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
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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
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
The viral genome
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.
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 .
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
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 .
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
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.
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There are 5 comments on this article so far. Add your comment
|Sandy Lunoe Comment left 12th September 2012 15:03:58|
Excellent article. However, I beg to differ on one point:
In the section for HPV Vaccines it is stated "They are prophylactic, that is, they prevent cervical cancer but do not cure existing infections".
It has not yet been shown that the HPV vaccines prevent cervical cervical cancer.
|Meryl Dorey Comment left 12th September 2012 22:10:40|
I agree - this is an excellent article and it deserves widespread media attention but we know that the mainstream media will ignore this just as it ignores anything else that questions the safety or effectiveness of vaccines.
What I am confused about - and I have always wondered this - is why the KNOWN contamination of all vaccines never mentioned. Why are fragments of DNA from the HPV virus all of a sudden dangerous when contamination with monkey, dog, horse and human viruses in all other vaccines not a matter for concern (the only potential exception is the HepB vaccine but considering the fact that its production is not that different to the production of HPV vaccines, there may also be contamination there)?
We are not allowed to use xenotransplantation for fear of mixing viruses and bacteria from animal species into the human genome - but we have been doing this for over 200 years through the practice of vaccination (originally the pus and blood of cows) without anyone asking the obvious question - could this practice not only be seeding the human race with new and potentially deadly diseases (eg HIV, Ebola, etc), but can it also be changing our genetic makeup - perhaps not for the better?
These are questions that need to be asked and looking at the HPV DNA contamination of Gardasil and Cervarix is a great place to start. I'm just saying it is not the finish line - if we are concerned about contamination, we need to be concerned about ALL contamination.
Australian Vaccination Network, Inc.
|Todd Millions Comment left 13th September 2012 11:11:36|
This has being a persistent problem since -'variation',was brought back too england from turkey in the 1600's.The weakened small pox pus,produced by keeping it in a bound up walnut shell,between a womens breasts(as was bread dough and beer starter) for 'a moon',was pressed under the skin too cause a mild smallpox infection that children would survive,undisfigured.
Too strong a starter strain,or other contaminates were a risk.Sometimes producing epidemics that this was meant too prevent.Thus cow pox was seen as a great improvement.
Later,my grandparents were very concerned about the mercury based preservitive that the polio vaccines required,all others at the time were preserved with salt,and multigenreationa rat studies had already indicated pup imparment by the mercury based stabilizers.Add too this the contamination by shabby manufacuting standards health/pharmawhore canaduh is now NOT noticing...
These DNA/RNA contaminates,may be of some use in tracking how the moded fragments travel through the body.Which may be of use to track the same on GM crops.Something health and ag athourities are being extemely slow and very able NOT doing.
|joe cummins Comment left 13th September 2012 21:09:28|
In reply to Sandy Lunoe. Meryl Dorey and Todd Millions: Your comments are valuable and appreciated. Describing HPV vaccines as being prophylactic is used to differentiate them from therapeutic vaccines which are being developed to treat ongoing cervical cancers. It has not yet been proven that prophylactic vaccines prevent cancer. However, it has been found that HPV is found in 99.7% of cervical cancers. It has been shown that the prophylactic vaccines greatly reduce HPV infections of the types for which the vaccines are designed. For that reason it is probably safe to assume that the absence of virus infection will mean that cervical cancers will not be found. However, time alone will tell weather cervical cancers will be reduced by vaccination.
The special hazard of DNA and RNA contamination in vaccines is that fragments of DNA from HPV have been shown to be present in the chromosomes cervical cancer cells. Finding DNA fragments from the virus in the vaccine may pose the threat that the DNA fragments will be introduced into the chromosomes of human cells leading those cells to become cancerous. Some small RNA fragments may also act make human cells cancerous. The HPV L1 gene DNA identified in the contaminated vaccines are not likely able to cause human cells to become cancerous but fuller measurements on all of the DNA fragments in the vaccine is essential because manufacture of the vaccine involves plasmids ( small chromosomes) containing the L1 gene with flanking genes for transcription of the L1 gene from the virus coat used to make up the vaccine along with genes for propagating the plasmid in yeast or insect cells. Some of those genes are known to produce proteins or RNA that promote cancer in human cells (oncogenes).
One aspect of the DNA contamination of vaccines was very disturbing. Initially the manufacturer claimed that the vaccine was free of DNA. When contaminating DNA was independently discovered in vaccines the manufacturer acknowledged that DNA contamination was expected because of the manufacturing process. Both FDA and World Health Organization (WHO) accepted conclusions of the manufacturer without comment or fuller effort to examine the contaminating DNA. The 'regulatory' agencies further presumed that DNA fragments less than 300 bases long were safe for contamination even though they should have been aware that RNA oncogenes copied from DNA are much smaller than 300 bases long! To conclude, we must insist that the DNA contaminants in the HPV vaccines be full sequenced and identified before many more girls and young men are vaccinated. The regulators must not be allowed further cover up.
|Sandy Lunoe Comment left 19th September 2012 23:11:26|
As someone who is obviously serious about scientific truth, I am sure you will agree it is simply not acceptable to expose a healthy population of young women to the inherent risks of vaccination on the chance that a few cases of cervical cancer may be averted. This is particularly true when you consider the fact that normal cervical screening practices, already proven as a safe and effective means to control this disease, are still required.