SARS-CoV-2 ORF8 regulates immune response
And other ways to Rome ! And N fragments.
In case you don’t follow me on Twitter, this is where I wrote for most of this afternoon (including the previous MonkeyPox piece).
First and foremost, you need to understand the entire SARS-CoV-2 structure before anything else here and in the near future makes sense.
Layed out here would be the entire virus in a 2-dimensional representation. For most of the pandemic almost the entire attention is aimed at the S (Spike) protein, for obvious reasons, and after much research, we came to understand the M, E, and N better, but all the other proteins, from all the NSP’s to the Accessory proteins were elusive. Each and every single one of these has multiple functions and purposes.
I intended to cover it in one massive post, but I rather fit it into other pieces, and the overall research and cover the other proteins soon enough. Here we will cover the ORF8, and boy that is a good one (not near as nasty as ORF6 though lol).
SARS-CoV-2 ORF8 is a viral cytokine regulating immune responses
Administration of recombinant ORF8 protein to hamsters also induced lymphocyte infiltration into the lungs. Similar pro-inflammatory cytokine production was observed in primary human monocytes treated with recombinant ORF8 protein. Furthermore, we demonstrate that the serum ORF8 protein levels are correlated well with clinical markers of inflammation. These results demonstrated that the ORF8 protein is a viral cytokine of SARS-CoV-2 involved in the in the immune dysregulation observed in COVID-19 patients, and that the ORF8 protein could be a novel therapeutic target in severe COVID-19 patients.
It is known that some viruses, such as poxvirus and herpesvirus, possess proteins that mimic cytokines to modulate immune response…
There was no difference in viral titer in the lungs of hamsters infected with WT and ORF8-knockout SARS-CoV-2. However, lymphocyte infiltration into the lungs and subsequent lung damage were less severe in the lungs of hamsters infected with the ORF8- knockout SARS-CoV-2 than in the lungs of those infected with WT SARS-CoV-2 (Fig. 1C). IL-6, CCL3, CCL5 and CXCL10 mRNA expression levels were significantly reduced in the lungs of hamsters infected with the ORF8-knockout SARS-CoV-2.
If you don’t recall what knockout means, it means removing that specific protein from something, so a SARS-CoV-2 with ORF-8 knockout means they engineered the virus without that protein. In this case, it lead to a very different immune response between the virus with and without ORF8, in which the one with this protein had significantly higher levels of inflammatory markers, and infiltration of lymphocytes, meaning there were immune cells in the tissue (lung here) causing lung damage.
When we analyzed the expression of MHC class II on lung cells from on SARS-CoV-2-infected hamsters, MHC class II expression on lung cells from WT SARS-CoV-2-infected hamsters was down-regulated, but not that from ORF8-knockout SARS-CoV-2-infected hamsters.
These results indicated that the ORF8 protein produced by SARS-CoV-2 infection is involved not only in the inflammatory response and subsequent lung damage, but also in the suppression of the acquired immune response via decreased MHC class II expression.
To understand how this is meaning, and to understand future pieces, you need to understand what is MHC. Here is a short but nice explanation. The short of it, the virus suppresses your acquired immune response by lowering the amount of these molecules.
SARS-CoV-2 ORF8 regulates immune responses
When monocytes from the PBMCs of healthy donors were stimulated with recombinant ORF8 protein, inflammatory cytokine, particularly high levels of IL-6 and IL-8, production was also observed. It has been reported that SARS-CoV2 ORF8 physically interacts with IL-17 receptor A (IL-17RA). Consistent with these previous reports, IL-6 and IL-8 production induced by ORF8 protein were reduced with anti-IL-17RA antibody treatment.
IFN-γ regulates the expression of MHC class II molecules on almost all cells including monocytes. We examined whether ORF8 protein could bind to IFN-γ receptor (IFNGR). IL-17RA, IFNGR, and IL-4 receptor (IL-4R) were expressed on HEK293T, and examined the binding of ORF8 protein to these receptors. We found that in addition to IL-17RA, ORF8 protein bound to IFNGR but not to IL-4R. Next, we analyzed the effect of ORF8 protein on IFN-γ-induced MHC class II expression. MHC class II expression on THP-1 cells was enhanced by IFN-γ treatment, and this enhancement was attenuated by treatment with ORF8 protein. These results suggested that the ORF8 protein is involved in down-regulation of MHC class II through the modulation of IFN-γ signaling.
When bone marrow-derived immune cells (that is what PBMCs are, also super-important) were put together with ORF8, the production of high levels of IL-6, and IL-8, and the author refers to an older paper (that I missed !!!) where the ORF8 was proposed, and later tested to bind to the Interleukin-17 Receptor A, and by blocking this receptor, the levels of both of the previous cytokines were diminished. Binding to a receptor means you are activating that pathway, meaning the ORF8 itself not only leads to an inflammatory response, but to Th17. Anyone who reads this Substack is most likely familiar with that by now.
Interferon Gamma (γ) is an important member of the Interferon family, and it is in a class of its own (Type II). Too much or too little at the wrong times, and you get disease progression, disease enhancement, and the atypical inflammatory response we often see with Covid (and the vaccines). And here they progressed from other authors' work and tested if ORF8 could bind to other important receptors, not only IL-17RA, and it did. To the Interferon-Gamma Receptor (IFNGR), further tests elucidated how the ORF8 is lowering the expression of MHC II molecule, by interfering with Interferon Gamma signaling. This is important because IFN-gamma is what keeps tumors, viruses, and other things in check, also it is what sets off apoptosis (cell death), it is a house cleaner in a simplistic way.
They further tested and found that ORF8 is a secretory protein, which was surprising to me. A secretory protein is a protein produced outside the cell, and well… secreted, and sent away, which makes sense, to disrupt the cell mechanism and signaling.
If it was not clear from the start, this is solely about the viral infection.
This part of this piece will also help you understand how SARS-CoV-2 can affect your immune response towards other diseases and infections, at least superficially, we will cover all the other proteins soon.
And since we mentioned Th17, of course, I gotta do this. The following paper is in regards to severe patients.
Multi-omics integrated analysis reveals a specific phenotype of CD8+ T cell may contribute to immunothromosis via Th17 response in severe and critical COVID-19
This paper is highly complex if you go to analyze all the different classes of T cells, the signaling, and the network analysis of interaction and signaling, if you have a good knowledge of immunology and proteins, as a clinician or scientist I advise you to dig in or to learn. Pen and paper are a must if you are learning.
Here the authors found a specific subset (phenotype) of CD8 cells is a critical component of going from mild/moderate disease to severe/critical, and one of the markers is a decrease in T-cells (reduction, and exhaustion), followed by senescent CD8 cells, this is a dysfunctional state (more common with aging) in which the cells stop its proper function, and increases the production of inflammation. Here they argue that this mechanism in specific subsets of CD8 cells is what drives the immune-mediated coagulation, damage, and dysfunction of the immune system in severe/critical cases.
Cases in which these changes can persist for months. Why is this important ? Well, you should read this.
Everything the virus does, 80% of the time, the Spike Protein will do elsewhere. This is more evidence that most of the damage and a portion of the long-term dysfunction done by the Spike Protein is mediated by the differentiation into a Th17 immune state, you can find how/why that is meaningful inside the piece above.
As the last one. I finally have evidence about one of my (insane) observations/idea.
The paper itself is really good, where the authors find that the N protein is cleavaged (fancy biology word for broken into smaller pieces), and the virus uses this protein in a time-dependent manner to antagonize (a term for lowering the levels of something) Interferon, which you are aware by now why and how this is important.
But the meaningful part to me was the “N fragments” still being present and having a physiological effect, it isn’t a novel concept, but also not very much accepted. And the basis for part of my argument for Long Covid dysfunction, is the presence of certain viral fragments “stuck” in the body/cells, affecting the immune expression, and causing a low-grade, long-term inflammatory response. Leading to this news article sent by a friend.
People who talk to me directly, at least a few, will know that one of my (insane) ideas for over a year was “Can the Spike Protein ALONE bind to receptors ?” Because that would explain a looooooot of things, it is not merely the body breaking down the protein and eliciting an immune response, but the entire protein binding to a receptor, and eliciting an entirely different (more inflammatory, and damaging) response.
Even “the science” now wonders if that is the case. Taking the ORF8 paper I am now a little less concerned about publicly expressing some of my more outrageous (from current dogma) observations. Over a year ago I started the meme of Vaccine AIDS (Reverse AIDS) by saying you should get intimate with the term Reverse AIDS, you are going to need it.
You should get intimate with the term “Non-canonical functions of…” you are going to need it.
A very big thank you to all supporters here and those who use KoFi =) !
Huge Thank you!
Those BASTARDS, and they knew all the time!
Like I said before, John Paul, at least all that AIDS research didn't go to waste, and no they weren't trying to cure a lifestyle, but weaponize it!
I was wrong then, and I freely admit it now. This is so much worse than we could have even conceived decade(s) ago.
Those motherf-ckers and yes, the GULAG would be too good for them.
Damn them, damn them all to hell.
Fantastic job, sir, really outstanding!