Did Pfizer/BioNTech insert additional translational regions into their COVID vaccines? (Correction)

A new lawsuit from biotech firm Promosome against Moderna raises some serious questions about what modifications were made to the COVID vaccine's sequences.

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*Additional sentence inserted below for more context, as well as minor corrections.

Correction: In digging deeper into the actual spike sequence it does appear that the spike contains many of these secondary initiation sites. Because of this finding the below remarks are incorrect in some regard. It’s possible that the remaining secondary initiation sites seen in Pfizer/BioNTech’s vaccine may be due to codon optimization, but the number found in Pfizer/BioNTech’s vaccine is still far lower than the number found in the native spike. It is still interesting to see that there are still higher numbers of secondary initiation sites relative to Moderna, which is something worth considering.

A correction can now be found in the following post:

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On Tuesday Biotech firm Promosome LLC issued a lawsuit against Moderna over patent infringements related to technology used by Moderna for their COVID mRNA vaccines.

The lawsuit appears to allege that Moderna was made aware of Promosome’s intellectual property when president of Moderna Dr. Stephen Hoge took a visit to Promosome’s facilities and was made aware of this IP. It appears that Promosome was hoping to enter into a business partnership with Moderna, which never materialized. Interestingly, at some point so Promosome showed Moderna how they could utilize Promosome’s patented methodology on some of Moderna’s mRNA-related research, supposedly providing Moderna insight into how to use this technology for themselves.

Now, the question here is answering what exactly this new technology is.

This patent appears to relate to optimization of mRNA for higher protein synthesis. In this case, it’s a modification of codons in such a way that it removes many secondary start codon regions within the sequence, as noted in the lawsuit as well as the actual patent:

By all accounts, this is an actual revelation in determining what modifications have occurred within the sequence of the spike protein, and it provides some crucial insights into a distinction between adverse reactions between even the Pfizer/BioNTech mRNA vaccines and Moderna’s.

In order get a better grasp of what has occurred with the mRNA vaccines, it’s important to explain what start codons are.

In order to produce a protein a ribosome needs to know where to bind on a sequence. For many genes transcribed into mRNA there exists a region known as the start codon, identified by the presence of the AUG codon. This codon is easy to remember- remember that school starts in AUGust- at least, that’s how we were taught to remember this codon in school.

AUG is the only codon to encode for the amino acid methionine, and it serves as a starting point recognition for ribosomes, making it the first amino acid translated in a majority of genes.

Structure of methionine. Note that the name reveals the constituents on the sidechain, as meth refers to a methyl group (CH3) and thio refers to a sulfur group.

Methionine is usually recognized as being the first amino acid to be translated, although it serves a broader purpose than just that.

As noted by Aledo, J. C.¹ methionine is critical for hydrophobic interactions, including with aromatic amino acids, and even adds additional flexibility to protein structures:

Methionine, a sulfur‐containing amino acid, is often thought to be a generic hydrophobic residue. In fact, this is the way most Biochemistry textbooks describe this molecule. Not surprisingly, many biochemists regard this amino acid as one functionally replaceable with another hydrophobic residue. Although this might be the case for some methionine positions in some proteins, we should realize that methionine is a unique proteionogenic amino acid, remarkable in many regards. For instance, unlike other hydrophobic residues such as valine, leucine or isoleucine, the side chain of methionine is unbranched providing ample flexibility.

This extra‐flexibility is not an idle property. When several methionines are arranged on one side of an amphiphilic α‐helix, these flexible residues provide a malleable nonpolar surface that can adapt itself to peptide binding partners of varied sequence. This seems to be the case of calmodulin and the signal recognition particle 54 kDa subunit. These proteins share two relevant features: (a) they contain structural domains that are unusually rich in methionine residues, and (b) they are able to interact, in a specific way, with many protein partners that are diverse in sequence among them.

Thus, methionine serves more than just a starting position for translation, and for that reason methionine can be found dispersed within certain proteins, with some proteins being methionine-rich.

However, the problem comes with the fact that ribosomes may not be able to differentiate AUG as a start codon or AUG as being within a sequence to encode for methionine. In that regard, ribosomes may mistakenly take any region with an AUG codon as being an initiation region for translation. This is what is meant by the “secondary initiation codons” in Promosome’s patent, as ribosomes may mistake downstream AUG codons for regions to start translating, creating what are called cryptic peptides as they aren’t made up of all of the amino acids found in the original mRNA sequence.

Now, it’s important to remember that this incorrect recognition of secondary initiation codons can occur through two mechanisms:

1. Downstream AUG codons encoding for methionine may be mistaken as starting points (as stated above), or

2. Two adjacent codons may result in the formation of an AUG codon, creating an out-of-frame codon sequence akin to a frameshift mutation. For instance, a serine with the codon UCA that is adjacent to another codon UGU for cysteine would result in a sequence of UC-AUG-U. A ribosome may then improperly bind to the middle to that AUG sequence and begin translation there, shifting the entire downstream amino acid sequence.

Interestingly, Promosome’s lawsuit goes into detail about these two concepts. As an aside, it’s very interesting to see this sort of language within a lawsuit that is more technical and gets into some of the more finer details of molecular biology.

In any case, the important concept here is that regions of AUG may lead to improper translations, formation of cryptic proteins, and reduced production of the intended protein.

Thus, Promosome’s answer to this conundrum, as noted within their patent and lawsuit, is to create modifications within the specific mRNA sequence in order to reduce AUG regions, allowing for the actual start codon to be targeted by ribosomes.

And this appears to be the reason for Promosome’s lawsuit against Moderna, as Promosome alleges that Moderna used its patented methodology in the formation of their mRNA vaccines, essentially reducing the number of AUG regions within their spike’s sequence.

This practice can be termed “translation initiation fidelity”, as the intent of this process is to increase the number of correct proteins formed while reducing the number of cryptic peptides.

This term “translation initiation fidelity” apparently appeared in one of Moderna’s presentations with respect to their vaccines, and may have served as an indication that Promosome’s patented methodology was being used by Moderna.

As the lawsuit alleges in Points 12 and 13 of the Introduction & Nature of the Action, starting on Page 6:

Thus, it appears that Moderna modified some of the spike’ protein’s mRNA sequence in order to remove some of these AUG-rich regions using Promosome’s alleged approach, allowing for selection of the authentic starting region over secondary regions.

For now, I would argue that people read the lawsuit for additional information. I haven’t read it in full, and as much as we should be critical of these companies I do appreciate the technical language in the lawsuit which adds a ton of necessary context.

Distinction between Pfizer/BioNTech and Moderna’s vaccines

When coming across this lawsuit I noticed that several mainstream outlets were incorrectly alleging that Promosome was suing both Moderna and Pfizer/BioNTech.

This doesn’t appear to be the case, as any mention of Pfizer within Promosome’s lawsuit references the lawsuit between Moderna and Pfizer. I haven’t come across any supposed lawsuits between Promosome and Pfizer/BioNTech as of the time of this post.

Now, this raises a very interesting thing to consider- if Promosome hasn’t filed a lawsuit against Pfizer/BioNTech, does that mean that Pfizer/BioNTech’s mRNA vaccine doesn’t have the same “translation initiation fidelity” augmentations as Moderna’s vaccine does?

Answering this question requires examining differences between the two sequences.

The two sequences were made available online in 2021, with a GitHub link to the sequence provided here.

Keep in mind that the sequences provided on GitHub are through experimental sequencing, and don’t appear to be provided by the vaccine manufacturers, so there are likely to be errors with the sequences provided.

Nonetheless, it serves as one of the only avenues to examining the actual makeup of the sequences.

Here, if we posit that Moderna has allegedly utilized Promosome’s “translation initiation fidelity” process and Pfizer/BioNTech has not, we may infer that Moderna’s sequence should have fewer AUG regions relative to Pfizer/BioNTech.

When looking at the two sequences, this does in fact appear to be the case.

If we exclude the authentic start codon and regions within the stop codon there’s an apparent discrepancy in the number of secondary initiation regions. Note that most sequence databases use the DNA sequence, and not RNA. In this case, the codon to look for is not AUG but ATG as there will be no uridine.

If we compare the two sequences for secondary initiation regions we get the following:

• 29 in Pfizer/BioNTech’s sequence

• 12 in Moderna’s

Thus, it seems quite apparent that this sequence appears far more frequently in Pfizer/BioNTech’s vaccine.

It’s interesting that Moderna’s vaccine still contains several of these AUG-like regions. It’s possible that AUG-like regions still exist for the two conundrums above: either there is no replacement for methionine and thus AUG must be used, or an overlap between two adjacent sequences unfortunately leads to the formation an AUG-like region.

The former would be easy to assess by way of seeing if the AUG regions in Moderna’s sequence are actually associated with a codon and not out-of-frame overlaps, although I must admit that would be quite an endeavor to undertake so I’ll only speculate on that matter for now.

Regardless, it does suggest that Pfizer/BioNTech’s vaccine is far more saturated with these secondary initiation regions, and may infer something with respect to these vaccines and adverse reactions.

The actual rate of adverse reactions between Pfizer/BioNTech and Moderna have been up for debate. There’s been an argument that Moderna’s vaccine should, hypothetically, lead to more adverse reactions due to containing a higher dose of mRNA, with some evidence such as the Cho, et al.² piece from yesterday may infer. However, some evidence tends to contradict Moderna being more correlated with adverse reactions, as some reports seem to suggest that Pfizer/BioNTech’s vaccine may be associated with more cases of thrombotic adverse events.³ Note that although Cho, et al. notes a higher rate of adverse reactions per 100,000 people with Moderna’s vaccine, the reported cases of adverse reactions occurred largely among Pfizer/BioNTech recipients. It’s important to consider difference in how adverse reactions are reported.

In any given case, what’s important to consider here is that the AUG-rich regions of Pfizer/BioNTech’s vaccine means that there are greater chances of translations for off-site, cryptic peptides relative to Moderna’s vaccine. Non-fully formed peptides can have the potential to be cytotoxic, and can possibly induce autoimmunity by way of exposing immunogenic epitopic regions that may not be present in the fully formed protein.

Keep in mind that this issue may be made worse by virtue of codon optimization, as the use of synonymous amino acids with different codons may induce secondary AUG-rich regions within the sequence and therefore may lead to formation of cryptic peptides.

Interestingly, Promosome’s lawsuit even raises this concern (found on Page 19; emphasis mine):

The consequences of binding to a secondary initiation codon, then, would include reduced expression of the full-length protein and the potential creation of dangerous cryptic peptides. The latter consequence would be exacerbated by codon optimization, because while substituting synonymous codons preserves the intended codon sequence of the primary reading frame, it completely changes outof-frame codons read when elongation begins at out-of-frame secondary initiation codons. This means that codon optimization can cause the body to produce novel cryptic peptides.

Now, at this point I was going to leave the information be. However, I decided to look at the spike protein’s sequence, and there seems to be more to this mystery.

Did Pfizer’s codon optimization lead to secondary initiation site insertions?

Again, I was going to leave this post with a bit of speculation, but I became curious about these AUG-rich regions featured in Pfizer/BioNTech’s vaccine.

That is, I was curious whether these were found in the original spike protein and Moderna removed them via Promosome’s patented methodology, or whether Pfizer’s use of codon optimization secondarily lead to the insertion of these AUG-regions.

The answer here, lies in the original spike protein’s sequence.

If we take a look at the sequence, using either UniProt’s sequence or the sequence on NIH’s website we can see that, aside from the start codon, the original spike appears to have 13 methionine residues. This may affirm the fact that the codons found in Moderna’s vaccine may actually encode for methionine and not be out-of-frame codons.

Note that both UniProt and NIH’s website use the amino acid sequence and not the DNA sequence. However, given that AUG is the only codon to code for methionine this issue solves itself as there can’t be any other explanation for these AUG/ATG regions in the vaccines. Note that the letter for methionine is M.

I’ll include the amino acid sequence from NIH below since it’s much easier to follow. Note that in my assessment I excluded the M from the start codon.

So Moderna’s sequence differs only by one methionine residue, and that may be due to modifications or due to an incorrect sequence being uploaded.

But what can we glean from this information?

There’s actually two regions that I found interesting in the above sequence:

• the 3 Methionine residues nearby one another near the end of the sequence,

• as well as the M-Q-M amino acid sandwich found in the 8th to last line.

I find these regions interesting because they actually help to validate the fact that the ATG regions found in the vaccine sequences are related to methionine.

For instance, in both of the vaccine sequences there’s a 9-nucleotide sequence of ATG-CAG-ATG which can be seen below from Pfizer/BioNTech’s sequence:

CAG encodes for the amino acid glutamine which has the letter Q. Thus, the M-Q-M sequence in the spike protein confirms this region.

The 3 methionine residues are bit trickier to find since they are separated by 3 amino acids each. However, since they are at the end the search isn’t made too difficult. Note the string of amino acids below in the sequence supposedly from Moderna:

We can confirm that this sequence is the same as the original spike’s as CTG codes for leucine (L) and TGC codes for cysteine (C), thus making up the LCC amino acid sequence found in between the middle and last methionine residues. Interestingly, Pifzer/BioNTech’s vaccine appears to use the codon TGT for the first cysteine residue.

Hopefully I haven’t lost anyone yet. However, the point of this exercise is to note that there are two distinct regions that can serve as a frames of reference.

That is, in both the spike protein’s sequence and Moderna’s vaccine there should only be two methionine residues (i.e. ATG/AUG⁴ regions) that exist between the M-Q-M sequence as well as the 3-methionine residues at the end.

However, if we take a look at Pfizer/BioNTech’s vaccine do we get two ATG/AUG regions?

No, in fact there appears to be 6 ATG/AUG regions in between the two methionine regions noted above, suggesting that an additional 4 secondary initiation regions were inserted into Pfizer'/BioNTech’s vaccine.

But why exactly are they there?

Since we know that Pfizer has done codon optimization for their vaccine, I’m led to believe that the addition of these secondary initiation regions are a consequence of codon optimization as remarked on in Promosome’s lawsuit.

Note that codon optimization should not lead to formation of different amino acids, so the ATG/AUG regions in this case are not likely to encode for methionine given the original sequence only carried two methionine residues in this region of the sequence. Rather, they are likely due to adjacent codons leading to these secondary initiation regions (i.e. the second mechanism listed above).

*This means that in Pfizer/BioNTech’s vaccine there could be up to 16 secondary initiation sites inserted into the vaccine’s sequence.

Perhaps this means that Promosome’s lawsuit may not go through, given that Moderna doesn’t seem to have modified their sequence all-too differently compared to the original spike, although this is based on the sequence uploaded onto GitHub.

However, this does raise a serious question as to whether the fragments of amino acids seen with these vaccines could be due to these secondary initiation sites. It appears that Pfizer/BioNTech’s approach for codon optimization may have led to nearly doubling the possible number of sites which ribosomes may secondarily bind to, leading to formation of cryptic peptides that may be cytotoxic and immunogenic in nature.

Note that Pfizer/BioNTech’s sequence strangely includes two ATG sequences within the stop codon, as well as several within the 3’-UTR region (note that these regions weren’t included in the 29 ATG sequences cited).

Overall, this raises a serious question of whether some of these adverse reactions seen are a consequence of codon optimization and insertion of these secondary initiation sites.

Could it be possible that some of the cryptic peptides formed from these secondary sites may be causing some of the adverse reactions seen?

More investigation will be required to answer such a question, but one approach to figuring out this question may lie in the fact that the out-of-frame use of these secondary initiation sites may code for completely different amino acids relative to the original spike. That is, proteomic investigations can search for strangely encoded proteins and try to trace their sequence back to possible sequences in Pfizer/BioNTech’s vaccine, using these secondary initiation sites as a frame of reference.

Overall, this tells us that there’s more to the vaccine mystery than we were initially led to believe.

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1. Aledo J. C. (2019). Methionine in proteins: The Cinderella of the proteinogenic amino acids. Protein science : a publication of the Protein Society, 28(10), 1785–1796. https://doi.org/10.1002/pro.3698

2. Jae Yeong Cho and others, COVID-19 vaccination-related myocarditis: a Korean nationwide study, European Heart Journal, 2023;, ehad339, https://doi.org/10.1093/eurheartj/ehad339

3. Tobaiqy, M., MacLure, K., Elkout, H., & Stewart, D. (2021). Thrombotic Adverse Events Reported for Moderna, Pfizer and Oxford-AstraZeneca COVID-19 Vaccines: Comparison of Occurrence and Clinical Outcomes in the EudraVigilance Database. Vaccines, 9(11), 1326. https://doi.org/10.3390/vaccines9111326

4. Note that I am using ATG and AUG interchangeably here. They both encode methionine but it depends on whether one looks at a DNA or RNA sequence. Because the sequences uploaded to GitHub are DNA sequences I have used ATG, although please not that in any case we are referring to AUG regardless.