The studies listed below are peer reviewed papers (preprints are also included if deemed relevant) on the topic of vaccine contamination, relating to the Covid mRNA vaccines and mRNA|DNA and SV40 contamination risk. Please check back from time to time as this list is expanded with the release of new papers.
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Papers are listed by date, the most recent appearing at the top of the page. Click to expand for a full text link, author details, correspondence and abstract. Where a paper is published in multiple journals, the link provided is to a full text version. If we have missed important validated research, please log in and use the comment box below to send us a link. Acceptance of submitted links is at the discretion of the editors.
Please note that on occasion, a retracted study will still be listed. This is an editorial decision, based on the reasons for the papers retraction. Clear attempts to discredit research with a view to censorship does not warrant retraction. These papers are highlighted in red and where possible PDF versions exist on our servers.
RNA:DNA Hybrids Survive Digestion in mRNA Vaccine Manufacturing
Study Details
| FULL TEXT LINK: View Paper |
| PUBLICATION DATE: Jan, 2026 |
| PUBLICATION: Journal of Independent Medicine, Research Gate |
| AUTHORS |
| Kevin McKernan, Charles Rixey, Jessica Rose |
| CORRESPONDENCE TO |
| [email protected] |
| DOI: 10.71189/JIM/2026/V02N01A04 |
| PMID: PREPRINT |
| ABSTRACT |
| The process of mRNA vaccine manufacturing relies on proper DNA digestion following an in-vitro transcription reaction to remove residual contaminating DNA from the plasmid backbone from the process. To assess the quality and quantity of potential DNA impurities in mRNA vaccines, we analyzed unopened, cold-chain compliant vaccine lots for residual DNA contamination using quantitative PCR (qPCR), RNase A/Qβubit fluorometry, and Oxford Nanopore sequencing from two Pfizer and three Moderna vials. We compared spike-protein amplicons and plasmid-vector amplicons to distinguish between DNA contaminant as double stranded DNA (dsDNA) versus RNA:DNA hybrids, qPCR assays revealed more than a 100-fold discrepancy in quantitation between dsDNA with RNA:DNA hybrids consistent with uneven DNase I digestion efficiency during mRNA vaccine manufacturing. Indeed, treatment of vaccines with DNase I-XT resulted in 100-1000X higher degradation of spike DNA, particularly in plasmid regions that form RNA:DNA hybrids. Together these results indicate that residual DNA testing which relies on a single qPCR for dsDNA fails to accurately quantify impurities, and that treating vaccine preparations with DNase I-XT during the manufacturing process may improve the quality by reducing contamination due to RNA:DNA hybrids. |
Hyperstimulatory N⁶-methyladenine (m6A) in residual SV40 plasmid DNA in mRNA vaccines.
PAPER DETAILS
| FULL PREPRINT TEXT LINK: View Paper |
| PUBLICATION DATE: October, 2025 |
| PUBLICATION: Zenodo |
| AUTHORS |
| Kevin McKernan |
| CORRESPONDENCE TO |
| [email protected] |
| DOI: 10.5281/zenodo.17272426 |
| PMID: PREPRINT |
| ABSTRACT |
| Many mRNA vaccine production pipelines rely on Escherichia coli to replicate plasmid DNA templates used in the in vitro transcription of modified RNA. However, E. coli DNA methylation patterns differ substantially from those of humans. In E. coli, DNA methylation is primarily mediated by DNA adenine methyltransferase (Dam), which introduces N⁶-methyladenine (m6A) within GATC motifs, whereas human methylation occurs predominantly at cytosines in CpG dinucleotides. Some E. coli strains also express Dcm methyltransferase, which methylates CCWGG sequences (CC[A/T]GG), further distinguishing bacterial from mammalian epigenetic marks. Cytosolic DNA that lacks CpG methylation can potently activate Toll-like receptor 9 (TLR9), while m6A-modified DNA has been shown to stimulate the cGAS–STING pathway, leading to the induction of type I interferons and other inflammatory mediators. Because the Pfizer mRNA vaccine plasmids are propagated in E. coli, and residual plasmid DNA has been detected in finished vaccine material, it is likely that this DNA bears bacterial-type methylation patterns that could be immunostimulatory through TLR9 and cGAS–STING signaling. To investigate this possibility, we applied Oxford Nanopore sequencing to examine the methylation status of plasmid DNA present in Pfizer lot FL8095. |
Dental biofilms contain DNase I-resistant Z-DNA and G-quadruplexes but alternative DNase overcomes this resistance
STUDY DETAILS
| FULL TEXT LINK: View Paper |
| PUBLICATION DATE: May, 2025 |
| PUBLICATION: Nature, NPJ Biofilms and Microbiomes |
| AUTHORS |
| Dominique C. S. Evans, Mathilde F. Kristensen, Gabriel Antonio S. Minero, Lorena G. Palmén, Inge Knap, Manish K. Tiwari, Sebastian Schlafer, Rikke L. Meyer |
| CORRESPONDENCE TO |
| [email protected], [email protected] |
| DOI: 10.1038/s41522-025-00694-x |
| PMID: 40389511 |
| ABSTRACT |
| We demonstrate successful transfection of nucleoside-modified mRNA (modRNA) biologicals into HEK293 cells and show robust levels of spike proteins over several days of cell culture. Secretion into cell supernatants occurred predominantly via extracellular vesicles enriched for exosome markers. We further analyzed RNA and DNA contents of these vials and identified large amounts of DNA after RNase A digestion in all lots with concentrations ranging from 32.7 ng to 43.4 ng per clinical dose. This far exceeds the maximal acceptable concentration of 10 ng per clinical dose that has been set by international regulatory authorities. Gene analyses with selected PCR primer pairs proved that residual DNA represents not only fragments of the DNA matrices coding for the spike gene, but all genes from the plasmid including the SV40 promoter/enhancer and the antibiotic resistance gene. |
Confirmation of the presence of vaccine DNA in the Pfizer anti-COVID-19 vaccine
Study Details
| FULL TEXT LINK: View Paper |
| PUBLICATION DATE: May, 2025 |
| PUBLICATION: HAL |
| AUTHORS |
| Didier Raoult |
| CORRESPONDENCE TO |
| [email protected] |
| HAL ID: hal-04778576 |
| PMID: None |
| ABSTRACT |
| The rapid production of messenger RNA (mRNA)-based vaccines has been chosen as the most suitable strategy to fight the COVID-19 pandemic. Three studies reported the presence of DNA in significant amounts in Pfizer mRNA vaccines. We aimed to confirm the presence of this residual DNA. Vaccine plasmid DNA quantification using the Qubit fluorometer on a vaccine vial showed it was 216 ng/dose on average and approximately 24 times greater, reaching 5,160 ng/dose on average, after treatment with Triton-X-100. In addition, we obtained by next-generation sequencing the sequence of the complete plasmid DNA vaccine matrix (7,824 base pairs) with high coverage (98.3%) and sequencing depths (mean, 4,181-4,389 reads), indicating the presence of the plasmid DNA in high copy number. These results calls for systematic quality control of mRNA-based vaccine batches by national health authorities to assess the copy number and nature of DNA present in the different batches of vaccines placed on the market, notably regarding the putative risk of DNA integration after delivery into cells. |
Quantitative Analysis of Nucleic Acid Content in Spikevax (Moderna) and BNT162b2 (Pfizer) COVID-19 Vaccine Lots
Study Details
| FULL TEXT LINK: View Paper |
| PUBLICATION DATE: April, 2025 |
| PUBLICATION: Herald Open Access |
| AUTHORS |
| Richard M Fleming, PhD, MD, Peter Kotlár, MD, Sona Pekova, MD and PhD |
| CORRESPONDENCE TO |
| [email protected] |
| DOI: 10.24966/AVS-7397/100124 |
| PMID: Not indexed |
| ABSTRACT |
| Background: The emergence of mRNA-based COVID-19 vaccines represented a significant advancement in public health response during the pandemic. However, questions have arisen regarding the consistency of their nucleic acid content, the presence of undeclared genomic material, and the continued use of outdated viral sequences. Methods: In this study, 17 lots of Spikevax (Moderna) and 7 lots of BNT162b2 (Pfizer) vaccines were analyzed for nucleic acid content using multiplex quantitative Real-Time PCR. Samples were assessed for mRNA identity, quantity, homogeneity, and the presence of undeclared nucleic acids, including DNA elements. The stability of nucleic acid content in expired vaccine lots stored at -80°C was also examined. Results: Quantitative analysis confirmed the presence of mRNA sequences consistent with the Spikevax and BNT162b2 vaccines. However, variations in nucleic acid quantity were observed across lots. DNA sequences, including Escherichia coli genomic fragments, were detected in some samples. Despite the evolution of circulating SARS-CoV-2 variants, both vaccine types retained the original “Wuhan” S protein sequence. Expired vaccine samples exhibited reduced nucleic acid integrity. No evidence of SV40 was identified. Conclusions: The presence of undeclared DNA sequences and variability in nucleic acid content across lots underscores the need for enhanced quality control in vaccine manufacturing. The presence also raises InflammoThrombotic Immunologic Response (ITIR) concerns. Regulatory oversight should address the potential risks associated with genetic material inconsistencies to ensure vaccine safety and efficacy. |
Quantification of residual plasmid DNA and SV40 promoter-enhancer sequences in Pfizer/BioNTech and Moderna modRNA COVID-19 vaccines from Ontario, Canada
Study Details
| FULL TEXT LINK: View Paper |
| PUBLICATION DATE: April, 2025 |
| PUBLICATION: Taylor and Francis, Autoimmunity |
| AUTHORS |
| David J. Speicher, Jessica Rose, Kevin McKernan |
| CORRESPONDENCE TO |
| [email protected] |
| DOI: 10.1080/08916934.2025.2551517 |
| PMID: 40913499 |
| ABSTRACT |
| For some of the COVID-19 vaccines, the drug substances released to market were manufactured differently than those used in clinical trials. Manufacturing nucleoside-modified mRNA (modRNA) for commercial COVID-19 vaccines relies on RNA polymerase transcription of a plasmid DNA template. Previous studies identified high levels of plasmid DNA in vials of modRNA vaccines, suggesting that the removal of residual DNA template is problematic. Therefore, we quantified the DNA load in a limited number of Pfizer-BioNTech and Moderna COVID-19 modRNA vaccine vials using two independent methods. Total DNA and specific DNA targets were quantified by Qubit fluorometry and quantitative polymerase chain reaction (qPCR), respectively on 32 vials representing 16 unique vaccine lots. RNase A treatment was used to assess the impact of RNA crosstalk in DNA fluorometry. A preliminary assessment of DNA fragment length and DNase I sensitivity were also performed. Total DNA ranged 371–1,548 ng/dose and 1,130–6,280 ng/dose in Pfizer and Moderna products, respectively. Specific DNA of multiple plasmid DNA targets ranged 0.22–7.28 ng/dose for Pfizer, and 0.01–0.78 ng/dose for Moderna. The SV40 promoter-enhancer-ori (0.25–23.72 ng/dose) was only detected in Pfizer vials. Oxford Nanopore sequencing of one vial found mean and maximum DNA fragment lengths of 214 bp and 3.5 kb, respectively. These data demonstrate the presence of 1.23 × 108 to 1.60 × 1011 plasmid DNA fragments per dose encapsulated in lipid nanoparticles. Using fluorometry, total DNA in all vials tested exceeded the regulatory limit for residual DNA set by the US Food & Drug Administration (FDA) and the World Health Organization (WHO) by 36–153-fold for Pfizer and 112–627-fold for Moderna after accounting for nonspecific binding to modRNA. When tested by qPCR, all Moderna vials were within the regulatory limit, but 2/6 Pfizer lots (3 vials) exceeded the regulatory limit for the SV40 promoter-enhancer-ori by 2-fold. The presence of the SV40 promoter-enhancer element in Pfizer vials raises significant safety concerns. This study emphasizes the importance of methodological considerations when quantifying residual plasmid DNA in modRNA products, considering increased LNP transfection efficiency, and cumulative dosing presents significant and unquantified risks to human health. |
BioNTech RNA-Based COVID-19 Injections Contain Large Amounts Of Residual DNA Including An SV40 Promoter/Enhancer Sequence
Study Details
| , FULL TEXT LINK: View Paper |
| PUBLICATION DATE: Dec, 2024 |
| PUBLICATION: Public Health Policy Journal, Research Gate |
| AUTHORS |
| Ulrike Kämmerer, Verena Schulz, Klaus Steger |
| CORRESPONDENCE TO |
| [email protected] |
| DOI: None issued |
| PMID: None issued |
| ABSTRACT |
| BNT162b2 RNA-based COVID-19 injections are specified to transfect human cells to efficiently produce spike proteins for an immune response. Methods: We analyzed four German BNT162b2 lots applying HEK293 cell culture, immunohistochemistry, ELISA, PCR, and mass spectrometry. Results: We demonstrate successful transfection of nucleoside-modified mRNA (modRNA) biologicals into HEK293 cells and show robust levels of spike proteins over several days of cell culture. Secretion into cell supernatants occurred predominantly via extracellular vesicles enriched for exosome markers. We further analyzed RNA and DNA contents of these vials and identified large amounts of DNA after RNase A digestion in all lots with concentrations ranging from 32.7 ng to 43.4 ng per clinical dose. This far exceeds the maximal acceptable concentration of 10 ng per clinical dose that has been set by international regulatory authorities. Gene analyses with selected PCR primer pairs proved that residual DNA represents not only fragments of the DNA matrices coding for the spike gene, but of all genes from the plasmid including the SV40 promoter/enhancer and the antibiotic resistance gene. Conclusion: Our results raise grave concerns regarding the safety of the BNT162b2 vaccine and call for an immediate halt of all RNA biologicals unless these concerns can be dispelled. |
Understanding the impact of in vitro transcription byproducts and contaminants
Study Details
| FULL TEXT LINK: View Paper |
| PUBLICATION DATE: July, 2024 |
| PUBLICATION: Frontiers |
| AUTHORS |
| Robin Lenk, Werner Kleindienst, Gábor Tamás Szabó, Markus Baiersdörfer, Gábor Boros, Jason M. Keller, Azita J. Mahiny, Irena Vlatkovic |
| CORRESPONDENCE TO |
| [email protected] |
| DOI: 10.3389/fmolb.2024.1426129 |
| PMID: 39050733 |
| ABSTRACT |
| The success of messenger (m)RNA-based vaccines against SARS-CoV-2 during the COVID-19 pandemic has led to rapid growth and innovation in the field of mRNA-based therapeutics. However, mRNA production, whether in small amounts for research or large-scale GMP-grade for biopharmaceutics, is still based on the In Vitro Transcription (IVT) reaction developed in the early 1980s. The IVT reaction exploits phage RNA polymerase to catalyze the formation of an engineered mRNA that depends on a linearized DNA template, nucleotide building blocks, as well as pH, temperature, and reaction time. But depending on the IVT conditions and subsequent purification steps, diverse byproducts such as dsRNA, abortive RNAs and RNA:DNA hybrids might form. Unwanted byproducts, if not removed, could be formulated together with the full-length mRNA and cause an immune response in cells by activating host pattern recognition receptors. In this review, we summarize the potential types of IVT byproducts, their known biological activity, and how they can impact the efficacy and safety of mRNA therapeutics. In addition, we briefly overview non-nucleotide-based contaminants such as RNases, endotoxin and metal ions that, when present in the IVT reaction, can also influence the activity of mRNA-based drugs. We further discuss current approaches aimed at adjusting the IVT reaction conditions or improving mRNA purification to achieve optimal performance for medical applications. |
Methodological Considerations Regarding the Quantification of DNA Impurities in the COVID-19 mRNA Vaccine Comirnat
Study Details
| FULL PREPRINT TEXT LINK: View Paper |
| PUBLICATION DATE: May, 2024 |
| PUBLICATION: MDPI |
| AUTHORS |
| Brigitte König and Jürgen O. Kirchner |
| CORRESPONDENCE TO |
| [email protected], [email protected] |
| DOI: 10.3390/mps7030041 |
| PMID: 38804335 |
| ABSTRACT |
| DNA impurities can impact the safety of genetically engineered pharmaceuticals; thus, a specific limit value must be set for them during marketing authorisation. This particularly applies to mRNA vaccines, as large quantities of DNA templates are used for their production. Furthermore, when quantifying the total DNA content in the final product, we must observe that, in addition to the mRNA active ingredient, DNA impurities are also encased in lipid nanoparticles and are therefore difficult to quantify. In fact, the manufacturer of the mRNA vaccine Comirnaty (BioNTech/Pfizer) only measures DNA impurities in the active substance by means of a quantitative polymerase chain reaction (qPCR), whose DNA target sequence is less than just 1% of the originally added DNA template. This means that no direct DNA quantification takes place, and compliance with the limit value for DNA contamination is only estimated from the qPCR data using mathematical extrapolation methods. However, it is also possible to dissolve the lipid nanoparticles with a detergent to directly measure DNA contamination in the final product by using fluorescence spectroscopic methods. Experimental testing of this approach confirms that reliable values can be obtained in this way. |
DNA fragments detected in monovalent and bivalent Pfizer/BioNTech and Moderna modRNA COVID-19 vaccines from Ontario, Canada: Exploratory dose response relationship with serious adverse events.
Study Details
| FULL PREPRINT TEXT LINK: View Paper |
| PUBLICATION DATE: October, 2023 |
| PUBLICATION: OSF |
| AUTHORS |
| David J. Speicher, Jessica Rose, L. Maria Gutschi, David Wiseman, Kevin McKernan |
| CORRESPONDENCE TO |
| [email protected] |
| DOI: 10.31219/osf.io/mjc97 |
| PMID: PREPRINT |
| ABSTRACT |
| In vitro transcription (IVT) reactions used to generate nucleoside modified RNA (modRNA) for SARS-CoV-2 vaccines currently rely on an RNA polymerase transcribing from a DNA template. Production of modRNA used in the original Pfizer randomized clinical trial (RCT) utilized a PCR-generated DNA template (Process 1). To generate billions of vaccine doses, this DNA was cloned into a bacterial plasmid vector for amplification in Escherichia coli before linearization (Process 2), expanding the size and complexity of potential residual DNA and introducing sequences not present in the Process 1 template. It appears that Moderna used a similar plasmid-based process for both clinical trial and post-trial use vaccines. Recently, DNA sequencing studies have revealed this plasmid DNA at significant levels in both Pfizer-BioNTech and Moderna modRNA vaccines. These studies surveyed a limited number of lots and questions remain regarding the variance in residual DNA observed internationally. |
High spontaneous integration rates of end-modified linear DNAs upon mammalian cell transfection
Study Details
| FULL TEXT LINK: View Paper |
| PUBLICATION DATE: April, 2023 |
| PUBLICATION: PubMed |
| AUTHORS |
| Samuel Lim, R Rogers Yocum, Pamela A Silver, Jeffrey C Way |
| CORRESPONDENCE TO |
| [email protected], [email protected] |
| DOI: 10.1038/s41598-023-33862-0 |
| PMID: 37100816 |
| ABSTRACT |
| In gene therapy, potential integration of therapeutic transgene into host cell genomes is a serious risk that can lead to insertional mutagenesis and tumorigenesis. Viral vectors are often used as the gene delivery vehicle, but they are prone to undergoing integration events. More recently, non-viral delivery of linear DNAs having modified geometry such as closed-end linear duplex DNA (CELiD) have shown promise as an alternative, due to prolonged transgene expression and less cytotoxicity. However, whether modified-end linear DNAs can also provide a safe, non-integrating gene transfer remains unanswered. Herein, we compare the genomic integration frequency upon transfection of cells with expression vectors in the forms of circular plasmid, unmodified linear DNA, CELiDs with thioester loops, and Streptavidin-conjugated blocked-end linear DNA. All of the forms of linear DNA resulted in a high fraction of the cells being stably transfected-between 10 and 20% of the initially transfected cells. These results indicate that blocking the ends of linear DNA is insufficient to prevent integration. |
