DNA Contamination in HPV vaccines
ISIS Report 12/09/12
DNA Contamination in HPV vaccines
A serious safety issue that should not be swept under the regulatory carpet Professor Joe Cummins
Please circulate widely and repost, but you must give the URL of the original and preserve all the links back to articles on our website. If you find this report useful, please support ISIS by subscribing to our magazine Science in Society, and encourage your friends to do so. Or have a look at the ISIS bookstore for other publications
When the Human Papilloma Virus (HPV) vaccine Gardasil was recently found to be contaminated with DNA, the US Food and Drug Administration (FDA) lost no time in declaring that the DNA was not a contaminant but a harmless by-product of vaccine production. I disagree; that extraneous DNA is potentially harmful. It should also be noted that the safety and efficacy of HPV vaccines have been controversial from the start (see  The HPV Vaccine Controversy and other articles in the series, SiS 41).
HPV establishes productive infections only in keratinocytes of the skin or mucous membranes. While the majority of the known HPV types cause no symptoms in most people, some types can cause warts (verrucae), while others can lead to cancers of the cervix, vulva, vagina, penis, oropharynx and anus.
Recently, HPV has been linked to an increased risk of cardiovascular disease. In addition, HPV 16 and 18 infections are strongly associated with an increased risk of developing throat cancer. Worldwide in 2002, an estimated 561 200 new cancer cases (5.2 %) were attributable to HPV, making HPV one of the most important infectious causes of cancer, and cervical cancer is the second most common cancer in women worldwide. In 2008, there were an estimated 529 000 new cases of cervical cancer and 274 000 deaths; more than 85 % of the deaths in developing countries, where it accounts for 13 % of all female cancers .
The viral genome
The HPV genome consists of 8 genes coding for proteins and a non-protein-coding region with regulatory genes. The genes are distinguished as early and late functioning in virus development. The early genes include those involved in virus replication and transcription along with the oncogenes for cancer development. The late genes encode the two structural proteins L1 and L2 of the virus capsid. HPV infects the basal cells of the cervical epithelium when it is damaged in some way. The viral genome becomes established in the basal cells as an episome (an independently replicating nuclear micro-chromosome). The episome replicates in tandem with the chromosomes of the cell and forms virus particles. The complete virus particles are in the outermost cells of the epithelium and the viruses are spread as the cells slough off from the epithelium. Some virus proteins function as oncoproteins, transforming the epithelial cells to a precancerous state. HPV infection is necessary but not sufficient for cancer formation, however. In high grade lesions and cancer, an episome is integrated into the cell chromosome. Integration disrupts a viral transcription regulatory protein that controls the production of the cancer proteins, leading to their continual and enhanced production  (Recombinant Cervical Cancer Vaccines, SiS 29). HPV integration into human chromosomes is non-random; with integration hot spots in chromosome regions homologous to the oncogene E5 of HPV or the structural protein L2 . Women with cervical cancer have been found with viral chromosomes integrated completely or partially as chromosome fragments, or as independent episomes. Partially integrated HPV was most prevalent in women with cancer while complete virus integration was about half as frequent and the episomal form rare. The cancer- causing integration breaks the HPV chromosome at the E1/E2 region, causing a loss of that region. This in turn results in loss of control of the cancer genes E6 and E7. The E7 cancer gene produces a protein that inactivates the retinoblastoma gene – a cancer suppressor gene - of the host cell, thereby promoting cancer . (Retinoblastoma is an inherited cancer of the eye caused by loss of the retinoblastoma gene.) The main lesson is that fragmentation or breakage of the HPV DNA is an important factor in cancer progression of the host cell.
The viral genes have a complex transcription pattern. There is a single promoter for all of the early genes. The early promoter initiates production of a large pre-messenger RNA from which messages containing exons and introns are then spliced to generate each of the early proteins. The other viral promoter initiates production of pre-messenger for structural proteins L1 and L2, which also contain exons and introns, and are similarly spliced prior to translation of the messenger RNA into protein. There are early and late polyadenylation (poly A) signals for the large pre-messenger RNA transcripts. Gene expression of HPV is tightly coupled to the developmental status of the host cells .
Micro RNAs are very small (around 22 nucleotides in chains) non-coding regulatory gene products of cells. Micro RNAs are altered in a number of human cancers and one is significantly elevated in HPV anal cancer . A cluster of micro RNAs was found associated with HPV head and neck cancers . The natural history of HPV cancers shows a complex pattern of gene transcription and micro RNAs are implicated in the development of HPV cancers.
HPV vaccines have been deployed worldwide since 2006. Two vaccines have been commercialized: Gardasil, manufactured by Merck and Cervarix, manufactured by GlaxoSmithKline. They are prophylactic, that is, they prevent cervical cancer but do not cure existing infections, and are based on the L1 virus-like particles to achieve immunity against HPV. The L1 protein is capable of self-assembly to form empty virus like particles that activate the human immune system to form antibodies. The HPVs targeted by the vaccines are “high risk” types 16 and 18 and “low risk” types 6 and 11. The two commercial HPV vaccines are both made using genetically modified (GM) microbes in a laboratory. Gardasil protects against all four HPV types because it contains virus like particles with mixtures of the four subunit proteins, and is called a tetravalent vaccine. The four L1 proteins are manufactured using GM baker’s yeast. Cervarix protects against the HPV types 16 and 18, and is a bivalent vaccine, and is manufactured using GM baculovirus (a soil-born insect virus) in cultured insect cells .
The vaccine consists of the four Monovalent Bulk Adsorbed Products (MBAPs), one for each of the four human papillomavirus (HPV) types. The active components in each MBAP are virus-like particles (VLPs) made up of the recombinant major capsid (L1) protein for that HPV type, produced in recombinant S. cerevisiae. The pGAL110 yeast expression vector was used for all four HPVL1 proteins. The L1 genes were derived by a direct cloning protocol. However, the coding sequence for HPV11L1 was synthetically rebuilt based on HPV6L1 nucleotide sequences that supported good VLP expression in yeast. Polymerase chain reaction (PCR) was used to subclone the L1 genes into the yeast expression vector pGAL110, which contains the yeast GAL1-GAL10 promoter and the yeast ADH1 terminator (ADH1t) for transcription termination and polyadenylation. The pGAL110-related yeast expression vectors for each of the four HPV types were used to transform the recombinant S. cerevisiae .
Gardasil DNA contamination
In 2011, Gardasil was found to be contaminated with recombinant HPV DNA in all of the lots of vaccine marketed in the United States, Australia, New Zealand, Spain, France and Poland. One of the DNA fragments identified was a gene fragment from the HPV capsid protein L1 . Sane Vax, a girl age 13, was found to have blood containing HPV DNA two years after Gardasil inoculation . Other DNA contaminants were not specifically identified in the many contaminated vaccine samples but are presumably DNA fragments from the genetically modified yeast used to produce the vaccine protein. Even though the FDA and the vaccine manufacturer had earlier claimed that Gardasil contained no DNA they later changed their tune, and are now claiming that DNA HPV L1 gene in the vaccine is not a contaminant but it is a normal consequence of vaccine production. The remaining DNA fragments are presumed to be safe . The World Health Organization (WHO) had earlier claimed that DNA fragments shorter than 200 base pairs should be presumed safe . It appears that the Merck Corporation, FDA and WHO are closing ranks to claim that the DNA contaminant in vaccines should be presumed safe without any evidence.
The DNA contaminants in Gardasil such as L1 gene fragments and the probable yeast DNA fragments such as the GAL1-GAL10 promoter and the ADH1 terminator flanking the synthetic L1 gene used to produce the vaccine, may pose no threat to the victims of Gardasil vaccination. But it is totally unacceptable to presume that they are safe for human vaccination without experimental evidence or evaluation. Indeed, short DNA fragments can be incorporated into the human genome. Although the yeast used to produce the vaccine does not have the small regulatory RNA employed by most organisms from bacteria to humans, it does contain 247 small open reading frames including 22 short DNA sequences specifying peptides involved in cell growth or damage and growth in the presence of DNA damage and replication arrest. At least one yeast gene product inactivates the cancer suppressor gene p53 and in that way promotes cancer in multicellular organisms . The integration of the L1 and/or yeast genes may enhance the chances of acquiring cancer in numerous tissues of the body. It has been known for many years that ingested DNA may be covalently linked to mammalian DNA in blood cells, liver cells, spleen macrophages and T cells , and horizontal gene transfer and recombination via circulating nucleic acids is now well known  (Intercommunication via Circulating Nucleic Acids, SiS 42) .
Cervarix contains recombinant C-terminally truncated (shortened) major capsid L1 proteins of HPV types 16 and 18 as active ingredients. The L1 proteins of HPV-16 and HPV-18 are separately produced using a recombinant baculovirus expression system and the insect cell line Hi-5 Rix4446 derived from Trichoplusia. After extracting the L1 proteins and further purification, they are assembled separately as VLPs. The VLPs of each HPV type are formulated with the AS04 adjuvant system composed of aluminium hydroxide and 3-O-desacyl-4.-monophosphoryl lipid A (MPL). The MPL immunostimulant is a detoxified derivative of the lipopolysaccharide of the gram negative bacterium Salmonella minnesota R595 strain. Host cell proteins (HCP), DNA, and infectious recombinant baculovirus DNA are potential impurities removed in the preparation process. Other impurities such as lipids or carbohydrates are present only in negligible trace amounts .
Cervarix DNA contamination?
Cervarix’s manufacturer maintains that the vaccine is not contaminated with DNA or other products from the baculovirus vector or the insect cells. The baculovirus, Autographa californica nucleopolyhedrovirus (AcMNPV) for which the complete genome sequence has been determined, has a circular, double-stranded, super-coiled DNA genome of approximately 130 kilobases packaged in a rod-shaped nucleocapsid. These nucleocapsids can be extended lengthways and thus the virus genome can effectively accommodate large insertions of foreign DNA. Such insertions of foreign genes into the AcMNPV genome has resulted in production of baculovirus expression vectors; recombinant viruses genetically modified to contain a foreign gene of interest, which can be expressed in insect cells under the control of a baculovirus gene promoter . Baculovirus infects and is viable in human cells. Baculoviruses mediate gene expression in a wide array of vertebrate cells including those of humans  and numerous baculovirus genes are expressed in human cells [21, 22]. Baculoviruses contain two genes that prevent apoptosis and in that way facilitate progress of cancer cells . Baculoviruses contain small DNA genes coding for micro RNAs with 8 viral and 64 cellular targets including interference with the host immune defence machinery . There is clear evidence that the baculovirus vector DNA harbours genes detrimental to humans. It is imperative that DNA and RNA along with proteins from baculovirus and insect cells should not contaminate Cervarix vaccine.
DNA contamination of HPV vaccines is a serious problem, and not a normal or acceptable consequence of recombinant vaccine production as claimed by FDA. The false claims of FDA put into serious question not only Gardasil but also Cervarix. A truly independent agency is urgently required to undertake studies on the content of contaminating DNA and of RNA in the two vaccines.
- Cummins J and Ho MW. The HPV vaccine controversy. Science in Society 41, 24-26, 2009,
- Retrospective International Survey and HPV Time Trends Study Group Human papillomavirus genotype attribution in invasive cervical cancer: a retrospective cross-sectional worldwide study. Lancet Oncology 2010, 11, 1048-56.
- Cummins J. Recombinant Cervical Cancer Vaccines. Science in Society 29, 20, 2006.
- Schmitz M, Driesch C, Jansen L, Runnebaum IB, Dürst M.Non-Random Integration of the HPV Genome in Cervical Cancer. PLoS One 2012, 7, e39632
- Šepetienė A, Gudlevičienė Ž, Bumbulienė Ž, Drąsutienė GS, Didžiapetrienė J. HPV16 integration in Lithuanian women with cervical neoplasia. Centr Eur J Med. 2011, 6, 205–212.
- Zheng ZM, Baker CC. Papillomavirus genome structure, expression, and post-transcriptional regulation. Frontiers in Biosciences 2006, 11, 2286-302.
- Myklebust MP, Bruland O, Fluge Ø, Skarstein A, Balteskard L, Dahl O. MicroRNA-15b is induced with E2F-controlled genes in HPV-related cancer. British Journal of Cancer 2011, 105, 1719-25
- Lajer CB, Garnæs E, Friis-Hansen L, Norrild B, Therkildsen MH, Glud M, Rossing M, Lajer H, Svane D, Skotte L, Specht L, Buchwald C, Nielsen FC The role of miRNAs in human papilloma virus (HPV)-associated cancers: bridging between HPV-related head and neck cancer and cervical cancer. British Journal of Cancer 2012, 106, 1526-34.
- Cummins J, Ho MW. The HPV Vaccine Controversy. Science in Society 41, 2009, 24
- Europe, the Middle East and Africa (EMEA) EU regulatory agency for the evaluation of medicinal products Silgard, INN-Human Papillomavirus Vaccine [Types 6, 11, 16, 18] 2006http://www.ema.europa.eu/docs/en_GB/document_library/EPAR_-_Scientific_Discussion/human/000732/WC500051547.pdf
- SaneVax,Inc.SANE Vax to FDA: Recombinant HPV DNA found in multiple samples of Gardasil August29,2011http://sanevax.org/sane-vax-to-fda-recombinant-hpv-dna-found-in-multiple-samples-of-gardasil/
- Business Wire SANE Vax, Inc. Reports Human Papillomavirus (HPV) DNA Contamination in Gardasil™ To FDA: Requests Public Safety Investigation Sept. 6,2011http://www.businesswire.com/news/home/20110906005422/en/SANE-Vax-Reports-Human-Papillomavirus-HPV-DNA
- US Food and Drug Administration FDA Information on Gardasil – Presence of DNA Fragments Expected, No Safety Risk Page Last Updated: 10/21/2011http://www.fda.gov/BiologicsBloodVaccines/Vaccines/ApprovedProducts/ucm276859.htm
- World Health Organization WHO Study Group on Cell Substrates for Production of Biologicals 5.3. Approaches in reducing risk of cell DNA - Discussion 2007http://www.who.int/biologicals/publications/meetings/areas/vaccines/cells/Cells.FINAL.MtgRep.IK.26_Sep_07.pdf
- Kastenmayer JP, Ni L, Chu A, Kitchen LE, Au WC, Yang H, Carter CD, Wheeler D, Davis RW, Boeke JD, Snyder MA, Basrai MA. Functional genomics of genes with small open reading frames (sORFs) in S. cerevisiae. Genome Research 2006, 16, 365-73.
- Schubbert R, Renz D, Schmitz B, Doerfler W. Foreign (M13) DNA ingested by mice reaches peripheral leukocytes, spleen, and liver via the intestinal wall mucosa and can be covalently linked to mouse DNA. Proc Natl Acad Sci U S A1997, 94, 961-6.
- Ho MW. Intercommunication via circulating nucleic acids. Science in Society 42, 46-48, 2009.
- Europe, the Middle East and Africa (EMEA) EU regulatory agency for the evaluation of medicinal products SCIENTIFIC DISCUSSION 2. Quality aspects 2007 http://www.ema.europa.eu/docs/en_GB/document_library/EPAR_ Scientific_Discussion/human/000721/WC500024636.pdf
- Haines,F,Possee,R,King,L Baculovirus Expression Vectors 2005http://www.expressiontechnologies.com/pdf/BEV%20Paper.pdf
- Airenne,K,Makkonen,K,Mähönen.A.Ylä-Herttuala,S. Chapter 12 Baculoviruses Mediate Efficient Gene Expression in a Wide Range of Vertebrate Cells Otto-Wilhelm Merten and Mohamed Al-Rubeai (eds.), Viral Vectors for Gene Therapy: Methods and Protocols, Methods in Molecular Biology, Vol. 737, DOI 10.1007/978-1-61779-095-9_12, © Springer Science+Business Media, LLC 2011
- Fujita R, Matsuyama T, Yamagishi J, Sahara K, Asano S, Bando H. Expression of Autographa californica multiple nucleopolyhedrovirus genes in mammalian cells and upregulation of the host beta-actin gene. Journal of Virology2006, 80, 2390-5.
- Liu CY, Wang CH, Wang JC, Chao YC. Stimulation of baculovirus transcriptome expression in mammalian cells by baculoviral transcriptional activators. Journal of Genetic Virology 2007, 88, 2176-84.
- Clem RJ, Hardwick JM, Miller LK. Anti-apoptotic genes of baculoviruses Cell Death & Differentiation 1996, 3, 9-16.
- Singh J, Singh CP, Bhavani A, Nagaraju J. Discovering microRNAs from Bombyx mori nucleopolyhedrosis virus .Virology 2010, 407, 120-8