A Study by a Leading Expert in Sequencing Methods for DNA and RNA finds that COVID mRNA Shots Contain Excessive Quantities of Bacterial DNA that Cause Severe Health Risks

by Michael Palmer, MD and Jonathan Gilthorpe, PhD

From [HERE] Recent studies by Kevin McKernan, a leading expert in sequencing methods for DNA and RNA, have revealed that batches of the modified mRNA vaccines produced by both Pfizer and Moderna contain a high proportion of contaminating bacterial DNA. In all, the DNA accounts for up to 20-35% of the nucleic acids contained in each of the vaccine batches. These alarmingly high concentrations far exceed the levels deemed safe by standard-setting organizations such as the European Medicines Agency (EMA). This document summarizes the evidence of that DNA contamination and discusses what possible health risks it implies to the recipients of the vaccines.

1. The role of DNA in the manufacture of mRNA vaccines

1.1. General background

Most readers will be aware that

1. the synthetic RNAs contained in the COVID-19 mRNA vaccines encode the SARS-CoV-2 spike protein;

2. in living mammalian cells, the instructions for building a given protein molecule are stored as a gene within the DNA inside the nucleus;

3. to build a given protein molecule, the cell first transcribes its gene into RNA and modifies the two ends of this molecule to form messenger RNA (mRNA). The mRNA is then transported from the nucleus to the cytoplasm, where it induces the cell’s protein factories—the ribosomes—to translate the mRNA’s nucleotide sequence into the corresponding amino acid sequence and assemble the protein.

1.2. Steps in the manufacture of mRNA vaccines

Since the spike protein is a large molecule, so is the mRNA which encodes it. The total chemical synthesis of large mRNA molecules is not practical at scale. Therefore, in order to obtain the mRNA molecule encoding spike, the process by which cells produce their own mRNAs is mimicked in vitro. This involves the following steps:

1. A DNA copy of the gene for the spike protein is inserted into a bacterial plasmid. This is a ring-shaped, double-stranded DNA molecule which can exist in a bacterial cell independently of the cell’s own chromosomal DNA, and which can also be copied and passed on to both daughter cells when that cell divides.

2. The recombinant (artificial) plasmid carrying the spike protein gene is introduced into a cell of the bacterial species Escherichia coli (E. coli ). Since E. coli cells divide very rapidly, this one cell can within a short time be grown up to a very large number of cells. Each of these progeny cells will contain their own inherited copies of the plasmid, and therefore of the spike protein gene.While there is a certain chance of the plasmid being lost from some of the offspring during successive cell divisions, we can enforce its maintenance by giving it a selectable marker, which ensures that only those cells which retain the plasmid will survive. With the plasmids used by both Pfizer and Moderna, this selection marker is a gene which endows the host cells with resistance to the antibiotic kanamycin. To apply the selection, the bacteria are simply grown in the presence of kanamycin.

3. After growing up a sufficient number of bacterial cells in a nutrient broth containing kanamycin, these cells are broken up and the plasmid DNA is purified from the other bacterial cell components.

4. The ring-shaped plasmid molecules are converted to linear form using a restriction enzyme, which cleaves both strands of the DNA molecule at a specific, unique site which is located downstream of the spike protein gene. This step is needed to prevent the formation of RNA molecules that are overly long and might have undesired effects in vivo.The linearized DNA molecules can be purified from remaining circular ones, but in what manner and how efficiently this may be done in the production of Pfizer’s and Moderna’s vaccines is not public knowledge.

5. An RNA polymerase is used, in the presence of the necessary nucleoside building blocks and cofactors, to copy the spike protein gene from the DNA version on the linearized plasmid into the mRNA version. Both Pfizer and Moderna employ the T7 RNA polymerase, which is derived from the eponymous bacteriophage. This enzyme binds to a cognate promotersequence likewise derived from T7 that has been engineered into the plasmid upstream of the gene for the spike protein. This interaction between polymerase and promoter initiates the transcription.At this stage, the synthetic nucleoside N-methyl-pseudouridine (mψU) is incorporated into the artificial RNA instead of the natural uridine nucleoside. When delivered in the form of a vaccine, RNA modified in this manner is less stimulatory to the innate immune system than is RNA containing the natural uridine. It is also more efficiently translated into protein, and under certain conditions more resistant to degradation [1]. Both Pfizer’s and Moderna’s mRNA vaccines contain mψU instead of uridine.

6. The two ends of each RNA molecule are coupled enzymatically to certain moieties that are also found at these positions within natural mammalian mRNAs, and which enhance its biological activity and stability in vivo.

This steps provide a functional mRNA which is capable of instructing the cells’ ribosomes to produce the spike protein. However, at this stage the product is not yet pure—all of the bacterially derived template DNA is still present. The latter should not be included in the final drug product, because it poses health risks to the recipients (see Section 4). To get rid of this DNA, another enzyme called DNase is added. This should break up the DNA into smaller fragments, which can then be removed from the much larger RNA molecules by filtration and other purification techniques. In the final step, the mRNA is combined with a lipid mixture in order to package it into lipid nanoparticles (LNPs), which induce human cells to take up the mRNA molecule and to make the spike protein.

2. What did we know previously about the DNA contamination problem?

In a nutshell, very little. The FDA’s assessment reports on both vaccines [2,3] do not mention the issue at all. The European Medicines Agency (EMA) assessment report on the Pfizer vaccine mentions that “The robustness of the DNase digestion step is not considered comprehensively demonstrated” [4, p. 17]. Similar language is used in the EMA report on the Moderna vaccine [5, p. 19f]. However, based on this sparse information alone, it is impossible to say whether the problem was considered serious, and what remedies were required by the regulator, if any.

3. Independent evidence about DNA contamination of mRNA products

As of April 3, 2023, Kevin McKernan has described his recent findings in three articles on his Substack site [6–8]. The experiments described in the first two reports were carried out on samples of newly introduced “bivalent” vaccines from Pfizer and Moderna. These preparations resemble the previous “monovalent” ones in their chemical composition, i.e. they should contain highly pure mRNA, complexed with a mixture of lipid (fat-like) molecules into mRNA /lipid nanoparticles. The only difference between the two varieties is that the bivalent vaccines contain a mixture of two mRNAs encoding two antigenic variants of the spike protein. This has no bearing on the technical problem of DNA contamination as such. We note, however, that the extent of DNA contamination may vary between production batches, and that only a small number of batches has so far been characterized in this regard.

3.1. McKernan’s first report

In an initial study [6], McKernan characterized both the RNA and the DNA contained in the mRNA vaccines.

3.1.1. Extraction and direct characterization of nucleic acids from the vaccines

The first step consisted in stripping away the lipids in order to obtain the pure nucleic acids. The solvent-based method that he used does not discriminate between DNA and RNA—if both are present, both will be recovered. The extracted nucleic acids were separated according to size. This revealed not only the expected regular, full-length spike mRNA species, but also smaller fragments, which had been noted previously both by the regulators and in work published by one of the manufacturers [9]. More surprisingly, RNA species larger than the full-length mRNA were also found. These species remain uncharacterized.

3.1.2. Amplification of the extracted nucleic acids

As a preparatory step for determining the exact nucleotide sequences of the extracted nucleic acids, they were amplified by PCR methods. In the case of the RNA, PCR was preceded by reverse transcription into DNA using a dedicated enzyme (reverse transcriptase). Since this study’s primary goal was to study the RNA rather than the DNA, this amplification step was biased against DNA through the addition of actinomycin D, which under the given experimental conditions selectively inhibits DNA synthesis. Accordingly, relatively low amounts of DNA were recovered in the amplified sample. Nevertheless, in case of the Pfizer vaccine, the amount of DNA determined to be present already exceeded EMA’s arbitrarily decided limit for the maximal permissible proportion of DNA per RNA.

3.1.3. DNA sequencing results

With both Pfizer’s and Moderna’s products, DNA sequences of complete DNA plasmids were obtained, although some ambiguity remained in the case of the Moderna plasmids. The features of the plasmid sequences will therefore be discussed in connection with McKernan’s second study, which used more and more pure DNA for sequencing and therefore provided more reliable results.

3.2. McKernan’s second report

The second study [7] focused on quantifying and characterizing the DNA contamination that was qualitatively detected in the first one.

3.2.1. Plasmid DNA contained in the mRNA vaccines is competent to propagate in bacterial cells

In the first experiment, it was determined whether the plasmid DNA whose presence had been inferred from the previous sequencing results is indeed biologically functional, to the extent that it can be introduced into and persist within bacterial cells. To this end, nucleic acids were again extracted from the vaccine samples. These nucleic acids were mixed with a suspension of E. coli cells that had been rendered competent for DNA uptake.

After inducing these cells to take up the DNA and giving them some time to recover, they were spread onto Petri dishes filled with solidified growth medium containing kanamycin. As noted earlier, kanamycin will kill any E. coli cells that do not contain a resistance gene to it. Therefore, the observed growth of bacterial colonies on those Petri dishes confirmed that some cells had indeed acquired resistance to kanamycin by taking up and propagating the plasmids. This was observed with both the Pfizer and the Moderna vaccine samples.

In this context, we should note that only circular plasmid molecules, but not linearized ones, can be efficiently introduced into bacterial cells. The success of this experiment therefore suggests that some of the plasmid molecules had escaped the linearization step (step 4 in Section 1.2) and made it all the way through the production process in the circular form which exists in bacterial cells. On the other hand, since the number of bacterial colonies observed in this experiment was not high, it is likely that most of the DNA had indeed been linearized. Because the biological hazards of foreign DNA within our own body may vary depending on whether it is linear or circular, the likely presence of both forms in the vaccines is worth noting. The exact proportions of circular and linear DNA in the mixtures remain to be determined.

3.2.2. The abundance of contaminating DNA

The second major finding of this study is the quantitation by PCR of both DNA and mRNA contained in the vaccine samples. As you may be aware, in a PCR reaction, a chosen segment of a nucleic acid sequence is reduplicated by enzymatic synthesis in several successive reaction cycles. From the number of cycles (or doublings) necessary to reach a certain threshold concentration, we can calculate how many copies of the target sequence were present at the outset.

In these experiments, the chosen experimental format was multiplex PCR, i.e. two target sequences were amplified in a single reaction mixture. One of these targets was within the spike protein gene, and it thus should be present both on the plasmid DNA molecules and on the spike mRNA molecules transcribed from them. In order to include the mRNA molecules in this amplification, PCR was again preceded by reverse transcription.

The other target sequence was within the kanamycin resistance gene, which should be present only on the plasmid DNA. By comparing the number of cycles required for each of the two targets to cross the threshold, it was determined that up to 35% of the total nucleic acid contained in the vaccines is in fact DNA. For comparison, the EMA has stipulated that DNA should not amount to more than 0.033% of the total nucleic acids.

3.2.3. Determination of plasmid DNA sequences

The plasmids that had originally been contained in the vaccines and then been introduced into bacterial cells (see Section 3.2.1) were again isolated from those bacterial cultures, and their complete DNA sequences were determined. Such sequences were provided in full in McKernan’s first study [6], but he indicated that he was still working on corroborating and refining the sequencing data. Meanwhile, the functional features of the plasmid DNA found in the Pfizer vaccine samples are shown in Figure 1. They will be discussed in connection with the risk assessment.