Victoria and colleagues have identified the contamination of live viral vaccines for use in healthy children, with viral nucleic acids; the findings have since been confirmed by the vaccine manufacturers and the data reported to the FDA. Contaminating nucleic acids include retroviral sequences from the producer chicken and primate cells. Specifically, Avian leucosis virus (ALV) was present as RNA in viral particles while simian retrovirus (SRV) was present as genetically defective DNA. Rotarix, an orally administered rotavirus vaccine, contained nucleic acids from porcine circovirus-1 (PCV1), virus.[1] Since this report, a second rotavirus vaccine (RotaTeq) has been shown to contain nucleic acids from both PCV1 and PCV2, a pathogen in pigs that is associated with wasting and immunodeficiency. The circumstances in which PCV2 induces disease are discussed below.
Based upon the initial report1 Rotarix, containing PCV1 (which is not known to be a pathogen in pigs), was temporarily suspended by FDA. Following the discovery of both pathogenic and non-pathogenic PCV strains, the FDA advisory committee in an emergency public session on Friday May 7, 2010, recommended the continued use of both Rotarix and RotaTeq in the US childhood immunization schedule.
Recent investigations associated with gene therapy and vaccines leave little doubt that naked and free nucleic acids are readily taken up by the cells of all species including human beings, and may become integrated into the cell’s genetic material. There is also abundant evidence that the extraneous nucleic acids (ENA) taken up can have significant and harmful biological effects including cancers in mammals.[2]
Because they can be incorporated into the genetic material of the host or microbes, for example, colonizing the host intestinal tract, these ENA can be greatly amplified, and at the same time introduced into foreign genomes where recombination with host genes and the genes of the host’s viral pathogens may readily occur. Environmental contamination with large amounts of free or potentially free ENA will be released unregulated and unmonitored for their presence and effects.
Many assumptions about the safety of naked DNA have been proven false in light of the appropriate scientific investigations.
1. It has been argued that rapid degradation of naked DNA would reduce their biohazard potential. It is now known that naked or free DNA persists in all natural environments and is readily taken up by cells of all organisms. Of particular relevance to orally administered rota virus vaccines, DNA persists in the digestive tract of mammals[3], where it may be taken up and incorporated by the resident microbes, and by the cells of the mammalian host.
As noted by Ho et al2 in their review, viral DNA fed to mice is found to reach white blood cells, spleen and liver cells via the intestinal wall, to become incorporated into the mouse cell genome. When fed to pregnant mice, the viral DNA ends up in cells of the wall[4]. The authors remark that "The consequences of foreign DNA uptake for mutagenesis and oncogenesis have not yet been investigated." Recent developments in gene therapy demonstrate how readily naked nucleic acids can gain access to practically every type of human cells and cells of model mammals.
2. It has been argued that subgenomic viral nucleic acids are not infectious: one of the key findings is that naked viral DNA is more infectious and have a wider host range than the intact virus. Human T-cell leukaemia viral DNA formed complete viruses when injected into the bloodstream of rabbits[5].
3. It was argued that PCV1 and 2 are not infectious in humans. This is irrelevant since host range cannot be predicted from the behavior of the intact virus and modifications to viral genomes can have unexpected effects on virulence and the host range[6]. For example, naked DNA from the human polyomavirus BK (BKV) gave a full-blown infection when injected into rabbits, despite the fact that the intact BKV virus is not infectious[7]. This hazard is particularly relevant to PCV 2, which is already known to cause an AIDS-like disease in pigs.
4. Naked DNA can also trigger autoimmune reactions, in which the body’s immune system attack and kill its own tissues and cells. New research shows that any fragment of double-stranded DNA or RNA introduced into cells can induce these reactions which are responsible for many diseases[8].
5. Insertion mutagenesis – mutations in the host gene resulting from insertions of non-host genetic material - is now found to be associated with a range of cancers, including lung[9], breast[10], colon[11] and liver[12] cancers. Finally, unintended modification of germ-cells may result from exposure to naked and free DNA[13] [50].
The hazards of horizontal gene transfer
Horizontal gene transfer (HGT) refers to the direct uptake and incorporation of genetic material from unrelated species, in this instance from adventitious viral contaminants in live viralvaccines into a human host or a host-related bacteria such as those colonizing the gut. HGT is uncontrollable. Unlike chemical pollutants which break down and become diluted out, nucleic acids are infectious, they can invade cells and genomes, to multiply, mutate and recombine indefinitely.
Potential hazards of HGT of naked/free nucleic acids include:
· Generation of new viruses that cause disease
· Generation of new bacteria that cause diseases
· Spreading drug and antibiotic resistance genes among the viral and bacterial pathogens, making infections untreatable
· Random insertion into genomes of cells resulting in harmful effects including cancer
· Reactivation of dormant viruses, present in all cells and genomes, which may cause diseases
· Multiplication of ecological impacts due to all the above
It is relevant to the issue of regulatory oversight that while the technology to detect these adventitious agents and their “cryptic” consequences was not available until relatively recently, boththe dangers of generating new viruses and bacteria that can cause diseases, and spreading drug and antibiotic resistance among the pathogens, were foreseen by the pioneers of geneticengineering. That was why they called for a moratorium in the Asilomar Declaration of 1975. But commercial pressures cut the moratorium short, and guidelines were set up based on assumptions, every one of which has been invalidated by scientific findings since[14].
The presence of dormant and relict viral sequences in the human and other animal genomes has been known for at least 20 years[15]. These include human retroviral sequences that have been identified in live viral vaccines grown in human cells. In addition Victoria et al1 have confirmed the presence of viral particle-associated avian leucosis virus in the MMR vaccine. The combination of three RNA viruses with the enzymatic machinery to convert RNA into complementary DNA (cDNA) that is then capable of causing all the aforementioned problems with naked/free DNA, presents a particularly worrying biohazard. Not only are endogenous viruses such as HRV able to exert this effect on RNA vaccine viruses, as shown by Klennerman and Zinkernagel for lymphocytic choriomeningitis virus (LCMV)[16], but also viral transgenes have been found to recombine with defective viruses such as HRV, to generate infectious recombinants[17]. In turn, recombination between exogenous and endogenous viral sequences are associated with animal cancers[18] .
PCV Type 2: presence and pathogenesis
PCV Type 2 is a lymphotropic virus that infects primary lymphoid tissues[19]. Its detection in exposed (vaccinated) children should be focused on these tissues. They are available in intestinal biopsies from children with a variety of conditions including autism. They are also available from rhesus macaques exposed to the current vaccine schedule as part of ongoing safety studies. These tissues should be screened using the same metagenomic and pan-microbial array technology used by Victoria et al to identify adventitious sequences in vaccines.
The pathogenic potential of PCV Type 2 to cause an AIDS-like disease in pigs is unleashed when there is simultaneous immune system activation (e.g. concurrent vaccination) in these animals[20]. Thus, the concurrent ingestion of rotavirus vaccine and PCV Type 2 DNA sequences provides a high-risk scenario for disease – delayed or otherwise - in humans.
[1] Viral nucleic acids in live-attenuated vaccines: detection of 1 minority variants and an adventitious virus. Joseph G. Victoria, Chunlin Wang, Morris S. Jones, Crystal Jaing, Kevin McLoughlin , Shea Gardner, Eric L. Delwart. J. Virol. doi:10.1128/JVI.02690-09
[2] Ho MW, Ryan A, Cummins J, Traavik T. Unregulated Hazards - ‘Naked’ and ‘Free’ Nucleic Acids
http://www.i-sis.org.uk/naked.shtml
[3] Schubbert, R., Lettmann, C. and Doerfler, W. (1994). Ingested foreign (phage M13) DNA survives transiently in the gastrointestinal tract and enters the bloodstream of mice. Molecular and General Genetics 242, 495-504; Schubbert, R., Rentz, D., Schmitzx, B. and Doerfler, W. (1997). Foreign (M13 DNA ingested by mice reaches peripheral leukocytes, spleen and liver via the intestinal wall mucosa and can be covalently linked to mouse DNA. Proc. Nat. Acad. Sci. USA 94, 961-6.
[4] Doerfler, W. and Schubbert, R. (1998). Uptake of foreign DNA from the environment: the gastroinestinal tract and the placenta as portals of entry, Wien Klin Wochenschr. 110, 40-44
[5] Zhaqo,T., Robinson, M., Bowers, F. and Kindt,T. Infectivity of chimeric human T-cell leukaemia virus type I molecular clones assessed by naked DNA inoculation. Proc. Natnl. Acad Sci. USA 93,6653-8 (1996).
[6] Ho, M.W., Traavik, T., Olsvik, R., Tappeser, B., Howard, V., von Weizsacker, C. and McGavin, G. (1998b). Gene Technology and Gene Ecology of Infectious Diseases. Microbial Ecology in Health and Disease 10, 33-59; Traavik, T. (1999b). An orphan in science: Environmental risks of genetically engineered vaccines. Reported to the Directorate of Nature Management, Norway.
[7] Rekvig, O.P. Fredriksen, K., Brannsether, B., Moens, U., Sundsfjord, A. and Traavik, T., Antibodies to eucaryotic, including autologous, native DNA are produced during BK virus infection, but not after immunization with non-infectious BK DNA. Scand. J. Immunol. 36, 487-495 (1992).
[8] 45. Suzuki, K., Mori, A., Ishii, K.J., Singer, D.S., Klinman, D.M., Krause, P.R. and Kohn, L.D. (1999). Activation of target-tissue immune-recognition molecules by double-stranded polynucleotides. Proc. Natl. Acad. Sci. USA 96, 2285-90
[9] Fong KM et al (1997) FHIT and FRA3B 3p14.2 allele loss are common in lung cancer and preneoplasic bronchial lesions and are associated with cancer related FHIT cDNA splicing abberations. Cancer Res. (CNF), 57 (11) ; 2256-67.
[10] Asch HL (1996) Comparative expression of the LINE-1 p40 protein in human breast carcinomas and normal breast tissues. Oncol. Res (BBN) 8 (6): 239-47.
[11] Miki Y. (1992). Disruption of the ARC gene by retrotransposal insertion of L1 sequence in a colon cancer. Cancer Res (CNF), 52 (3):643-5
[12] Buendia, M.A. (1992). Mammalian hepatitis B viruses and primary liver cancer. Semin. Cancer Biol. 3, 309-20.
[13] Verdier, F. and Descotes, J. (1999). Preclinical safety evaluation of human gene therapy products. Toxicological Sciences 47, 9-15; Jane, S.M., Cunningham, J.M. and Vanin, E.F. (1998). Vector development: a major obstacle in human gene therapy. Annals of Medicine 30, 413-5.; Spadafora, C. (1998). Sperm cells and foreign DNA: a controversial relation. BioEssays 20, 955-64.
[14] Ho, M.W., Traavik, T., Olsvik, R., Tappeser, B., Howard, V., von Weizsacker, C. and McGavin, G. (1998b). Gene Technology and Gene Ecology of Infectious Diseases. Microbial Ecology in Health and Disease 10, 33-59; Traavik, T. (1999b). An orphan in science: Environmental risks of genetically engineered vaccines. Reported to the Directorate of Nature Management, Norway.
[15] See note 14 above
[16] Klennerman P and Zinkernagel R. A non-retroviral RNA virus persists in DNA form. Nature 1997;390:298-301
[17] Greene, A.E. and Allison, R.F. (1994). Recombination between viral RNA and transgenic plant transcripts. Science 263, 1423-5; Wintermantel, W.M. and Schoelz, J.E. (1996). Isolation of recombinant viruses between cauliflower mosaic virus and a viral gene in transgenic plants under conditions of moderate selection pressure. Virology 223, 156-64.
[18] See note 14 above.
[19] Choi C, Chae C, Clark EG. Porcine postweaning multisystemic wasting syndrome in Korean pig: detection of porcine circovirus 2 infection by immunohistochemistry and polymerase chain reaction. Journal of Veterinary Diagnostic Investigation, 2000;12:151-153
[20]S. Krakowka, J. A. Ellis, F. McNeilly, S. Ringler, D. M. Rings and G. Allan Activation of the Immune System is the Pivotal Event in the Production of Wasting Disease in Pigs infected with Porcine Circovirus-2 (PCV-2) Vet Pathol2001 38: 31
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