Understanding mRNA

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jujubeen66
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I'm trying to understand how mRNA propagates or not throughout the body. We have been lead to believe the vaccine's mRNA protein coder permeates every cell in the body eventually, but explanations of mRNA technology do NOT support this, instead they appear to say that only the small localized muscle cells at the injection site will absorb the vaccine mRNA, which will encode and produce its protein on the surface of those cells only. Passing immune system cells will recognize this protein and prepare to acknowledge it as foreign. The minor reactions to the initial shots are the mild immune system response. Is this how it works? I cannot find a single article that explains this satisfactorily.
 

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I think you’re right about the beginnings.

You‘re given the shot in the arm. The cells within the vicinity absorb the mRNA encapsulated in an liposome wrapper known as transfection.

The infected cell machinery uses the mRNA, like it does with any internally generated mRNA, to manufacture the so-called protein spike that is the hallmark of the SARS Cov2 virus.

The cell then exports the protein spike to its cell wall exposing it to the outside world.

Immune cells spot the foreign body and mount a response to neutralize it by generating antibodies to attach to it.

Once attached, the antibodies have marked the cell for destruction by killer T-cells and the immune system in doing so learns to neutralize the threat throughout the body.
 
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jim mcnamara
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The mRNA in one or more liposomes goes into one cell. The commandeered cell makes lots of spike proteins. The immune system detects foreign spike protein, cleans up the mess, and the cell dies.

One cell, one liposome, lots of spike proteins. Not every cell in the body is affected.

So 500 liposomes transfect 500 cells. Because the dose is in the muscle tissue most of the transfections occur at the point of the injection. Some cells even get more than one liposome attached. So 500 liposomes might only get into 450 cells, for example.

Other remote cells are indirectly affected -but no mRNA from the injection is involved.

Example:
There are lymph nodes in your armpit. A help! call goes out from the immune system's cells that got awakened by the spike protein arrival. That call is answered in part of the immune system that lives in your armpit. It then creates new, additional special immune cells. These guys can get into really complicated biochemical "cascades".

Biochemical cascade, also known as a signaling cascade or signaling pathway, is a series of chemical reactions that occur within a biological cell when initiated by a stimulus.

Vaccine mRNA getting inside the lymph node cells does not really happen. And is not part of the hugely complex stuff in your armpit.

Does that help?
 
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One difference between virus and vaccine I'd like to bring out more.

A virus infects a cell and makes it produce more virus, which infects other cells and so on and so on. The immune system learns about the virus and confers immunity post-infection. Well, if the person survives.

The mRNA infects a cell and makes it produce the spike protein on the Covid virus. And then that's it. The immune system learns about the protein and thereby the virus and confers immunity post-vaccination.
 
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jujubeen66
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OK, so the mRNA cannot bounce around and enter other cells. That sounds right although it could encounter blood vessels and thereby blood cells but you're implying that the mRNA/lipid structure confines all its production to whichever cell it enters, exhausts itself and dies there. I have read that some mRNA structures can be coded to look for new playing fields after a certain level of production and that the lipid encasement ensures a significally longer survival for the mRNA transport system, otherwise it is especially fragile. In studies in Nature Neuroscience it was reported that the current mRNA tech produced the spike protein in mouse brains. How would this happen, since they obviously didn't inject the brains specifically. It was also found throughout their bodies in major organs, same question. If the spike protein is localized to the injection site why are ACE2 receptors constantly mentioned? It doesn't seem like muscle tissue would contain many of them anyway?
Thanks for your concise answers everyone.
 
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jujubeen66
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Also Jim, in a previous reply to another similar question in the forum you said that the spike protein factory cells DON'T die, I can't understand why they would as the spike protein doesn't really affect them individually, or does it? I realize this is pretty controversial material and you will need to tread lightly. Perhaps a more complex analogy will help.
 
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1. The mRNA isn't alive.
2. Of course the spike protein gets into the bloodstream. It wouldn't do to have just your upper arm immune.
 
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Ygggdrasil
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Of course the spike protein gets into the bloodstream. It wouldn't do to have just your upper arm immune.

The spike protein does not get into the bloodstream.

Here's a decent text description of how mRNA vaccines work that goes into a bit more detail than others: https://scopeblog.stanford.edu/2020/12/22/how-do-the-new-covid-19-vaccines-work/

I'll also add a bit more explanation to clarify some issues in the thread:

The mRNA vaccines consists of an mRNA molecule encapsulated in a lipid nanoparticle shell. The vaccine gets injected intramuscularly into the arm. While the lipid nanoparticle shell does help it get into cells, it does not get into all different types of cells equally. There is a type of immune cell called dendritic cells (DCs) whose job it is to surveil the body for potential pathogens, take them up into the cell (via endocytosis), break the pathogen up into small fragments (antigens), and present those antigens to other immune cells (like T-cells and B-cells) to active those cells and start the adaptive immune response.

Because of their role in recognizing foreign particles, the DCs are the main type of cell taking up the mRNA vaccines. Inside the DCs, the mRNA causes the cell to produce the spike protein, some of which goes to the surface of the cell and some of which gets cut up and presented on antigen-presenting MHC molecules. The process of taking up the mRNA (likely through specific lipids in the nanoparticle shell) helps activate the DCs, which causes them to migrate to lymph nodes so that they can present their antigens to immature T-cells and B-cells. Interactions between the DCs and these immune cells will activate the T-cells and B-cells to cause them to replicate and spread throughout the body to activate the cellular and humoral arms of the adaptive immune response.

So, it is neither the mRNA nor the spike protein that gets spread throughout the body. Rather, the mRNA likely gets take up by DCs (which is the important part) as well as some cells in the local area of injection and maybe some other cells scattered throughout the body (if some, say, does get into the blood stream). However, it is the uptake of the mRNA by the DCs and their downstream activation of other immune cells that allow the replication of the T- and B-cells and for these immune cells to spread throughout the bloodstream and provide body-wide protection against the SARS-CoV-2 virus.

Another good reference for the science behind mRNA vaccines (though focused on speaking to researchers working in the area): https://www.nature.com/articles/nrd.2017.243
 
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Ygggdrasil
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I have read that some mRNA structures can be coded to look for new playing fields after a certain level of production and that the lipid encasement ensures a significally longer survival for the mRNA transport system, otherwise it is especially fragile.
It would be helpful if you provided a link to where you read this in order for us to clarify.

In studies in Nature Neuroscience it was reported that the current mRNA tech produced the spike protein in mouse brains. How would this happen, since they obviously didn't inject the brains specifically. It was also found throughout their bodies in major organs, same question.
It would be helpful if you could provide link(s) to the study(s).

If the spike protein is localized to the injection site why are ACE2 receptors constantly mentioned? It doesn't seem like muscle tissue would contain many of them anyway?
In the SARS-CoV-2 coronavirus, the spike protein attaches to ACE2 receptors on the cells that it infects. However, mRNA vaccines get into cells via the lipid nanoparticles, so the spike protein is not involved in entry of the vaccines into cells (in fact, the mRNA vaccines don't have any spike protein, just mRNA that contains instructions on how to make the spike protein). Therefore, ACE2 receptors are largely irrelevant to how the mRNA vaccines function.
 
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Another vaccine virus distinction is that with virus the immune may identify other structures to target whereas the vaccine provides only the spike protein as the target.
 
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jujubeen66
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Okay this is getting better. Thanks Yggdrasil (love that moniker) for a more in depth explanation. I check out your links and I'm going to run my new understanding past you to see if I get it.
The true target of the mRNA structure is the dendritic cell because it is responsible for informing the core immune system of the antigen spike protein. Exogenous mRNA is encased in a lipid molecule to hide it from the immune system initially which would assault it if it were recognized in a free state. Once in the cytoplasm, the mRNA strands find ribosome, pass on the protein code, and the ribosomes begin churning out the proteins. The cell actually does chew up some of the proteins produced, but displays the bulk of the production on its cell surface for the benefit of the immune system.
Am I solid so far? it sounds like dendritic cells could be totally covered in this mess.
It says the mRNA is a copy of the DNA strands coding for the protein in the coronavirus- but there are lots of those I understand. Also, that piece says that non-dendritic cells can take up the mRNA as well and produce the spike protein, but what would they do with it, since muscle cells aren't immune cells? I would imagine that at the beyond microscopic level of circulation where veins and arteries are nearly one, it would be possible that mRNA vehicles could be taken up there as well.
That article also said mRNA cannot penetrate the nucleus, but it didn't say why not. If the lipid capsule shields the mRNA structure from detection by the immune system, what prevents it from tricking the endoplasmic reticulum ( did I get that right?)
Also, the Stanford piece always used RNA and not mRNA, they are quite different correct? that was confusing, and even worse once I got to dsRNA!
The Nature article was a treasure trove but it will take more time to digest and I don't have the background to crunch it all but some material stood out quickly. IVT= in vivo transcription? I don't think it was decoded. They explain that RNA is a copied DNA template and one assumes that the segment of the covid virus that codes for the protein was copied. I understand though that RNA doesn't always copy completely and often leaves out material that it deems superfluous.
The mRNA degrades quickly, although the lipid case does prolong its durability yes? once it is gone, can/do the ribosomes continue to make the proteins or is the presence of the coded mRNA crucial? Can the dendritic cell ever be covered with too many proteins such that they might free float in interstitial fluid and actually attach to ACE2 receptor cells elsewhere? ( no one mentions this) If, in fact, the cell is covered in whole and protein fragments, isn't a certain level of fraying possible?
The Nature article is very complex and I would love to have someone hold my hand through it sometime, but until then I'll just ask for clarification on some vocab: what is a cap structure? when they say the cap measures 5' how long is that exactly? What is a poly A tail- I heard a podcast somewhere that claimed that this was a structure that limited the life of the mRNA strands, true?
Finally, the Nature article indicated that the accompanying adjuvants play a rather crucial role in ensuring that the proteins are expressed strongly and recognized by the immune system and that some of these could result in the production of Interferon. Isn't that a good immune response? It sounded negative in the article.
You're all terrific for helping me out in understanding this. Thanks.
 
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jujubeen66
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Oh, in reference to my missing links. I've read about the fragility of the mRNA strands in several places and didn't write them down. The suggestion of a new playing field was from a podcast I no longer have a link for.
The Nature Neuroscience piece was posted on the Children's Health Defense website under the title Could Spike proteins cause blood clots. I mean we already know the J&J one does so that's not debatable, but my interest lies in the ability of the proteins to travel through the body. Check it out and correct my misunderstandings- I'd appreciate it.
Originally it was explained that the PCR tests required swabs that contained high levels of ACE2receptors because that's where the covid, or that protein was located. I have read in odd places that they were still in play.
 
  • #13
TeethWhitener
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Okay this is getting better. Thanks Yggdrasil (love that moniker) for a more in depth explanation. I check out your links and I'm going to run my new understanding past you to see if I get it.
Well, I'm no @Ygggdrasil, but I'm awake, so I might as well take a crack at it. NB--I have no idea what your level of expertise is, so this might be a little basic.
The true target of the mRNA structure is the dendritic cell because it is responsible for informing the core immune system of the antigen spike protein.
This isn't strictly true. Given a mode of transfection (in this case cationic liposome-based transfection), each cell type has a certain transfection efficiency. In (roughly) lay terms, what that means is that each cell type (dendritic, other immune, muscle cell, fat cell, etc.) will be transfected (i.e., mRNA from the liposome will make its way into a given cell's cytoplasm) with a certain efficiency--sometimes the transfection will succeed; sometimes it will fail. In vitro, a good optimized transfection efficiency may be on the order of 50-70%: 50-70% of cells will have non-native genetic information introduced into them. I don't know tons about in vivo transfection efficiency, but I imagine it'll be lower. Living things are complicated.
Exogenous mRNA is encased in a lipid molecule to hide it from the immune system initially which would assault it if it were recognized in a free state.
mRNA is encased in a liposome (think of it as a nanoscopic soap bubble made up of molecules that look a lot like the molecules that make up the membrane of your cells). This is less to hide it from the immune system and more to hide it from an enzyme called RNAse which is...just everywhere and a huge pain in the butt. RNAse's entire job is to chew up RNA into its monomeric pieces. It's very good at its job, and it's expressed both inside cells and outside of cells. In fact, RNA research labs have to take special pains to exclude the presence of RNAse in their experiments. The liposome in the vaccine has two functions: 1) it gives the mRNA some breathing room and a buffer against encountering RNAse, thus increasing the likelihood it'll find its way into a cell, and 2) the liposome itself, being cationic, will associate well with the anionic nucleic acid and, since its molecules share similiarities with your cell membranes, it can integrate more effectively with the cell and deposit its mRNA payload more efficiently into the cytoplasm of your cells.
Once in the cytoplasm, the mRNA strands find ribosome, pass on the protein code, and the ribosomes begin churning out the proteins.
This is all fine.
The cell actually does chew up some of the proteins produced, but displays the bulk of the production on its cell surface for the benefit of the immune system.
Am I solid so far?
Close enough.
it sounds like dendritic cells could be totally covered in this mess.
Maybe. The adaptive immune system is wildly complicated, so I'll let this be for the moment.
It says the mRNA is a copy of the DNA strands coding for the protein in the coronavirus- but there are lots of those I understand.
There are lots of proteins that make up SARS-CoV-2, and there has been some talk of using mRNA to code for multiple proteins so that variants (whose structural proteins tend to mutate more slowly than the spike protein) can be caught more easily with boosters.
Also, that piece says that non-dendritic cells can take up the mRNA as well and produce the spike protein, but what would they do with it, since muscle cells aren't immune cells?
All of the body's cells contain ribosomes. @Ygggdrasil's point was that, ultimately, it's the immune cells that need to recognize foreign proteins to kickstart the process of adaptive immunity. But if muscle cells express antigens on their surfaces, the immune system will still find out about it eventually: immune cells constantly circulate throughout the body, bouncing off other cells and occasionally attaching to foreign invader-jerks (by recognizing antigens--foreigners--bound to a protein known as the major histocompatibility complex--non-foreigner--and signaling that something is amiss).
I would imagine that at the beyond microscopic level of circulation where veins and arteries are nearly one, it would be possible that mRNA vehicles could be taken up there as well.
Sure, why not?
That article also said mRNA cannot penetrate the nucleus, but it didn't say why not. If the lipid capsule shields the mRNA structure from detection by the immune system, what prevents it from tricking the endoplasmic reticulum ( did I get that right?)
The liposome that encases the mRNA becomes part of the lipid cell membrane when it deposits its mRNA payload into the cytoplasm. At that point, the mRNA is naked in the cytoplasm; there is no packaging that will send it through the nuclear membrane.
Also, the Stanford piece always used RNA and not mRNA, they are quite different correct? that was confusing, and even worse once I got to dsRNA!
There are lots of RNA's. It's admittedly confusing. mRNA is specifically what gets translated by the ribosome into proteins. The ribosome knows it's chewing on mRNA and not another version of RNA due to the presence of what's called a polyadenine (or polyA) tail. Basically, all native mRNA is post-transcriptionally modified (modified after being synthesized from DNA) by tagging it with a tail made of lots of adenines in a row. The ribosome recognizes this polyA tail, binding to it and beginning the translation of the mRNA into a protein. The vaccine takes advantage of this by sticking a polyA tail onto the mRNA.
The Nature article was a treasure trove but it will take more time to digest and I don't have the background to crunch it all but some material stood out quickly. IVT= in vivo transcription? I don't think it was decoded.
Not at work right now so I don't have journal access.
They explain that RNA is a copied DNA template and one assumes that the segment of the covid virus that codes for the protein was copied. I understand though that RNA doesn't always copy completely and often leaves out material that it deems superfluous.
I'm not sure this is accurate the way you're thinking about it. The ribosome will translate whatever mRNA you give it. There are (rare) errors, but it would be helpful to know where you're getting this from.
The mRNA degrades quickly, although the lipid case does prolong its durability yes?
Yes
once it is gone, can/do the ribosomes continue to make the proteins or is the presence of the coded mRNA crucial?
The presence of the mRNA is crucial. The ribosome can't operate without mRNA in its binding pocket.
Can the dendritic cell ever be covered with too many proteins such that they might free float in interstitial fluid and actually attach to ACE2 receptor cells elsewhere? ( no one mentions this)
Haha, well I admit, I'd never thought of this before. But no, typically overexpression of a protein doesn't extend to this degree. I suppose it's possible, albeit extremely unlikely, that a spike protein may escape its host cell and attach to another ACE2 receptor somewhere. But at any rate, things attach to ACE2 receptors all the time (namely angiotensin, the A in ACE2). The problem with COVID is that it uses the ACE2 receptor to enter a cell and hijack its protein production machinery to produce more COVID.
If, in fact, the cell is covered in whole and protein fragments, isn't a certain level of fraying possible?
Not sure what this means.
The Nature article is very complex and I would love to have someone hold my hand through it sometime,
Will get to it at work at some point.
but until then I'll just ask for clarification on some vocab: what is a cap structure? when they say the cap measures 5' how long is that exactly?
I'll have to read the article. 5' and 3' usually refer to the (non-equivalent) ends of a nucleic acid strand (wiki to the rescue: https://en.wikipedia.org/wiki/Directionality_(molecular_biology))
What is a poly A tail- I heard a podcast somewhere that claimed that this was a structure that limited the life of the mRNA strands, true?
See above.
Finally, the Nature article indicated that the accompanying adjuvants play a rather crucial role in ensuring that the proteins are expressed strongly and recognized by the immune system and that some of these could result in the production of Interferon. Isn't that a good immune response? It sounded negative in the article.
I'd have to read the article to see for myself.
You're all terrific for helping me out in understanding this. Thanks.
OK, @Ygggdrasil I'm totally taking credit for this. :wink:
 
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jujubeen66
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Excellent TW, a lot was covered here. I want to say how much I appreciate the rapid responses on the forum; it's so helpful. We're not out of the woods yet. I have no background in this material, just a natural interest in science and so I try to master what I can and, particularly, what's hot at the moment. I've always loved microbiology but I wasn't strong mathematically, so I didn't think I'd survive a science career. I taught AP French for 18 years and I'm retired now. I do remember a lot of the basics so I'm not lost. Let's continue...
Picking up where the liposome blanket gets the mRNA into the cytoplasm. Clearly the liposome blanket disintegrates and ultimately becomes part of the cell membrane. This exposes the mRNA to the local ribosomes but also to RNAse then, which goes to work ripping it apart? or can the ribosomes connect with the mRNA while it is still encased? Do any of the adjuvants in the vaccine neutralize the RNAse to maximize spike protein production time?

Are the current mRNA structures self-amplifying?

Also, mRNA is generic then in the sense that it doesn't have to have any genetic connection to your specific DNA, just a single strand of an RNA sequence coding the protein you want? And the poly A tail is also generic as in it works on everyone for all ribosomes?


In a Hopkins/Bloomberg study/report entitled Technologies to Address Global Catastrophic Biological Risks ( sounds awful) on pg. 47 they talk about using cytomegalovirus as a vector for mass inoculation of vaccine material. I'm assuming this would spread via air and breath droplets or could it be engineered for simple skin to skin contact? I can't imagine how they could get exogenetic mRNA to stay active in saliva or breath droplets and I guess it would just enter through epithelial cells. I realize they're working on it and don't have it, it's just fascinating.


One last thing. On pg. 51 of the same report it sounds as if the positive mRNA is replicated by a viral replicase which creates first a neg mRNA copy from which a duplicate positive is mass produced. Is this mRNA copying going on while the ribosomes are making the protein or is this something else? Check out the article when you can. I understand the "ase" suffix indicates an enzyme, is this RNAse they're talking about? I do wish scientific vocab was a bit more consistent. Thanks team.

Oh, is it possible to print this thread? I'm taking notes but I'd love to have a hard copy. I've attached the Bloomberg report.
 
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jujubeen66
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I thought I attached the report, but I don't see it. Need directions for that move I guess.
 
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Oh, I was an ultrasound tech for 14 years prior to the French- quite the contrast, don't you think?!
 
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Ygggdrasil
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To add to @TeethWhitener's excellent response:
Exogenous mRNA is encased in a lipid molecule to hide it from the immune system initially which would assault it if it were recognized in a free state.
As mentioned above, RNA is fairly prone to degradation from enzymes like RNases, and the lipid molecule help protect the RNA from degradation from those enzymes. The RNA is also chemically modified (normally, RNA contains for letters, A, U, C and G, but in the vaccines, we use the nucleotide N1-methyl-pseudouridine (m1Ψ) instead of U. The substitution of m1Ψ for U helps to prevent the innate immune system from attacking the RNA and to boost the translation of the RNA into protein. (For a technical reference see: https://pubs.acs.org/doi/10.1021/acscentsci.1c00197). See a section below for more description of the innate immune response.

That article also said mRNA cannot penetrate the nucleus, but it didn't say why not. If the lipid capsule shields the mRNA structure from detection by the immune system, what prevents it from tricking the endoplasmic reticulum ( did I get that right?)
Transport between the nucleus and cytoplasm is highly regulated. Only macromolecule with special tags are able to transit from the cytoplasm to the nucleus, and the vaccine mRNA lacks any of those special tags. Here's a good textbook chapter on the regulation of transport between the nucleus and cytoplasm: https://www.ncbi.nlm.nih.gov/books/NBK26932/

The Nature article was a treasure trove but it will take more time to digest and I don't have the background to crunch it all but some material stood out quickly. IVT= in vivo transcription? I don't think it was decoded. They explain that RNA is a copied DNA template and one assumes that the segment of the covid virus that codes for the protein was copied. I understand though that RNA doesn't always copy completely and often leaves out material that it deems superfluous.
Yes, IVT stands for in vitro transcription. When you say that "RNA doesn't always copy completely and often leaves out material that it deems superfluous," are you talking about the process of splicing? Splicing does not occur for the type of IVT reactions used to produce the mRNA for the vaccines.

The Nature article is very complex and I would love to have someone hold my hand through it sometime, but until then I'll just ask for clarification on some vocab: what is a cap structure? when they say the cap measures 5' how long is that exactly? What is a poly A tail- I heard a podcast somewhere that claimed that this was a structure that limited the life of the mRNA strands, true?
The cap is a special nucleotide-like molecule at the 5' end of the mRNA molecule that helps identify the RNA as an mRNA molecule, protect the mRNA for degradation and aids in the translation of the mRNA. Here's a decent article written for non-scientists explaining the different parts of the mRNA as they relate to the mRNA vaccine: https://berthub.eu/articles/posts/reverse-engineering-source-code-of-the-biontech-pfizer-vaccine/

Finally, the Nature article indicated that the accompanying adjuvants play a rather crucial role in ensuring that the proteins are expressed strongly and recognized by the immune system and that some of these could result in the production of Interferon. Isn't that a good immune response? It sounded negative in the article.
There are two broad categories of immune responses. The first response is the innate immune response. This is the first line of defense against pathogens and relies on patter recognition receptors to recognize common molecular features of pathogens (e.g. molecules that resemble bacterial cell walls, or foreign DNA or RNA molecules that look like they could be coming from viruses). The adaptive immune response comes later and involves the production of antibodies by B-cells and the elimination of infected cells by T-cells. Vaccines help to establish an adaptive immune response.

In the case of the mRNA vaccines, early research showed that injecting unmodified mRNA molecules into the cells activated the cells' innate immune response -- in other words, the vaccine RNA triggered the cells to think that they were being infected with a virus. Now, some stimulation of the immune response is good as this contributes to activation of the DCs, but too much stimulation of the the innate immune response will cause the cells taking up the mRNA to just die. Using m1Ψ modified mRNAs to reduce activation of the innate immune response was a key advancement that helped to make mRNA vaccines possible (see this PF thread for more discussion: https://www.physicsforums.com/threads/messenger-rna-mrna-not-just-for-coronavirus-vaccines.1000153/).

The Nature Neuroscience piece was posted on the Children's Health Defense website under the title Could Spike proteins cause blood clots. I mean we already know the J&J one does so that's not debatable, but my interest lies in the ability of the proteins to travel through the body. Check it out and correct my misunderstandings- I'd appreciate it.

I assume you are talking about this article:

The S1 protein of SARS-CoV-2 crosses the blood–brain barrier in mice
https://www.nature.com/articles/s41593-020-00771-8

In this case, the researchers injected free spike proteins into the bloodstreams of mice, which is not what is happening for the vaccine (all of the spike protein produced by the vaccine should be tethered to the surface of the cells producing the spike). Furthermore, the article does not directly address the question of blood clots. All the article says is that the spike proteins can make their way into the brain. This is suggestive that maybe the virus could get into brain through similar means, but the article does not address this question. Similarly, the article does not address what problems the spike protein or virus could be causing in the brain (such as blood clots). As with a lot of science journalism, the website made a giant leap from what was actually shown in the study to something is not supported by the data presented in the article.

How the adenoviral vaccines (like J&J and AstraZeneca) cause blood clotting has been established, and it does not seem to involve the spike protein (e.g. similar blood clotting issues have not been seen with the mRNA vaccines). (see my post here for more discussion: https://www.physicsforums.com/threads/oxford-vaccine-clotting.1001834/post-6481805)

Originally it was explained that the PCR tests required swabs that contained high levels of ACE2receptors because that's where the covid, or that protein was located. I have read in odd places that they were still in play.
No, ACE2 receptors are not involved in the PCR tests.
 
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Ygggdrasil
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Picking up where the liposome blanket gets the mRNA into the cytoplasm. Clearly the liposome blanket disintegrates and ultimately becomes part of the cell membrane. This exposes the mRNA to the local ribosomes but also to RNAse then, which goes to work ripping it apart? or can the ribosomes connect with the mRNA while it is still encased?
Most of the really bad RNases that RNA researchers like me worry about chewing up our RNA are extracellular. There are RNases inside the cell, but the 5' cap and 3' poly(A) tail prevent the RNases from acting on mRNAs before they can be translated by the ribosome (though eventually, the RNAs will be degraded by those RNases). The mRNAs would need to be released from the liposomes before they could be read by the ribosomes.

Do any of the adjuvants in the vaccine neutralize the RNAse to maximize spike protein production time?
The mRNA vaccines do not contain any traditional adjuvants (though some parts of the lipid shell could have adjuvant-like effects). The modified nucleotides on the mRNA (m1Ψ), however, help to boost translation of the mRNA into spike protein.

Are the current mRNA structures self-amplifying?
No

Also, mRNA is generic then in the sense that it doesn't have to have any genetic connection to your specific DNA, just a single strand of an RNA sequence coding the protein you want? And the poly A tail is also generic as in it works on everyone for all ribosomes?
As described in the article I linked in my previous post, the mRNA structure is fairly modular. You can switch the coding region of the mRNA to code for another protein (such as a flu protein to make a flu vaccine). The poly(A) tail is also fairly generic and is mostly similar to the poly(A) tails found on most other mRNAs.

In a Hopkins/Bloomberg study/report entitled Technologies to Address Global Catastrophic Biological Risks ( sounds awful) on pg. 47 they talk about using cytomegalovirus as a vector for mass inoculation of vaccine material. I'm assuming this would spread via air and breath droplets or could it be engineered for simple skin to skin contact? I can't imagine how they could get exogenetic mRNA to stay active in saliva or breath droplets and I guess it would just enter through epithelial cells. I realize they're working on it and don't have it, it's just fascinating.
That technology is very far away from being used. However, that type of vaccine is not based on mRNA technology (rather, they are trying to create a live virus to use for vaccination).

One last thing. On pg. 51 of the same report it sounds as if the positive mRNA is replicated by a viral replicase which creates first a neg mRNA copy from which a duplicate positive is mass produced. Is this mRNA copying going on while the ribosomes are making the protein or is this something else? Check out the article when you can. I understand the "ase" suffix indicates an enzyme, is this RNAse they're talking about? I do wish scientific vocab was a bit more consistent. Thanks team.
Yes, the -ase suffix generally denotes an enzyme. The enzyme responsible for copying an RNA molecule is an RNA-dependent RNA polymerase (RdRp), named such because these enzymes catalyze the polymerization of ribonucleotides into RNA. The RdRp for the SARS-CoV-2 coronavirus is actually a complex consisting of three proteins encoded by the NSP12, NSP7, and NSP8 genes. RNase enzymes are enzymes that catalyze the degradation of RNA.

Copying of the RNA cannot occur at the same time as translation of the mRNA. Various different viruses use different tricks for separating pools of RNAs to use as templates for replication vs mRNAs for translation vs genomes for making new viruses. I have not studied coronaviruses enough to give an answer off of the top of my head, but there is probably some spatial segregation that goes on to separate the RNA replication from the pool of RNAs undergoing translation (e.g. see https://www.nature.com/articles/s41579-020-0406-z for a summary of some research on this issue).
 
  • #19
jujubeen66
Gold Member
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Thanks Yggy (!) almost home. 2 final questions/comments.

My friend, who works in a major hospital laboratory where they run the PCR tests told me that the techs were told that the swabbing had to include ACE2 receptors and that's why they were swabbing so high up in the nose, although that never made any sense because the cheeks contain plenty of ACE2 receptors I've read elsewhere.

and finally: what is Luciferase? I have read/heard in podcasts that it is part of the mRNA/lipid package.

After this I'll give you all a break.
 
  • #20
Ygggdrasil
Science Advisor
Insights Author
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My friend, who works in a major hospital laboratory where they run the PCR tests told me that the techs were told that the swabbing had to include ACE2 receptors and that's why they were swabbing so high up in the nose, although that never made any sense because the cheeks contain plenty of ACE2 receptors I've read elsewhere.
The CDC has published instructions for its RT-qPCR test here: https://www.fda.gov/media/134922/download

ACE2 is not mentioned in the document, and it is not one of the genes tested for. The test does contain a control test for a human gene, but this gene is RNase P.

and finally: what is Luciferase? I have read/heard in podcasts that it is part of the mRNA/lipid package.
Luciferases are a class of enzymes that create bioluminescence (e.g. in fireflies): https://en.wikipedia.org/wiki/Luciferase

They are often used in scientific research as reporter genes (which, for example, would allow one to track activity of a gene by seeing cells with that gene glow under certain circumstances). Luciferase genes and luciferase enzymes are not part of the mRNA/lipid package for any of the mRNA vaccines.

The components of the Pfizer vaccine are explained here: https://www.technologyreview.com/20...-the-ingredients-of-pfizers-covid-19-vaccine/
 
  • #21
jujubeen66
Gold Member
10
1
thanks for all your help. Till next time.
 

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