Quantitative Analysis of Nucleic Acid Content in Spikevax (Moderna) and BNT162b2 (Pfizer) COVID-19 Vaccine Lots

Infectious processes, including that caused by Severe Acute Respiratory Syndrome Coronavirus number 2 (SARS-CoV-2) result in an InflammoThrombotic Immunologic Response (ITIR) Disease (ITIRD) [1,2], in this instance Coronavirus Disease first described in 2019 (COVID-19). While medical management is possible [3], enhancement of immunity can be acquired through the administration of vaccines [4,5]. 

The development of mRNA-based COVID-19 vaccines, such as Spikevax (Moderna) and BNT162b2 (Pfizer), has been pivotal in mitigating the global pandemic. These vaccines rely on modified messenger RNA (mRNA) molecules to instruct cells to produce the SARS-CoV-2 spike (S) protein, triggering an immune response. 

While mRNA vaccines underwent rigorous clinical evaluation, emerging concerns have prompted further examination of their nucleic acid content and batch consistency. In particular, discrepancies in genomic material, including the presence of undeclared DNA sequences, have raised questions about manufacturing integrity and potential biological risks. 

This study was conducted in response to a formal request by Peter Kotlár, MD, Plenipotentiary for the Government of the Slovak Republic, to assess the nucleic acid content in multiple lots of Spikevax and BNT162b2 vaccines. The analysis aimed to: 

• Identify the nucleic acids present in the vaccine preparations.
• Quantify nucleic acid content across multiple lots.
• Evaluate batch homogeneity.
• Determine whether undeclared genetic material was present.
• Assess potential biological implications of unexpected nucleic acids.
• Examine the effects of expired storage conditions on nucleic acid stability.
• The findings provide insights into vaccine composition, stability, and manufacturing practices, with implications for public health policy and regulatory oversight.

Terminology 

Given the relative infancy of this field of genetic vaccines, we provide the following terminology. 

mRNA – messenger RNA; a nucleic acid to convey the genetic information contained in it into a protein by translation, DNA – deoxyribonucleic acid, in this project used in the sense of double-stranded DNA (dsDNA), Multiplex quantitative Real-Time PCR – Real-time PCR, allowing quantitative simultaneous detection of several molecular targets at the same time, Expression cloning vector – a circular double-stranded DNA molecule, which is used for subcloning, propagation and expression of a transgene. Transgene – a foreign, often artificially created, synthetic nucleic acid construct (typically DNA) intended for the purposes of genetic modification of a recipient cell or organism. 

GMO – genetically modified organism, and S protein – one of the SARS-CoV-2 proteins composing the viral spike which attaches to the surface of cells, the genetic sequence of which differs between individual variant strains of the virus. 

Sample collection and handling 

Twenty-four distinct vaccine lots (17 Spikevax, 7 BNT162b2) were provided under controlled conditions. Each lot contained 10 original, unopened vials stored at -80°C. Transport conditions were verified using an onboard thermometer to ensure continuous temperature control.

The package contained the following lots 

Spikevax (Moderna): MV1013A, 200023A, 200156A, 223049, 200090A, 200106A, 200100A,

3005885, 3005836, 000090A, 000058A, 3005241, 3005697, 3006272, MV1018A, 400012A,

400011A. 

BNT162b2 (Pfizer): FP9632, 1F1051A, 1LO84A, 1F1047A, 1F1059A, 1F1055A, PCB0020. 

Nucleic acid isolation 

Samples were thawed rapidly and processed using the QIAamp DNA Mini Kit (Qiagen, DE) following the manufacturer’s protocol. DNA and RNA were eluted in 50 µl of elution buffer. Reverse transcription was performed using the verso cDNA Kit (ThermoFisher Scientific, USA) at 47°C for 1 hour. All isolates were subsequently stored at -80°C. 

Analytical procedure used 

From each tested lot (containing 10 individual vials in labeled boxes), 5 original, unopened, unused vials of the product were used. The remaining unused vials were left intact as reference material at -80°C. The residues after removing the appropriate volume for analysis (see below) were secured with parafilm and returned to -80°C for further reference. 

DNA and RNA isolation 

DNA and RNA isolation was performed using a commercial isolation kit QIAamp DNA Mini Kit (Qiagen, DE) according to the manufacturer's instructions. Vials of individual lots intended for processing were removed from -80°C and quickly thawed in a stream of air at ambient temperature. 500 μl of each sample was pipetted into pre-prepared tubes with Proteinase K and Lysis Buffer. After incubation for 10 minutes at 60°C, the lysate was precipitated with 96°C ethanol and centrifuged through isolation columns. Isolation columns were then washed with buffers with different ethanol contents and the filters were air-dried. DNA and RNA were eluted into 50 μl of Elution Buffer. 

4 μl of the DNA/RNA isolate each was used for reverse transcription using the verso cDNA kit (ThermoFisher Scientific, USA) according to the manufacturer's instructions. Reverse transcription was performed at 47°C for 1 hour. 

DNA/RNA isolates were stored at -80°C, cDNA at -20°C. 

PCR analysis

Multiplex quantitative Real-Time PCR was performed using oligonucleotides specific to:

• mRNA for the S protein (manufacturer-declared molecular target)
• DNA Ori sequence (contained in expression cloning vectors)
• DNA ITS Escherichia coli (to detect possible contamination from bacterial DNA used in vector propagation)

Molecular targets for quantitative multiplex Real-Time PCR 

The following manufacturer-declared and manufacturer-non-declared molecular targets were

used to analyze the content of individual lots at the nucleic acid level: 

• mRNA for S protein, manufacturer-declared molecular target; sequence in Spikevax GenBank: OK120841; sequence in BNT162b2 GenBank: OR134577 

• DNA Ori contained in the expression cloning vector, GenBank: OR134577 

• DNA ITS Escherichia coli – Internal Transcribed Spacer in the 16S rDNA cassette, 

GenBank: AP027563, to assess the level of possible contamination of the samples with genomic DNA of Escherichia coli used for the propagation of expression vectors during the manufacturing processes. 

Genomic reference sequences were obtained from GenBank, including 

• Original Wuhan SARS-CoV-2 reference strain sequence (GenBank: MT192773) [6]
• Spikevax mRNA sequence (GenBank: OK120841) [7]
• BNT162b2 mRNA sequence (GenBank: OR134577) [8]

Nucleic acid identification 

PCR analysis confirmed the presence of mRNA sequences corresponding to the declared Spikevax and BNT162b2 vaccine profiles. Sequence alignments demonstrated significant differences between the vaccine mRNA sequences and the original Wuhan SARS-CoV-2 spike (S) protein mRNA sequence (GenBank: MT192773). These differences reflect intentional modifications made to enhance mRNA stability and translation efficiency.

Despite these engineered differences, the encoded S protein in both vaccines contained only two amino acid substitutions (K986P and V987P) relative to the Wuhan strain. This stabilization strategy is consistent with manufacturer documentation. 

Oligonucleotides for quantitative multiplex Real-Time PCR 

The mRNA sequences in Spikevax and BNT162b2 differ not only significantly from each other, but also, from the original SARS-CoV-2 mRNA sequence of the original circulating strain

“Wuhan” (GenBank MT192773). 

These differences were used to distinguish mRNAs from different sources. mRNAs can have different sequences and will still encode the same protein – the codons (triplets encoding amino acids) can differ for individual amino acids; at least at their third position; codon sequence heterogeneity for amino acids is high. 

At the protein level, the S protein in both Spikevax (Moderna) and BNT162b2 (Pfizer) is almost identical and differs from the S protein of the original SARS-CoV-2 “Wuhan” strain that circulated in March 2020 by only two amino acid substitutions, K986P (Lys986Pro) and V987P (Val987Pro). The results are shown in figures 1-5. 

Based upon the bioinformatic analysis a region was identified in the mRNA sequence of Spikevax and BNT162b2 that was sufficiently homologous to allow primers to be designed for amplification of both Spikevax and BNT162b2 S protein mRNAs, and at the same time sufficiently heterologous to allow fluorescently labeled hybridization probes to accurately discriminate between Spikevax and BNT162b2 S protein mRNAs by quantitative Real-Time PCR. The region that was selected is shown in figure 6.

Figure 6: Region near the 3’ end of the S protein mRNA of Spikevax (Query) and BNT162b2 (Subject) sequences targeted by quantitative Real-Time PCR. Common forward primer (red), common reverse primer (blue), probe-discriminating region (BNT162b2 in green - FAM, Spikevax in orange - ROX).

Primers and fluorescently labelled probe for quantitative Real-Time PCR detection of Escherichia coli genomic DNA (HEX) were designed to the internal transcribed spacer region (ITS) of the 16S rDNA cassette. The assay has been validated according to ISO 13485 and is routinely diagnostically used by the laboratory. 

All primers and fluorescently labeled hybridization probes used were custom synthesized by Eurofins Genomics, DE.

The sequences of the primers and fluorescently labeled hybridization probes used in the multiplex quantitative Real-Time PCR of mRNA and expression cassettes for S protein Spikevax, BNT162b2, expression cloning vector and Escherichia coli genomic DNA are shown in table 1.

Table 1: Primes and fluorescent labelling. 

Primers and the fluorescently labeled probe (Cy5) for quantitative Real-Time PCR of the expression cloning DNA vector map to the Ori promoter region of Pfizer bivalent expression vector BNT162b2 (GenBank OR134577) and their sequences are published elsewhere.

Design and validation of quantitative multiplex Real-Time PCR

Since the declared mRNA in both Spikevax and BNT162b2 is modified with pseudouridine and it is not known to what extent this modification was performed by the manufacturer, it was not possible to have it artificially synthesized, as is a common practice for construction of calibration curves based on precise knowledge of the sequence, sequence length, and mass of the synthesized target sequence. 

Therefore, we used an alternative method where the Spikevax cDNA and BNT162b2 cDNA were diluted in serial log dilutions to assess whether the difference in the amplification curves for a given assay corresponds to a difference of approximately 3 cycles (Ct), which is a parameter of a correctly designed test and optimal reaction efficiency of all reaction components. If the measured difference in Ct values in individual logarithmic dilutions is approximately 3 cycles, i) the measurement results can be considered valid and ii) it is possible to use a universal calibration curve, which we routinely employ for similar purposes in cases when quantitation of an unknown target is needed. 

The universal calibration curve was created by averaging calibration curves constructed using serial dilutions of synthetic, precisely defined genomic fragments of 50 microorganisms, including ssRNA viruses, dsRNA viruses, ssDNA viruses, dsDNA viruses, bacteria and fungi (molecular targets that the laboratory routinely diagnostically examines using quantitative Real-Time PCR). 

The universal calibration curve equation we used in this investigation was 

[Eq. 1] conc =10^(-0.257*Ct+11.897). 

Using this equation, we calculated the number of copies of the target sequence in a given volume using the obtained Ct values for each individual amplicon. 

Quantitative Real-Time PCR was performed using FastStart™ Taq DNA Polymerase, Roche, USA, with 2 mM MgCl2 final. Nucleotides were purchased from Sigma, DE. 

The temperature profile for amplification of all molecular targets in quantitative multiplex Real Time PCR was as follows: 94°C 5 min initial denaturation, then 50 cycles: 94°C for 20 sec, 57°C for 30 sec, then 72°C for 30 sec. 

These results for Moderna and Pfizer are shown in figures 7 & 8 respectively. 

Figure 7: Spikevax cDNA dilution experiment – concentrated cDNA and 4 serial log dilutions. 

Figure 8: BNT162b2 cDNA dilution experiment - concentrated cDNA and 4 serial log dilutions.

Presence of undeclared DNA sequences

A notable observation made during this initial validation was that the cDNA isolates were heavily contaminated with the expression cloning vector. 

These unexpected DNA sequences were identified in several vaccine lots. Analysis confirmed the presence of bacterial genomic DNA fragments from Escherichia coli, specifically sequences from the 16S rDNA internal transcribed spacer (ITS) region. These DNA elements are associated with bacterial vectors used during mRNA manufacturing. 

For the sake of completeness, we carried out serial log dilution curves of the DNA of the contaminating expression vector, which were measured using the quantitative multiplex Real-Time PCR test used, essentially as an incidental finding against the background of the primary goal - to validate the reaction efficiency of the detection of the manufacturer's declared target, i.e. mRNA for the S protein. 

These DNA amplicons also showed acceptable reaction efficiency, based on the measured Ct values in the dilution experiment as shown in figures 9 & 10. 

Figure 9: Spikevax cDNA dilution experiment – concentrated cDNA and 4 serial log dilutions.  Accidental co-amplification of contaminating genomic DNA of the expression vector.

Figure 10: BNT162b2 cDNA dilution experiment – concentrated cDNA and 4 serial log dilutions.  Accidental co-amplification of contaminating genomic DNA of the expression vector.

Oligonucleotides for quantitative Real-Time PCR of the Ori promoter expression cloning Sequencing of bivalent Moderna and Pfizer mRNA vaccines demonstrated nanogram to microgram quantities of expression vector dsDNA per dose. No evidence of SV40 was identified. 

Oligonucleotides for quantitative Real-Time PCR of Escherichia coli genomic DNA were validated based on the requirements of ISO 13 485 as a constituent of a commercial diagnostic kit. 

The oligonucleotides for quantitative Real-Time PCR cassettes for S protein (mRNA) in Spikevax and BNT162b2 preparations, newly used in this work, were verified by direct sequencing of obtained PCR products on an ABI 3500 capillary sequencer (ThermoFisher Scientific, USA). 

All 17 lots of Spikevax and all 7 lots of BNT162b2 were sequenced. Direct sequencing of PCR products was performed using the BigDye™ Terminator v3.1 Cycle Sequencing Kit (ThermoFisher Scientific, USA). Sequencing reactions were purified using the BigDye XTerminator™ Purification Kit (ThermoFisher Scientific, USA), according to the manufacturer’s recommended procedure. 

One typical example of a fragment of the chromatogram of the Spikevax S protein gene and one typical example of a fragment of the chromatogram of the BNT162b2 S protein gene are shown in figure 11. ABI 3500 sequencing data of all other lots are available upon request.   

Figure 11: Sanger sequencing chromatograms of obtained PCR products. In all cases, the sequenced PCR products showed 100% identity to the expected reference sequence. A 1_Moderna_S_protein_MV1013A_DNA; B – 86_Pfizer_S protein_FP9632_DNA.

Quantitative real-time PCR analysis of mRNA for S protein

At the mRNA (cDNA) level, it was found that the expression of the declared molecular target (S protein mRNA) varies across lots for both Spikevax and BNT162b2. 

For Spikevax (Moderna), as shown in figure 12, lots 200106A and MV1018A are characterized by one order of magnitude (10x) lower expression of S protein mRNA than the other tested lots. The blue dots in the graph indicate the S protein mRNA expression values for individual measurements (in the range of 10e9 to 10e10 copies of the target sequence/ml; analysis performed in quintuple for each lot). The orange dots in this case indicate the quantity of expression cloning vector DNA (in the range of 10e7 to 10e8 copies of the target sequence/ml; analysis performed in quintuple for each lot) present in the analyzed cDNAs. Similar findings are shown in figure 13, lots 1L084A and 1F1059A for Pfizer BNT162b2. 

Figure 12: The graph shows the mRNA expression level for S protein in Spikevax (Moderna), GenBank OK120841 - blue dots in quintuplicates for each tested lot. Orange dots serve as a reference; it is an admixture of double-stranded DNA of the expression cloning vector in the tested cDNAs. 

Figure 13: In BNT162b2 (Pfizer), the variation in mRNA expression for the declared S protein (GenBank OR134577) between individual lots is also noticeable – a difference of one order of magnitude (10x) is evident for lots 1L084A and 1F1059A – mRNA expression for S protein is shown in the graph by blue dots. The orange dots serve as a reference; it is an admixture of double-stranded DNA of the expression cloning vector in the tested cDNAs. Analysis of all lots was carried out in quintuples.

Quantitative Real-Time PCR analysis of DNA targets - S protein cassette and expression cloning vector promoter 

A significant finding is the high quantity of DNA present across all tested lots, both Spikevax (Moderna) and BNT162b2 (Pfizer). In all preparations, a very high Real Time PCR signal from the promoter of the cloning expression vector and from the 3' end of the cassette encoding the S protein was measured (10e7 – 10e9 copies/ml for Spikevax; 10e8 – 10e9 copies/ml for BNT162b2). Analysis of all lots was carried out in quintuples with the final results displayed in figures 14 & 15 respectively. 

Figure 14. Shows a high quantity of DNA for both the expression cloning vector and the DNA for the S protein cassette. Of note are lots 200023A, 200106A, MV1018A, where the ratios of vector vs cassette are reversed. It is also interesting that lots 3005697 and 400012A are quantitatively significantly outside the range of the other tested lots.

Figure 15: Shows a similar situation for BNT162b2 (Pfizer) preparations. In these preparations, the amount of expression cloning vector is higher, but what is more important is the horizontal heterogeneity within the blue dots, which represent quantitative S protein DNA cassette amplicons. Note the one order of magnitude (10x) difference between the lots. 

These data suggest that both assays might detect identical DNA construct, targeted at both its 5' end (promoter Ori) and its 3' end (bases 3184 to 3417 of the total 3880 bp of the complete coding sequence for the S protein). This most likely suggests that both Spikevax and BNT162b2 might contain the complete coding DNA sequence for the S protein cassette, together with some regulatory promoter sequences. 

The results demonstrate more than degraded or fragmented DNA, that might arise from instability of the stored preparations or during suboptimal manufacturing process. The combined signal originates very likely from a full-length DNA construct, which might be capable of encoding a full-length mRNA for the S protein. 

Within the tested lots of Spikevax (Moderna), the heterogeneity in the quantity of DNA coding for the S protein cassette and/or the cloning expression vector is remarkable. In three cases, the ratio of the amount of expression cloning vector and DNA cassette for the S protein is reversed, and the difference in quantity is of one order of magnitude. This indicates that the given lot might also contain another construct cloned in the given expression vector. It is possible it could represent an admixture of Omicron, although this could mean that its amount is very random across the individual lots, which would raise concerns regarding good manufacturing practice. 

We did not search for the Omicron sequence in this investigation. Rather, we focused on sequences that are clearly declared to be in the official content of the preparations – thus, we targeted the S protein mRNA [6], GenBank OK120841 (Spikevax, Moderna) [7]; GenBank OR134577 (BNT162b2, Pfizer) [8]. 

These differences in the quantity of DNA amplicons are clearly visible already in the raw data from the analyzer (RotorGene Q, Qiagen, Germany). Note the significantly different starts of the amplification curves. 

Figure 16 shows the raw data analyzed at the DNA level for the S protein cassette Spikevax and BNT162b2, as well as for the expression cloning vector, which, like the DNA cassette for the S protein, is present in all tested lots. 

 Figure 16: Te following graphic print screens show raw data for the DNA cassette for the S protein (Spikevax - ROX, BNT162b2 – FAM, expression cloning vector – Cy5). 

The summarized results of the mRNA and DNA quantities for the S protein cassette are shown in Table 2.

Table 2: Summarized mRNA and DNA quantities in the Moderna and Pfizer Vials.

Quantitative detection of genomic DNA of escherichia coli

The presence of Escherichia coli genomic DNA was also analyzed within the multiplex quantitative Real-Time PCR to verify whether the preparations were contaminated with GMO, typically used for the propagation of the cloning expression vector harboring the S protein cassette. 

A borderline quantity, in individual copies of the microorganism/ml of sample, was found in two lots of BNT162b2 (Pfizer), namely FP9632 and 1F1047A. 

The data is not shown, though they can be provided upon request.

Homogeneity assessment 

While there was a 28% batch-to-batch variability in nucleic acid content, there was no significant intra-lot inconsistencies observed. Each set of vials within a given lot showed consistent mRNA and DNA profiles. 

Impact of expired storage conditions 

Analysis of expired vaccine samples stored at -80°C indicated partial degradation of nucleic acid content. While mRNA fragments remained detectable, their integrity was reduced, potentially compromising vaccine effectiveness.

This research study conducted Molecular analysis of the following lots of Moderna and Pfizer COVID genetic vaccines: 

Spikevax (Moderna): MV1013A, 200023A, 200156A, 223049, 200090A, 200106A, 200100A, 3005885, 3005836, 000090A, 000058A, 3005241, 3005697, 3006272, MV1018A, 400012A, 400011A, and 

BNT162b2 (Pfizer): FP9632, 1F1051A, 1LO84A, 1F1047A, 1F1059A, 1F1055A, PCB0020. 

Analysis of the samples using quantitative multiplex Real-Time PCR revealed: 

• Inter-individual heterogeneity between individual lots is remarkable, with a difference in the quantity of declared mRNA of up to 10x.
• All Spikevax (Moderna) and BNT162b2 (Pfizer) lots tested contain a significant admixture of DNA, which is most likely the complete cassette for S protein cloned in an expression vector carrying intact regulatory sequences. Thus, expression of full-length mRNA for S protein from this DNA cassette cannot be excluded.
• The Real-Time PCR measured quantity of DNA in all preparations is comparable to the quantity of the only officially declared item, which is mRNA for S protein. This suggests that it is therefore not DNA “contamination”, but rather a regular admixture, undeclared by either manufacturer.
• Based on the Real-Time PCR measurement of the quantity of DNA present, another degree of heterogeneity can be observed between individual lots, where the possibility that other DNA construct(s), which identity is currently unknown, might also be present in some lots. 

The presence of Escherichia coli DNA sequences raises questions about manufacturing quality control. While residual DNA from bacterial propagation systems is not uncommon in recombinant DNA products, its detection in these vaccines suggests incomplete purification during production. Regulatory standards such as those outlined by the European Medicines Agency (EMA) and the U.S. Food and Drug Administration (FDA) set limits on allowable residual DNA [4,5]. The observed DNA content may exceed these limits and pose theoretical risks related to genomic integration or immunological responses. 

Furthermore, the observed variability in mRNA content may affect dose consistency. mRNA vaccines rely on precise nucleic acid delivery to ensure effective antigen expression. Deviations of 20–30% across lots, as observed in this study, may lead to inconsistent immune responses and altered clinical outcomes. 

The continued use of the Wuhan strain’s S protein sequence in both vaccines highlights a notable concern. Since early 2020, SARS-CoV-2 variants with significantly altered spike protein structures have dominated viral circulation. The use of an outdated sequence may reduce the vaccines’ protective efficacy, particularly against newer variants with substantial antigenic drift. 

The findings regarding mRNA degradation in expired samples emphasize the importance of strict adherence to storage protocols. Even with ultra-cold storage conditions, prolonged storage beyond designated expiration dates resulted in partial mRNA degradation, potentially impairing vaccine performance. 

The above data shows a high degree of heterogeneity between individual Spikevax (Moderna) and BNT162b2 (Pfizer) preparations, measured by the quantity of declared mRNA for S protein. The individual lots differ in the quantity of the target mRNA sequence by 1 order of magnitude (10x).

In all preparations tested, a high quantity of double-stranded DNA (cloning expression vector, DNA cassette for S protein) was identified. Interestingly, the quantity of DNA was comparable to the quantity of mRNA declared by the manufacturer. 

Such a high quantity of DNA clearly cannot be considered mere "contamination" during the manufacturing process. In the case of “a contamination”, the quantity of the contaminating DNA would be expected many orders of magnitude lower, in the range of approximately 10e2 or 10e3 copies/ml. A random “contamination” of the Spikevax and BNT162b2 preparations with double-stranded DNA during the manufacturing process can therefore be excluded. 

Moreover, unremarkable, borderline, contamination with genomic DNA of Escherichia coli (GMO used for large-scale production of cloning vectors) was found only in two cases of the BNT162b2 preparation (Pfizer), in individual units of copies of the target microorganism / ml.  This might be considered a negligible finding in comparison to the large quantities of DNA of cloning vector and S cassette found in all lots tested. No SV40 was identified. 

The presence of such high quantities of DNA in all the lots tested implies that it might not result from some contamination during an inadequate manufacturing process but rather might be considered a regular (though not officially declared) constituent of all lots tested, present in quantities (almost) identical to the quantity of mRNA for the S protein. 

Both Pfizer and Moderna identified the only oligonucleotide materials within their vaccines as being mRNA. The presence of double-stranded DNA, or any other DNA, in the Moderna and Pfizer preparations, was not declared by either the manufacturers.

This study highlights key findings regarding the oligonucleotide content of Spikevax (Moderna) and BNT162b2 (Pfizer) COVID-19 vaccines. The presence of these genetic sequences also raises InflammoThrombotic Immunologic Response (ITIR) concerns [1]. While declared mRNA sequences were confirmed, variability in nucleic acid content and the presence of undeclared DNA sequences underscore the need for improved quality control in vaccine manufacturing. 

Key recommendations include: 

• Enhanced Purification Protocols: Manufacturers should review DNA removal processes to minimize residual contamination and reduce InflammoThrombotic Immunologic Response (ITIR).
• Stricter Lot Testing: Additional oversight is recommended to ensure batch-to-batch consistency in nucleic acid content.
• Genomic Updates: Given the ongoing evolution of SARS-CoV-2 variants, vaccine designs should align with contemporary circulating strains.
• Removal of contaminated genetic vaccines from the market.
• Stricter oversight by regulatory agencies including the FDA, EMA and ŠÚKL. 

Continued vigilance in vaccine production and regulatory oversight is crucial to ensuring public confidence in mRNA and DNA vaccine technology and maximizing global immunization efforts.

This study was carried out in response to a formal request by Slovakian Prime Minister Mr. Robert Fico in and through the office of the Plenipotentiary for the Slovak Republic to assess the nucleic acid content in multiple lots of Spikevax and BNT162b2 vaccines.

No Institutional Review Board required.

The authors declare no conflict-of-interest.

Further data is available if determined appropriate by submission request including but not limited to the individual(s) and institution(s), purpose/intent of request, and pertinent relevant requested information as deemed applicable by the authors. https://www.flemingmethod.com/

This research was carried out following Slovakian Prime Minister Mr. Robert Fico’s appointment of Dr. Kotlár as plenipotentiary and a directive to assess Slovakia’s response to the COVID pandemic in addition to a request for transparent research and publication by Secretary of HHS, Mr. Robert F. Kennedy, Jr.

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3. Fleming RM, Fleming MR (2021) FMTVDM Quantitative Nuclear Imaging finds Three Treatments for SARS-CoV-2. Biomed J Sci Tech Res 33: 26041-26083. https://biomedres.us/fulltexts/BJSTR.MS.ID.005443.php
4. European Medicines Agency (2011) Guideline on the Use of DNA in Vaccines and Gene Therapy Products: EMA/CHMP/BWP/814397/2011. European Medicines Agency.
5. S. Food and Drug Administration (2010) Guidance for Industry: Characterization and Qualification of Cell Substrates and Other Biological Materials Used in the Production of Viral Vaccines for the Prevention and Treatment of Infectious Diseases. U.S. Food and Drug Administration, USA.
6. GenBank Database. SARS-CoV-2 Wuhan-Hu-1 Reference Genome. GenBank Accession: MT192773.
7. GenBank Database. Spikevax (Moderna) mRNA Sequence. GenBank Accession: OK120841.
8. GenBank Database (2024) BNT162b2 (Pfizer) mRNA Sequence. GenBank Accession: OR134577.