614G mutation on the S protein.Go to:

8. The Spike Protein and the Conspiracy Theory

As previously mentioned, the additional polybasic furin cleavage site 1 (Figure 2A) has been a lasting source of conspiracy theory that SARS-CoV-2 is man-made. Advocates of the conspiracy theory assume that scientists have ignored or refused to address their legit concerns. In this review, two points are made. First, the evidence for a natural origin of SARS-CoV-2 is accumulating, albeit at a rate slower than desired. Second, the reasons behind the conspiracy theory have been seriously considered by scientists and have been deemed to be not strong reasons.

There are three main reasons for the conspiracy theory, all involving the polybasic furin cleavage site (Figure 2A). First, the furin cleavage site has not been observed in any close relatives of SARS-CoV-2 in nature. A somewhat similar furin cleavage site was present at a roughly homologous site in S protein of the murine hepatitis virus [45] and in a few alphacoronaviruses [2,8,29]. However, it is not clear how SARS-CoV-2 could gain it from these remote relatives. While recombination might be a possibility, there is hardly any sequence homology between SARS-2-S and its homologues in the murine hepatitis virus or alphacoronaviruses at sequences flanking the cleavage site, therefore, a recombination origin of the cleavage site is tenuous at present. An insertion at the same site was found in a bat-derived coronavirus [92], but the inserted sequence was different and could not function as a furin cleavage site. A novel bat-derived coronavirus (RmYN02) was reported to have an insertion bearing a weak semblance to the polybasic furin cleavage site in Figure 2A [92], suggesting the possibility of a natural origin of the polybasic furin cleavage site. However, the sequence homology between RmYN02 and SARS-2-S is low, and it is not clear if the insertion in RmYN02 is real or an artefact of alignment. Therefore, if one cannot offer a plausible hypothesis of natural origin of the polybasic site, it is easy to fall back on the hypothesis of artificial origin. This reminds us of the period of time before Darwin, i.e., when the origin of species cannot be fully explained, it is easy to fall back to the theory of a creator.

The second reason for the conspiracy theory is associated with the feasibility of creating such a polybasic site and a need to create such a site for testing certain biological hypotheses. Some background information arising from SARS-S is needed to understand this reason. The roughly homologous RNA segment in SARS-S is a weak cleavage site, likely cleaved by transmembrane serine protease TMPRSS2 [93]. R667 in SARS-S (immediately upstream of the site 1 cleavage in Figure 2A) is required for cleavage by TMPRSS2 [93]. The site can also be cleaved by trypsin, and processing of SARS-S by trypsin enhances viral infectivity [34,45,94]. Because trypsin and trypsin-like proteases are strongly tissue restricted (Figure 2C), the site is typically not cleaved in SARS-S [24]. It is natural for one to hypothesize that adding a furin cleavage site would allow the site to be efficiently cleaved in nearly all tissues, potentially enhancing SARS-CoV infection and broadening its cell tropism. Indeed, introducing a furin cleavage site at the S1 and S2 boundary of SARS-S has increased cell-cell fusion (syncytium formation) and viral infectivity [34]. This result suggests that the additional polybasic furin cleavage site may have contributed significantly to the efficiency of SARS-CoV-2 in infecting human. Host cells, in response to viral infection, may reduce furin activities [8].

In short, given the seemingly sudden appearance of the additional furin cleavage site that cannot be readily explained by a hypothesis of natural origin, and the fact that virologists have already experimented with adding a furin cleavage site at this specific location and learned the consequence of enhanced viral infectivity and cell-cell fusion, the claim that the polybasic furin cleavage site in SARS-2-S has been experimentally inserted is not too far-fetched. However, the global collaboration among scientists, in general, and virologists, in particular, has created scientific communities that are far more closely knit than before. While it is possible to create a viral pathogen, it is extremely unlikely for a laboratory to create SARS-CoV-2 without being noticed.

The third reason is that the 12 nt insertion encoding the polybasic furin cleavage site carries two CpG dinucleotides. Such CpG dinucleotides are very rare in SARS-CoV-2 [95], and particularly rare in SARS-2-S. Why would such CpG rarity contribute to the conspiracy theory? Mammalian zinc finger antiviral protein (ZAP, gene name ZC3HAC1) targets CpG dinucleotides in viral RNA to mediate RNA degradation and inhibit viral replication [96]. The ZAP-mediated RNA degradation is cumulative [96], as shown by the following experiment. When CpG dinucleotides were experimentally added to individual viral segment 1 or 2, the inhibitory effect of ZAP was weak. However, when the same CpG dinucleotides were added to both segments 1 and 2, the ZAP inhibition effect was strong [96]. This implies that only mRNA sequences of sufficient length would be targeted by ZAP (i.e., S1ab, and 1a mRNAs in SARS-CoV and SARS-CoV-2). SARS-CoV-2 and its closest relatives from bat (RaTG13) and pangolin exhibit the strongest genomic CpG deficiency among all betacoronaviruses [95], presumably to evade ZAP-mediated host defense. The S gene is particularly CpG-deficient as measured by two indices, ICpG [95,97] and ln (NCG/NGC) (Table 1), where NCG and NGC are the numbers of CpG and GpC dinucleotides in the S gene. ICpG < 1, or ln (NCG/NGC) < 0, means CpG deficiency.

Table 1

Genomic CpG deficiency in the coding sequence encoding the spike proteins, measured by two indices: ICpG = (PC*PG/PCG) and ln (NCG/NGC). The expectation of no CpG deficiency is 1 for ICpG and 0 for ln (NCG/NGC).

Sequence NameLengthICpGNCGNGCLn (NCG/NGC)
NC_045512_SARS-CoV-238190.217929137−1.5527
MN996532_Bat_RaTG1338070.275337140−1.3307
pangolin/EPI_ISL_410721/201937950.285738139−1.2969
MG772933_Bat_SARS-like37380.361850148−1.0852
MG772934_Bat_SARS-like37350.369750142−1.0438
NC_004718_SARS37650.367352174−1.2078

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Because of this ZAP-mediated selection against CpG, SARS-CoV-2 and its close relatives encode most of arginine residues by the two AGR codons, instead of the four CGN codons. The S gene encodes 42 arginine residues, with only 12 (28.57%) encoded by the four CGN codons in contrast to 30 encoded by the two AGR codons. The two arginine residues in the polybasic furin cleavage site are encoded by the rare CGN codons, which seems unnatural in this context. However, the probability of randomly picking up two arginine codons that happen to be both CGN codons is not extremely low (i.e. =0.28572 = 0.0816).

One way to dispel the conspiracy theory is to find a set of viral lineages in wildlife that would allow reconstruction of a plausible evolutionary path leading to the origin of the polybasic furin cleavage site. The “missing link” that would satisfy conspiracy theorists is still to be found. However, there is no guarantee that it will be found because nature is not obliged to preserve all what she has created.Go to:

9. Conclusions

In summary, although much is known about the S protein in coronaviruses, the temporal and spatial changes of S during synthesis, glycosylation, cleavage, membrane fusion, and viral entry remain poorly defined. It is also important to keep in mind that the S-mediated cell entry is only one step in the viral infection cycle and naturally cannot explain all differences in virulence among betacoronaviruses. For example, MERS viruses found in Africa exhibit reduced replicative capability and are typically not pathogenic relative to the prototypic and highly pathogenic Arabian MERS-CoV strain. However, the two are not different in their efficiency in gaining host cell entry [98], pointing to differences in other parts of the viruses that may contribute to their differences in pathogenicity.Go to:

Acknowledgments

The author thanks D. Gray, J. Mennigen, Y. Wei, and Z. Xie for discussion.Go to:

Funding

This research was funded by a Discovery Grant from the Natural Science and Engineering Research Council (NSERC, RGPIN/2018-03878) of Canada. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.Go to:

Institutional Review Board Statement

Not applicable.Go to:

Informed Consent Statement

Not applicable.Go to:

Data Availability Statement

Not applicable.Go to:

Conflicts of Interest

The author declares no conflict of interest.Go to:

Footnotes

Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.Go to:

References

1. Zhou P., Yang X.-L., Wang X.-G., Hu B., Zhang L., Zhang W., Si H.-R., Zhu Y., Li B., Huang C.-L., et al. A pneumonia outbreak associated with a new coronavirus of probable bat origin. Nature. 2020;579:270–273. doi: 10.1038/s41586-020-2012-7. [PMC free article] [PubMed] [CrossRef] [Google Scholar]2. Hoffmann M., Kleine-Weber H., Pöhlmann S. A Multibasic Cleavage Site in the Spike Protein of SARS-CoV-2 Is Essential for Infection of Human Lung Cells. Mol. Cell. 2020;78:779–784.e775. doi: 10.1016/j.molcel.2020.04.022. [PMC free article] [PubMed] [CrossRef] [Google Scholar]3. Hoffmann M., Kleine-Weber H., Schroeder S., Krüger N., Herrler T., Erichsen S., Schiergens T.S., Herrler G., Wu N.H., Nitsche A., et al. SARS-CoV-2 Cell Entry Depends on ACE2 and TMPRSS2 and Is Blocked by a Clinically Proven Protease Inhibitor. Cell. 2020;181:271–280.e278. [PMC free article] [PubMed] [Google Scholar]4. Ou X., Liu Y., Lei X., Li P., Mi D., Ren L., Guo L., Guo R., Chen T., Hu J., et al. Characterization of spike glycoprotein of SARS-CoV-2 on virus entry and its immune cross-reactivity with SARS-CoV. Nat. Commun. 2020;11:1620. [PMC free article] [PubMed] [Google Scholar]5. Li F., Li W., Farzan M., Harrison S.C. Structure of SARS coronavirus spike receptor-binding domain complexed with receptor. Science. 2005;309:1864–1868. doi: 10.1126/science.1116480. [PubMed] [CrossRef] [Google Scholar]6. Lu G., Wang Q., Gao G.F. Bat-to-human: Spike features determining ‘host jump’ of coronaviruses SARS-CoV, MERS-CoV, and beyond. Trends Microbiol. 2015;23:468–478. doi: 10.1016/j.tim.2015.06.003. [PMC free article] [PubMed] [CrossRef] [Google Scholar]7. Hulswit R.J.G., de Haan C.A.M., Bosch B.J. Coronavirus Spike Protein and Tropism Changes. In: Ziebuhr J., editor. Advances in Virus Research. Volume 96. Academic Press; Cambridge, MA, USA: 2016. pp. 29–57. [PMC free article] [PubMed] [Google Scholar]8. Coutard B., Valle C., de Lamballerie X., Canard B., Seidah N.G., Decroly E. The spike glycoprotein of the new coronavirus 2019-nCoV contains a furin-like cleavage site absent in CoV of the same clade. Antivir. Res. 2020;176:104742. [PMC free article] [PubMed] [Google Scholar]9. Walls A.C., Tortorici M.A., Bosch B.-J., Frenz B., Rottier P.J.M., DiMaio F., Rey F.A., Veesler D. Cryo-electron microscopy structure of a coronavirus spike glycoprotein trimer. Nature. 2016;531:114–117. doi: 10.1038/nature16988. [PMC free article] [PubMed] [CrossRef] [Google Scholar]10. Kirchdoerfer R.N., Cottrell C.A., Wang N., Pallesen J., Yassine H.M., Turner H.L., Corbett K.S., Graham B.S., McLellan J.S., Ward A.B. Pre-fusion structure of a human coronavirus spike protein. Nature. 2016;531:118–121. doi: 10.1038/nature17200. [PMC free article] [PubMed] [CrossRef] [Google Scholar]11. Walls A.C., Park Y.J., Tortorici M.A., Wall A., McGuire A.T., Veesler D. Structure, Function, and Antigenicity of the SARS-CoV-2 Spike Glycoprotein. Cell. 2020;181:281–292.e286. doi: 10.1016/j.cell.2020.02.058. [PMC free article] [PubMed] [CrossRef] [Google Scholar]12. Wrapp D., Wang N., Corbett K.S., Goldsmith J.A., Hsieh C.-L., Abiona O., Graham B.S., McLellan J.S. Cryo-EM structure of the 2019-nCoV spike in the prefusion conformation. Science. 2020;367:1260. doi: 10.1126/science.abb2507. [PMC free article] [PubMed] [CrossRef] [Google Scholar]13. Lan J., Ge J., Yu J., Shan S., Zhou H., Fan S., Zhang Q., Shi X., Wang Q., Zhang L., et al. Structure of the SARS-CoV-2 spike receptor-binding domain bound to the ACE2 receptor. Nature. 2020;581:215–220. doi: 10.1038/s41586-020-2180-5. [PubMed] [CrossRef] [Google Scholar]14. Shang J., Ye G., Shi K., Wan Y., Luo C., Aihara H., Geng Q., Auerbach A., Li F. Structural basis of receptor recognition by SARS-CoV-2. Nature. 2020;581:221–224. doi: 10.1038/s41586-020-2179-y. [PMC free article] [PubMed] [CrossRef] [Google Scholar]15. Tian X., Li C., Huang A., Xia S., Lu S., Shi Z., Lu L., Jiang S., Yang Z., Wu Y., et al. Potent binding of 2019 novel coronavirus spike protein by a SARS coronavirus-specific human monoclonal antibody. Emerg. Microb. Infect. 2020;9:382–385. doi: 10.1080/22221751.2020.1729069. [PMC free article] [PubMed] [CrossRef] [Google Scholar]16. Xia X. DAMBE5: A comprehensive software package for data analysis in molecular biology and evolution. Mol. Biol. Evol. 2013;30:1720–1728. doi: 10.1093/molbev/mst064. [PMC free article] [PubMed] [CrossRef] [Google Scholar]17. Lai M.M., Cavanagh D. The molecular biology of coronaviruses. Adv. Virus Res. 1997;48:1–100. [PMC free article] [PubMed] [Google Scholar]18. Chakraborti S., Prabakaran P., Xiao X., Dimitrov D.S. The SARS coronavirus S glycoprotein receptor binding domain: Fine mapping and functional characterization. Virol. J. 2005;2:73. doi: 10.1186/1743-422X-2-73. [PMC free article] [PubMed] [CrossRef] [Google Scholar]19. Xiao X., Feng Y., Chakraborti S., Dimitrov D.S. Oligomerization of the SARS-CoV S glycoprotein: Dimerization of the N-terminus and trimerization of the ectodomain. Biochem. Biophys. Res. Commun. 2004;322:93–99. doi: 10.1016/j.bbrc.2004.07.084. [PMC free article] [PubMed] [CrossRef] [Google Scholar]20. Beniac D.R., deVarennes S.L., Andonov A., He R., Booth T.F. Conformational reorganization of the SARS coronavirus spike following receptor binding: Implications for membrane fusion. PLoS ONE. 2007;2:e1082. doi: 10.1371/journal.pone.0001082. [PMC free article] [PubMed] [CrossRef] [Google Scholar]21. Madu I.G., Belouzard S., Whittaker G.R. SARS-coronavirus spike S2 domain flanked by cysteine residues C822 and C833 is important for activation of membrane fusion. Virology. 2009;393:265–271. doi: 10.1016/j.virol.2009.07.038. [PMC free article] [PubMed] [CrossRef] [Google Scholar]22. Mitsuki Y.Y., Ohnishi K., Takagi H., Oshima M., Yamamoto T., Mizukoshi F., Terahara K., Kobayashi K., Yamamoto N., Yamaoka S., et al. A single amino acid substitution in the S1 and S2 Spike protein domains determines the neutralization escape phenotype of SARS-CoV. Microbes Infect. 2008;10:908–915. doi: 10.1016/j.micinf.2008.05.009. [PMC free article] [PubMed] [CrossRef] [Google Scholar]23. Ng O.W., Keng C.T., Leung C.S., Peiris J.S., Poon L.L., Tan Y.J. Substitution at aspartic acid 1128 in the SARS coronavirus spike glycoprotein mediates escape from a S2 domain-targeting neutralizing monoclonal antibody. PLoS ONE. 2014;9:e102415. doi: 10.1371/journal.pone.0102415. [PMC free article] [PubMed] [CrossRef] [Google Scholar]24. Song H.C., Seo M.Y., Stadler K., Yoo B.J., Choo Q.L., Coates S.R., Uematsu Y., Harada T., Greer C.E., Polo J.M., et al. Synthesis and characterization of a native, oligomeric form of recombinant severe acute respiratory syndrome coronavirus spike glycoprotein. J. Virol. 2004;78:10328–10335. doi: 10.1128/JVI.78.19.10328-10335.2004. [PMC free article] [PubMed] [CrossRef] [Google Scholar]25. Jin D.Y., Zheng B.J. Roles of spike protein in the pathogenesis of SARS coronavirus. Hong Kong Med. J. 2009;15:37–40. [PubMed] [Google Scholar]26. Xia X. Translation Control of HAC1 by Regulation of Splicing in Saccharomyces cerevisiaeInt. J. Mol. Sci. 2019;20:2860. doi: 10.3390/ijms20122860. [PMC free article] [PubMed] [CrossRef] [Google Scholar]27. Chow K.Y., Yeung Y.S., Hon C.C., Zeng F., Law K.M., Leung F.C. Adenovirus-mediated expression of the C-terminal domain of SARS-CoV spike protein is sufficient to induce apoptosis in Vero E6 cells. FEBS Lett. 2005;579:6699–6704. doi: 10.1016/j.febslet.2005.10.065. [PMC free article] [PubMed] [CrossRef] [Google Scholar]28. Shajahan A., Supekar N.T., Gleinich A.S., Azadi P. Deducing the N- and O- glycosylation profile of the spike protein of novel coronavirus SARS-CoV-2. Glycobiology. 2020;30:981–988. doi: 10.1093/glycob/cwaa042. [PMC free article] [PubMed] [CrossRef] [Google Scholar]29. Andersen K.G., Rambaut A., Lipkin W.I., Holmes E.C., Garry R.F. The proximal origin of SARS-CoV-2. Nat. Med. 2020;26:450–452. doi: 10.1038/s41591-020-0820-9. [PMC free article] [PubMed] [CrossRef] [Google Scholar]30. Fagerberg L., Hallström B.M., Oksvold P., Kampf C., Djureinovic D., Odeberg J., Habuka M., Tahmasebpoor S., Danielsson A., Edlund K., et al. Analysis of the human tissue-specific expression by genome-wide integration of transcriptomics and antibody-based proteomics. Mol. Cell. Proteom. 2014;13:397–406. doi: 10.1074/mcp.M113.035600. [PMC free article] [PubMed] [CrossRef] [Google Scholar]31. Sun X., Tse L.V., Ferguson A.D., Whittaker G.R. Modifications to the Hemagglutinin Cleavage Site Control the Virulence of a Neurotropic H1N1 Influenza Virus. J. Virol. 2010;84:8683. doi: 10.1128/JVI.00797-10. [PMC free article] [PubMed] [CrossRef] [Google Scholar]32. Kido H., Okumura Y., Takahashi E., Pan H.-Y., Wang S., Yao D., Yao M., Chida J., Yano M. Role of host cellular proteases in the pathogenesis of influenza and influenza-induced multiple organ failure. Biochim. Biophys. Acta Proteins Proteom. 2012;1824:186–194. doi: 10.1016/j.bbapap.2011.07.001. [PubMed] [CrossRef] [Google Scholar]33. Wei Y., Silke J.R., Aris P., Xia X. Coronavirus genomes carry the signatures of their habitats. PLoS ONE. 2020;15:e0244025. [PMC free article] [PubMed] [Google Scholar]34. Belouzard S., Chu V.C., Whittaker G.R. Activation of the SARS coronavirus spike protein via sequential proteolytic cleavage at two distinct sites. Proc. Natl. Acad. Sci. USA. 2009;106:5871–5876. doi: 10.1073/pnas.0809524106. [PMC free article] [PubMed] [CrossRef] [Google Scholar]35. Hsieh C.-L., Goldsmith J.A., Schaub J.M., DiVenere A.M., Kuo H.-C., Javanmardi K., Le K.C., Wrapp D., Lee A.G., Liu Y., et al. Structure-based design of prefusion-stabilized SARS-CoV-2 spikes. Science. 2020;369:1501. doi: 10.1126/science.abd0826. [PMC free article] [PubMed] [CrossRef] [Google Scholar]36. Simmons G., Gosalia D.N., Rennekamp A.J., Reeves J.D., Diamond S.L., Bates P. Inhibitors of cathepsin L prevent severe acute respiratory syndrome coronavirus entry. Proc. Natl. Acad. Sci. USA. 2005;102:11876. doi: 10.1073/pnas.0505577102. [PMC free article] [PubMed] [CrossRef] [Google Scholar]37. Bosch B.J., Bartelink W., Rottier P.J.M. Cathepsin L Functionally Cleaves the Severe Acute Respiratory Syndrome Coronavirus Class I Fusion Protein Upstream of Rather than Adjacent to the Fusion Peptide. J. Virol. 2008;82:8887. doi: 10.1128/JVI.00415-08. [PMC free article] [PubMed] [CrossRef] [Google Scholar]38. Burkard C., Verheije M.H., Wicht O., van Kasteren S.I., van Kuppeveld F.J., Haagmans B.L., Pelkmans L., Rottier P.J., Bosch B.J., de Haan C.A. Coronavirus cell entry occurs through the endo-/lysosomal pathway in a proteolysis-dependent manner. PLoS Pathog. 2014;10:e1004502. doi: 10.1371/journal.ppat.1004502. [PMC free article] [PubMed] [CrossRef] [Google Scholar]39. Kirschke H. Chapter 410—Cathepsin L. In: Rawlings N.D., Salvesen G., editors. Handbook of Proteolytic Enzymes. 3rd ed. Academic Press; Cambridge, MA, USA: 2013. pp. 1808–1817. [Google Scholar]40. Jaimes J.A., André N.M., Chappie J.S., Millet J.K., Whittaker G.R. Phylogenetic Analysis and Structural Modeling of SARS-CoV-2 Spike Protein Reveals an Evolutionary Distinct and Proteolytically Sensitive Activation Loop. J. Mol. Biol. 2020;432:3309–3325. doi: 10.1016/j.jmb.2020.04.009. [PMC free article] [PubMed] [CrossRef] [Google Scholar]41. Matsuyama S., Nagata N., Shirato K., Kawase M., Takeda M., Taguchi F. Efficient Activation of the Severe Acute Respiratory Syndrome Coronavirus Spike Protein by the Transmembrane Protease TMPRSS2. J. Virol. 2010;84:12658. doi: 10.1128/JVI.01542-10. [PMC free article] [PubMed] [CrossRef] [Google Scholar]42. Hoffmann M., Hofmann-Winkler H., Pöhlmann S. Priming Time: How Cellular Proteases Arm Coronavirus Spike Proteins. In: Böttcher-Friebertshäuser E., Garten W., Klenk H.D., editors. Activation of Viruses by Host Proteases. Springer International Publishing; Cham, Switzerland: 2018. pp. 71–98. [Google Scholar]43. Glowacka I., Bertram S., Müller M.A., Allen P., Soilleux E., Pfefferle S., Steffen I., Tsegaye T.S., He Y., Gnirss K., et al. Evidence that TMPRSS2 activates the severe acute respiratory syndrome coronavirus spike protein for membrane fusion and reduces viral control by the humoral immune response. J. Virol. 2011;85:4122–4134. doi: 10.1128/JVI.02232-10. [PMC free article] [PubMed] [CrossRef] [Google Scholar]44. Kleine-Weber H., Elzayat M.T., Hoffmann M., Pöhlmann S. Functional analysis of potential cleavage sites in the MERS-coronavirus spike protein. Sci. Rep. 2018;8:16597. doi: 10.1038/s41598-018-34859-w. [PMC free article] [PubMed] [CrossRef] [Google Scholar]45. Simmons G., Reeves J.D., Rennekamp A.J., Amberg S.M., Piefer A.J., Bates P. Characterization of severe acute respiratory syndrome-associated coronavirus (SARS-CoV) spike glycoprotein-mediated viral entry. Proc. Natl. Acad. Sci. USA. 2004;101:4240–4245. doi: 10.1073/pnas.0306446101. [PMC free article] [PubMed] [CrossRef] [Google Scholar]46. Matsuyama S., Ujike M., Morikawa S., Tashiro M., Taguchi F. Protease-mediated enhancement of severe acute respiratory syndrome coronavirus infection. Proc. Natl. Acad. Sci. USA. 2005;102:12543. doi: 10.1073/pnas.0503203102. [PMC free article] [PubMed] [CrossRef] [Google Scholar]47. Peng G., Sun D., Rajashankar K.R., Qian Z., Holmes K.V., Li F. Crystal structure of mouse coronavirus receptor-binding domain complexed with its murine receptor. Proc. Natl. Acad. Sci. USA. 2011;108:10696. doi: 10.1073/pnas.1104306108. [PMC free article] [PubMed] [CrossRef] [Google Scholar]48. Williams R.K., Jiang G.S., Holmes K.V. Receptor for mouse hepatitis virus is a member of the carcinoembryonic antigen family of glycoproteins. Proc. Natl. Acad. Sci. USA. 1991;88:5533. doi: 10.1073/pnas.88.13.5533. [PMC free article] [PubMed] [CrossRef] [Google Scholar]49. Xia X. DAMBE7: New and improved tools for data analysis in molecular biology and evolution. Mol. Biol. Evol. 2018;35:1550–1552. doi: 10.1093/molbev/msy073. [PMC free article] [PubMed] [CrossRef] [Google Scholar]50. Gui M., Song W., Zhou H., Xu J., Chen S., Xiang Y., Wang X. Cryo-electron microscopy structures of the SARS-CoV spike glycoprotein reveal a prerequisite conformational state for receptor binding. Cell Res. 2017;27:119–129. doi: 10.1038/cr.2016.152. [PMC free article] [PubMed] [CrossRef] [Google Scholar]51. Zakhartchouk A.N., Sharon C., Satkunarajah M., Auperin T., Viswanathan S., Mutwiri G., Petric M., See R.H., Brunham R.C., Finlay B.B., et al. Immunogenicity of a receptor-binding domain of SARS coronavirus spike protein in mice: Implications for a subunit vaccine. Vaccine. 2007;25:136–143. doi: 10.1016/j.vaccine.2006.06.084. [PMC free article] [PubMed] [CrossRef] [Google Scholar]52. He Y., Zhou Y., Liu S., Kou Z., Li W., Farzan M., Jiang S. Receptor-binding domain of SARS-CoV spike protein induces highly potent neutralizing antibodies: Implication for developing subunit vaccine. Biochem. Biophys. Res. Commun. 2004;324:773–781. doi: 10.1016/j.bbrc.2004.09.106. [PMC free article] [PubMed] [CrossRef] [Google Scholar]53. He Y., Zhou Y., Siddiqui P., Jiang S. Inactivated SARS-CoV vaccine elicits high titers of spike protein-specific antibodies that block receptor binding and virus entry. Biochem. Biophys. Res. Commun. 2004;325:445–452. doi: 10.1016/j.bbrc.2004.10.052. [PMC free article] [PubMed] [CrossRef] [Google Scholar]54. Du L., Zhao G., Chan C.C., Sun S., Chen M., Liu Z., Guo H., He Y., Zhou Y., Zheng B.J., et al. Recombinant receptor-binding domain of SARS-CoV spike protein expressed in mammalian, insect and E. coli cells elicits potent neutralizing antibody and protective immunity. Virology. 2009;393:144–150. doi: 10.1016/j.virol.2009.07.018. [PMC free article] [PubMed] [CrossRef] [Google Scholar]55. Du L., Zhao G., He Y., Guo Y., Zheng B.J., Jiang S., Zhou Y. Receptor-binding domain of SARS-CoV spike protein induces long-term protective immunity in an animal model. Vaccine. 2007;25:2832–2838. doi: 10.1016/j.vaccine.2006.10.031. [PMC free article] [PubMed] [CrossRef] [Google Scholar]56. Du L., Zhao G., Lin Y., Sui H., Chan C., Ma S., He Y., Jiang S., Wu C., Yuen K.Y., et al. Intranasal vaccination of recombinant adeno-associated virus encoding receptor-binding domain of severe acute respiratory syndrome coronavirus (SARS-CoV) spike protein induces strong mucosal immune responses and provides long-term protection against SARS-CoV infection. J. Immunol. 2008;180:948–956. [PMC free article] [PubMed] [Google Scholar]57. Zhang X., Wang J., Wen K., Mou Z., Zou L., Che X., Ni B., Wu Y. Antibody binding site mapping of SARS-CoV spike protein receptor-binding domain by a combination of yeast surface display and phage peptide library screening. Viral Immunol. 2009;22:407–415. [PubMed] [Google Scholar]58. Cao Z., Liu L., Du L., Zhang C., Jiang S., Li T., He Y. Potent and persistent antibody responses against the receptor-binding domain of SARS-CoV spike protein in recovered patients. Virol. J. 2010;7:299. [PMC free article] [PubMed] [Google Scholar]59. Prabakaran P., Gan J., Feng Y., Zhu Z., Choudhry V., Xiao X., Ji X., Dimitrov D.S. Structure of severe acute respiratory syndrome coronavirus receptor-binding domain complexed with neutralizing antibody. J. Biol. Chem. 2006;281:15829–15836. doi: 10.1074/jbc.M600697200. [PMC free article] [PubMed] [CrossRef] [Google Scholar]60. Chen W.H., Du L., Chag S.M., Ma C., Tricoche N., Tao X., Seid C.A., Hudspeth E.M., Lustigman S., Tseng C.T., et al. Yeast-expressed recombinant protein of the receptor-binding domain in SARS-CoV spike protein with deglycosylated forms as a SARS vaccine candidate. Hum. Vaccines Immunother. 2014;10:648–658. [PMC free article] [PubMed] [Google Scholar]61. Du L., Ma C., Jiang S. Antibodies induced by receptor-binding domain in spike protein of SARS-CoV do not cross-neutralize the novel human coronavirus hCoV-EMC. J. Infect. 2013;67:348–350. [PMC free article] [PubMed] [Google Scholar]62. Zhu Z., Chakraborti S., He Y., Roberts A., Sheahan T., Xiao X., Hensley L.E., Prabakaran P., Rockx B., Sidorov I.A., et al. Potent cross-reactive neutralization of SARS coronavirus isolates by human monoclonal antibodies. Proc. Natl. Acad. Sci. USA. 2007;104:12123–12128. [PMC free article] [PubMed] [Google Scholar]63. Elshabrawy H.A., Coughlin M.M., Baker S.C., Prabhakar B.S. Human monoclonal antibodies against highly conserved HR1 and HR2 domains of the SARS-CoV spike protein are more broadly neutralizing. PLoS ONE. 2012;7:e50366. [PMC free article] [PubMed] [Google Scholar]64. He Y., Li J., Jiang S. A single amino acid substitution (R441A) in the receptor-binding domain of SARS coronavirus spike protein disrupts the antigenic structure and binding activity. Biochem. Biophys. Res. Commun. 2006;344:106–113. doi: 10.1016/j.bbrc.2006.03.139. [PMC free article] [PubMed] [CrossRef] [Google Scholar]65. Poh W.P., Narasaraju T., Pereira N.A., Zhong F., Phoon M.C., Macary P.A., Wong S.H., Lu J., Koh D.R., Chow V.T. Characterization of cytotoxic T-lymphocyte epitopes and immune responses to SARS coronavirus spike DNA vaccine expressing the RGD-integrin-binding motif. J. Med. Virol. 2009;81:1131–1139. doi: 10.1002/jmv.21571. [PMC free article] [PubMed] [CrossRef] [Google Scholar]66. Lin Y.S., Lin C.F., Fang Y.T., Kuo Y.M., Liao P.C., Yeh T.M., Hwa K.Y., Shieh C.C., Yen J.H., Wang H.J., et al. Antibody to severe acute respiratory syndrome (SARS)-associated coronavirus spike protein domain 2 cross-reacts with lung epithelial cells and causes cytotoxicity. Clin. Exp. Immunol. 2005;141:500–508. doi: 10.1111/j.1365-2249.2005.02864.x. [PMC free article] [PubMed] [CrossRef] [Google Scholar]67. White J.M., Delos S.E., Brecher M., Schornberg K. Structures and mechanisms of viral membrane fusion proteins: Multiple variations on a common theme. Crit. Rev. Biochem. Mol. Biol. 2008;43:189–219. doi: 10.1080/10409230802058320. [PMC free article] [PubMed] [CrossRef] [Google Scholar]68. Bosch B.J., Martina B.E., Van Der Zee R., Lepault J., Haijema B.J., Versluis C., Heck A.J., De Groot R., Osterhaus A.D., Rottier P.J. Severe acute respiratory syndrome coronavirus (SARS-CoV) infection inhibition using spike protein heptad repeat-derived peptides. Proc. Natl. Acad. Sci. USA. 2004;101:8455–8460. doi: 10.1073/pnas.0400576101. [PMC free article] [PubMed] [CrossRef] [Google Scholar]69. Broer R., Boson B., Spaan W., Cosset F.L., Corver J. Important role for the transmembrane domain of severe acute respiratory syndrome coronavirus spike protein during entry. J. Virol. 2006;80:1302–1310. doi: 10.1128/JVI.80.3.1302-1310.2006. [PMC free article] [PubMed] [CrossRef] [Google Scholar]70. Modis Y. Class II fusion proteins. Adv. Exp. Med. Biol. 2013;790:150–166. [PMC free article] [PubMed] [Google Scholar]71. Zeng X., Herndon A.M., Hu J.C. Buried asparagines determine the dimerization specificities of leucine zipper mutants. Proc. Natl. Acad. Sci. USA. 1997;94:3673. doi: 10.1073/pnas.94.8.3673. [PMC free article] [PubMed] [CrossRef] [Google Scholar]72. Yoshida H., Oku M., Suzuki M., Mori K. pXBP1(U) encoded in XBP1 pre-mRNA negatively regulates unfolded protein response activator pXBP1(S) in mammalian ER stress response. J. Cell. Biol. 2006;172:565–575. doi: 10.1083/jcb.200508145. [PMC free article] [PubMed] [CrossRef] [Google Scholar]73. Xia X. Beyond Trees: Regulons and Regulatory Motif Characterization. Genes. 2020;11:995. doi: 10.3390/genes11090995. [PMC free article] [PubMed] [CrossRef] [Google Scholar]74. Chambers P., Pringle C.R., Easton A.J. Heptad repeat sequences are located adjacent to hydrophobic regions in several types of virus fusion glycoproteins. J. Gen. Virol. 1990;71:3075–3080. doi: 10.1099/0022-1317-71-12-3075. [PubMed] [CrossRef] [Google Scholar]75. Basak S., Hao X., Chen A., Chrétien M., Basak A. Structural and biochemical investigation of heptad repeat derived peptides of human SARS corona virus (hSARS-CoV) spike protein. Protein Pept. Lett. 2008;15:874–886. doi: 10.2174/092986608785849173. [PubMed] [CrossRef] [Google Scholar]76. Xia S., Liu M., Wang C., Xu W., Lan Q., Feng S., Qi F., Bao L., Du L., Liu S., et al. Inhibition of SARS-CoV-2 (previously 2019-nCoV) infection by a highly potent pan-coronavirus fusion inhibitor targeting its spike protein that harbors a high capacity to mediate membrane fusion. Cell Res. 2020;30:343–355. doi: 10.1038/s41422-020-0305-x. [PMC free article] [PubMed] [CrossRef] [Google Scholar]77. Yuan Y., Cao D., Zhang Y., Ma J., Qi J., Wang Q., Lu G., Wu Y., Yan J., Shi Y., et al. Cryo-EM structures of MERS-CoV and SARS-CoV spike glycoproteins reveal the dynamic receptor binding domains. Nat. Commun. 2017;8:15092. doi: 10.1038/ncomms15092. [PMC free article] [PubMed] [CrossRef] [Google Scholar]78. Ni L., Zhu J., Zhang J., Yan M., Gao G.F., Tien P. Design of recombinant protein-based SARS-CoV entry inhibitors targeting the heptad-repeat regions of the spike protein S2 domain. Biochem. Biophys. Res. Commun. 2005;330:39–45. doi: 10.1016/j.bbrc.2005.02.117. [PMC free article] [PubMed] [CrossRef] [Google Scholar]79. Pallesen J., Wang N., Corbett K.S., Wrapp D., Kirchdoerfer R.N., Turner H.L., Cottrell C.A., Becker M.M., Wang L., Shi W., et al. Immunogenicity and structures of a rationally designed prefusion MERS-CoV spike antigen. Proc. Natl. Acad. Sci. USA. 2017;114:E7348. doi: 10.1073/pnas.1707304114. [PMC free article] [PubMed] [CrossRef] [Google Scholar]80. Corver J., Broer R., van Kasteren P., Spaan W. Mutagenesis of the transmembrane domain of the SARS coronavirus spike glycoprotein: Refinement of the requirements for SARS coronavirus cell entry. Virol. J. 2009;6:230. doi: 10.1186/1743-422X-6-230. [PMC free article] [PubMed] [CrossRef] [Google Scholar]81. Liao Y., Zhang S.M., Neo T.L., Tam J.P. Tryptophan-dependent membrane interaction and heteromerization with the internal fusion peptide by the membrane proximal external region of SARS-CoV spike protein. Biochemistry. 2015;54:1819–1830. doi: 10.1021/bi501352u. [PubMed] [CrossRef] [Google Scholar]82. Petit C.M., Chouljenko V.N., Iyer A., Colgrove R., Farzan M., Knipe D.M., Kousoulas K.G. Palmitoylation of the cysteine-rich endodomain of the SARS-coronavirus spike glycoprotein is important for spike-mediated cell fusion. Virology. 2007;360:264–274. doi: 10.1016/j.virol.2006.10.034. [PMC free article] [PubMed] [CrossRef] [Google Scholar]83. Petit C.M., Melancon J.M., Chouljenko V.N., Colgrove R., Farzan M., Knipe D.M., Kousoulas K.G. Genetic analysis of the SARS-coronavirus spike glycoprotein functional domains involved in cell-surface expression and cell-to-cell fusion. Virology. 2005;341:215–230. doi: 10.1016/j.virol.2005.06.046. [PMC free article] [PubMed] [CrossRef] [Google Scholar]84. Millet J.K., Kien F., Cheung C.Y., Siu Y.L., Chan W.L., Li H., Leung H.L., Jaume M., Bruzzone R., Peiris J.S., et al. Ezrin interacts with the SARS coronavirus Spike protein and restrains infection at the entry stage. PLoS ONE. 2012;7:e49566. [PMC free article] [PubMed] [Google Scholar]85. Anderson E.J., Rouphael N.G., Widge A.T., Jackson L.A., Roberts P.C., Makhene M., Chappell J.D., Denison M.R., Stevens L.J., Pruijssers A.J., et al. Safety and Immunogenicity of SARS-CoV-2 mRNA-1273 Vaccine in Older Adults. N. Engl. J. Med. 2020;383:2427–2438. doi: 10.1056/NEJMoa2028436. [PMC free article] [PubMed] [CrossRef] [Google Scholar]86. Jackson L.A., Anderson E.J., Rouphael N.G., Roberts P.C., Makhene M., Coler R.N., McCullough M.P., Chappell J.D., Denison M.R., Stevens L.J., et al. An mRNA Vaccine against SARS-CoV-2—Preliminary Report. N. Engl. J. Med. 2020;383:1920–1931. doi: 10.1056/NEJMoa2022483. [PMC free article] [PubMed] [CrossRef] [Google Scholar]87. Denison M.R., Graham R.L., Donaldson E.F., Eckerle L.D., Baric R.S. Coronaviruses: An RNA proofreading machine regulates replication fidelity and diversity. RNA Biol. 2011;8:270–279. doi: 10.4161/rna.8.2.15013. [PMC free article] [PubMed] [CrossRef] [Google Scholar]88. Ferron F., Subissi L., Silveira De Morais A.T., Le N.T.T., Sevajol M., Gluais L., Decroly E., Vonrhein C., Bricogne G., Canard B., et al. Structural and molecular basis of mismatch correction and ribavirin excision from coronavirus RNA. Proc. Natl. Acad. Sci. USA. 2018;115:E162–E171. doi: 10.1073/pnas.1718806115. [PMC free article] [PubMed] [CrossRef] [Google Scholar]89. Robson F., Khan K.S., Le T.K., Paris C., Demirbag S., Barfuss P., Rocchi P., Ng W.-L. Coronavirus RNA Proofreading: Molecular Basis and Therapeutic Targeting. Mol. Cell. 2020;79:710–727. doi: 10.1016/j.molcel.2020.07.027. [PMC free article] [PubMed] [CrossRef] [Google Scholar]90. Korber B., Fischer W.M., Gnanakaran S., Yoon H., Theiler J., Abfalterer W., Hengartner N., Giorgi E.E., Bhattacharya T., Foley B., et al. Tracking Changes in SARS-CoV-2 Spike: Evidence that D614G Increases Infectivity of the COVID-19 Virus. Cell. 2020;182:812–827.e819. doi: 10.1016/j.cell.2020.06.043. [PMC free article] [PubMed] [CrossRef] [Google Scholar]91. Yurkovetskiy L., Wang X., Pascal K.E., Tomkins-Tinch C., Nyalile T.P., Wang Y., Baum A., Diehl W.E., Dauphin A., Carbone C., et al. Structural and Functional Analysis of the D614G SARS-CoV-2 Spike Protein Variant. Cell. 2020;183:739–751.e738. doi: 10.1016/j.cell.2020.09.032. [PMC free article] [PubMed] [CrossRef] [Google Scholar]92. Zhou H., Chen X., Hu T., Li J., Song H., Liu Y., Wang P., Liu D., Yang J., Holmes E.C., et al. A Novel Bat Coronavirus Closely Related to SARS-CoV-2 Contains Natural Insertions at the S1/S2 Cleavage Site of the Spike Protein. Curr. Biol. 2020;30:2196–2203.e3. doi: 10.1016/j.cub.2020.05.023. [PMC free article] [PubMed] [CrossRef] [Google Scholar]93. Reinke L.M., Spiegel M., Plegge T., Hartleib A., Nehlmeier I., Gierer S., Hoffmann M., Hofmann-Winkler H., Winkler M., Pöhlmann S. Different residues in the SARS-CoV spike protein determine cleavage and activation by the host cell protease TMPRSS2. PLoS ONE. 2017;12:e0179177. [PMC free article] [PubMed] [Google Scholar]94. Yao Y.X., Ren J., Heinen P., Zambon M., Jones I.M. Cleavage and Serum Reactivity of the Severe Acute Respiratory Syndrome Coronavirus Spike Protein. J. Infect. Dis. 2004;190:91–98. doi: 10.1086/421280. [PMC free article] [PubMed] [CrossRef] [Google Scholar]95. Xia X. Extreme genomic CpG deficiency in SARS-CoV-2 and evasion of host antiviral defense. Mol. Biol. Evol. 2020;37:2699–2705. doi: 10.1093/molbev/msaa094. [PMC free article] [PubMed] [CrossRef] [Google Scholar]96. Takata M.A., Gonçalves-Carneiro D., Zang T.M., Soll S.J., York A., Blanco-Melo D., Bieniasz P.D. CG dinucleotide suppression enables antiviral defence targeting non-self RNA. Nature. 2017;550:124–127. doi: 10.1038/nature24039. [PMC free article] [PubMed] [CrossRef] [Google Scholar]97. Lobry J.R. Origin of replication of Mycoplasma genitaliumScience. 1996;272:745–746. doi: 10.1126/science.272.5262.745. [PubMed] [CrossRef] [Google Scholar]98. Kleine-Weber H., Pöhlmann S., Hoffmann M. Spike proteins of novel MERS-coronavirus isolates from North- and West-African dromedary camels mediate robust viral entry into human target cells. Virology. 2019;535:261–265. doi: 10.1016/j.virol.2019.07.016. [PMC free article] [PubMed] [CrossRef] [Google Scholar]

‘We Made a Big Mistake’ — COVID Vaccine Spike Protein Travels From Injection Site, Can Cause Organ Damage

Authors:  Children’s Health Defense

COVID vaccine researchers had previously assumed mRNA COVID vaccines would behave like traditional vaccines. The vaccine’s spike protein — responsible for infection and its most severe symptoms — would remain mostly in the injection site at the shoulder muscle or local lymph nodes.

But new research obtained by a group of scientists contradicts that theory, a Canadian cancer vaccine researcher said last week.

“We made a big mistake. We didn’t realize it until now,” said Byram Bridle, a viral immunologist and associate professor at University of Guelph, Ontario. “We thought the spike protein was a great target antigen, we never knew the spike protein itself was a toxin and was a pathogenic protein. So by vaccinating people we are inadvertently inoculating them with a toxin.”

Bridle, who was awarded a $230,000 grant by the Canadian government last year for research on COVID vaccine development, said he and a group of international scientists filed a request for information from the Japanese regulatory agency to get access to Pfizer’s “biodistribution study.”

Biodistribution studies are used to determine where an injected compound travels in the body, and which tissues or organs it accumulates in.

“It’s the first time ever scientists have been privy to seeing where these messenger RNA [mRNA] vaccines go after vaccination,” Bridle said in an interview with Alex Pierson where he first disclosed the data. “Is it a safe assumption that it stays in the shoulder muscle? The short answer is: absolutely not. It’s very disconcerting.”

The Sars-CoV-2 has a spike protein on its surface. That spike protein is what allows it to infect our bodies, Bridle explained. “That is why we have been using the spike protein in our vaccines,” Bridle said. “The vaccines we’re using get the cells in our bodies to manufacture that protein. If we can mount an immune response against that protein, in theory we could prevent this virus from infecting the body. That is the theory behind the vaccine.”

“However, when studying the severe COVID-19, […] heart problems, lots of problems with the cardiovascular system, bleeding and clotting, are all associated with COVID-19,”  he added. “In doing that research, what has been discovered by the scientific community, the spike protein on its own is almost entirely responsible for the damage to the cardiovascular system, if it gets into circulation.”

When the purified spike protein is injected into the blood of research animals, they experience damage to the cardiovascular system and the protein can cross the blood-brain barrier and cause damage to the brain, Bridle explained.

The biodistribution study obtained by Bridle shows the COVID spike protein gets into the blood where it circulates for several days post-vaccination and then accumulates in organs and tissues including the spleen, bone marrow, the liver, adrenal glands and in “quite high concentrations” in the ovaries.

“We have known for a long time that the spike protein is a pathogenic protein, Bridle said. “It is a toxin. It can cause damage in our body if it gets into circulation.”

A large number of studies have shown the most severe effects of SARS-CoV-2, the virus that causes COVID, such as blood clotting and bleeding, are due to the effects of the spike protein of the virus itself.

A recent study in Clinical and Infectious Diseases led by researchers at Brigham and Women’s Hospital and the Harvard Medical School measured longitudinal plasma samples collected from 13 recipients of the Moderna vaccine 1 and 29 days after the first dose and 1-28 days after the second dose.

Out of these individuals, 11 had detectable levels of SARS-CoV-2 protein in blood plasma as early as one day after the first vaccine dose, including three who had detectable levels of spike protein. A “subunit” protein called S1, part of the spike protein, was also detected.

Spike protein was detected an average of 15 days after the first injection, and one patient had spike protein detectable on day 29 –– one day after a second vaccine dose –– which disappeared two days later.

The results showed S1 antigen production after the initial vaccination can be detected by day one and is present beyond the injection site and the associated regional lymph nodes.

Assuming an average adult blood volume of approximately 5 liters, this corresponds to peak levels of approximately 0.3 micrograms of circulating free antigen for a vaccine designed only to express membrane-anchored antigen.

In a study published in Nature Neuroscience, lab animals injected with purified spike protein into their bloodstream developed cardiovascular problems. The spike protein also crossed the blood-brain barrier and caused damage to the brain.

It was a grave mistake to believe the spike protein would not escape into the blood circulation, according to Bridle. “Now, we have clear-cut evidence that the vaccines that make the cells in our deltoid muscles manufacture this protein — that the vaccine itself, plus the protein — gets into blood circulation,” he said.

Bridle said the scientific community has discovered the spike protein, on its own, is almost entirely responsible for the damage to the cardiovascular system, if it gets into circulation.

Once in circulation, the spike protein can attach to specific ACE2 receptors that are on blood platelets and the cells that line blood vessels, Bridle said. “When that happens it can do one of two things. It can either cause platelets to clump, and that can lead to clotting — that’s exactly why we’ve been seeing clotting disorders associated with these vaccines. It can also lead to bleeding,” he added.

Both clotting and bleeding are associated with vaccine-induced thrombotic thrombocytopenia (VITT). Bridle also said the spike protein in circulation would explain recently reported heart problems in vaccinated teens.

Stephanie Seneff, senior research scientists at Massachusetts Institute of Technology, said it is now clear vaccine content is being delivered to the spleen and the glands, including the ovaries and the adrenal glands, and is being shed into the medium and then eventually reaches the bloodstream causing systemic damage.

“ACE2 receptors are common in the heart and brain,” she added. “And this is how the spike protein causes cardiovascular and cognitive problems.”

Dr. J. Patrick Whelan, a pediatric rheumatologist, warned the U.S. Food and Drug Administration (FDA) in December mRNA vaccines could cause microvascular injury to the brain, heart, liver and kidneys in ways not assessed in safety trials.

In a public submission, Whelan sought to alert the FDA to the potential for vaccines designed to create immunity to the SARS-CoV-2 spike protein to instead cause injuries.

Whelan was concerned the mRNA vaccine technology utilized by Pfizer and Moderna had “the potential to cause microvascular injury (inflammation and small blood clots called microthrombi) to the brain, heart, liver and kidneys in ways that were not assessed in the safety trials.”

13 ways that the SARS-CoV-2 spike protein causes damage

Authors: Posted on January 13, 2022 by Jesse Santiano, M.D.

The SARS-CoV-2 virus has four structural proteins. The spike, membrane, envelop, and nucleocapsid proteins. The spike protein protrudes from the middle of the coronavirus and attaches to the ACE2 receptor of cells to start the process of cell entry, replication, and infection. The two major parts of the spike protein are the S1 and S2 subunit. The S1 has the receptor-binding domain.

For easier reading, this review starts with what happens after the COVID jabs, soluble spike proteins, and what it takes to have a normal blood vessel. Then I will enumerate how the spike protein damages the body.

What happens after the COVID shots?

COVID vaccination aims to produce an immune response against the spike protein in the form of neutralizing antibodies so that in future SARS-CoV-2 exposures, COVID-19 will be prevented.

The injected messenger RNA provides instructions to the cells on making the spike proteins. Once the spike protein is produced, it migrates to the outside of the cell to be anchored on the cells’ outer surface, where the immune system will recognize it and develop an immune response to it. (antibodies, T cells, B cells).

Soluble spike proteins

Ideally, the whole spike protein should stay attached to the outside of the cells. Sometimes incomplete spike proteins are produced in the form of spike peptides. As shown below, they are also presented to the immune system by cells outside the surface with an anchoring protein called the Major Histocompatibility Complex (MHC).

Anchoring to the cells is critical because once the spike protein or its pieces in the form of peptides become soluble or float in the bloodstream, they induce inflammation and clot formation in the arteries and capillaries. Scientists have found several ways that it happens.

First is that enzymes called metalloproteinases can cut the MHC1 at their bases.[1] Free-floating MHCs are found in patients with systemic lupus erythematosus SLE and cancers.[4][5]

The second is that errors can happen while RNA splicing occurs inside the nucleus. This results in variant spike proteins that are soluble.[2] Soluble S1 subunits were observed among recipients of the Moderna shots.[3

You can read more about it in this article: SARS-CoV-2 spike proteins detected in the plasma following Moderna shots.

Third, are exosomes released from cells containing MHCs with the spike proteins. [6] T-cells can interact with the spike proteins in the exosomes and cause inflammation [7]. Immunogenic spike proteins inside exosomes were demonstrated after Pfizer injection[8].

 Donor Blood Can Have Spike Protein Exosomes

The normal blood vessel

All organs in the body need an adequate blood supply, and blood vessels have to be in pristine working conditions for that to happen. They should be distensible to allow greater blood flow during exertion, smooth inside to prevent blood clot formation, and have working mechanisms to repair themselves and dissolve blood clots that may form.

All that work falls on the endothelial cells that line the inner wall of the blood vessels, andI talked about them at The Magical Endothelium. Any injury to the endothelium can elicit an inflammatory response and clot formation leading to organ dysfunctions like heart attacks, strokes, and deaths. 

Blood clots always start small, and once they develop, they initiate a chain reaction that promotes a more extensive clot. The good thing is that the body can do fibrinolysis, a built-in mechanism to dissolve clots.

13 ways Spike Proteins cause disease

The following are how the spike proteins and their S1 subunit can cause damage. They can work together and have four results, inflammationthrombosis or clot formation, auto-immunity, and amyloid formation.

Any foreign protein inside the body can elicit inflammation. That is why parts of the spike proteins in the form of their S1 subunits or shorter fragments are enough to cause damage.[9][10].

Inflammation and thrombosis

The S1 subunit activates Toll-like receptor 4 (TLR4) signaling to induce pro-inflammatory responses. It happens with the spike protein in COVID-19 [10] and the S1 subunits.[12]

The spike protein triggers cell signaling events that promote pulmonary vascular remodeling and pulmonary arterial hypertension (PAH), and other cardiovascular complications [13]Source: Suzuki and Gychka. Vaccines 2

The spike S1 initiates inflammatory responses from tumor-necrosis factor-alpha (TNF-α) and interleukin-6 (IL-6) to initiate a cytokine storm syndrome in the lungs. [14].

Spike proteins cause vascular leaks by degrading the barrier of the endothelium. [15] The leak may explain the proliferation of lymphocytes seen by German pathologists in the organs of deceased patients who died after the shots.

Spike proteins affect the cardiac pericytes, the cells that “supervise” the endothelial cells responsible for maintaining the smoothness of the blood vessels. [16]. Study shows spike proteins affect cardiac pericytes and explain why soccer players collapse

Spike proteins downregulate the ACE2 and impair endothelial function [17]

The S1 produces blood clots resistant to the body’s fibrinolysis and hospital clot-buster medications [18]. That’s why some have their limbs amputated after the shots. One woman had both legs and hands amputated. There’s a list here.

The spike protein can cause inflammation by activating the alternate complement pathway. [20]

Long COVID-Syndrome

  1. The S1 Proteins can persist in CD16+ Monocytes up to 15 Months Post-Infection and vaccination to induce chronic inflammation. This explains the symptoms of the Long COVID Syndrome. [19]

Amyloids formation and interaction

  1. Amyloids are fibrillar proteins. They are most commonly associated with neurodegenerative diseases like dementia. However, they can also form in the heart and lungs and make them rigid and form blood clots resistant to dissolution. [21The SARS-CoV-2 spike protein can form amyloids seen in lung, blood, and nervous system disorders
  2. The S1 protein contains heparin-binding sites that attract amyloids to initiate amyloid protein aggregation. Amyloid formation leads to neurodegeneration like Parkinson’s Disease, Alzheimer’s’ disease, and Frontal lobe dementia. [22]

Molecular mimicry

12. Molecular mimicry happens if protein sequences in the spike protein and peptides have similarities to human proteins. Antibodies made for those viral proteins may also attack the host proteins.[24][25][26].

This leads to several autoimmune diseases like immune thrombocytopenia (low platelet counts) [23], autoimmune liver diseasesGuillain-Barré syndromeIgA nephropathyrheumatoid arthritis, and systemic lupus erythematosus[27]

Cancer and Immune Deficiency

  1. Spike proteins impair DNA damage repair and result in ineffective antibodies and damaged tumor-suppressor genes like the BRCA1 and 53BP1 that lead to cancers. BRCA1 damage is associated with breast, ovarian, and prostate cancers.

53BP1 loss of function in tumor tissues is elated to tumor occurrence, progression, and poor prognosis in human malignancies.[30]

Parting thoughts

The disease-causing part of the SARS-CoV-2 virus is the spike protein, and it is present in COVID-19 and the COVID injections. Prevention and early treatment are possible for COVID-19. Once you have the shot, there is no way to control the spike protein.

It is unclear why not all have the adverse effects or die. What is sure is that there are over one million reported adverse effects on VAERS, and more than one hundred thousand have been killed. Vaccine-induced deaths in the U.S. and Europe are way higher than the VAERS reports!

This list is not all-inclusive, and I probably missed some. Indeed, more will be discovered in the future, and I don’t want my body to find out. Do you?

Don’t Get Sick!

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Related:

  1. Blood Vessel Damaging Proteins of the SARS-CoV-2
  2. Cerebral Thrombosis after the Pfizer Covid-19 Vaccine
  3. The High Risk of Deadly Brain Clots in the J & J COVID Vaccine
  4. This Study shows a Ten-Fold Risk of Developing Blood Clots after the COVID Vaccines.
  5. You got the COVID shot and found that others developed blood clots. Now what?
  6. Platelet Changes Causes Blood Clots in COVID-19
  7. Unidentified Foreign Bodies in the Vaccines Form Clots
  8. Retinal complications after COVID shots
  9. U.K. Study of COVID-19 shots and Excess Rates of Guillain-Barré Syndrome
  10. mRNA Vaccination Increases the Risk of Acute Coronary Syndrome
  11. German Analysis: The Higher the Vaccination Rate, the Higher the Excess Mortality
  12. Anti-Idiotype Antibodies against the Spike Proteins may Explain Myocarditis

References:

  1. Rijkers GT, Weterings N, Obregon-Henao A, et al. Antigen Presentation of mRNA-Based and Virus-Vectored SARS-CoV-2 VaccinesVaccines (Basel). 2021;9(8):848. Published 2021 Aug 3. doi:10.3390/vaccines9080848
  2. Kowarz E, Krutzke L, Reis J, et al. “Vaccine-Induced Covid-19 Mimicry” Syndrome: Splice reactions within the SARS-CoV-2 Spike open reading frame result in Spike protein variants that may cause thromboembolic events in patients immunized with vector-based vaccines. Research Square; 2021. DOI: 10.21203/rs.3.rs-558954/v1
  3. Ogata AF. et al. Circulating SARS-CoV-2 Vaccine Antigen Detected in the Plasma of mRNA-1273 Vaccine Recipients [published online ahead of print, 2021 May 20]. Clin Infect Dis. 2021;ciab465. doi:10.1093/cid/ciab465
  4. Hervier B, Ribon M, Tarantino N, Mussard J, Breckler M, Vieillard V, Amoura Z, Steinle A, Klein R, Kötter I, Decker P. Increased Concentrations of Circulating Soluble MHC Class I-Related Chain A (sMICA) and sMICB and Modulation of Plasma Membrane MICA Expression: Potential Mechanisms and Correlation With Natural Killer Cell Activity in Systemic Lupus Erythematosus. Front Immunol. 2021 May 3;12:633658. doi: 10.3389/fimmu.2021.633658. PMID: 34012432; PMCID: PMC8126610.
  5. Salih, Helmut & Goehlsdorf, Dennis & Steinle, Alexander. (2006). Salih HR, Goehlsdorf D, Steinle A. Release of MICB molecules by tumor cells: mechanism and soluble MICB in sera of cancer patients. Hum Immunol 67: 188-195. Human immunology. 67. 188-95. 10.1016/j.humimm.2006.02.008.
  6. Edgar JR. Q&A: What are exosomes, exactly?BMC Biol. 2016;14:46. Published 2016 Jun 13. doi:10.1186/s12915-016-0268-z
  7. Raposo G, Nijman HW, Stoorvogel W, Liejendekker R, Harding CV, Melief CJ, Geuze HJ. B lymphocytes secrete antigen-presenting vesicles. J Exp Med. 1996 Mar 1;183(3):1161-72. doi: 10.1084/jem.183.3.1161. PMID: 8642258; PMCID: PMC2192324.
  8. Bansal et al. Cutting Edge: Circulating Exosomes with COVID Spike Protein Are Induced by BNT162b2 (Pfizer–BioNTech) Vaccination prior to Development of Antibodies: A Novel Mechanism for Immune Activation by mRNA Vaccines. J Immunol November 15, 2021, 207 (10) 2405-2410
  9. Nuovo, G.J. et al. (2021) Endothelial cell damage is the central part of COVID-19 and a mouse model induced by injection of the S1 subunit of the spike protein. Ann. Diagn. Pathol. 51, 151682, https://doi.org/10.1016/j.anndiagpath.2020.151682
  10. Gu, T. et al. (2020) Cytokine signature induced by SARS-CoV-2 spike protein in a mouse model. Front. Immunol. 11, 621441,
  11. Aboudounya MM, Heads RJ. COVID-19 and Toll-Like Receptor 4 (TLR4): SARS-CoV-2 May Bind and Activate TLR4 to Increase ACE2 Expression, Facilitating Entry and Causing Hyperinflammation. Mediators Inflamm. 2021 Jan 14;2021:8874339. doi: 10.1155/2021/8874339. PMID: 33505220; PMCID: PMC7811571.
  12. Shirato K, Kizaki T. SARS-CoV-2 spike protein S1 subunit induces pro-inflammatory responses via toll-like receptor 4 signaling in murine and human macrophages. Heliyon. 2021 Feb 2;7(2):e06187. doi: 10.1016/j.heliyon.2021.e06187. PMID: 33644468; PMCID: PMC7887388. https://pubmed.ncbi.nlm.nih.gov/33644468/
  13. Suzuki YJ, et al. SARS-CoV-2 Spike Protein Elicits Cell Signaling in Human Host Cells: Implications for Possible Consequences of COVID-19 VaccinesVaccines (Basel). 2021;9(1):36. Published 2021 Jan 11. doi:10.3390/vaccines9010036
  14. Cao, X. et al. (2021) Spike protein of SARS-CoV-2 activates macrophages and contributes to induction of acute lung inflammation in male mice. FASEB J. 35, e21801
  15. Biering et al. SARS-CoV-2 Spike triggers barrier dysfunction and vascular leak via integrins and TGF-β signaling. bioRxiv 2021.12.10.472112
  16. Avolio E et al.  The SARS-CoV-2 Spike protein disrupts human cardiac pericytes function through CD147 receptor-mediated signaling: a potential non-infective mechanism of COVID-19 microvascular disease. Clin Sci (Lond). 2021 Dec 22;135(24):2667-2689. doi: 10.1042/CS20210735. PMID: 34807265; PMCID: PMC8674568.
  17. Lei, Y. et al. SARS-CoV-2 Spike Protein Impairs Endothelial Function via Downregulation of ACE 2. Circulation Research. 2021;128:1323–1326
  18. Grobbelaar, L.M. et al. (2021) SARS-CoV-2 spike protein S1 induces fibrin(ogen) resistant to fibrinolysis: implications for microclot formation in COVID-19. Biosci. Rep. 41 (8)
  19. Patterson B. et al. Persistence of SARS CoV-2 S1 Protein in CD16+ Monocytes in Post-Acute Sequelae of COVID-19 (PASC) Up to 15 Months Post-Infection.bioRxiv 2021.06.25.449905
  20. Yu J, Yuan X, Chen H, Chaturvedi S, Braunstein EM, Brodsky RA. Direct activation of the alternative complement pathway by SARS-CoV-2 spike proteins is blocked by factor D inhibition. Blood. 2020.
  21. Nyström et al. Amyloidogenesis of SARS-CoV-2 Spike Protein. bioRxiv 2021.12.16.472920. 
  22. Idrees D, Kumar V. SARS-CoV-2 spike protein interactions with amyloidogenic proteins: Potential clues to neurodegeneration. Biochem Biophys Res Commun. 2021 May 21;554:94-98. doi: 10.1016/j.bbrc.2021.03.100. Epub 2021 Mar 24. PMID: 33789211; PMCID: PMC7988450
  23. Nunez-Castilla, J et al. Spike mimicry of thrombopoietin may induce thrombocytopenia in COVID-19. bioRxiv 2021.08.10.455737
  24. Ehrenfeld M et al. Covid-19 and autoimmunity. Autoimmun Rev. 2020;19:102597.
  25. Kanduc D, Shoenfeld Y. On the molecular determinants of the SARS-CoV-2 attack. Clin Immunol. 2020;215. 
  26. Vojdani A, Kharrazian D. Potential antigenic cross-reactivity between SARS-CoV-2 and human tissue with a possible link to an increase in autoimmune diseases. Clin Immunol. 2020;217:108480
  27. Chen Y, Xu Z, Wang P, Li XM, Shuai ZW, Ye DQ, Pan HF. New-onset autoimmune phenomena post-COVID-19 vaccination. Immunology. 2021 Dec 27. doi: 10.1111/imm.13443. Epub ahead of print. PMID: 34957554.
  28. Jiang H, Mei YF. SARS-CoV-2 Spike Impairs DNA Damage Repair and Inhibits V(D)J Recombination In VitroViruses. 2021;13(10):2056. Published 2021 Oct 13. doi:10.3390/v13102056
  29. BRCA1 gene. Medline [website]
  30. Mirza-Aghazadeh-Attari M, et al.  53BP1: A key player of DNA damage response with critical functions in cancer. DNA Repair (Amst). 2019 Jan;73:110-119. doi: 10.1016/j.dnarep.2018.11.008. Epub 2018 Nov 20. PMID: 30497961.

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