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Questions on the Pfizer vaccine
Posted by: Ca Bob
Date: November 11, 2020 05:51PM
OK, so I understand mRNA pretty well. In fact you would consider me something of an expert on it. Maybe that's why I have these questions, and I wonder if one of you could explain things to me/us.

1) I understand that the mRNA vaccine would get sucked up into cells (presumably lots of them) and will code for the spike protein (or part of it) and that protein will be synthesized on the normal polysomal machinery. So far so good. But will the engineered mRNA carry some export sequence so that the newly synthesized protein is exported from the cells and into the extracellular matrix and/or the circulation? This would seem to make sense if you want the circulating immune cells to make contact with the spike protein and generate an immune response. Note: I ask this question because it seems really obvious, yet the news stories seem to miss it entirely.

It seems that you would very much want to avoid a situation where the viral protein remains in the cell or (even worse) is presented on the cell surface. That would be a bad thing. I wonder how the manufacturer can be sure that neither of these is the case.

2) Can the vaccine target itself to a particular type of cells? What happens to standard vaccines that are injected into the arm muscle? Do they gradually get absorbed into the circulation? If not, are they found by elements of the immune system that are migrating through the tissue and not necessarily in the circulation? Can you engineer a vaccine so that it's active element is preferentially transferred to the circulation and therefore meets up with the circulating immune cells and/or antibodies?

3) This one is kind of inside-baseball: In human cells (at least those in tissue culture), mRNAs have half-lives that vary from a few minutes to (typically) about a day. Some last even longer, particularly if it is a global mRNA in specialized cells. It seems to me that you would like for the vaccine mRNA to be degraded eventually so that it quietly disappears from the cells it has transfected. Is there any information on how this is supposed to work in these new vaccine prototypes?



Edited 1 time(s). Last edit at 11/11/2020 05:59PM by Ca Bob.
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Re: Questions on the Pfizer vaccine
Posted by: lost in space
Date: November 11, 2020 05:57PM
I tried Google translate on this and it blew up.



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Re: Questions on the Pfizer vaccine
Posted by: PeterB
Date: November 11, 2020 07:39PM
Quote
Ca Bob
OK, so I understand mRNA pretty well. In fact you would consider me something of an expert on it. Maybe that's why I have these questions, and I wonder if one of you could explain things to me/us.

1) I understand that the mRNA vaccine would get sucked up into cells (presumably lots of them) and will code for the spike protein (or part of it) and that protein will be synthesized on the normal polysomal machinery. So far so good. But will the engineered mRNA carry some export sequence so that the newly synthesized protein is exported from the cells and into the extracellular matrix and/or the circulation? This would seem to make sense if you want the circulating immune cells to make contact with the spike protein and generate an immune response. Note: I ask this question because it seems really obvious, yet the news stories seem to miss it entirely.

It seems that you would very much want to avoid a situation where the viral protein remains in the cell or (even worse) is presented on the cell surface. That would be a bad thing. I wonder how the manufacturer can be sure that neither of these is the case.

2) Can the vaccine target itself to a particular type of cells? What happens to standard vaccines that are injected into the arm muscle? Do they gradually get absorbed into the circulation? If not, are they found by elements of the immune system that are migrating through the tissue and not necessarily in the circulation? Can you engineer a vaccine so that it's active element is preferentially transferred to the circulation and therefore meets up with the circulating immune cells and/or antibodies?

3) This one is kind of inside-baseball: In human cells (at least those in tissue culture), mRNAs have half-lives that vary from a few minutes to (typically) about a day. Some last even longer, particularly if it is a global mRNA in specialized cells. It seems to me that you would like for the vaccine mRNA to be degraded eventually so that it quietly disappears from the cells it has transfected. Is there any information on how this is supposed to work in these new vaccine prototypes?

I'll tackle some of these, but I am NOT an expert in the RNA field ... I know others here are.

1) My understanding is that the mRNA has been designed to be taken up by cells by way of the lipid nanoparticles, but I can't imagine that the uptake is that specific to particular cell types. Presumably one wants the mRNA to be targeted to antigen-presenting cells, which will then "present" the spike protein to the B- and T-cells. So ideally the mRNA must incorporate cell-specific sequence signals that permit translation into protein only in certain cell types.

2) See #1, and I also presume that this is the purpose of adjuvants.

3) I presume that this the purpose of the nucleoside modifications that they've made to the mRNA, so as to make it more stable, persistent, and resistant to breakdown (and therefore translated more frequently). One would assume that the mRNA will eventually break down, but I think this is a legitimate concern.

[www.phgfoundation.org]
[sitn.hms.harvard.edu]
[www.statnews.com]
[www.ncbi.nlm.nih.gov]
[www.nature.com]




Freya says, 'Hello from NOLA, baby!' (Laissez bon temps rouler!)
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Re: Questions on the Pfizer vaccine
Posted by: Marc Anthony
Date: November 11, 2020 08:29PM
1. mRNA is the export sequence. Coded mRNA exits the nucleus and gets translated into a protein within the cytoplasm. You want the cell to present proteins on the surface, for recognition as self or non-self and so that other cells are recruited in response.

2. Vaccines don’t target specific cells; their general purpose is to present molecules for pattern recognition. A mRNA vaccine causes the cell to produce these, rather than virus. All vaccine is eventually absorbed and the immune system is in both serum and tissues, but slow absorption is better—"depot effect."

3. All non-self cells should eventually be inactivated, escorted out of the body, and/or summarily destroyed.



Le poète doit vivre beaucoup, vivre dans tous les sens. - Verlaine
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Re: Questions on the Pfizer vaccine
Posted by: PeterB
Date: November 11, 2020 08:59PM
Quote
Marc Anthony
1. mRNA is the export sequence. Coded mRNA exits the nucleus and gets translated into a protein within the cytoplasm. You want the cell to present proteins on the surface, for recognition as self or non-self and so that other cells are recruited in response.

2. Vaccines don’t target specific cells; their general purpose is to present molecules for pattern recognition. A mRNA vaccine causes the cell to produce these, rather than virus. All vaccine is eventually absorbed and the immune system is in both serum and tissues, but slow absorption is better—"depot effect."

3. All non-self cells should eventually be inactivated, escorted out of the body, and/or summarily destroyed.

Marc, some questions:

1) Shouldn't this mRNA go directly to the cytosol for translation? Or were you just referring to mRNAs in general? And I think the concern is, if cells are presenting viral proteins on the cell surface, specifically APC's ... what if the viral proteins are also being expressed on the surface of other cell types, then you'd be targeting your own cells for destruction?

2) I would assume that there IS some cell-specificity in that they've apparently programmed these mRNAs for immunogenicity ... probably at the protein targeting level.

3) I think the issue is ... does anyone know for sure that the mRNA eventually completely disappears? By modifying the mRNA to make it more stable and resistant to degradation, you increase the risk of unexpected consequences of that mRNA being expressed in cell types it shouldn't, and though the claim is that mRNA cannot convert to DNA, I wonder what might happen in a person who receives such a vaccine and is also infected with a retrovirus.




Freya says, 'Hello from NOLA, baby!' (Laissez bon temps rouler!)
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Re: Questions on the Pfizer vaccine
Posted by: Marc Anthony
Date: November 11, 2020 11:11PM
Hi, Peter. Yes. I was speaking generally from transcription. Any cell that's making viral proteins should be marked for destruction. Your own cells die all the time, a few here or there to rid an invader would be collateral damage. Without a reservoir of cells, macrophages would likely clear any leftover mRNA vaccine instruction.



Le poète doit vivre beaucoup, vivre dans tous les sens. - Verlaine
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Re: Questions on the Pfizer vaccine
Posted by: Ca Bob
Date: November 12, 2020 01:43AM
This is an interesting discussion. Yes, mRNAs normally get exported from the nucleus to the cytoplasm because they are coded by the nuclear DNA and that is where chromosomal transcription takes place. But this is an artificial mRNA that is made in a factory, and the quickest way to get it into cellular cytoplasm is to let it soak through the cell membrane. So thanks to you all for clarifying that this vaccine uses a lipid coating to allow it to penetrate the cell. When we have done this sort of thing in the lab, we either inject lipid coated materials into the muscle or give it intravenously.

What's curious to me is how little detail we are getting of the pathway that the signal takes, starting with a vaccine and presumably ending as the spike protein floating in the blood stream. You raise another interesting question, namely -- is this vaccine designed so that the cells which express the mRNA are going to be destroyed by the immune system? Or will those cells simply export some spike protein, degrade what's left of the vaccine mRNA, and continue to live? The former results in a hole in the muscle of your arm, at least for a while.

This stuff has gone through multiple layers of review, so I'm assuming these questions have all been asked and answered, but I would prefer that the kinds of questions that a college sophomore could ask will be answered clearly and publicly.
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Re: Questions on the Pfizer vaccine
Posted by: neophyte
Date: November 12, 2020 06:49AM
These are the concerns I have too. I suspect there may be proprietary issues limiting the amount of public info released about each mRNA vaccine construct. To wit:

1) It would be useless to translate a mRNA that creates only a cytosolic protein. There must be an encoded amino acid sequence that results in its transport to the cell surface.

2) It could be potentially dangerous to have that protein remain embedded in the plasmalemma. If the immune system mounts a response to that protein (which is the intent anyway), the resulting cell lysis will release a lot of other cellular material not usually found extra-cellularly. This material may also become antigenic, and create a potential autoimmune response. If some of this material also creates an antigenic complex with "normal" cell surface proteins, such an autoimmune response could be wide-spread.

3) In view of 2), an amino acid cleavage site in the protein most likely is engineered into the mRNA sequence, to allow the release of the protein once it is extruded through the plasmalemma. This cleavage site is not present in the native viral spike protein because it remains bound to the viral phospholipid membrane. Hence a proprietary mRNA construct is created by a vaccine manufacturer, which also protects their investment via patent.

4) There are probably other factors that need to be considered when engineering a biochemical such as this which requires the cooperation of multiple biological pathways to bring about a desired effect. I'll bet some unintended medical consequences will evolve in some unlucky people.

5) I'll wait for a simple spike protein vaccine to be brought to market. I expect one to be available in less than six months.
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Re: Questions on the Pfizer vaccine
Posted by: PeterB
Date: November 12, 2020 08:00PM
Quote
Ca Bob
This is an interesting discussion. Yes, mRNAs normally get exported from the nucleus to the cytoplasm because they are coded by the nuclear DNA and that is where chromosomal transcription takes place. But this is an artificial mRNA that is made in a factory, and the quickest way to get it into cellular cytoplasm is to let it soak through the cell membrane. So thanks to you all for clarifying that this vaccine uses a lipid coating to allow it to penetrate the cell. When we have done this sort of thing in the lab, we either inject lipid coated materials into the muscle or give it intravenously.

What's curious to me is how little detail we are getting of the pathway that the signal takes, starting with a vaccine and presumably ending as the spike protein floating in the blood stream. You raise another interesting question, namely -- is this vaccine designed so that the cells which express the mRNA are going to be destroyed by the immune system? Or will those cells simply export some spike protein, degrade what's left of the vaccine mRNA, and continue to live? The former results in a hole in the muscle of your arm, at least for a while.

This stuff has gone through multiple layers of review, so I'm assuming these questions have all been asked and answered, but I would prefer that the kinds of questions that a college sophomore could ask will be answered clearly and publicly.

1) Presumably the mRNA is getting "coated" or encased by the lipid nanoparticles, such that the nanoparticle is fusing with the cell surface and enabling the mRNA to get into the cytoplasm to get translated. It's also possible that the mRNA is making it to the rough ER for translation, and then the antigen is getting trafficked to the cell surface via the endocytotic pathway.

2) Regarding the pathway, there were a couple of diagrams in the papers I cited above that show the pathways involved. Here are a few:







As I've mentioned (and you pointed out as well), one major concern is ... what about non-APC's that express the viral protein, one would assume that they are targeted for destruction as having a foreign protein on their cell surface. That is, unless (as I suggested) the mRNA contains some information targeting or limiting expression/translation to particular cell types. That information can be encoded in the mRNA in either the 5' or 3' untranslated regions.

3) One would indeed hope that all these questions have been asked and answered, but... it's one thing to do things in cell culture or even an animal model, it's another to do it in a human being.

Quote
neophyte
These are the concerns I have too. I suspect there may be proprietary issues limiting the amount of public info released about each mRNA vaccine construct. To wit:

1) It would be useless to translate a mRNA that creates only a cytosolic protein. There must be an encoded amino acid sequence that results in its transport to the cell surface.

2) It could be potentially dangerous to have that protein remain embedded in the plasmalemma. If the immune system mounts a response to that protein (which is the intent anyway), the resulting cell lysis will release a lot of other cellular material not usually found extra-cellularly. This material may also become antigenic, and create a potential autoimmune response. If some of this material also creates an antigenic complex with "normal" cell surface proteins, such an autoimmune response could be wide-spread.

3) In view of 2), an amino acid cleavage site in the protein most likely is engineered into the mRNA sequence, to allow the release of the protein once it is extruded through the plasmalemma. This cleavage site is not present in the native viral spike protein because it remains bound to the viral phospholipid membrane. Hence a proprietary mRNA construct is created by a vaccine manufacturer, which also protects their investment via patent.

4) There are probably other factors that need to be considered when engineering a biochemical such as this which requires the cooperation of multiple biological pathways to bring about a desired effect. I'll bet some unintended medical consequences will evolve in some unlucky people.

5) I'll wait for a simple spike protein vaccine to be brought to market. I expect one to be available in less than six months.

The proprietary aspect is a little concerning; I haven't checked for it, but I'm sure the patent application is available somewhere.

1) Probably done either though targeting the mRNA to rough ER as I mentioned above, and/or protein sorting signals within the protein itself.

2) That's the concern I mentioned above -- if the spike protein being expressed is locked into an "open" conformation, as mentioned in the article Sarcany posted awhile back in the thread I wrote about this issue, then it's possible the protein could bind the person's own ACE2 receptors ... and then if antibodies are made against the ACE2 receptor, you've got a serious problem -- an autoimmune disease that could take years to show up.

3) See my comment about the patent application above. Presumably the protein is being destroyed after being presented by the APC.

4) See my comment under #2. I also think a big problem is that -- you're assuming how the immune system works and will respond, when in fact it's FAR more complicated than really any of us know. (For example, we don't know all the interactions, etc.) Tinkering around with things like this might lead to lots of unforeseen consequences. Also worth considering: NOT everyone's immune system works or will respond the the same way! My own B-cells and probably also T-cells do not behave normally.

5) Bingo, and that's also why I raised the issue -- months ago -- which Sarcany answered, of why not to simply immunize with dead (inactivated) virus... that's likely to give you a much broader, presumably much more robust protection, since you're making antibodies against numerous antigens in the virus. Apparently that's one of the vaccines being developed by the Chinese.




Freya says, 'Hello from NOLA, baby!' (Laissez bon temps rouler!)



Edited 1 time(s). Last edit at 11/12/2020 08:01PM by PeterB.
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Re: Questions on the Pfizer vaccine
Posted by: pdq
Date: November 13, 2020 08:31AM
Quote
PeterB
why not to simply immunize with dead (inactivated) virus...

Probably several reasons, one of which I guess is that it’s trickier, takes longer, and is more potentially dangerous growing up enough virus than churning out mRNA or loading/modifying a previously used viral vector.

Wikipedia has some details. Interestingly, it looks like the two Chinese vaccines in development are both inactivated-Covid vaccines.
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Re: Questions on the Pfizer vaccine
Posted by: PeterB
Date: November 13, 2020 12:13PM
Quote
pdq
Quote
PeterB
why not to simply immunize with dead (inactivated) virus...

Probably several reasons, one of which I guess is that it’s trickier, takes longer, and is more potentially dangerous growing up enough virus than churning out mRNA or loading/modifying a previously used viral vector.

Wikipedia has some details. Interestingly, it looks like the two Chinese vaccines in development are both inactivated-Covid vaccines.

Already noted in one of my posts above. I'm sure it might be harder to make an inactivated virus vaccine, but there might be a lot of advantages of doing that, since as I said -- the immune response will be broader, likely more robust, and more protective. The problem with a lot of the vaccines under development right now is that they only target the spike protein, and if the virus mutates in any way, it's possible that such a vaccine would rapidly become ineffective.

Edit: and as an addendum to that -- it's currently thought that a lot of the reason that we don't have persistent immunity to this particular virus is some of the other viral proteins that may be interfering with certain aspects of our immune system ... so any vaccine that targets these other proteins as well might be substantially more effective and give longer-lasting immunity. I hope the Chinese are willing to share their vaccines with us...




Freya says, 'Hello from NOLA, baby!' (Laissez bon temps rouler!)



Edited 1 time(s). Last edit at 11/13/2020 12:16PM by PeterB.
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