Additional follow up on DNA contamination of COVID-19 injectable products (there's more coming too)

There are circular plasmids (DNAs) as contaminants...

The saga of work being done in the lab context to confirm or deny the presence of DNA contamination in the Pfizer and Moderna shots continues. Unlike ‘bored’ meetings held behind closed doors, lab procedures take time and care (also behind closed doors, but for other reasons), and no matter how much you might want to, you can’t hurry love. Or, lab results. ‘Tis better to be smart and cautious, than first.

…The results continue to roll in like a glassy 1 meter wave at day break in a place that no one knows about.

Anandamide’s newest piece is entitled: “Sequencing of RNase A treated Pfizer bivalent vaccines reveals paired-end sequencing evidence of circular plasmids and an inter-vial 72bp variation in the SV40 promoter” and it confirms the presence of circular DNAs, as opposed to linear DNAs, as contaminants in the tested vials. By the way, if you want to watch an amazing documentary about the origins of the name Anandamide, please do. It’s called The Scientist, and it’s very relevant to the crushing blows that real science sustains, to this day.

To summarize the current situation, Kevin’s team sequenced both the RNA and DNA in samples of Moderna and Pfizer injectable products. They used an RNA-Seq kit but since this method favors RNA sequencing, the realized it might actually underestimate DNA contamination levels. Since they wanted to assess the amount of DNA contamination and quantify the ratio of linear to circular plasmid DNA, they went one step further and RNase A-treated the samples (this gets rid of any RNA in the samples) and sequenced the remaining DNA.

By the way, whenever you see ‘-ase’ tagged onto a word, it means that you’re dealing with an enzyme which is (not simply :D) a biological catalyst of a chemical reaction. So RNase just acts to break up RNA into little RNA bits. Kind of like when you cook spaghetti too long and it breaks up into smaller bits when you take it out of the pot. Sort of. The real question is: is it still spaghetti?

The following screenshot is from this latest article and shows the Pfizer version of the plasmid vector on the left and their RNase A sequencing and assembly-produced vector on the right.

The first thing I noticed was that the Pfizer disclosed plasmid contains the poly-A tail in the plasmid sequence. As a point of interest, this saves the step of having to add it after mRNA production however, it is problematic because bacteria don't support long stretches of As (anything longer than 80 AAs is too many). Longer poly-As can cause recombination events to occur which is bad for clinical material production. Also, if you wanted to extend the poly-A tail, you can use a poly-A polymerase afterwards, but this costs a lot of money to do. It could also cause problems in terms of the different distributions of poly-A tail lengths that would arise which, of course, would have to be measured and monitored, ie: reproducibility of poly-A tail lengths between batches using qualitative (and quantitative) methods. We all know how good the quality control is with regard to these products, so if they did add poly-As postfactum, I am sure they maintained consistency across the board at all the different manufacturing sites. Sarcasm detector ‘sploding. Here’s the screenshot.

Here are some definitions that I probably should have already provided:

In genetics, a promoter is a sequence of DNA to which proteins bind to initiate transcription of a single RNA transcript from the DNA downstream of the promoter.¹ It controls gene expression.

SV40 is an abbreviation for Simian Virus 40, a polyomavirus that is found in both monkeys and humans. It is a mammalian promoter with high expression driving ability with close affinity with RNA polymerase and can direct the massive synthesizing of mRNA. It improves the gene expression level of many host cells.² It promotes expression of particular genes in mammals.

The origin of replication (ORI or ori) is a particular sequence in a genome at which replication is initiated.³ It's where replication starts.

KanR is a kanamycin resistance gene cassette.⁴ It confers kanamycin resistance.

Back to the nitty gritty.

The orientation of the paired end reads (they point outward - seen in green) also provides strong evidence that the plasmid is circular. Referring to the original EMA document entitled: “Rapporteur Rolling Review critical assessment report - Quality aspects” dated November 19, 2020, where Pfizer were told that they had to address some serious problems, on page 32 of the downloadable document provided by Sasha, it clearly states the following:

No descriptions of the analytical methods used for the control of the linear DNA template nor evidence regarding their qualification/validation have been provided (OC).

This is just one of the many things that were not provided, by the way. Here’s the document. Please read it. I added page numbers.

Rapporteurs Rolling Review Report Quality Covid 19 Mrna Vaccine Biontec

16.2MB ∙ PDF file

Download

Download

Kevin gets deeper into some of these other issues raised by the EMA that relate to potential places where dsDNA or dsRNA (which could act as an adjuvant - bonus points from a vaccine point of view) contamination could occur. He explains the disparities between what should have been - according to the EMA report, and what was/is in reality really well, so I leave it to the reader to read and assess.

By the way, on the subject of contamination, as a point of interest, purifying the mRNA is really expensive and really important. It ensures the removal of things like dsRNA, truncated mRNA products, residual DNA, etc., and if the purification was really rigorous, I would imagine the problems with contamination that we are witnessing would be much reduced if not eliminated. That might be a stretch but I always like to give benefit of the doubt.

NB: My next Substack is going to be all about how the mRNA production line works. I need to do some reading on this so it won’t be published tomorrow.

Here’s something they found that is wildly concerning. Kevin writes:

Sampling just two Pfizer vials delivers two different expression vectors? These vials are from the same lot. The inserts are 100% identical and unrelated to bivalent vaccines. I wonder how many more plasmids are out there?

There are different expression vector types findable even within batches. Well I wish I could say that I am surprised by this, but I am not. The sheer number of places in the production line of mRNA where things could go wrong tells the story of this probability. Even if everything is done ‘right’ and checks and balances are made, this is still BIOLOGY: she strives for variability! The question for me is: why so many expression vectors appearing in the first place?

Last point I want to put emphasis on from this latest work is on the SV40 promoter. Kevin writes:

A known SV40 promoter improvement is evident in 1/2 of the vials from the same lot of vaccine.

Kevin’s full conclusions to date from this recent updated (and ongoing) analysis:

Conclusions

1. Paired end reads support the existence of circular plasmids in the Pfizer bivalent vaccines. This is supported by transformation in kanamycin sensitive E.coli. The exact ratio of circular versus linear DNA is still being evaluated but the levels of DNA contamination exceed the EMA specifications by several orders of magnitude.

2. EMA documentation reveals regulators are unsatisfied with the molecular methods used to evaluate linearization. There are concerns over non specific CT values (<32) in the PCR methods used for quantification. Additional concerns are voiced over the variation in DNase I activity and methods used to evaluate its performance.

3. A long poly A tract is evident in the vectors reducing the odds of off target poly adenylation. The 10bp linker isn’t well resolved with Illumina sequencing and the two vectors also differ in this artifactual regard.

4. A known SV40 promoter improvement is evident in 1/2 of the vials from the same lot of vaccine.

Summary

There is stuff in the vials that shouldn’t be there with regard to DNA contamination that came from bad manufacturing - probably could have been ‘solved’ at the purification step. The EMA knows about these inadequacies and instructed Pfizer to deal with it. They are trying their very best with their limited resources after injecting this stuff into billions of people. Circular DNAs are bad news in this context because they are replication competent. The poly-A tail is encoded in the plasmid, which is efficient from a production point of view. Great. But what about the issues mentioned above? The implications of the promoter enhancement or improvement remains to be seen but safe to say, pumpin’ up the jam of the continued expression of spike genes and prolonged spike expression might not end up being as popular as Technotronic’s tune.

1. https://en.wikipedia.org/wiki/Promoter_(genetics)

2. https://en.wikipedia.org/wiki/SV40

3. https://en.wikipedia.org//wiki/Origin_of_replication

4. https://www.addgene.org/117122/