Adventitious Viral Genomes in Vaccines but Not in
Vaccinees
It is a pleasant change to write about viruses that might
have emerged but haven't. In this issue, Hussain and colleagues at the
Centers for Disease Control and Prevention, the U.S. Department of
Agriculture, and Harvard University report that recipients of measles,
mumps, and rubella (MMR) vaccine show no evidence of infection by endogenous
avian retroviruses, even though viral genomes and reverse transcriptase
activity have been detected in vaccine preparations. Influenza, yellow
fever, and MMR vaccines are usually prepared in embryonated eggs or in
cultures of chick embryo fibroblasts (CEF). These fibroblasts contain and
express endogenous retroviral genomes (1). In any vaccine, adventitious
agents in the cellular substrate may contaminate the biological product. In
live, attenuated vaccines, such contaminants are not inactivated, and
endogenous retroviruses by their very nature as Mendelian transmitted
genomes are particularly difficult to eliminate. Endogenous retrovirus
release also has ramifications for pharmaceutical proteins made in cell
substrates (e.g., monoclonal antibodies) and for xenotransplantation (2,3).
Some 45 years ago, it was found that apparently healthy hens could transmit
avian leukosis virus (ALV) vertically in eggs (4); later it was demonstrated
that live virus vaccines made in CEF were contaminated with infectious ALV
(5). However, no increased risk for cancer was found in yellow fever
vaccinees with the longest presumed exposure to ALV (6). Nevertheless,
vaccine manufacturers were soon required to use eggs or CEF from leukosis-free
flocks. To screen for ALV infection, a complement fixation for ALV (COFAL)
antigen test was devised, and through pioneering work in the 1960s, the
existence of endogenous retroviruses came to light because many ALV-free
birds were COFAL positive (7-9).
As a graduate student at the time, I observed that CEF of COFAL-positive
embryos complemented envelope-defective Rous sarcoma virus, yielding
pseudotype viruses with xenotropic properties. The endogenous virus was
genetically transmitted in chickens but was infectious for other hosts such
as quail and pheasant. Many copies of partial or complete ALV genomes were
located in chicken DNA (1). We showed that ALV had colonized the host germ
line of red jungle fowl before domestication to become chickens but after
divergence of the genus Gallus into distinct species. Even so, it proved
possible in the 1970s to breed white leghorns free of endogenous ALV
genomes; such chickens are now being introduced by Merck as preferred
substrates for vaccine production.
A second class of endogenous avian retroviral genome (EAV), discovered in
1985 (10), is present in all breeds of chicken and cannot be eliminated. EAV
can release noninfectious virus particles containing active reverse
transcriptase; and this is the genome most commonly found in MMR and other
vaccines (Hussain et al., this issue; 11). The major retroviral pathogen of
meat-strain chickens is an infectious recombinant between ALV gag and pol
genes and an env gene related to EAV (12). This virus has not been observed
to infect human cells.
May we assume, therefore, that chicken cell substrate vaccines are safe?
With biological products, as with crossing the street, there is no such
thing as absolute safety. The paper by Hussain et al. is reassuring, and I
agree with the authors that no change in current U.S. policies (or WHO
policies, for that matter) is warranted, and the public should continue to
enjoy the benefit of the vaccine. However, it may be useful to probe the
possibility of interaction between endogenous avian viruses and the
infectious components of MMR. We showed that vesicular stomatitis virus (VSV)
could assemble its glycoprotein G on avian retrovirus virions and vice versa
(13). Indeed, VSV G protein has become an envelope of choice for retroviral
vectors developed for gene therapy. By analogy, the assembly of the
hemagglutinin and fusion glycoproteins of measles or mumps viruses might
confer a human host range on endogenous ALV or EAV particles. The possible
generation of such pseudotypes or phenotypically mixed virions in vaccines
may be worthy of investigation.
In addition, with ultrasensitive techniques, such as polymerase chain
reaction (PCR) gene amplification, we can detect viral genomes and reverse
transcriptase activity more readily in vaccine preparations. Virtually all
vertebrates studied, including humans, carry endogenous retroviral genomes
as part of their natural genetic constitution (1,14). Therefore, almost any
cell substrate for vaccine production (avian, rodent, or primate) is likely
to contain and express (at low level) endogenous retroviral genomes.
Vaccine contamination by adventitious viruses in the cellular substrate has,
of course, occurred before. In one instance, the discovery of SV40 in rhesus
macaque kidney cultures (15) soon led to the adoption of cynomolgus macaque
and later African green monkey (AGM) kidneys as the preferred substrate for
polio vaccines. That was, perhaps, a near escape as AGMs are now known to
frequently harbor a strain of simian immunodeficiency virus (SIV) that
luckily does not appear to infect humans. Following the potential exposure
of millions of polio vaccinees to SV40, no evidence was found of increased
cancer incidence (16). More recently, it has been reported that SV40 is
present in some human cancers (17). Cases include pediatric tumors in
patients born long after SV40 was eliminated from polio vaccines.
Ironically, it was the misguided attention of regulatory groups on
hypothetical oncogenic DNA that led to vaccine contamination by adventitious
oncogenic viruses in the first place. Fear of oncogenic DNA made tumor cell
lines taboo as cellular substrates for vaccine production. Despite all we
have learned about oncogenes and tumor suppressor genes in multistep
progression to cancer, the possible trace of "oncogenic" DNA in vaccines
prepared in established cell lines remained of greater concern to regulators
than adventitious infections in primary cells. It is high time to reevaluate
the relative risks, so it is heartening that the Food and Drug
Administration held a workshop last year to begin that process.
Robin A. Weiss
University College London, London, UK
References
1.. Coffin J. Endogenous viruses. In: Weiss RA, Teich NM, Varmus HE, Coffin
J, editors. RNA tumor viruses. New York: Cold Spring Harbor Laboratory
Press;1982.p. 1109-203.
2.. Weiss RA. Retroviruses produced by hybridomas. N Engl J Med
1982;307:1587.
3.. Patience C, Takeuchi Y, Weiss RA. Infection of human cells by an
endogenous retrovirus of pigs. Nature Medicine 1997;3:282-6.
4.. Burmester BR, Gentry RF, Waters NF. The presence of the virus of
visceral lymphomatosis in embryonated eggs of normal appearing hens. Poultry
Sci 1955;34:609-17.
5.. Dougherty RM, Harris RJ, Biggs PM, Payne LN, Goffe AP, Churchill AE, et
al. Contaminant viruses in two live virus vaccines produced in chick cells.
J Hyg 1966;64:1-7.
6.. Waters TD, Anderson PS, Beebe GW, Miller RW. Yellow fever vaccination,
avian leukosis virus, and cancer risk in man. Science 1972;177:76-7.
7.. Dougherty RM, DiStefano HS. Lack of relationship between infection with
avian leukosis virus and the presence of COFAL antigen in chick embryos.
Virology 1966;29:586-95.
8.. Dougherty RM, DiStefano HS, Roth FK. Virus particles and viral antigens
in chicken tissues free of infectious avian leukosis virus. Proc Natl Acad
Sci U S A 1967;58:808-17.
9.. Payne LN, Chubb RC. Studies on the nature and genetic control of an
antigen in normal chick embryos which reacts in the COFAL test. J Gen Virol
1968;3:379-91.
10.. Dunwiddie C, Faras AJ. Presence of retrovirus reverse
transcriptase-related gene sequences in avian cells lacking endogenous avian
leukosis viruses. Proc Natl Acad Sci U S A 1985;82:5097-101.
11.. World Health Organization. Reverse transcriptase activity in
chicken-cell derived vaccine. Wkly Epidemiol Rec 1998;73:209-12.
12.. Bai J, Payne LN, Skinner MA. HPRS-103 (exogenous avian leukosis virus,
subgroup J) has an env gene related to those of endogenous elements EAV-0
and E51 and an E element found previously only in sarcoma viruses. J Virol
1995;69:779-84.
13.. Weiss RA, Boettinger DE, Love D. Phenotypic mixing between vesicular
stomatitis virus and avian RNA tumor viruses. Cold Spring Harbor Symp Quant
Biol 1975;39:913-8.
14.. Patience C, Wilkinson DA, Weiss RA. Our retroviral heritage. Trends
Genet 1997;13:116-20.
15.. Sweet BH, Hilleman MR. The vacuolating virus, SV40. Proc Soc Exp Biol
Med 1960;105:420-7.
16.. Nathanson N, Shah K. Human exposure to SV40: review and comment. Am J
Epidemiol 1976;103:1-12.
17.. Butel JS, Lednicky JA. Cell and molecular biology of simian virus 40:
implications for human infections and disease. J Natl Cancer Inst
1999;91:119-34.

http://news.bmn.com/news/story?day=030919&story=1
However, both Falsey and Crowe point out that the high rate of hMTP
infections means that it may be a good target for vaccine development.
(Crowe is a member of the scientific advisory board for the company that
owns the intellectual property rights to develop such a vaccine.)
Metapneumovirus infection rates higher than expected
18 September 2003 9:00 GMTby Rabiya S. Tuma Metapneumovirus, a respiratory
pathogen identified just two years ago, is the second most common cause of
pediatric respiratory hospitalization, according to PCR analysis of samples
collected since the mid-1970s. "For many years we thought we knew the
dominant causes of viral respiratory infections, even though about half of
the cases didn't yield a laboratory diagnosis. We just thought our tests
weren't sensitive enough," said James Crowe, a virologist and pediatrician
at Vanderbilt University in Nashville, Tennessee, who led the new study.Then
in 2001, researchers in The Netherlands discovered a previously undetected
pathogen in patients with RSV-like symptoms. The virus is closely related
to an avian pathogen metapneumovirus and was named human metapneumovirus
(hMTP).Although scientists immediately recognized that the virus was
relatively common, with most children having antibodies against it by the
time they were five, the question remained what fraction of severe
respiratory infections were due to hMTP. To find out, Crowe and colleagues
turned to a large sample bank that they have established over the past three
decades.The physicians are both virologists and primary care pediatricians.
Each year they enroll about 40 children as close to birth or before as
possible and then follow them through the first five years of life. The
clinicians encourage the parents to bring the children in even for colds and
minor illnesses and with each of these visits samples are collected and some
portion of them stored at -80 degrees Centrigrade for later study.When the
team performed RT-PCR on 687 patient samples collected during visits due to
lower respiratory infections, they found that 49 were positive for hMTP.At
12%, it is the second most frequent cause of lower respiratory infections,
says Crowe. Only respiratory syncytial virus (RSV) is more common. Influenza
and parainfluenza are both less common."I am surprised it is as high as it
is," Crowe told BioMedNet News.Similarly, when researchers at the University
of Rochester Medical
Center in New York analyzed patient samples collected over the previous
several years, they found that 10% of elderly patients hospitalized for
lower respiratory infections had hMTP, says Ann Falsey, a professor of
infectious disease at the university."It remains to be determined how
serious hMTP infections are in adults," said Falsey. "We need to do good
studies with controls to nail down how important it is." For example,
she suggests checking all hospitalized patients, regardless of what they are
in for, for the virus. It may be that hMTP is not the underlying cause of
the respiratory illness in adults.However, both Falsey and Crowe point out
that the high rate of hMTP infections means that it may be a good target for
vaccine development. (Crowe is a member of the scientific advisory board for
the company that owns the intellectual property rights to develop such a
vaccine.)Interestingly, Falsey's group saw a substantial fluctuation in the
rate of hMTP infections in adults, whereas Crowe's group saw more
consistency between years. Both studies point to a late winter peak in the
rate of infections though, with hMTP rates increasing in January and
February, just as the rates of RSV begin to decline for the year.So if the
virus is so prevalent and has been common for decades, why wasn't it
detected previously? Crowe says that it simply doesn't grow well in culture
and thus when samples were taken from patients and plated out on the four
cell types typically used to test for RSV, the hMTP virus didn't grow to
detectable levels."After having studied respiratory illnesses for years, I'd
say we don't know what causes many of them. hMTP is an important part of the
puzzle, but there are many pathogens yet to be discovered," said Falsey.The
results of both studies were presented in September at the Interscience
Conference on Antimicrobial Agents and Chemotherapy (ICAAC) in Chicago.

What Is Coming Through That Needle?
The Problem of Pathogenic Vaccine Contamination
Benjamin McRearden
In recent times mankind is experiencing a situation never
previously encountered, that being the threat of release of pathogens
intended to kill or disable large numbers of people. That danger has
prompted certain health agencies to prepare for possible mass vaccination
of the populace. The purpose of this report is to examine the existing
scientific evidence of pathogenic contaminants in vaccines. This summary,
while making no claim of being a complete review of the subject, will
point out sufficient examples and illustrations of contamination with
bacteria, viruses, and their components, so as to enable the reader to
make a more informed decision regarding accepting a vaccination (or
forcing others to receive one). It is presented in a format intended for
the public, their physicians, and their agency or governmental
representatives, and may be freely copied in its entirety.
If you as an individual are too busy to read this brief summary in
one sitting, please be aware there is ample evidence in the scientific
literature that serious viruses, bacteria; or components and toxins there
from; as well as foreign animal or cancer-related proteins and DNA are
finding their way into the commercial vaccines intended for humans, pets,
and agricultural animals. If you are interested in the short and
long-term health of yourself and those you care about, or serve as a
public servant or medical advisor, you do owe it to yourself to be
informed.
In the production of viral vaccines on a commercial scale, the
virus of concern must be reproduced in large quantities. Viruses cannot
survive or reproduce without being introduced into cells that nourish
them, which enables the viral reproductive activity. In that sense all
viruses can be considered parasitic on other cells. Living cell types
commonly used to reproduce viruses in the lab include monkey kidney
cells, chicken embryos, as well as other animal and human cells. These
cells must also be nourished with food, and are most often fed with a
nutrient mix containing in large part, bovine (cow) calf serum (usually,
serum extracted from fetal calf blood). This product can carry many types
of bovine blood-borne viruses, and is one of the primary sources of
vaccine contaminants. A journal article states, "a potential risk
associated with the production and use of biological products is viral
contamination. This contamination may be present in the source material,
e.g. human blood, human or animal tissues, cell banks, or introduced in
the manufacturing process through the use of animal sera..."(1)
Bovine viruses
The viruses and other agents that can contaminate bovine calf serum
are numerous. One of the most prominent is a pestivirus called bovine
viral diarrhea virus (2). More specifically, we see in several scientific
journal sources these types of statements: "contamination of a vaccine as
a consequence of infection of fetal calf serum"(3); "many batches of
commercially available serum are contaminated with viruses such as BVD"
[bovine viral diarrhea] (4); "virus was isolated from 332 of 1,608
(20.6%) lots of raw fetal calf serum obtained specifically for the Center
and 93 of 190 (49%) lots of commercially available fetal calf serum (5);
"agents most frequently detected in CCL's [continuous cell lines] have
been bovine viral diarrhea virus and mycoplasma. Our laboratory has
consistently found that the source of bovine viral diarrhea contamination
of CCLs has been the use of contaminated fetal bovine cell culture
enrichment serum"(6); and finally, "In conclusion, most commercially
available bovine sera are contaminated with BVDV and, although there is
no evidence that the virus is infectious, bovine sera should be screened
for this virus.for the development or production of vaccine."(7)
Can this virus cause infection or disease in humans? New evidence
shows this is possible, as researchers have found a new strain that was
isolated from human cells, and it is very closely related to the bovine
strains (8). One study finds that an alarming 75% of all laboratory cell
lines examined were contaminated with pestivirus strains; of these, all
of the bovine cell lines were contaminated with one of three possible
BVDV strains; cell lines from other animal sources including primates,
sometimes contained one of these BVDV strains (9).
There is now heightened concern that this virus and others can
cross species lines, creating new strains as they adapt to their new
hosts, and this would include passage of the virus to and from humans.
Whether the human strain of BVDV causes overt illness is uncertain,
because physicians may be uninformed and not even be looking for this
virus. It may be useful however, to compare the infection patterns in
cattle. They can be persistently infected at a low level for their entire
life with a non-pathogenic strain of the virus. Under these conditions,
they consistently create and shed virus into the surrounding environment,
which then infects other animals. The virus can nonetheless become lethal
to the animal if it mutates, with the new form also causing "visible cell
damage and death" in cultured conditions (10). The animal succumbs to
gradual or acute deterioration of the gastrointestinal mucous lining,
which produces diarrhea and its eventual demise. However, mutated virus
is not always necessary to provoke debilitating illness and death, and
ordinary virus can be isolated from the cow's pancreas, adrenal glands,
and pituitary glands (11); the virus has also been documented as causing
serious pulmonary illness (12). A study describes an outbreak of disease
among goats due to a vaccine contaminated with a bovine pestivirus;
oddly, these animals experienced reproductive failure and lesions to the
central nervous system (13). So, can these disease symptoms in varied
organs and tissues also occur in humans when they carry this virus short
or long-term?
A cursory examination of the literature indicates this may be
occurring. One revealing study tells us "faeces from children under 2
years old who had gastroenteritis that could not be attributed to
recognised enteric pathogens were examined.for Pestivirus antigens. Such
antigens were detected in 30 of 128 episodes of gastroenteritis.The
diarrhoeal disease in children excreting Pestivirus antigens resembled
that in other children except that it was more commonly associated with
signs and symptoms of respiratory inflammation."(14) There are also
concerns regarding a pattern of pestivirus infection in infacts born with
microcephaly, a condition wherein the head or cranial capacity is
unusually small (15, 16).
Scientists from the USDA National Veterinary Services Laboratory
describe the situation quite clearly, and give an indication of the
seriousness of the problem: "The high frequency of virus and antibody
detection in individual animal or small pool samples suggests that any
large pool of unscreened sera will be contaminated. Infection of cell
cultures with BVDV can lead to interference with the growth of other
viruses. Vaccine produced on contaminated cells may in turn be
contaminated, leading to seroconversion or disease in the vaccine. The
safety, purity, and efficacy of viral vaccines require BVDV testing of
ingredients, cell substrates and final product."(17) And here is a
similar statement from a New York Blood Center: "Bovine viral diarrhea
virus, whose small virion size does not allow 100% assurance of its
removal by filtration, may potentially contaminate every lot of
commercially produced fetal bovine serum."(18)
In reality though, how much of this particular viral contaminant
has trickled into humans? Well, in spite of manufacturers and regulatory
agencies claiming efficacy of their testing procedures, one 2001 study
found 13% of human MMR, polio, or Streptococcus pneumoniae vaccines
tested positive for pestivirus RNA (19). And another researcher observes,
"serum antibodies against BVDV have been detected in approximately 30% of
human population who had no contact with potentially infected
animals."(16) Also, "pestiviruses adapted to human cell cultures may be
harmful because serious BVDV infections in humans have been frequently
suggested.The BVDV persistently infected in cell cultures used for
vaccine productions have been shown to be a source of contamination in
live virus vaccines. It is, therefore, prerequisite to examine pestivirus
contamination in cell cultures to avoid secondary infections in humans as
well as in animals."(20)
Continuous immortal cell lines
This same scientist brings up another important issue. Because many
medical-use biological products (including vaccines) are now being
cultured or produced on what is called "continuous" cell lines (i.e.,
these are cell cultures consisting of "immortal" or cancerous types of
cells because they have no limits on how many times they can divide),
there is concern that viral contamination of these cell lines with a
pathogen like bovine viral diarrhea virus, could spread cancer-promoting
material into the human recipient. How could this happen? Briefly, it
works like this. The virus (which in this case has a single strand of RNA
for its genome) is capable of incorporating RNA from the cells in which
it has been cultured, into its own genome. If any contaminant RNA virus
is present in a culture that contains immortal cancerous cells, this
virus can easily mutate to include unwanted oncogenic material, which can
then get passed into the biological product intended for human medical
use (16).
Were you aware that biological products, including some common
vaccines (for instance, polio and rabies), are being produced on
"continuous" immortal cell lines? Manufacturers, scientists, and agencies
will often assure us that these cells themselves are not "tumorigenic",
i.e., they do not cause tumors per se. A closer look however, shows this
is not always the case. While lab culturing may indicate that these types
of cells are not immediately changing to overt tumor cells, it is now
well-known in the scientific community that after these cells have been
repeatedly cultured a certain number of times, something causes them to
convert to a cancerous state (21).
This journal article summary addresses the issue in regards to Vero
cells, which is a continuous cell line coming from the African green
monkey, and is commonly used in vaccine production. It states, "One of
the current criteria for evaluating the acceptability of cell lines for
use in vaccine production is lack of tumorigenicity. Vero cells represent
an example of a class of cells known as continuous cell lines. They were
derived from African green monkey kidney, and their growth properties and
culture characteristics have many advantages over other cell substrates
for use in vaccine production. We have tested Vero cells for
tumorigenicity in nude mice and in a human muscle organ culture system,
and found a significant increase in their tumorigenic potential with
increasing passage numbers. Cells at passage 232 and higher produced
nodules in all nude mice inoculated."(22) [The term "passage" in this
context means the number of times a cell line has been cultured].
There is another very important issue reported in studies that is
evidently being largely ignored as regards long-term vaccine effects and
safety. There is obvious evidence that in the lab, continuous immortal
cell lines react differently between one type of animal species and
another (21, 23). As an example, tissue from one species will allow the
immortal cell to induce a cancerous change more quickly, in comparison to
tissue from a different species. These results then beg the following
questions. How extensively have these continuous cell lines been tested
on human tissues, and would the results vary from one type of tissue to
another? And what happens over the long term.if an immortal cell from a
vaccine culture makes its way into the final vaccine product, does it
keep dividing in the human body? Another scenario might suggest the
tumor-promoting portion of its DNA inserting into a viral genome, which
then gets injected into the body. what happens at that point?
Furthermore, given the evidence that closely-related animal species
(as an example, various species of monkeys) react differently to immortal
cells, do we also need to consider that any one vaccine intended for all
humans might ultimately react differently among the various races, ethnic
groups, and sexes? And what are the effects of the vaccine contaminants
on persons with immune depression, on the elderly, or on infants?
A letter from the FDA to vaccine manufacturers dated as recently as
March 2001 shows that this issue regarding immortal cell lines is still
of concern. It states, "In general, CBER [Center for Biologics Evaluation
and Research] currently views Vero cells as an acceptable substrate for
viral vaccines, but has residual concerns.CBER recommends that all
products derived from Vero cells be free of residual intact Vero cells.
If your manufacturing process does not include a validated filtration
step or other validated procedure to clear residual intact Vero cells
from the product, please incorporate such a procedure into your
manufacturing process."(24) It is now 16 years after the WHO gave a
go-ahead (in 1986) to use continuous cell lines for vaccine production
(25), and yet there are very basic safety questions not resolved by the
manufacturers, agencies, and scientific community, much less the finer
details (26, 27). One 1991 study reports: "Cell substrate DNA was shown
to be an abundant contaminant in the clarified preparations of the Sabin
type 1, 2 and 3 poliovaccines produced on a continuous cell line"(28).
Another indicates that immortal cell lines showed 100-times greater
number of DNA recombination events compared to normal cells (29). As one
researcher states, "Using neoplastic cell lines as substrates for vaccine
development could inadvertently result in viral-viral or viral-cellular
interactions whose biological consequences are unclear.viral-viral and
viral-cellular interactions can result in the generation of new
retroviruses with pathological consequences."(30). We note the term "neoplastic"
means the quality of having an abnormal growth characteristic.
There is an even stronger statement dating back to 1990. A
scientist in the field writes, "The present concern is for safety of
vaccines made using transformed or neoplastic mammalian cells that may
contain endogenous contaminating viruses or integrated gene sequences
from oncogenic viruses. There is also concern for use of plasmid vectors
employing promoter elements from oncogenic viruses. The principal concern
for safety lies with retention of residual DNA in the vaccine, especially
since induction of cancer is a single-cell phenomenon, and a single
functional unit of foreign DNA integrated into the host cell genome might
serve to induce cell transformation as a single event or part of a series
of multifactorial events. Current proposed standards for vaccines would
permit contamination with up to 100 pg [picograms] of heterologous DNA
per dose. This is equivalent to about 10(8) 'functional lengths' of DNA.
Total safety would seem to require complete absence of DNA from the
product."(31)
Please note that 10(8) means 10 to the power of 8, or 100,000,000
"functional lengths" of DNA are allowed per dose of vaccine. Is there
something wrong with this picture? How long will the general public be
subjected to these vaccine products that according to this information,
are nowhere near safe?
It has taken, for instance, approximately forty years for the
scientific community to finally acknowledge that we have a serious
problem as a result of the contamination of polio vaccines with simian
virus 40 (SV40) in the late 1950s-early 1960s. There has been previous
evidence of some human brain and other tumors containing this virus (32,
33), but the medical community has been slow to acknowledge a definitive
link between SV40 and cancer in humans. However, two independent research
teams have recently found this virus present in 43% of cases of non-Hodgkins
lymphoma (34, 35). Another study found it present in 36% of brain tumors,
16% of healthy blood cell samples, and 22% of healthy semen samples (36).
And strangely, SV40 has now been found to infect children (37).
Considering that children of this era, are not supposed to be receiving
the virus via the vaccine contamination route, this would therefore imply
that SV40 is being transmitted from one human to another, in ways not
previously known.
Other simian viruses may also be contaminating the (Vero) monkey
cell lines used for vaccine production. One example from the literature
cites the contamination presence of SV20, which is a oncogenic simian
adenovirus (38).
Simply put, are we in a state of denial that vaccines are
ultimately transmitting viruses, DNA, and proteins into humans from
foreign animal sources (and possibly unhealthy human sources), and that
this may be strongly contributing to the incredible upsurge in cancers
and serious chronic diseases? Are these foreign animal genes altering
your DNA? Furthermore, given that viral presence can sometimes take years
to manifest actual disease symptoms, and then considering the tendencies
of health-related agencies and corporations towards short-term solutions
and profits, will we ever truly know the long-term consequences until it
is too late?
Other bovine viruses
Another contaminating virus found in the calf serum used for
vaccine production is bovine polyoma virus (polyoma viruses are strongly
associated with cancer); one pertinent article is titled "Bovine polyoma
virus, a frequent contaminant of calf serum"(39). Other contaminants
include a virus from the parvovirus family (40); another study cites
"virus-like particles" and "mycoplasma-like agents" in 68% and 20% of the
samples, respectively (41); and yet another mentions the presence of
infectious bovine rhinotracheitis virus (aka bovine herpes virus 1), and
parainfluenza-3 virus in addition to the common BVDV (42). An interesting
report from 1975 not only affirms the presence of these viruses in calf
serum, and mentions the additional presence of bovine enterovirus-4, but
also tells us that 25% of serum lots that were pre-tested by the
suppliers and "considered to be free of known viral contaminants" were
actually contaminated with bovine viruses (43). It should be obvious that
any bovine blood-borne virus (including serious retroviruses such as
bovine leukemia virus, bovine visna virus, and bovine immunodeficiency
virus) could ultimately end up in human or animal vaccines via the use of
calf serum in the manufacturing process.
Contamination of calf serum with certain bovine herpes viruses, and
the possible implication for human health, deserves a bit of scrutiny. It
is known that bovine herpesvirus-1 replicates easily in a human embryo
cell line called WI-38 (44). It is also known that bovine herpesvirus-4
is quite "persistent" in calf serum, and has a wide host range, including
human cells (45). In fact, this particular virus strongly replicates in
two human embryonic cell lines, WI-38 and MRC-5, enough so to prompt one
author to give these details and a warning: "PCR [polymerase chain
reaction] detected a 10,000-times-higher level of BHV-4 [bovine
herpesvirus-4] DNA. the supernatant indicated a 100-fold increase of
infectious particles. Since this is the first bovine (human herpes virus
8 and Epstein-Barr virus related) herpes virus which replicates on human
cells in vitro, the danger of possible human BHV-4 infection should not
be ignored." (46)
The clincher to this possible contamination, is that these same
human cell lines WI-38 and MRC-5 are two of the most common human cell
lines used to manufacture viral vaccines, (for example - rubella,
chickenpox, smallpox) and these cell lines are of course, commonly
nurtured with calf serum.
Contaminants from chicken sources
Some viral vaccines are produced by growing the virus in chicken
eggs. Common human vaccines manufactured by this method include
influenza, mumps, measles, yellow fever, and others. Like the vaccines
that include bovine-source materials, those derived from chicken embryo
culture are plagued with some very serious viral contamination problems.
Avian leukosis virus (aka avian leukemia virus or ALV) is a
retroviral pathogen that infects large segments of the modern poultry
industry, is present in commercial chickens and eggs, and thus exposes
humans on a consistent basis (47). An interesting virus in the sense that
it can be considered a "parent", it easily transforms into a dizzying
array of related viruses by hijacking one of numerous cancer-related gene
segments from its host, and inserting it into its own genome.
Furthermore, it has the additional capability of inserting itself into
the host (including human) genome, hiding out so to speak, and causing
cancerous cell transformation from that location. There is now much
scientific literature available that describes the various active
mechanisms of this and other cancer-associated viruses (48). Viruses that
originate from the "parent" avian leukosis virus, include the potent Rous
sarcoma virus, Rous-associated viruses, avian myeloblastosis virus, avian
myelocytoma virus, avian erythroblastosis virus, Fujinami sarcoma virus,
etc. One group of researchers studying the mechanism of ALV writes,
"Serial passaging of a retrovirus that does not carry an oncogene on such
cultures leads with a high frequency to the emergence of new viruses that
have transduced oncogenes."(49). In other words, given the right growth
conditions, ALV can easily transform into other closely related viruses
that are known to be cancer-related.
Just how common is this avian leukosis virus in viral vaccines? The
first evidence of contamination came to light in the 1960s when yellow
fever vaccine was found to contain it (50). Since that time, it is common
knowledge in the industry that this virus (or components thereof) still
linger in human and animal vaccines (51). Indeed, the respected Fields
Virology text (year 2001 edition) states, "At the present time, vaccines
produced by some of the world's 12 manufacturing institutes are
contaminated with avian leukosis virus"(52). One point that researchers
in this field do agree upon, are the presence of ALV, avian endogenous
virus, avian reticuloendotheliosis virus (another poultry retrovirus),
and also an enzyme called reverse transcriptase (a component of
retroviruses) in final vaccine products intended for human use,
especially the mumps, measles, yellow fever, and influenza vaccines (53,
54, 55). What they do not agree upon are the effects on humans in terms
of transmission, infection, and possible subsequent disease. A recent
study coming out of the U.S. CDC (Centers for Disease Control), which
analyzed frozen blood serum samples from children that had received MMR
vaccinations, reports no avian viral presence in these samples (56).
And yet, we see reports from other researchers that make us
question the results of that study. As is often the case with viruses,
some strains will show particular affinities for certain types of tissues
or growth conditions, and ALV is no exception (57). One researcher makes
the effort to explain, "Because of the difficulty in infecting mammalian
cells in vitro with these viruses, it is generally held that they do not
infect humans.Our results show that exposed poultry workers and subjects
with no occupational exposure to these viruses have antibodies in their
sera specifically directed against ALSV [Avian leucosis/sarcoma viruses].
Further investigation into whether these findings mean that virus has
been integrated into the human genome is needed, to assess the public
health implications of these results."(58). He also explains in another
article, that given the known behavior of these viruses in mammalian
cellular culture, a blood serum test will not always provide the correct
evidence of viral presence in the human body (47). In other words, does
the virus (or viral antibodies) need to be actively present in the blood
stream at the time of the blood draw? What if the viral particles have
retreated into other tissues? Thus the CDC study mentioned above may not
have presented an accurate assessment of viral presence, or long-term
effects from the numerous ALV-associated "offspring" viruses. Considering
that ALV can for example, easily capture the human "erbB" oncogene (59),
and that erbB as well as the oncogene called myc are strongly associated
with common forms of human breast cancer, it seems that the issue of ALV
vaccine contamination would deserve a high level of attention! (By the
way, the general reader should not feel intimidated by the abbreviations
associated with oncogenes.erb refers to "erythroblastosis", and myc
refers to myelocytomatosis, which are the names of two ALV-associated
offspring viruses). A well-known microbiology text reinforces these
concepts by teaching, "Proto-oncogenes become incorporated into
retroviral genomes with surprising ease." (60)
Toxin contamination
The unintentional presence of bacterial-source toxins (called
"endotoxins" or "exotoxins") in human and veterinary vaccines has been
recognized for many years. Such toxins are originally present in source
materials, or are produced as a result of bacterial infection during the
manufacturing process (61, 62). The various methods used in attempts to
eliminate viruses and bacteria from vaccines are simply not effective in
the removal of these problematic toxic proteins (63). Several observers
have expressed concern that the presence of endotoxin may be a source of
severe adverse reactions seen in some individuals after receiving a
vaccine (61, 64). Some vaccines, such as those for diphtheria and
tetanus, are specifically created to induce a protective mechanism in the
body against the bacterial toxin; however, vaccines prepared from
bacteria can contain appreciable and potentially dangerous lingering
amounts of toxin, despite the steps used during manufacture to decrease
the toxic potency, as described in this comment: "Vaccines composed of
gram-negative bacteria contain endotoxin in considerable amounts. This
may result in adverse effects after vaccination of sensitive animals."
(65). It has also been reported that bacterial toxin contamination
residing in calf serum, can cause breaks in the DNA of human cells (66).
Bacterial contamination - nanobacteria
Nanobacteria is a recently discovered pathogen that infects humans.
Now considered to be the smallest existing bacterial form known to
science, it escapes through common filtering processes, and can easily
invade other cells and cause cell death. Nanobacteria also are classed as
"pleomorphic", that is, they have the ability the change physical form. A
human variety of this pathogen has been found to cause or be associated
with a host of disease conditions, only a few of which include
atherosclerosis, coronary artery / heart disease, kidney stones and
kidney disease, arthritis, MS, alzheimers, some cancers, and other
conditions (67).
Since this species of bacteria is specific to mammals, and must be
lab-cultured in mammalian blood or serum, it is not surprising that this
variety of nanobacterium has been isolated as a contaminant from bovine
calf serum, other mammaliam bio-products, and vaccines. One study reports
that 100% of serum of cattle in a US herd showed antigens to
nanobacteria, and cites another report from Europe that, "more than 80%
of commercial bovine serum lots contain Nanobacterium" (68). Obviously,
any vaccines that must incorporate mammalian products during production
(which would include cow, monkey, or human cells, blood or serum), will
be prone to nanobacterial contamination. This was indeed verified when a
group of researchers found that 2 out of 3 lots of inactivated polio
vaccine, and 3 out of 6 lots of veterinary vaccines were contaminated
with nanobacteria. They also point out that the bacteria could be coming
from calf serum and contaminated culture cell lines (69). Any reasoning
person with a basic knowledge of vaccine production can deduce that
nanobacteria have undoubtedly been infecting humans in a fairly
widespread manner via vaccination procedures. One might also wonder
whether it has contributed to the current prevalence of atherosclerosis
and generalized heart disease.
Bacterial contamination - mycoplasmas and related forms
If there is any one type of bacterial contamination in vaccines
that warrants particular attention, it would be mycoplasmas. These small
organisms have a structure not characteristic of most forms of bacteria,
i.e., they usually contain a thin outer membrane as compared to the more
complex walls of common bacterial forms. They are described as being
capable of slipping through filtration procedures, and can transfer to
other media through the air or via routine handling in the lab (70). One
source states that "less than 10% of laboratories actually test for
infection/contamination regularly".that mycoplasmas are "influencing
almost every aspect of cell biology".and that labs "which do not test for
mycoplasma probably harbour contaminated cell lines and may even have
their entire stocks contaminated, as mycoplasma spreads readily along
cell lines via regents and media, the operator and the work surface"
(71). They are resistant to certain types of antibiotics used to kill
other bacteria (70, 72), and are subject to changing form under varying
physiological or biochemical conditions (73).
The journal and industry literature is filled with references to
the problems of mycoplasma contamination in cell cultures and vaccines.
Various studies cite corrupted cell lines ranging in occurrence from 5%
to 87% (71, 72, 74, 75, 76), and as we now know, once this pathogen is in
the cell culture being used to make the vaccine, it is liable to end up
in the final product (77, 78, 79,80). One author states, "Mycoplasma
contaminants can be considered important not only because of their role
as pathogens but also because they may indicate that insufficient care
has been taken during vaccine manufacture or quality control." (81).
Species of mycoplasmas that have polluted the cell cultures include
Mycoplasma hominis, M. fermentans (implicated in Gulf War illness), M.
arginini, M. hyorhinis, M. orale, M. pirum, M. pneumoniae, and
Acholeplasma laidlawii (75, 76, 82). Any reputable company that sells
tissue or cell culture material, also must test for and sell kits to
detect mycoplasmas (72, 75, 76, 83, 84).
Mycoplasmas and associated variant forms have long been associated
with many disease processes, including cancer, chronic illnesses such as
chronic fatigue syndrome, fibromyalgia, arthritis, Gulf War Illness, and
many others (73, 85, 86). It would be impossible to cite all the
pertinent references in this short report, on this vast arena of
microbiology that is often ignored by much of the medical community,
sometimes with tragic consequences. Mycoplasmas without question have the
capability of altering cell membranes and their antigens, disrupting DNA,
and altering cellular metabolism both in vitro and in vivo (70, 71, 72,
73, 86).
Cross-contamination of cell lines
As we recall that all viral vaccines can only be produced with the
use of cells, the purity of the cell lines an important issue. The most
famous example of many cell lines becoming contaminated from outside
sources, occurred when the famous and extremely fastidious HeLa cancer
cells started showing up in labs across the world in the 1960s. The
phenomenon is well-documented (87, 88, 89, 90), and is even the subject
of an entire book (91). One study from 1976 cited a litany of
contamination in all primary and continuous cell lines that were examined
- many viruses were found, as well as HeLa cells (92). As the years
progress, the reports continue to come in: one from 1984, for instance,
tells of inter- and intra-species cell cross-contamination, that 35% of
all cell lines were corrupted, and that most of these lines were
(originally) cells of human origin (93).
Let's fast-forward to 1999. A study in Germany finds that the
problem is continuing, if not worsening. In a survey of human cell lines,
the most common cross-contaminants came from "classic tumor cell lines";
that these polluted lines had been unknowingly used in "several hundred"
projects which generated potentially false reports; and that they
considered it a "grave and chronic problem demanding radical measures"
(94).
The situation is such that several scientists were prompted to
write a letter to the respected journal "Nature" in January 2000, calling
for immediate action to institute procedures that would verify the purity
of cells used for research and production of biological products, ensure
freedom from mycoplasma, and include biohazard information (95). (Did I
hear that correctly - cells can be considered a biohazard)? Has anything
changed since then to remedy the situation? There is another report from
Jan. 2002, that two major cell lines used in research projects actually
turned out to be HeLa cells (96).
I ask the reader to now recall information from earlier in this
report, that there are proposals being considered to produce vaccines and
other biological products using distinctly cancerous cell lines,
including HeLa (25). Does this seem reasonable, especially since the
current lines are already dangerously tainted with HeLa and possibly
other cancerous cells? Please remember the 100,000,000 allowable pieces
of cell-source DNA allowed per dose of vaccine (and this does not include
the viral contaminants). Anyone care for a small, under-the-skin serving
of human cancer-cell-component soup? With maybe a few monkey cell
fragments for garnish, and viruses for flavor?
Additional points to consider
There are several issues the public and medical community may want
to be aware of concerning safe administration of vaccines. The human and
animal body has normal barriers that help to protect against infiltration
by foreign agents, among them are the skin, the respiratory and
intestinal mucous linings, and the blood-brain barrier. The puncture of
skin by a needle breaches that barrier. A group of researchers states,
"Virus contamination of bioproducts such as vaccines, blood products or
biological material used in surgery and for transplantations also is more
hazardous because the application of contaminating virus usually occurs
by circumvention of the natural barrier systems of the body.virus
contamination of bioproducts should be considered as a hazard no matter
which method has been used for its detection." (97). Of even more
concern, is the administration of vaccines nasally (through the nose), or
accidental passage via that route (98). Fields Virology text (2001) says,
"The olfactory tract has long been recognized as an alternative pathway
to the CNS [central nervous system].olfactory neurons.are unprotected by
the blood brain barrier." While that writer particularly addresses the
flavivirus family [i.e., "intranasal inoculation of flaviviruses may
result in lethal encephalitis" (99)], this pattern of potential danger
may deserve further attention than it currently receives, especially if
there ever is consideration to use a method of nasal inoculation for mass
vaccination of the public or military, and there may be contaminating
viruses or toxins in a vaccine that have an affinity for nerve cells and
tissues.
Mass immunization programs often use jet injectors to save the time
and inconvenience associated with needles and syringes. However, a study
published in July 2001, found that the four injectors tested had the
capability of transferring tiny amounts of fluid and blood (and thus,
viruses such as hepatitis B and C, HIV, etc.) from one recipient to the
next (100). Numerous other articles confirm the danger, and question the
safety of these devices, including one study that reported an outbreak of
hepatitis B associated with use of a jet injector (101, 102).
Some of the newest types of vaccines are called "subunit" and
"naked DNA" vaccines. Without going into the intricacies of their
production, they involve techniques used in genetic engineering. Subunit
vaccines generally will insert a viral or bacterial DNA section into the
DNA from yeast, which is allowed to reproduce in large quantities. The
protein intended for inclusion in the vaccine is then separated from the
yeast cells. In the case of naked DNA vaccines, the viral or DNA gene is
first reproduced, then spliced into a plasmid (which is essentially free
DNA, widely used in recombinant technology), reproduced in bacteria or
cells, and then separated from them for inclusion in the vaccine.
Recombinant gene vaccines can also be produced via these methods - for
instance, hepatitis B is now an exclusively recombinant vaccine (103,
104)
One of the major concerns with these methods is the
unpredictability and interaction of the final vaccine product with the
proteins or DNA of the host. A document from the FDA states: "Genetic
toxicity: Integration of the plasmid DNA vaccine into the genome of the
vaccinated subjects is an important theoretical risk to consider in
preclinical studies. The concern is that an integrated vaccine may result
in insertional mutagenesis through the activation of oncogenes or
inactivation of tumor suppressor genes. In addition, an integrated
plasmid DNA vaccine may result in chromosomal instability through the
induction of chromosomal breaks or rearrangements." (105). Another group
advises, "Research findings in gene therapy and vaccine development show
that naked/free nucleic acids constructs are readily taken up by the
cells of all species including human beings. These nucleic acid
constructs can become integrated into the cell's genome and such
integration may result in harmful biological effects, including cancers."
(106). And to reiterate the danger of tumorigenic cell lines, a
researcher says, "More recently, recombinant DNA technology has expanded
beyond bacterial cells to mammalian cells, some of which may also be
tumorigenic." (107).
It seems obvious that there needs to be a new and open dialog
regarding vaccines among the regulatory agencies, manufacturers, research
and medical community, and the public. Many have been ridiculed for
refusing vaccination for themselves or their children, but considering
the occurrences of short-term adverse events and questionable efficacy
(108), possible long-term health damage, and now also facing the
potential of wide-ranging loss of civil liberties (109), is it so
surprising that many are questioning what the actual benefits are
surrounding most vaccination protocols? Are the cases of damaged
children, non-functional adults, the huge increases in cancer rates,
immune and chronic diseases to be simply and blindly accepted by the
public as "tolerable losses"?
As a citizen with a right to good health, please be advised of the
following issues. Vaccine quality in the U.S. relies for the most part,
on manufacturers reporting to the FDA. Here is a relevant statement from
the CDC: "Manufacturers are required to submit the results of their own
tests for potency, safety, and purity for each vaccine lot to the FDA.
They are also required to submit samples of each vaccine lot to FDA for
testing. However, if the sponsor describes an alternative procedure which
provides continued assurance of safety, purity and potency, CBER may
determine that routine submission of lot release protocols (showing
results of applicable tests) and samples is not necessary." (110) Yes,
this is the scope of the quality-control protocol that oversees a market
worth billions of dollars, yet allowing all these contaminants into the
vaccines.
It may be helpful to have an idea of the scope of the operation to
understand what we are dealing with here. We are advised that
"Large-scale cell culture operations for biotechnology products use
millions of litres of complex media and gases as well as huge quantities
of organic and inorganic raw materials. These raw materials must always
be assumed to contain contamination by adventitious agents" (111). And
because there is a potentially large number of animal and human viruses
(or viral segments) that could be entering into the final vaccine
products, it would take a equally large bank of molecular probes, as well
as frequent, wide-spread testing, to screen for presence of these
contaminating agents. This would obviously add time and expense for the
manufacturers. What needs to be decided is this - is the effort and cost
involved in cleaning up these admittedly filthy medical products, worth
the resultant benefit to the public health? And since certain animal
products are necessary for the production of vaccines, it may also be
necessary to clean house at several levels, including the agricultural
sector. It is no secret for instance, that commercial chicken flocks
raised for meat and eggs are often carrying infectious avian leucosis
virus, mentioned earlier in this report (112, 113, 114)
For the record, the smallpox vaccine ordered by the U.S. government
from Aventis is being produced on two types of continuous cell lines, the
human embryonic MRC-5 and the green monkey Vero cells (115). We might
also be advised of one researcher's thoughts, that "normal embryo and
foreskin cells presumably represent a state in development which is
genetically unstable, rendering them considerably more susceptible to
malignant transformation." (116). Are remnants of these types of cells
something we want injected into our bodies?
The decision you make in accepting or refusing a vaccination can be
a very personal one, but whatever you decide, do try to be informed of
the true benefits and risks. Nobody should be forced to submit to any
medical procedure, especially one of questionable value.
References / Notes
[Items with a PMID number will usually have abstracts available to
read. Go to the PubMed website:
http://www4.ncbi.nlm.nih.gov/entrez/query.fcgi and enter the accession
number into the search box.]
1. Trijzelaar B. Regulatory affairs and biotechnology in Europe:
III. Introduction into good regulatory practice--validation of virus
removal and inactivation. Biotherapy 1993; 6(2):93-102. PMID 8398576.
2. Vilcek S. Identification of pestiviruses contaminating cell
lines and fetal calf sera. Acta Virol 2001 Apr;45(2):81-6. PMID 11719986.
3. Barkema HW, Bartels CJ, van Wuijckhuise L, Hesselink JW,
Holzhauer M, Weber MF, Franken P, Kock PA, Bruschke CJ, Zimmer GM.
Outbreak of bovine virus diarrhea on Dutch dairy farms induced by a
bovine herpesvirus 1 marker vaccine contaminated with bovine virus
diarrhea virus type 2. Tijdschr Diergeneeskd 2001 Mar 15;126(6):158-65.
PMID 11285633.
4. Rolleston WB. Bovine serum: reducing the variables through the
use of donor herds. Dev Biol Stand 1999;99:79-86. PMID 10404879.
5. Bolin SR, Matthews PJ, Ridpath JF. Methods for detection and
frequency of contamination of fetal calf serum with bovine viral diarrhea
virus and antibodies against bovine viral diarrhea virus. : J Vet Diagn
Invest 1991 Jul;3(3):199-203. PMID 1655059.
6. Erickson GA, Landgraf JG, Wessman SJ, Koski TA, Moss LM.
Detection and elimination of adventitious agents in continuous cell
lines. Dev Biol Stand 1989;70:59-66. PMID 2759356.
7. Yanagi M, Bukh J, Emerson SU, Purcell RH. Contamination of
commercially available fetal bovine sera with bovine viral diarrhea virus
genomes: implications for the study of hepatitis C virus in cell
cultures. J Infect Dis 1996 Dec;174(6):1324-7. PMID 8940226.
8. Giangaspero M, Harasawa R, Verhulst A. Genotypic analysis of the
5'-untranslated region of a pestivirus strain isolated from human
leucocytes. Microbiol Immunol 1997;41(10):829-34. PMID 9403511.
9. Harasawa R, Mizusawa H. Demonstration and genotyping of
pestivirus RNA from mammalian cell lines. Microbiol Immunol
1995;39(12):979-85. PMID 8789057.
10. Brock, KV. Pathogenesis of BVDV Infections.
http://www.vetmed.auburn.edu/~brockkv/path.htm and
http://www.vetmed.auburn.edu/~brockkv/terms.htm
11. Stoffregen B, Bolin SR, Ridpath JF, Pohlenz J. Morphologic
lesions in type 2 BVDV infections experimentally induced by strain
BVDV2-1373 recovered from a field case. Vet Microbiol 2000 Nov
15;77(1-2):157-62. PMID 11042409.
12. Meehan JT, Lehmkuhl HD, Cutlip RC, Bolin SR. Acute pulmonary
lesions in sheep experimentally infected with bovine viral diarrhoea
virus. J Comp Pathol 1998 Oct;119(3):277-92. PMID 9807729.
13. Loken T, Krogsrud J, Bjerkas I. Outbreaks of border disease in
goats induced by a pestivirus-contaminated orf vaccine, with virus
transmission to sheep and cattle. J Comp Pathol 1991 Feb;104(2):195-209.
PMID 1650802.
14. Yolken R, Dubovi E, Leister F, Reid R, Almeido-Hill J,
Santosham M. Infantile gastroenteritis associated with excretion of
pestivirus antigens. Lancet 1989 Mar 11;1(8637):517-20. PMID 2564059.
15. Potts BJ, Sever JL, Tzan NR, Huddleston D, Elder GA. Possible
role of pestiviruses in microcephaly. Lancet 1987 Apr 25;1(8539):972-3.
16. Harasawa R. Latent Risk in Bovine Serums Used for
Biopharmaceutic Production. http://www.asmusa.org/pcsrc/sum02.htm
17. Levings RL, Wessman SJ. Bovine viral diarrhea virus
contamination of nutrient serum, cell cultures and viral vaccines. Dev
Biol Stand 1991;75:177-81. PMID 1665461.
18.
http://www.nybloodcenter.org/PatentsAndLicensing/SDTechnology.htm
19. Giangaspero M, Vacirca G, Harasawa R, Buttner M, Panuccio A, De
Giuli Morghen C, Zanetti A, Belloli A, Verhulst A. Genotypes of
pestivirus RNA detected in live virus vaccines for human use. J Vet Med
Sci 2001 Jul;63(7):723-33. PMID 11503899.
20. Harasawa R, Mizusawa H. Detection of Pestiviruses from
Mammalian Cell Cultures by the Polymerase Chain Reaction. Proceedings of
3rd Internet World Congress on Biomedical Sciences 1996.12.9-20 Riken,
Tsukuba, Japan.
http://www.3iwc.riken.go.jp/CONGRESS/SYMPO/SBB0202/AK0111/TIT.HTM
21. Contreras G, Bather R, Furesz J, Becker BC. Activation of
metastatic potential in African green monkey kidney cell lines by
prolonged in vitro culture. In Vitro Cell Dev Biol 1985
Nov;21(11):649-52. PMID 4066602.
22. Levenbook IS, Petricciani JC, Elisberg BL. Tumorigenicity of
Vero cells. J Biol Stand 1984 Oct;12(4):391-8. PMID 6526826.
23. Furesz J, Fanok A, Contreras G, Becker B. Tumorigenicity
testing of various cell substrates for production of biologicals. Dev
Biol Stand 1989;70:233-43. PMID 2759351.
24. Letter to Sponsors Using Vero Cells as a Cell Substrate for
Investigational Vaccines. Department of Health and Human Services, Public
Health Service, Food and Drug Administration, Division of Vaccines and
Related Products Applications, March 12, 2001.
www.fda.gov/cber/ltr/vero031301.htm
25. U.S. Dept. of Health and Human Services, Public Health Service,
Food and Drug Administration, Center for Biologics Evaluation and
Research. Evolving Scientific and Regulatory Perspectives on Cell
Substrates for Vaccine Development.
http://www.fda.gov/cber/minutes/0907evolv.txt
26. Lewis AM Jr. Developing an approach to evaluate the use of
neoplastic cells as vaccine substrates. Dev Biol (Basel) 2001;106:37-42;
discussion 42-3. PMID 11761251.
27. Purcell DF. Pathogenesis of replication competent retroviruses
derived from mouse cells in immuno suppressed primates: implications for
use of neoplastic cells as vaccine substrates. Dev Biol (Basel)
2001;106:187-98; discussion 199, 253-63. PMID 11761231.
28. Amosenko FA, Svitkin YV, Popova VD, Terletskaya EN, Timofeev
AV, Elbert LB, Lashkevich VA, Drozdov SG. Use of protamine sulphate for
elimination of substrate DNA in polio vaccines produced on continuous
cell lines. Vaccine 1991 Mar;9(3):207-9. PMID 1645900.
29. Thyagarajan B, McCormick-Graham M, Romero DP, Campbell C.
Characterization of homologous DNA recombination activity in normal and
immortal mammalian cells. Nucleic Acids Res 1996 Oct 15;24(20):4084-91.
PMID 8918816 (full text article available free at this link).
30. Ruscetti SK. Generation of mink cell focus-inducing
retroviruses: a model for understanding how viral-viral and
viral-cellular interactions can result in biological consequences. Dev
Biol (Basel) 2001;106:163-7; discussion 167-8, 253-63. PMID 11761228.
31. Hilleman MR. History, precedent, and progress in the
development of mammalian cell culture systems for preparing vaccines:
safety considerations revisited. J Med Virol 1990 May;31(1):5-12. PMID
2198327.
32. Butel JS, Lednicky JA. Cell and molecular biology of simian
virus 40: implications for human infections and disease. J Natl Cancer
Inst 1999 Jan 20;91(2):119-34. PMID 9923853.
33. Arrington AS, Lednicky JA, Butel JS. Molecular characterization
of SV40 DNA in multiple samples from a human mesothelioma. Anticancer Res
2000 Mar-Apr;20(2A):879-84. PMID 10810370.
34. Vilchez RA, Madden CR, Kozinetz CA, Halvorson SJ, White ZS,
Jorgensen JL, Finch CJ, Butel JS. Association between simian virus 40 and
non-Hodgkin lymphoma. Lancet 2002 Mar 9;359(9309):817-23. PMID 11897278.
35. Shivapurkar N, Harada K, Reddy J, Scheuermann RH, Xu Y, McKenna
RW, Milchgrub S, Kroft SH, Feng Z, Gazdar AF. Presence of simian virus 40
DNA sequences in human lymphomas. Lancet 2002 Mar 9;359(9309):851-2. PMID
11897287.
36. Bu X, Zhang X, Zhang X, et Al. A study of simian virus 40
infection and its origin in human brain tumors. Zhonghua Liu Xing Bing
Xue Za Zhi 2000 Feb;21(1):19-21. PMID 11860751.
37. Butel JS, Jafar S, Wong C, Arrington AS, Opekun AR, Finegold
MJ, Adam E. Evidence of SV40 infections in hospitalized children. Hum
Pathol 1999 Dec;30(12):1496-502. PMID 10667429.
38. von Mettenheim AE. Studies on simian viruses as possible
contaminants of inactivated virus vaccines. I. Direct and serologic
detection of simian adenovirus SV20. Zentralbl Bakteriol [Orig A] 1975
Jul;232(2-3):131-40. PMID 1179876.
39. Schuurman R, van Steenis B, Sol C. Bovine polyomavirus, a
frequent contaminant of calf serum. Biologicals 1991 Oct;19(4):265-70.
PMID 1665699.
40. Nettleton PF, Rweyemamu MM. The association of calf serum with
the contamination of BHK21 clone 13 suspension cells by a parvovirus
serologically related to the minute virus of mice (MVM). Arch Virol
1980;64(4):359-74. PMID 7396725.
41. Fong CK, Gross PA, Hsiung GD, Swack NS. Use of electron
microscopy for detection of viral and other microbial contaminants in
bovine sera. J Clin Microbiol 1975 Feb;1(2):219-24. PMID 51855.
42. Erickson GA, Bolin SR, Landgraf JG. Viral contamination of
fetal bovine serum used for tissue culture: risks and concerns. Dev Biol
Stand 1991;75:173-5. PMID 1665460.
43. Kniazeff AJ, Wopschall LJ, Hopps HE, Morris CS. Detection of
bovine viruses in fetal bovine serum used in cell culture. In Vitro 1975
Nov-Dec;11(6):400-3. PMID 172434.
44. Michalski FJ, Dietz A, Hsiung GD. Growth characteristics of
bovine herpesvirus 1 (infectious bovine rhinotracheitis) in human diploid
cell strain WI-38. Proc Soc Exp Biol Med 1976 Feb;151(2):407-10. PMID
175382.
45. Egyed L. Bovine herpesvirus type 4: a special herpesvirus
(review article). Acta Vet Hung 2000;48(4):501-13. PMID 11402667.
46. Egyed L. Replication of bovine herpesvirus type 4 in human
cells in vitro. J Clin Microbiol 1998 Jul;36(7):2109-11. PMID 9650976.
47. Johnson ES. Poultry oncogenic retroviruses and humans. Cancer
Detect Prev 1994;18(1):9-30. PMID 8162609.
48. For example, see Nevins JR, "Cell Transformation by Viruses",
in Knipe DM et al (ed.), 2001. Fields Virology (4th ed), Vol. I, chapter
10, p.245-283. Lippincott. Also see Joklik WK, "Tumor Viruses", in Joklik
WK et al, 1992. Zinsser Microbiology (20th ed), chapter 59, p.869-905.
Appleton & Lange.
49. Felder MP, Eychene A, Laugier D, Marx M, Dezelee P, Calothy G.
Steps and mechanisms of oncogene transduction by retroviruses. Folia Biol
(Praha) 1994;40(5):225-35. PMID 7895853.
50. Harris RJ, Dougherty RM, Biggs PM, Payne LN, Goffe AP,
Churchill AE, Mortimer R. Contaminant viruses in two live virus vaccines
produced in chick cells. J Hyg (Lond) 1966 Mar;64(1):1-7. PMID 4286627.
51. Payne LN, Biggs PM, Chubb RC, Bowden RS. Contamination of
egg-adapted canine distemper vaccine by avian leukosis virus. Vet Rec
1966 Jan 8;78(2):45-8. PMID 4285488.
52. Knipe DM et al (ed.) 2001. Fields Virology (4th ed), Vol. I,
p.1103. Lippincott.
53. Johnson JA, Heneine W. Characterization of endogenous avian
leukosis viruses in chicken embryonic fibroblast substrates used in
production of measles and mumps vaccines. J Virol 2001 Apr;75(8):3605-12.
PMID 11264350.
54. Maudru T, Peden KW. Analysis of a coded panel of licensed
vaccines by polymerase chain reaction-based reverse transcriptase assays:
a collaborative study. J Clin Virol 1998 Jul 24;11(1):19-28. PMID
9784140.
55. Tsang SX, Switzer WM, Shanmugam V, Johnson JA, Goldsmith C,
Wright A, Fadly A, Thea D, Jaffe H, Folks TM, Heneine W. Evidence of
avian leukosis virus subgroup E and endogenous avian virus in measles and
mumps vaccines derived from chicken cells: investigation of transmission
to vaccine recipients. J Virol 1999 Jul;73(7):5843-51. PMID 10364336.
56. Hussain AI, Shanmugam V, Switzer WM, Tsang SX, Fadly A, Thea D,
Helfand R, Bellini WJ, Folks TM, Heneine W. Lack of evidence of
endogenous avian leukosis virus and endogenous avian retrovirus
transmission to measles, mumps, and rubella vaccine recipients. Emerg
Infect Dis 2001 Jan-Feb;7(1):66-72. PMID 11266296. Full article text
available at www.cdc.gov/ncidod/eid/vol7no1/hussain.htm
57. Arshad SS, Howes K, Barron GS, Smith LM, Russell PH, Payne LN.
Tissue tropism of the HPRS-103 strain of J subgroup avian leukosis virus
and of a derivative acutely transforming virus. Vet Pathol 1997
Mar;34(2):127-37. PMID 9066079.
58. Johnson ES, Overby L, Philpot R. Detection of antibodies to
avian leukosis/sarcoma viruses and reticuloendotheliosis viruses in
humans by western blot assay. Cancer Detect Prev 1995;19(6):472-86. PMID
8925516.
59. Raines MA, Maihle NJ, Moscovici C, Crittenden L, Kung HJ.
Mechanism of c-erbB transduction: newly released transducing viruses
retain poly(A) tracts of erbB transcripts and encode C-terminally intact
erbB proteins. J Virol 1988 Jul;62(7):2437-43. PMID 2897475.
60. Joklik WK, "Tumor Viruses", in Joklik WK et al, 1992. Zinsser
Microbiology (20th ed.), chapter 59, p.889. Appleton & Lange.
61. Geier MR, Stanbro H, Merril CR. Endotoxins in commercial
vaccines. Appl Environ Microbiol 1978 Sep;36(3):445-9. PMID 727776.
62. Kreeftenberg JG, Loggen HG, van Ramshorst JD, Beuvery EC. The
limulus amebocyte lysate test micromethod and application in the control
of sera and vaccines. Dev Biol Stand 1977;34:15-20. PMID 838139.
63. Sharma SK. Endotoxin detection and elimination in
biotechnology. Biotechnol Appl Biochem 1986 Feb;8(1):5-22. PMID 3548752.
64. Fumarola D, Panaro A, Palma R, Mazzone A. Endotoxic
contamination of biological products (ribosomal vaccines, viral vaccines
and interferon). G Batteriol Virol Immunol 1979 Jan-Jun;72(1-6):72-7.
PMID 95449.
65. Cussler K, Godau H, Gyra H. Investigation of the endotoxin
content of veterinary vaccines. ALTEX 1994;11(5):24-29. PMID 11178403.
66. Whitaker AM, Smith EM. Effect of bacterial toxins in serum on
the chromosomes of WI-38. Dev Biol Stand 1976 Dec 13-15;37:185-90. PMID
801471.
67. See "What are nanobacteria?" at
http://www.nanobaclabs.com/PageDisplay.asp?p1=6578
68. Breitschwerdt EB, Sontakke S, Cannedy A, Hancock SI, Bradley
JM. Infection with Bartonella weissii and detection of Nanobacterium
antigens in a North Carolina beef herd. J Clin Microbiol 2001
Mar;39(3):879-82. PMID 11230398. Full article text available at
http://jcm.asm.org/cgi/content/full/39/3/879?view=full&pmid=11230398
69. Nanobacteria detected in vaccines. NanoNews 2001 July;1(2).
Article available at
http://www.nanobaclabs.com/Files/Newsletter/JulyNANONEWS1.pdf
70. Cell Culture Contamination Example. Mycoplasma.
http://www.unc.edu/depts/tcf/mycoplasma.htm
71. Prasad E, Lim-Fong R. Mycoplasmas.
http://www2.provlab.ab.ca/bugs/biologos/9702mypl.htm
72. Mycoplasma Detection Kit.
http://www.atcc.org/Products/MycoplasmaDetectKit.cfm
73. Mattman LH, 2001. Cell wall deficient forms: stealth pathogens
(3rd ed.). CRC Press.
74. Uphoff CC, Drexler HG. Prevention of mycoplasma contamination
in leukemia-lymphoma cell lines. Hum Cell 2001 Sep;14(3):244-7. PMID
11774744.
75. Mycoplasma Detection and Elimination.
http://www.dsmz.de/mutz/mutzmyco.htm
76. Mycoplasma Detection Kit.
http://www.biovalley.fr/anglais/biology/mob_cc.htm
77. Kojima A, Takahashi T, Kijima M, Ogikubo Y, Tamura Y, Harasawa
R. Detection of mycoplasma DNA in veterinary live virus vaccines by the
polymerase chain reaction. J Vet Med Sci 1996 Oct;58(10):1045-8. PMID
8916012.
78. Kojima A, Takahashi T, Kijima M, Ogikubo Y, Nishimura M,
Nishimura S, Harasawa R, Tamura Y. Detection of Mycoplasma in avian live
virus vaccines by polymerase chain reaction. Biologicals 1997
Dec;25(4):365-71. PMID 9467032.
79. Benisheva T, Sovova V, Ivanov I, Opalchenova G. Comparison of
methods used for detection of mycoplasma contamination in cell cultures,
sera, and live-virus vaccines. Folia Biol (Praha) 1993;39(5):270-6. PMID
8206173.
80. Nicolson GL, Nass M, Nicolson N. Anthrax vaccine: controversy
over safety and efficacy. Antimicrobics and Infectious Disease Newsletter
(Elsevier Science) 2000. Article located at
http://www.flatlandbooks.com/anthrax.html
81. Thornton DH. A survey of mycoplasma detection in veterinary
vaccines. Vaccine 1986 Dec;4(4):237-40. PMID 3799018.
82. Kong F, James G, Gordon S, Zelynski A, Gilbert GL.
Species-specific PCR for identification of common contaminant mollicutes
in cell culture. Appl Environ Microbiol 2001 Jul;67(7):3195-200. PMID
11425741.
83. Mycoplasma testing by PCR.
http://locus.umdnj.edu/nia/qc/myco.html
84. Mycoplasma sp. Reagent Set.
http://www.euroclone.net/mol_biology/mycoplasma.htm
85. Macomber PB. Cancer and cell wall deficient bacteria. Med
Hypotheses 1990 May;32(1):1-9. PMID 2190063.
86. Baseman JB, Tully JG. Mycoplasmas: sophisticated, reemerging,
and burdened by their notoriety. Emerg Infect Dis 1997
Jan-Mar;3(1):21-32. PMID 9126441. Full text article available at
http://www.cdc.gov/ncidod/eid/vol3no1/baseman.htm
87. Gartler SM. Apparent Hela cell contamination of human
heteroploid cell lines. Nature 1968 Feb 24;217(5130):750-1. PMID 5641128.
88. Lavappa KS. Survey of ATCC stocks of human cell lines for HeLa
contamination. In Vitro 1978 May;14(5):469-75. PMID 566722.
89. Nelson-Rees WA, Daniels DW, Flandermeyer RR.
Cross-contamination of cells in culture. Science 1981 Apr
24;212(4493):446-52. PMID 6451928.
90. Gold M. The cells that would not die. Science 81 1981 April;
29-35.
91. Gold M, 1986. A Conspiracy of Cells: One Woman's Immortal
Legacy and the Medical Scandal It Caused. State University of New York
Press.
92. Demidova SA, Tsareva AA, Mikhailova GR, Perekrest VV, Gushchin
BV. Several methodologic problems in the control of cell cultures. Vopr
Virusol 1976 May-Jun;(3):371-9. PMID 983006.
93. Hukku B, Halton DM, Mally M, Peterson WD Jr. Cell
characterization by use of multiple genetic markers. Adv Exp Med Biol
1984;172:13-31. PMID 6328905.
94. MacLeod RA, Dirks WG, Matsuo Y, Kaufmann M, Milch H, Drexler
HG. Widespread intraspecies cross-contamination of human tumor cell lines
arising at source. Int J Cancer 1999 Nov 12;83(4):555-63. PMID 10508494.
95. Stacey GN. Cell contamination leads to inaccurate data: we must
take action now. Nature 2000 Jan 27;403(6768):356. PMID 10667765.
96. Kniss DA, Xie Y, Li Y, Kumar S, Linton EA, Cohen P, Fan-Havard
P, Redman CW, Sargent IL. ED(27) Trophoblast-like Cells Isolated from
First-trimester Chorionic Villi are Genetically Identical to HeLa Cells
Yet Exhibit a Distinct Phenotype. Placenta 2002 Jan;23(1):32-43. PMID
11869090.
97. Buttner M, Oehmig A, Weiland F, Rziha HJ, Pfaff E. Detection of
virus or virus specific nucleic acid in foodstuff or bioproducts--hazards
and risk assessment. Arch Virol Suppl 1997;13:57-66. PMID 9413526.
98. Monath TP, Cropp CB, Harrison AK. Mode of entry of a
neurotropic arbovirus into the central nervous system. Reinvestigation of
an old controversy. Lab Invest 1983 Apr;48(4):399-410. PMID 6300550.
99. Burke DS, Monath TP, "Flaviviruses", in Knipe DM et al (ed.),
2001. Fields Virology (4th ed), Vol. I, chapter 33, p.1057. Lippincott.
100. Hoffman PN, Abuknesha RA, Andrews NJ, Samuel D, Lloyd JS. A
model to assess the infection potential of jet injectors used in mass
immunisation. Vaccine 2001 Jul 16;19(28-29):4020-7. PMID 11427278.
101. Canter J, Mackey K, Good LS, Roberto RR, Chin J, Bond WW,
Alter MJ, Horan JM. An outbreak of hepatitis B associated with jet
injections in a weight reduction clinic. Arch Intern Med 1990
Sep;150(9):1923-7. PMID 2393323.
102. Brink PR, van Loon AM, Trommelen JC, Gribnau FW,
Smale-Novakova IR. Virus transmission by subcutaneous jet injection. J
Med Microbiol 1985 Dec;20(3):393-7. PMID 4068027.
103. McAleer WJ, Buynak EB, Maigetter RZ, Wampler DE, Miller WJ,
Hilleman MR. Human hepatitis B vaccine from recombinant yeast. Nature
1984 Jan 12-18;307(5947):178-80. PMID 6318124.
104. Hilleman MR. Yeast recombinant hepatitis B vaccine. Infection
1987 Jan-Feb;15(1):3-7. PMID 2437037.
105. Points to Consider on Plasmid DNA Vaccines for Preventive
Infectious Disease Indications. Food and Drug Administration, Center for
Biologics Evaluation and Research, Office of Vaccine Research and Review,
December 1996. Full article available at
http://www.fda.gov/cber/gdlns/plasmid.txt
106. Ho M, Ryan A, Cummins J, Traavik T. Slipping through the
regulatory net: 'Naked' and 'free' nucleic acids. TWN Biotechnology and
Biosafety Series No. 5, 2001. Available at
http://www.twnside.org.sg/title/biod5.htm
107. Petricciani JC. Safety issues relating to the use of mammalian
cells as hosts. Dev Biol Stand 1985;59:149-53. PMID 3891461.
108. Phillips A. Dispelling vaccination myths: an internationally
published, referenced report. 1998. Report available at
http://www.unc.edu/~aphillip/www/chf/myths/dvm1.htm For statistics
regarding adverse events, see the link at
http://www.unc.edu/~aphillip/www/chf/myths/dvm11.htm
109. See a discussion of issues surrounding proposed forced
smallpox vaccination at: Fisher, BL. Smallpox and forced vaccination:
what every American needs to know. The Vaccine Reaction, Winter 2002.
Article available at http://www.909shot.com/smallpoxspecialrpt.htm. The
entire text of the Model State Emergency Health Powers Act, currently
being considered by the various U.S. state governments is available at
http://www.publichealthlaw.net/MSEHPA/MSEHPA2.pdf
110. National Vaccine Program Office, Vaccine Fact Sheets: Vaccine
Product Approval Process. Article available at
http://www.cdc.gov/od/nvpo/fs_tableII_doc2.htm
111. Garnick RL. Raw materials as a source of contamination in
large-scale cell culture. Dev Biol Stand 1998;93:21-9. PMID 9737373.
112. Fadly AM, Smith EJ. Isolation and some characteristics of a
subgroup J-like avian leukosis virus associated with myeloid leukosis in
meat-type chickens in the United States. Avian Dis 1999
Jul-Sep;43(3):391-400. PMID 10494407.
113. Grunder AA, Benkel BF, Chambers JR, Sabour MP, Gavora JS,
Dickie JW. Characterization of four endogenous viral genes in
semi-congenic lines of meat chickens. Poult Sci 1999 Jun;78(6):873-7.
PMID 10438132.
114. Pham TD, Spencer JL, Johnson ES. Detection of avian leukosis
virus in albumen of chicken eggs using reverse transcription polymerase
chain reaction. J Virol Methods 1999 Mar;78(1-2):1-11. PMID 10204692.
115. http://www.worldnetdaily.com/news/article.asp?ARTICLE_ID=25538
116. Kopelovich L. Are all normal diploid human cell strains alike?
Relevance to carcinogenic mechanisms in vitro. Exp Cell Biol
1982;50(5):266-70. PMID 7141068.
Back to page