SARS-CoV-2 Spike protein is Not pro-inflammatory in Human Primary Macrophages?

Endotoxin Contamination negated or confounded many Spikeopathy studies

While there is no doubt the Synthetic SARS-CoV-2 Spike protein manufactured in jabbee cells, coded by synthetic mRNA in Covid19 jabs causes immense long-term damage to a generation, it can’t be responsible for the Adverse and sometimes Fatal reactions experienced in minutes to days after the jabs.

A most elegant proof of this was published by team¹ in Italy in early 2022 who obtained samples of Spike from commercial labs and tested them for inflammatory action on Human Macrophages (MΦ).

They concluded that a number of earlier studies were negated, or at least possibly confounded, by failure to take into account Endotoxin contamination. They said:

Indeed, in our hands, glycosylated truly LPS-free Spike has no inflammatory effects in human primary MΦ.

.. Our results militate against a relevant role of Spike per se in the cytokine storm of human infection played through a direct pro-inflammatory effect on MΦ. Apparently, Spike goes undetected by human MΦ only when it is glycosylated.

… LPS-free, glycosylated Spike (the form expressed in infected humans) caused no inflammation in human primary MΦ. Two (out of five) Spikes were contaminated with endotoxins ≥ 3 EU/ml and triggered inflammation. A non-contaminated non-glycosylated Spike produced in E. coli induced MΦ inflammation.

This extremely important finding shows that E. coli generated Spike is more toxic than that of the natural virus.

Here is their graphical abstract:

Studies confounded by the Endotoxin contamination

In 2006 it was reported² and still behind a paywall, that recombinant proteins used in experiments often contained “Dirty little secrets” - Endotoxin contamination.

Shirato and Kizaki reported in 2021 that Covid19 Spike caised inflammation of Human macrophages by interaction with TLR4.³

Chiok et al. study⁴ is also questioned.

Khan et al. proposed the TLR2 pathway for inflammation caused by Spike.⁵ They pointed the fact that Endotoxin could be a confounder of earlier studies, saying:

It appears that the recombinant S and E proteins used in those studies were generated in Escherichia coli. Thus, it is possible those recombinant proteins were contaminated by bacterial PAMPs. We used recombinant proteins produced in mammalian cell HEK293T, ensuring that the proteins are not contaminated by bacterial PAMPs. Using the endotoxin assay, we further confirmed that the recombinant S proteins are endotoxin-free.

Pantazi et al. were aware of the need to avoid E. coli cultures due to Endotoxin confounding hazard.⁶

Glycosylation prevents Spike Inflammation

Covid19 is a Stealthy virus, that evades your immune system with a coating of Glycans as determined by Crystallography, Enzyme Digestion and Mass Spectrometry.⁷

The Pfizer and Moderna Synthetic Spike coded by their synthetic mRNA will not have the same Glycan coating, making it highly inflammatory.

Spike in High Doses causes Coagulation ?

Zheng and coworkers found that Spike Protein expressed in HEK293 cells, with modifications at a similar concentration (~10 μg/mL) as the viral load in critically ill patients can cause directly blood coagulation and thrombosis in zebrafish model.⁸

However they used Endotoxin contaminated Spike. They used SARS-CoV-2 S protein, His Tag, Super stable trimer (MALS verified) certified with Endotoxin ( < 1.0 EU/μg)⁹ and SARS-CoV-2 (COVID-19) S protein RBD, Mouse IgG2a Fc Tag(MALS verified) ( < 1.0 EU/μg).¹⁰

The Lethal Dose to kill 50% (LD50) of Zebrafish Embryos by injection was determined to be 600 picogram by researchers in China in 2014.¹¹ The inflammatory response included the expected Cytokine Storm with expression of IL-1β, IL-6, and TNF-α and Macrophages and Neutrophils were both recruited to the Endotoxin injection site.

Zebrafish embryos injected with Endotoxin (Escherichia coli, ExPEC) suffer the same horrific damage as Covid19 Jabbees, including Tachycardia, Vascular Leakage, Oedema, Leukopenia and dysregulation of Clotting Cascades that could lead to problems like Disseminated Intravascular Coagulation (DIC), and Organ Failure.¹²

In 2017 Philip and coworkers in Canada performed elegant studies of Zebrafish damaged by Endotoxin.¹³ Looking at their Figure 6 note reduced microvascular circulation in Brain capillaries, largely in the central arteries, using hemoglobin staining (Figures 6A, B). Poor and defective circulation through the Dorsal Artery (DA) and Posterior Cardinal Vein (PCV) (Figure 6C), accompanied by narrowing of the DA (Figure 6D), increased Thrombocyte Clumping in the Common Cardinal Vein (CCV) and the caudal region of the embryo between the DA and the PCV compared with controls, which showed a few circulating thrombocytes in the caudal region of the zebrafish larvae (Figures 6E, F).

I expect this article will make some students of Inflammation “Go Crazy” or suffer Cognitive Dissonance.

I found that John Paul covered this topic in January 2023 but it was not evident without reading the subtitle “The Endotoxin Connection”.¹⁴

Update September 2023

John Paul just brought another dodgy publication¹⁵ to my attention.

The commercial SARS-CoV-2 spike protein S1 subunit¹⁶ that is not glycosylated was grown in E. coli, but has no Endotoxin test result at the supplier website or in downloadable documents there. This protein does not bind to ACE2.

Montezano and coworkers fail to mention Endotoxin, LPS or Lipopolysaccharide in their paper or its appendix.

Pity I was not invited to referee before publication. Nature used to be a quality journal.

The RayBiotech Spike contains a number of undesirable chemicals that could interfere with experiments apart from its unknown Endotoxin content that will induce Cytokine Storm.

"Filtered solution in 50 mM Tris-HCl, 500 mM NaCl, 600 mM arginine, 2 mM oxidized glutathione, 4 mM reduced glutathione, 0.2% PEG4000, 10% glycerol, 2 mM DTT, 20 mM Hepes (pH 9)."

DTT is Dithiothreitol.¹⁷

A recent study by Cao and coworkers is also at risk of Endotoxin contamination leading to false conclusions.¹⁸

They used Spike protein grown¹⁹ in ATCC Dulbecco's Modified Eagle's Medium (DMEM) growth medium with a specification Endotoxin ≤ 0.5 EU/mL.

A paper by Perico and coworkers²⁰ is also at risk due to Endotoxin content of the commercial spike from Miltenyi.²¹

A major narrative review of “Spikeopathy” was published by a group of Australian authors.²² They refer to a paper that shows many studies are fatally flawed if they use Endotoxin contaminated Spike protein.²³

April 2024 Update

Found another paper that will be confounded by Endotoxin in Plasmids.

Jiang and coworkers²⁴ describe a very lax approach to the contamination:

Plasmids of pGX-9501, pVAX1-S-WT, and pVAX1 were trans-formed into DH5a E. Coli, respectively. A single colony was undergone expansion in a one-liter flask for culturing in LB broth. Plasmids were extracted, purified by MaxPure Plasmid EF Giga Kit (Magen, China), dissolved in saline buffer at a final concentration at 1 mg/ml. The purity was measured by an agarose gel electrophoresis and a UV detector at a range of 1.8–2.0 OD260nm/280 nm. Endotoxin in those plasmids was below 30 EU/mg by LAL test.

Using Mice:

injected twice via the intramuscular route (i. m.) with 25 μg plasmid, and electroporation was followed at intervals of two weeks. Serum was collected 14 days after the second immunization.

So the injections delivered up to 1.5 EU Endotoxin per animal.

1. Cinquegrani G, et al. 2022. SARS‑CoV‑2 Spike protein is not pro‑inflammatory in human primary macrophages: endotoxin contamination and lack of protein glycosylation as possible confounders. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8749924/

2. Wakelin SJ, et al. 2006. “Dirty little secrets”—Endotoxin contamination of recombinant proteins. https://www.sciencedirect.com/science/article/abs/pii/S0165247806001209

3. Shirato K and Kizaki T. 2021. SARS-CoV-2 spike protein S1 subunit induces pro-inflammatory responses via toll-like receptor 4 signaling in murine and human macrophages. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7887388/

4. Chiok K et al. 2023 (original preprint June 2021). Proinflammatory Responses in SARS-CoV-2 and Soluble Spike Glycoprotein S1 Subunit Activated Human Macrophages. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10052676/

5. Khan S, et al. 2021. SARS-CoV-2 spike protein induces inflammation via TLR2-dependent activation of the NF-κB pathway. https://elifesciences.org/articles/68563

6. Pantazi I, et al. 2021. SARS-CoV-2/ACE2 Interaction Suppresses IRAK-M Expression and Promotes Pro-Inflammatory Cytokine Production in Macrophages. https://www.frontiersin.org/articles/10.3389/fimmu.2021.683800/full

7. Watanabe Y, et al. 2020. Vulnerabilities in coronavirus glycan shields despite extensive glycosylation. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7253482/

8. Zheng Y, et al. 2021. SARS-CoV-2 spike protein causes blood coagulation and thrombosis by competitive binding to heparan sulfate. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8553634/

9. https://www.acrobiosystems.com/P3349-SARS-CoV-2-S-protein-His-Tag-Super-stable-trimer-%28MALS-verified%29.html

10. https://www.acrobiosystems.com/P3146-SARS-CoV-2-%28COVID-19%29-S-protein-RBD-Mouse-IgG2a-Fc-Tag%28MALS-verified%29.html

11. Yang L-L, et al. Endotoxin Molecule Lipopolysaccharide-Induced Zebrafish Inflammation Model: A Novel Screening Method for Anti-Inflammatory Drugs. https://www.mdpi.com/1420-3049/19/2/2390

12. Barber AE. 2016. Similarly Lethal Strains of Extraintestinal Pathogenic Escherichia coli Trigger Markedly Diverse Host Responses in a Zebrafish Model of Sepsis. https://journals.asm.org/doi/full/10.1128/msphere.00062-16

13. Philip AM, et al. 2017. Development of a Zebrafish Sepsis Model for High-Throughput Drug Discovery. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5522968/

14. Circulating Spike in mRNA induced myocarditis

15. Montezano AC, et al. 2023. SARS-CoV-2 spike protein induces endothelial inflammation via ACE2 independently of viral replication. https://www.nature.com/articles/s41598-023-41115-3

16. https://www.raybiotech.com/recombinant-sars-cov-2-spike-protein-s1-subunit-230-01101

17. https://en.wikipedia.org/wiki/Dithiothreitol

18. Cao X, et al. 2023. The SARS-CoV-2 spike protein induces long-term transcriptional perturbations of mitochondrial metabolic genes, causes cardiac fibrosis, and reduces myocardial contractile in obese mice. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10281040/

19. Cao X, et al. 2021. Spike protein of SARS‐CoV‐2 activates macrophages and contributes to induction of acute lung inflammation in male mice. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8441663/

20. Perico L, et al. 2023. SARS-CoV-2 spike protein induces lung endothelial cell dysfunction and thrombo-inflammation depending on the C3a/C3a receptor signalling. https://www.nature.com/articles/s41598-023-38382-5

21. https://www.miltenyibiotec.com/AU-en/products/recombinant-sars-cov-2-spike-s1-hek.html

22. Parry, P.I.; Lefringhausen, A.; Turni, C.; Neil, C.J.; Cosford, R.; Hudson, N.J.; Gillespie, J. ‘Spikeopathy’: COVID-19 spike protein is pathogenic, from both virus and vaccine mRNA. Biomedicines 2023, 11, 2287. https://www.mdpi.com/2227-9059/11/8/2287

23. Weiming Ouyang, Tao Xie, Hui Fang, Chunling Gao, Tzanko Stantchev, Kathleen A. Clouse, Kun Yuan, Tongzhong Ju, and David M. Frucht. 2021. Variable Induction of Pro-Inflammatory Cytokines by Commercial SARS CoV-2 Spike Protein Reagents: Potential Impacts of LPS on In Vitro Modeling and Pathogenic Mechanisms In Vivo. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8305765/

24. Sheng Jianga,b, Shuting Wua, Gan Zhaoc, Yue Hec, Linlin Baod, Jiangning Liud, Chuan Qind, Jiawang Houc, Yuan Dingc, Alex Chengc, Brian Jiangc, John Wuc, Jian Yane, Laurent Humeaue, Ami Patellae, David B. Weinerf, Kate Brodericke, and Bin Wang. 2022. Comparison of Wild Type DNA Sequence of Spike Protein from SARS-CoV-2 with Optimized Sequence on The Induction of Protective Responses Against SARS-Cov-2 Challenge in Mouse Model. https://www.tandfonline.com/doi/full/10.1080/21645515.2021.2016201