Sequencing the Pfizer monovalent mRNA vaccines also reveals dual copy 72-bp SV40 Promoter

Figure 1. Depiction of the 72bp tandem repeat in the SV40 promoter of BNT162b2 vaccine.

Introduction

Using methods described earlier, we sequenced the Pfizer monovalent BNT162b2 vaccines and assembled these reads with Megahit. A 7,754 base pair spike encoded plasmid was assembled with a truncated Poly A tract and a previously described mis-assembly of the SV40 promoter region.

Results

This vector is nearly identical to the bivalent vectors recently assembled. The initial assembly also showed signs of a read coverage pile up across the 72bp enhancer in the SV40 promoter described in figure 5A and figure 5B of this preprint. This mis-assembly was also reproduced from sequence data derived from an independent group in Europe (Voor Waarheid) who has also sequenced BNT162b2 (personal communication and in prep).

Once we inserted an additional copy of the 72bp element into the Megahit constructed reference (shown in red in Figure 2), the coverage pile up disappeared. This implies that Megahit is struggling to assemble a 72bp tandem repeat with 105bp insert sizes. Both monovalent and bivalent Pfizer vaccines contain 2 copies of the 72bp Enhancer in the SV40 promoter.

Figure 2. Pfizer monovalent vaccine annotated with SnapGene. Highlighted region in blue with a Red vector line is the 72bp insert manually added to the assembly to test for more accurate read mapping.

>Pfizer_mono_k141_80

TTTTTTTTTTTTTTTTTTTTGCTAGCTCCAGGGTGTGGCTGGCACGAAATTGACCAACCCTGGGGTTAGTATAGCTTAGTTAAACTTTCGTTTATTGCTAAAGGTTAATCACTGCTGTTTCCCGTGGGGGTGTGGCTAGGCTAAGCGTTTTGAGCTGCATTGCTGCGTGCTTGGGAGGTGTCTGGAACTAGCAGAGGTGGTGAGTGGGGCAGGTGGAGGTGGGAGCATACCTGGGACCCGAGGTCGGGGGAGACTCGGGGTACCCAGGACGGGAAAGGGGCAGCTAGCATTGCGTGCATGCAGTACCAGCTCGAGTCATCATGTGTAGTGCAGTTTCACGCCCTTCAGCACGGGCTCAGAATCGTCCTCGTCGAACTTGCAGCAGCTGCCACAGCTACAACAGCCCTTCAGGCAGCTACAGCAGCTGGTCATGCAACACAGCATGATTGTGACCATCACGATGGCAATCAGTCCGGCGATAAAGCCCAGCCAGATGTACCAGGGCCACTTGATGTACTGCTCGTACTTCCCCAGTTCTTGCAGGTCGATCAGGCTCTCGTTCAGATTCTTGGCCACCTCGTTCAGCCGGTCGATCTCTTTCTGGATGTTCACGACGCTGGCATTGATTCCGCTGATATCGCCCAGGTCCACGTCGGGGCTTGTGTGGTTCTTAAAGTACTTGTCCAGTTCCTCTTTGAAGCTGTCCAGCTCGGGCTGCAGAGGGTCGTACACGGTATTGTTCACAATGCCGATCACGACGTCGCAGTTGCCAGACACGAAGGTGTTGTCGGTGGTGATGATCTGGGGCTCGTAGAAGTTCCGCTGTGTCACGAACCAATGGGTGCCGTTGGACACGAACACGCCTTCTCTAGGAAAGTGGGCTTTGCCGTCGTGGCAGATGGCTGGAGCGGTGGTGAAATTCTTCTCTTGAGCGGGCACATATGTCACGTGCAGAAACACCACGCCGTGAGGGGCAGACTGAGGGAAGCTCATCAGGTGGTAGCCCTTGCCGCAAAAGTCCACTCTCTTGCTCTGGCCCAGCACACACTCAGACATCTTGGTGGCGGCCAGATTGGCAGAGGCTCTAATCTCGGCGGCTCTGATCAGCTGCTGGGTCACGTATGTCTGGAGGCTCTGCAGTCTGCCTGTGATCAGTCTGTCGATCTGCACCTCGGCCTCAGGAGGGTCCAGTCTGCTCAGGATATCGTTCAGCACAGAGCTGATGGCGCCGAAGTTGGAGGACAGCTGCTTGACCAGGGTGTTCAGTGCCTGGGCATTCTGGTTGACCACGTCCTGCAGCTTTCCCAGGGCGCTTGCTGTGCTGCTCAGGCTGTCCTGGATCTTGCCGATGGCGCTGTTGAACTGGTTGGCGATCAGCTTCTGGTTCTCGTACAGCACATTCTGGGTCACTCCGATGCCGTTGAACCGGTAGGCCATCTGCATAGCAAAGGGGATCTGCAGAGCGGCGCCTGCTCCAAATGTCCAGCCGCTTGTGATTGTGCCGGCCAGCAGGGCAGATGTGTACTGGGCGATCATCTCATCGGTCAGCAGAGGAGGCAGCACTGTCAGTCCGTTAAACTTCTGGGCGCAAATCAGATCCCTGGCGGCAATGTCGCCCAGACAATCGCCATACTGCTTGATGAAGCCGGCGTCGGCCAGTGTCACTTTGTTGAACAGCAGGTCCTCGATGAAGCTCCGCTTGCTGGGCTTGCTAGGATCGGGCAGAATCTGGCTGAAATTGAAGCCGCCGAAGTCCTTGATAGGAGGGGTCTTGTAGATCTGCTTCACTTGGGCGAACACCTCTTGGGTGTTCTTGTCCTGTTCCACGGCGATCCCTGTCAGGGCTCTATTCAGCTGGGTGCAGAAGCTGCCGTACTGCAGCAGCAGGTTGGAGCACTCGGTGGAATCGCCGCAGATGTACATGGTGCAGTCCACGCTGGTCTTGGTCATGGACACAGGCAGGATCTCTGTGGTCACGCTGATGGTGAAGTTGGTGGGGATAGCGATAGAGTTGTTGGAGTAGGCCACGCTGTTCTCGGCGCCCAGAGACATTGTGTAGGCAATGATGCTCTGGCTGGCCACGCTTCTGGCTCTCCGAGGGCTGTTTGTCTGTGTCTGGTAGCTGGCGCAGATTCCAGCGCCGATGGGGATGTCGCACTCGTAGCTATTGTTCACGTGCTCGGCTCCGATCAGACAGCCGGCTCTGGTCTGAAACACATTGCTGCCGGTGGAGTACACCCGCCATGTAGGTGTCAGCTGATCGGCGTGAATGGCCACGGGCACTTCGGTACAGTTCACGTCCTGGTACAGCACTGCCACCTGATTGCTGGTGTTGGTGCCAGGGGTGATCACAGACACTCCGCCGAAGCTGCAAGGGGTGATGTCCAGGATTTCCAGTGTCTGGGGATCTCTAACGGCGTCTGTGGTATCGGCGATATCCCGGCCAAACTGCTGGAATGGCAGGAACTTCTTGTTGCTCTCTGTCAGCACGCCGGTGCCGGTCAGGCCGTTGAAGTTGAAGTTCACGCATTTGTTCTTCACGAGATTGGTGCTTTTCTTAGGGCCGCACACTGTGGCAGGGGCATGCAGCAGTTCGAAGCTCAGCACCACCACTCTGTAGGGCTGATAGCCCACGCCATTTGTGGGCTGAAAGCCGTAGGACTGCAGTGGGAAGTAGCAGTTGAAGCCTTCCACGCCGTTACAAGGGGTGCTGCCGGCCTGATAGATCTCGGTGGAGATGTCCCGCTCGAAGGGCTTCAGATTGGACTTCCGGAACAGCCGGTACAGGTAATTGTAGTTGCCGCCGACTTTGGAGTCCAGGTTGTTGCTGTTCCAGGCAATCACACAGCCGGTGAAGTCGTCGGGCAGCTTGTAGTTGTAGTCGGCGATCTTGCCTGTCTGTCCAGGGGCAATCTGCCGCACTTCATCTCCCCGGATCACGAAGCTGTCGGCGTACACGTTTGTGAAGCACAGGTCGTTCAGCTTGGTAGGGGACACGCCGTAGCACTTGAAGGTGCTGAAGCTGGCGGAGTTGTACAGCACGGAGTAGTCGGCCACGCAATTGCTGATCCGCTTCCGGTTCCAGGCGTACACAGAGGCGAATCTGGTGGCATTGAACACCTCGCCGAAGGGGCACAGATTGGTGATATTGGGGAACCGCACGATGGATTCGGTGGGCTGCACCCGGAAGTTGCTGGTCTGGTAGATGCCCTTTTCCACGGTGAAGGACTTCAGGGTGCACTTTGTCTCGCTCAGAGGATCCAGAGCACAATCCACGGCGTCGGTGATGGTGCCGTTCTCGTTGTACTTCAGCAGGAAGGTTCTAGGCTGCAGGTAGCCCACATAGTAAGCGGCGGCACCAGCTGTCCATCCGCTGCTGCTATCGCCAGGTGTCAGGTAGCTTCTGTGCAGGGCCAGCAGTGTCTGAAACCGGGTGATGTTGATGCCGATGGGCAGATCCACCAGGGGTTCCAGAGCAGAGAAGCCCTGAGGCAGATCCCGCACGAGGTTGATAGGGGTGTGCTTGCTGTAGATCTTGAAGTAGCCGTCGATGTTCTTAAACACGAACTCGCGCAGGTTCTTGAAGTTGCCCTGCTTGCCTTCCAGGTCCATCAGGAAAGGCTGGGACACGTACTCGAAGGTGCAGTTGTTGGCGCTGCTGTACACCCGGAACTCGCTTTCCATCCAGCTCTTGTTGTTCTTGTGGTAGTAGACGCCCAGGAAGGGGTCGTTGCAGAACTGGAACTCGCACACTTTGATGACCACGTTGGTGGCGTTGTTCACGATCAGCAGGCTCTGGGTCTTGCTGTCCAGTGTGGTGCCGAAGATCCAGCCTCTGATGATGTTGGACTTCTCGGTGCTGGCAAAGTACACCCCGTCGTTGAAGGGCAGCACGGGGTTGTCGAATCTCTTGGTGCCATTGGTGCCGGACACGTGGATGGCGTGGAACCAGGTCACGTTGCTGAAGAAAGGCAGGAACAGGTCCTGGGTAGAGTGCAGCACGCTGGATCTGAACACCTTGTCGGGGTAGTACACGCCTCTGGTAAAGCTGTTGGTGTAGGCTGGAGGCAGCTGTGTTCTGGTGGTCAGGTTCACACACTGGCTGGACACCAGAGGCAGCAGCACCAGGAACACGAACATGGTGGCGGGTTCTCTCTGAGTCTGTGGGGACCAGAAGAATACTAGTTTATTCTTATAGTGAGTCGTATTAATTAATAACTAATGCATGGCGGTAATACGGTTATCCACAGAATCAGGGGATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGAACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTATATATGAGTAACCTGAGGCTATGGCAGGGCCTGCCGCCCCGACGTTGGCTGCGAGCCCTGGGCCTTCACCCGAACTTGGGGGGTGGGGTGGGGAAAAGGAAGAAACGCGGGCGTATTGGCCCCAATGGGGTCTCGGTGGGGTATCGACAGAGTGCCAGCCCTGGGACCGAACCCCGCGTTTATGAACAAACGACCCAACACCGTGCGTTTTATTCTGTCTTTTTATTGCCGTCATAGCGCGGGTTCCTTCCGGTATTGTCTCCTTCCGTGTTTCAGTTAGCCTCCCCCTAGGGTGGGCGAAGAACTCCAGCATGAGATCCCCGCGCTGGAGGATCATCCAGCCGGCGTCCCGGAAAACGATTCCGAAGCCCAACCTTTCATAGAAGGCGGCGGTGGAATCGAAATCTCGTGATGGCAGGTTGGGCGTCGCTTGGTCGGTCATTTCGAACCCCAGAGTCCCGCTCAGAAGAACTCGTCAAGAAGGCGATAGAAGGCGATGCGCTGCGAATCGGGAGCGGCGATACCGTAAAGCACGAGGAAGCGGTCAGCCCATTCGCCGCCAAGTTCTTCAGCAATATCACGGGTAGCCAACGCTATGTCCTGATAGCGGTCCGCCACACCCAGCCGGCCACAGTCGATGAATCCAGAAAAGCGGCCATTTTCCACCATGATATTCGGCAAGCAGGCATCGCCATGGGTCACGACGAGATCCTCGCCGTCGGGCATGCTCGCCTTGAGCCTGGCGAACAGTTCGGCTGGCGCGAGCCCCTGATGTTCTTCGTCCAGATCATCCTGATCGACAAGACCGGCTTCCATCCGAGTACGTGCTCGCTCGATGCGATGTTTCGCTTGGTGGTCGAATGGGCAGGTAGCCGGATCAAGCGTATGCAGCCGCCGCATTGCATCAGCCATGATGGATACTTTCTCGGCAGGAGCAAGGTGAGATGACAGGAGATCCTGCCCCGGCACTTCGCCCAATAGCAGCCAGTCCCTTCCCGCTTCAGTGACAACGTCGAGCACAGCTGCGCAAGGAACGCCCGTCGTGGCCAGCCACGATAGCCGCGCTGCCTCGTCTTGCAGTTCATTCAGGGCACCGGACAGGTCGGTCTTGACAAAAAGAACCGGGCGCCCCTGCGCTGACAGCCGGAACACGGCGGCATCAGAGCAGCCGATTGTCTGTTGTGCCCAGTCATAGCCGAATAGCCTCTCCACCCAAGCGGCCGGAGAACCTGCGTGCAATCCATCTTGTTCAATCATGCGAAACGATCCTCATCCTGTCTCTTGATCGATCTTTGCAAAAGCCTAGGCCTCCAAAAAAGCCTCCTCACTACTTCTGGAATAGCTCAGAGGCCGAGGCGGCCTCGGCCTCTGCATAAATAAAAAAAATTAGTCAGCCATGGGGCGGAGAATGGGCGGAACTGGGCGGAGTTAGGGGCGGGATGGGCGGAGTTAGGGGCGGGACTATGGTTGCTGACTAATTGAGATGCATGCTTTGCATACTTCTGCCTGCTGGGGAGCCTGGGGACTTTCCACACCTGGTTGCTGACTAATTGAGATGCATGCTTTGCATACTTCTGCCTGCTGGGGAGCCTGGGGACTTTCCACACCCTAACTGACACACATTCCACAGCTGGTTCTTTCCGCCTCAGGATTCTTCCTTTTTCAATATTATTGAAGCATTTATCAGGGTTATTGTCTCATGAGCGGATACATATTTGAATGTATTTAGAAAAATAAACAAATAGGGGTTCCGCGCACATTTCCCCGAAAAGTGCCACCTGACGCGCCCTGTAGCGGCGCATTAAGCGCGGCGGGTGTGGTGGTTACGCGCAGCGTGACCGCTACACTTGCCAGCGCCCTAGCGCCCGCTCCTTTCGCTTTCTTCCCTTCCTTTCTCGCCACGTTCGCCGGCTTTCCCCGTCAAGCTCTAAATCGGGGGCTCCCTTTAGGGTTCCGATTTAGTGCTTTACGGCACCTCGACCCCAAAAAACTTGATTAGGGTGATGGTTCACGTAGTGGGCCATCGCCCTGATAGACGGTTTTTCGCCCTTTGACGTTGGAGTCCACGTTCTTTAATAGTGGACTCTTGTTCCAAACTGGAACAACACTCAACCCTATCTCGGTCTATTCTTTTGATTTATAAGGGATTTTGCCGATTTCGGCCTATTGGTTAAAAAATGAGCTGATTTAACAAAAATTTAACGCGAATTTTAACAAAATATTAACGCTTACAATTTACGCGTTAAGATACATTGATGAGTTTGGACAAACCACAACTAGAATGCAGTGAAAAAAATGCTTTATTTGTGAAATTTGTGATGCTATTGCTTTATTTGTAACCATTATAAGCTGCAATAAACAAGTTAACAACAACAATTGCATTCATTTTATGTTTCAGGTTCAGGGGGAGGTGTGGGAGGTTTTTTAAAGCAAGTAAAACCTCTACAAATGTGGTATGGCTGATTATGATCATGAACAGACTGTGAGGACTGAGGGGCCTGAAATGAGCCTTGGGACTGTGAATCTAAAATACACAAACAATTAGAATCAGTAGTTTAACACATTATACACTTAAAAATTGGATCTCCATTCGCCATTCAGGCTGCGCAACTGTTGGGAAGGGCGATGTTAAACGGCGGAGCTACCACACCGGTTGGAGCTCTTCTTTTTTTTTTTTTTTTTTTT

Figure 3. Vector with a single copy of the repeat result in noisier read mapping

Figure 4. Megahit misassembles the 72bp-72bp SV40 tandem repeat from 2 copies into 1 copy. Mapping reads back to such a truncated reference results in read coverage anomalies shown in Red rectangle. Inserting the additional 72bp copy to the reference and remapping the reads produces more accurate read coverage seen in figure 3.

This data leads to a CORRECTION- Previous substacks assumed this artifact to represent multiple vectors in one vial (heteroplasmic 72bp indel) of Pfizer bivalent vaccines. The more parsimonious solution to these data are that ALL Pfizer vaccines rely on a single vector that has an additional copy of this 72bp repeat that is known for nuclear localization and enhanced SV40 promoter performance. Megahit results with short reads and long indels should be scrutinized in more detail.

This has now been observed in Pfizer monovalent vaccines from two different continents and 2 Pfizer bivalent vaccines from the same lot. This artifact is also discussed in this preprint.

Conclusions

SV40 72bp enhancers and promoters are observed in both Pfizer monovalent and bivalent vaccines. This vector contamination has been observed in bivalent and monovalent vaccines and independently in the US and Europe.

References

We want to thank the Voor Waarheid group for sharing their RNA sequencing data of BNT162b2. Their data predates our generation of RNase-Seq data for the monovalent vaccine. Both datasets agree on the presence of vector DNA in the vaccines (personal communication and data sharing).

Sequence Data

Pfizer monovalent mRNA vaccine (BNT162b2) fasta file (Pfizer_mono_k141_80 with only one 72bp repeat)

Pfizer monovalent mRNA vaccine (BNT162b2) BAM file.

Pfizer monovalent mRNA vaccine (BNT162b2) BAM index file.

Pfizer monovalent mRNA vaccine (BNT162b2) Fastq file.