The mRNA plataform is the only "computer ready" plataform, it takes little fidgeting and mathematical equations to change the entire immunogenicity of the desired protein. They had to start mass testing to gather data to "fix" the kinds and bug somewhere I guess.
I abhor mRNA as a vaccine plataform, but I could infer their intent was based on forecasting biological threats, it is the only plataform "fast enough" for aggressive responses against, well, basically almost anything.
The biggest problem of the mRNA lies on its origins, at this point, everyone is aware it was intended as a "cancer vaccine", and it is precisely there the biggest hurdle lies and all the paradoxical effects come from. To get inside the cells without alerting the body, they literally can start "turbo cancer" at any age.
Ironic. A cancer vaccine that can induce aggressice oncogensis.
Based on your interesting writeup, may I ask some follow-up questions/comments, please?
Have you previously ever noticed that the SV40 promoter, or an undeclared ORI, was added by default or accidentally? In reading various articles, it is obvious that the SV40 promoter/enhancer has long been used sort of as a positive control for numerous experiments.
Theoretically, could it have been the case that the SV40 part was during the earlier phase of the Pfizer jab development (Process 2) used as a positive control but then forgotten to take out from their model plasmids? They may argue that "it was all about speed" and that taking out the SV40 part was "just an oversight."
Finally, I would like to encourage everyone who is able to do so to please always sequence the plasmids in question and not merely rely on any (purported) digital description of them alone. Especially since the contamination issue with the Pfizer jabs has made news, it seems to me that presence of any offensive genes etc in the plasmids could easily be erased ONLINE from their DIGITAL description. This would NOT mean that they are absent from the actual/real plasmid. This is a huge safety/security issue that is largely under-appreciated, please see my article in Biosafety& Health: https://pubmed.ncbi.nlm.nih.gov/33015604/. There is a inherent GAP between the physical/biological entity and their digitized/online DESCRIPTION. If someone wanted to, this gap could easily be exploited to ingress changes (additions, deletions) that you can only know if you check both components.
Not an oversight. A normal working FDA/EMA would say too risky, take it out unless suitably justified. Not lazy either. Quality be Design means you start with the end product in mind, the "safest" design. Intent to deceive in at the minimum. Intent to harm looks likely especially with the undeclared ORF. WHO/FDA guidelines specifically state to determine if these elements are present in the plasmid.
Thank you for highlighting the security issue which as we see is very real. Having said that, this plasmid issue crossed a red line that I have not yet seen, imho. It is the Food and Drug Act (of Canada) that adulteration (omission, addition etc) is contrary to the act, and the onus is on the manufacturer to prove otherwise. It is not a regulation or a guideline. Marketing and clinical trials are in the regulations which are not legally binding and are open to interpretation. This is my assessment of the matter and I see this as quite serious.
The rolling review by the EMA reviewers did mention that the plasmid map was lacking detail and requested more data. It is the requirement of the manufacturer to provide the data; the reviewers should not have to verify a sequence or say, the structure of a new drug. themselves.
You raise a fascinating question: "If plasmids are used for protein expression, why did we stop at mRNA?" I wonder if I may offer a suggestion of what I believe could have been (part of) the reasoning.
(1) All protein-based Covid vaccines triggered, to my knowledge, a lower antibody response than those for the mRNA "vaccines." Since Ab titer was, and unfortunately (!) still is, used as a proxy for protection, this was likely critical to determine the outcome of the Warp-speed vaccine race. What I find troubling is that it appears that much of the Ab response evoked by the mRNA vaccines is, however, attributable to the LNPs. I elaborate on this in my book - https://link.springer.com/book/10.1007/978-3-031-18903-6; please see especially: Ndeupen S, Qin Z, Jacobsen S, Bouteau A, Estanbouli H, Igyártó BZ (2021) The mRNALNP platform’s lipid nanoparticle component used in preclinical vaccine studies is highly inflammatory. Iscience 24(12):103479
(2) It has long been known that protein-based vaccines have their issues too, e.g. as you still need a vector to bring the stuff inside the target cells. By contrast, LNPs were, albeit wrongfully, depicted as inert biological carriers only, as if all they did was act as a "postal container" to carry the genetic payload.
(3) It has previously been known that there are no working vaccines against respiratory viruses. Seems like by promoting and selling the mRNA shots as a novel breakthrough-technology that the narrative was created that they would be the one-and-for-all solution.
Antibodies, schmantibodies is my answer when anyone bring up "but it protects you!" etc. There still is no accepted correlate of protection. Coronavac had to bow out because its unmodified mRNA product didn't make enough antibodies, so yes.
Pure protein doesn't invoke an immune response, of course. So what part of the mRNA is the adjuvant? The LNPs (Mueller thesis), Endotoxin (Geoff Pain's thesis), the dsRNA (my choice), or all that dsDNA?
Did the manufacturers keep the mRNA vaccines a little "dirty" on purpose?
Yes indeed, you are certainly right. The mRNA products intrinsically harbor additional issues that drive the schmantibody response (love that term!).
And you raise a good point. Seems to make sense: the manufacturers may have kept the vaccines a little "dirty" on purpose! The argument could indeed have been to get the highest Ab response as possible!!! Specifically, exo-RNAs had long been portrayed as "self-adjuvants" based on the argument that foreign RNAs (such as dsRNAs which mostly, but not exclusively, indicate a foreign invader) usually do trigger hefty immune responses. The ideas was that was a GOOD thing - well, just tame it a little with the psi or other modifications - but overall, the idea that you should MAXIMIZE the immune response (focusing on the Ab titer) was the clear goal. Sigh....
I agree with you that the dsRNAs play ONE key role. I spent several chapters in my book on them, as I argue these could lead to NUMEROUS underappreciated consequences, including genotoxicity dangers -- for these, I extended the work by Jack Heinemann "Should dsRNA treatments applied in outdoor environments be regulated?" DOI: 10.1016/j.envint.2019.05.050 to the context of the vaccine (INCLUDING what does dsRNAs could do to our microbiome).
That dsRNAs are a problem with the mRNA platforms has been long known; incidentally, however, some have previously recognized it as "a double-edged sword," leaving the door open for regulators for interpretation and which side they may have focused on (I don't want to continue to point to my book, but the dsRNA issue was initially the driving force for me to write a book to begin with, and then I realized there is so much more to it, including all the microRNA concerns, that it covered several chapters....).
And then, yes, totally agree, we have all the other horrific "by-products," formed during manufacturing and/or in vivo, which not only includes the dsRNAs and other "aberrant" genetic material (DNA-RNA hybrids, short RNAs with potentially regulatory function, etc). Many of those are not even the result of Process 2 --- which then adds so many other horrors, which likely act synergistically with the other, and there is not way one would be able to tell which is the worst for which patient in which tissue etc.
Overall, it seems such a complex and inhomogeneous mess that it would be next to impossible to find ONE culprit -- which e.g. the WHO-AEFI guidelines require so that regulators could admit that an AE was CAUSED by the products...
Thank you again for all your detailed and clear work!
Regarding the choice of vaccine platform/technology... if we took a veterinary approach to vaccination, then most of our human vaccines would be live attenuated vaccines (genetically weakened versions of the virus) because of high efficacy. The poultry industry has a huge problem with the deadly coronavirus IBV, which they fight with constant re-application of a live attenuated vaccine. (The vaccine also causes outbreaks of deadly IBV variants, similar to the situation with human oral polio vaccines.)
However, the Western countries will likely embrace mRNA for economic reasons. Patent protection and captured regulators will likely lead to a mRNA duopoly. Because the technology is complex, inherently risky (all vaccines have had safety issues), and prone to manufacturing issues... the Western regulators will love it because they wield the power to end a manufacturer's profits. This gives them leverage over pharma which is reciprocated with high-paying jobs once employees leave FDA CBER, CDC, EMA, etc. etc. VAERS staffing has also grown dramatically since the onset of the pandemic (driven by the decision to ask healthcare to report breakthrough infection hospitalizations)... the bureaucracy is now much bigger and partially contracted out to the private sector.
I need to do some more digging. n the pcDNA3.1 platform, the SV40 promoter sits in front of the neomycin gene which is useful for mammalian cells because normally mammalian cells die to neomycin / G418 / geneticin.
QUOTE: However, it is important to know that both ampicillin (Amp) or kanamycin (Kan) are completely ineffective for mammalian cells selection (they can only be used for bacteria). Mammalian cells selection requires use of puromycin, geneticin (G418), zeocin, hygromycin, or blasticidin.
However, the Pfizer plasmid has the SV40 promoter in front of an Ampicillin resistance gene. That gene is not useful in mammalian cells because 'ampicillin is completely ineffective for mammalian cells selection'. So presumably the SV40 promoter is there for bacteria such as the E Coli used for Pfizer's Process 2 as it works as a promoter in bacteria too.
Presumably the SV40 and AmpR promoters in front of the Ampicillin resistance gene affects the rate of that protein's production, which affects the reproduction speed of E Coli in Process 2. The E Coli may be further genetically modified to suit Process 2, so perhaps a tweak to Amp resistance expression was needed. Because it seems like a key variable in the production process, perhaps Pfizer is hesitant to change a variable in a finely tuned production process? ("Don't fix it if it ain't broke.")
The Process 2 plasmid uses the version of the SV40 promoter with 2 copies of the 72bp enhancer. I don't know what the enhancers do in the Process 2 E Coli.
This Delta plasmid map looks a lot like the plasmid map that Kevin McKernan sequenced from the Pfizer vials. That was my main point. Plus the entire reverse ORF is present in the map as well, which is something usual. Cant figure that out
The mRNA plataform is the only "computer ready" plataform, it takes little fidgeting and mathematical equations to change the entire immunogenicity of the desired protein. They had to start mass testing to gather data to "fix" the kinds and bug somewhere I guess.
I abhor mRNA as a vaccine plataform, but I could infer their intent was based on forecasting biological threats, it is the only plataform "fast enough" for aggressive responses against, well, basically almost anything.
The biggest problem of the mRNA lies on its origins, at this point, everyone is aware it was intended as a "cancer vaccine", and it is precisely there the biggest hurdle lies and all the paradoxical effects come from. To get inside the cells without alerting the body, they literally can start "turbo cancer" at any age.
Ironic. A cancer vaccine that can induce aggressice oncogensis.
Who would have thought?? LOL
Plus there are unusual features on the Pfizer plasmid. The kind of stuff that results in more questions than answers.
Based on your interesting writeup, may I ask some follow-up questions/comments, please?
Have you previously ever noticed that the SV40 promoter, or an undeclared ORI, was added by default or accidentally? In reading various articles, it is obvious that the SV40 promoter/enhancer has long been used sort of as a positive control for numerous experiments.
Theoretically, could it have been the case that the SV40 part was during the earlier phase of the Pfizer jab development (Process 2) used as a positive control but then forgotten to take out from their model plasmids? They may argue that "it was all about speed" and that taking out the SV40 part was "just an oversight."
Finally, I would like to encourage everyone who is able to do so to please always sequence the plasmids in question and not merely rely on any (purported) digital description of them alone. Especially since the contamination issue with the Pfizer jabs has made news, it seems to me that presence of any offensive genes etc in the plasmids could easily be erased ONLINE from their DIGITAL description. This would NOT mean that they are absent from the actual/real plasmid. This is a huge safety/security issue that is largely under-appreciated, please see my article in Biosafety& Health: https://pubmed.ncbi.nlm.nih.gov/33015604/. There is a inherent GAP between the physical/biological entity and their digitized/online DESCRIPTION. If someone wanted to, this gap could easily be exploited to ingress changes (additions, deletions) that you can only know if you check both components.
Thank you.
Not an oversight. A normal working FDA/EMA would say too risky, take it out unless suitably justified. Not lazy either. Quality be Design means you start with the end product in mind, the "safest" design. Intent to deceive in at the minimum. Intent to harm looks likely especially with the undeclared ORF. WHO/FDA guidelines specifically state to determine if these elements are present in the plasmid.
Thank you for highlighting the security issue which as we see is very real. Having said that, this plasmid issue crossed a red line that I have not yet seen, imho. It is the Food and Drug Act (of Canada) that adulteration (omission, addition etc) is contrary to the act, and the onus is on the manufacturer to prove otherwise. It is not a regulation or a guideline. Marketing and clinical trials are in the regulations which are not legally binding and are open to interpretation. This is my assessment of the matter and I see this as quite serious.
The rolling review by the EMA reviewers did mention that the plasmid map was lacking detail and requested more data. It is the requirement of the manufacturer to provide the data; the reviewers should not have to verify a sequence or say, the structure of a new drug. themselves.
As always, a very interesting writeup. Thank you!
You raise a fascinating question: "If plasmids are used for protein expression, why did we stop at mRNA?" I wonder if I may offer a suggestion of what I believe could have been (part of) the reasoning.
(1) All protein-based Covid vaccines triggered, to my knowledge, a lower antibody response than those for the mRNA "vaccines." Since Ab titer was, and unfortunately (!) still is, used as a proxy for protection, this was likely critical to determine the outcome of the Warp-speed vaccine race. What I find troubling is that it appears that much of the Ab response evoked by the mRNA vaccines is, however, attributable to the LNPs. I elaborate on this in my book - https://link.springer.com/book/10.1007/978-3-031-18903-6; please see especially: Ndeupen S, Qin Z, Jacobsen S, Bouteau A, Estanbouli H, Igyártó BZ (2021) The mRNALNP platform’s lipid nanoparticle component used in preclinical vaccine studies is highly inflammatory. Iscience 24(12):103479
(2) It has long been known that protein-based vaccines have their issues too, e.g. as you still need a vector to bring the stuff inside the target cells. By contrast, LNPs were, albeit wrongfully, depicted as inert biological carriers only, as if all they did was act as a "postal container" to carry the genetic payload.
(3) It has previously been known that there are no working vaccines against respiratory viruses. Seems like by promoting and selling the mRNA shots as a novel breakthrough-technology that the narrative was created that they would be the one-and-for-all solution.
Thank you.
Agree totally.
Antibodies, schmantibodies is my answer when anyone bring up "but it protects you!" etc. There still is no accepted correlate of protection. Coronavac had to bow out because its unmodified mRNA product didn't make enough antibodies, so yes.
Pure protein doesn't invoke an immune response, of course. So what part of the mRNA is the adjuvant? The LNPs (Mueller thesis), Endotoxin (Geoff Pain's thesis), the dsRNA (my choice), or all that dsDNA?
Did the manufacturers keep the mRNA vaccines a little "dirty" on purpose?
Excellent comments, thank you.
Yes indeed, you are certainly right. The mRNA products intrinsically harbor additional issues that drive the schmantibody response (love that term!).
And you raise a good point. Seems to make sense: the manufacturers may have kept the vaccines a little "dirty" on purpose! The argument could indeed have been to get the highest Ab response as possible!!! Specifically, exo-RNAs had long been portrayed as "self-adjuvants" based on the argument that foreign RNAs (such as dsRNAs which mostly, but not exclusively, indicate a foreign invader) usually do trigger hefty immune responses. The ideas was that was a GOOD thing - well, just tame it a little with the psi or other modifications - but overall, the idea that you should MAXIMIZE the immune response (focusing on the Ab titer) was the clear goal. Sigh....
I agree with you that the dsRNAs play ONE key role. I spent several chapters in my book on them, as I argue these could lead to NUMEROUS underappreciated consequences, including genotoxicity dangers -- for these, I extended the work by Jack Heinemann "Should dsRNA treatments applied in outdoor environments be regulated?" DOI: 10.1016/j.envint.2019.05.050 to the context of the vaccine (INCLUDING what does dsRNAs could do to our microbiome).
That dsRNAs are a problem with the mRNA platforms has been long known; incidentally, however, some have previously recognized it as "a double-edged sword," leaving the door open for regulators for interpretation and which side they may have focused on (I don't want to continue to point to my book, but the dsRNA issue was initially the driving force for me to write a book to begin with, and then I realized there is so much more to it, including all the microRNA concerns, that it covered several chapters....).
And then, yes, totally agree, we have all the other horrific "by-products," formed during manufacturing and/or in vivo, which not only includes the dsRNAs and other "aberrant" genetic material (DNA-RNA hybrids, short RNAs with potentially regulatory function, etc). Many of those are not even the result of Process 2 --- which then adds so many other horrors, which likely act synergistically with the other, and there is not way one would be able to tell which is the worst for which patient in which tissue etc.
Overall, it seems such a complex and inhomogeneous mess that it would be next to impossible to find ONE culprit -- which e.g. the WHO-AEFI guidelines require so that regulators could admit that an AE was CAUSED by the products...
Thank you again for all your detailed and clear work!
Regarding the choice of vaccine platform/technology... if we took a veterinary approach to vaccination, then most of our human vaccines would be live attenuated vaccines (genetically weakened versions of the virus) because of high efficacy. The poultry industry has a huge problem with the deadly coronavirus IBV, which they fight with constant re-application of a live attenuated vaccine. (The vaccine also causes outbreaks of deadly IBV variants, similar to the situation with human oral polio vaccines.)
However, the Western countries will likely embrace mRNA for economic reasons. Patent protection and captured regulators will likely lead to a mRNA duopoly. Because the technology is complex, inherently risky (all vaccines have had safety issues), and prone to manufacturing issues... the Western regulators will love it because they wield the power to end a manufacturer's profits. This gives them leverage over pharma which is reciprocated with high-paying jobs once employees leave FDA CBER, CDC, EMA, etc. etc. VAERS staffing has also grown dramatically since the onset of the pandemic (driven by the decision to ask healthcare to report breakthrough infection hospitalizations)... the bureaucracy is now much bigger and partially contracted out to the private sector.
The pcDNA platform has some marketing literature that explains the purpose of the SV40 promoter in that plasmid.
First, read about the application of COS cells.
https://en.wikipedia.org/wiki/COS_cells#Applications
Then see page 17 of the Thermo Fisher manual for the pcDNA3.1 platform/plasmid:
https://assets.thermofisher.com/TFS-Assets/LSG/manuals/pcdna3_1_man.pdf
https://www.thermofisher.com/order/catalog/product/V79020
SV40 early promoter and origin
Allows efficient, high-level expression of the neomycin resistance gene and episomal replication
in cells expressing SV40 large T antigen.
Let me think about this some more.
I need to do some more digging. n the pcDNA3.1 platform, the SV40 promoter sits in front of the neomycin gene which is useful for mammalian cells because normally mammalian cells die to neomycin / G418 / geneticin.
QUOTE: However, it is important to know that both ampicillin (Amp) or kanamycin (Kan) are completely ineffective for mammalian cells selection (they can only be used for bacteria). Mammalian cells selection requires use of puromycin, geneticin (G418), zeocin, hygromycin, or blasticidin.
https://altogen.com/best-antibiotic-use-stable-cell-selection-mammalian-cells/
However, the Pfizer plasmid has the SV40 promoter in front of an Ampicillin resistance gene. That gene is not useful in mammalian cells because 'ampicillin is completely ineffective for mammalian cells selection'. So presumably the SV40 promoter is there for bacteria such as the E Coli used for Pfizer's Process 2 as it works as a promoter in bacteria too.
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1181807/
Presumably the SV40 and AmpR promoters in front of the Ampicillin resistance gene affects the rate of that protein's production, which affects the reproduction speed of E Coli in Process 2. The E Coli may be further genetically modified to suit Process 2, so perhaps a tweak to Amp resistance expression was needed. Because it seems like a key variable in the production process, perhaps Pfizer is hesitant to change a variable in a finely tuned production process? ("Don't fix it if it ain't broke.")
The Process 2 plasmid uses the version of the SV40 promoter with 2 copies of the 72bp enhancer. I don't know what the enhancers do in the Process 2 E Coli.
This Delta plasmid map looks a lot like the plasmid map that Kevin McKernan sequenced from the Pfizer vials. That was my main point. Plus the entire reverse ORF is present in the map as well, which is something usual. Cant figure that out