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ISIS Report 07/01/09

Dangers of HPV Vaccine Production in Plants, Microbes, and Viruses

Widespread releases of hazardous transgenes and vaccines have the potential to create viruses more deadly than the ones the vaccines protect against

Prof. Joe Cummins and Dr. Mae-Wan Ho

Living Rainbow H2O. By Dr. Mae-Wan Ho Human papilloma virus (HPV) vaccines are already commercialised and promoted worldwide in a bid to protect young girls and women from cervical cancer [1, 2] (Recombinant Cervical Cancer Vaccines, SiS 29; The HPV Vaccine Controversy, SiS 41), while there is still major uncertainty over their efficacy and safety, especially in the long term. One obstacle to the adoption of the vaccines by developing countries is that the two available are very costly. There appears to have been a rush to create cheap oral HPV vaccines in transgenic plants, microbes and viruses that do not require refrigeration and can be distributed relatively inexpensively, but would involve widespread releases of hazardous transgenes and products into the open environment. Some of these are near commercialization, and regulators must be warned against the approval of such production methods unless and until strict containment and safeguards are put in place.  

HPV vaccines in crop plants 

The main concern over the vaccines produced in crop plants is that transgenes from tests sites or production farms can readily spread by pollen or by mechanical dispersal of seeds.  Debris from transgenic crops can also spread transgenes and vaccine proteins through contaminating surface and groundwater. Debris in the form of dust in the air can impact on the respiratory mucosa directly, with the potential of triggering acute and delayed immune reactions in humans and animals exposed. HPV vaccines have already been associated with various adverse acute immune reactions some of which resulted in death [2]. People subject to persistent exposure to the crop vaccine are likely to develop oral tolerance rendering them susceptible to virus infection [3] (Pharm Crops for Vaccines and Therapeutic Antibodies, SiS 24)..

There have been reports since 2006 that HPV virus and L1 proteins were produced in plants including transgenic potato, tobacco and a wild tobacco  N. benthamiana  [4]. HPV L1 virus-like particles were expressed in transgenic potatoes and these particles  were found to  immunize animals fed the potatoes. The  gene for the particle protein L1 had been  optimized for activity in potato by codon alterations. The full length message had a C-terminal signal sequence for nuclear localization of the protein and  production of the L1 protein is enhanced by removal of the signal sequence for nuclear localization. The oral immunization using transgenic potato had to be  enhanced  by ingesting LI protein produced  from insect cell (baculovirus) cultures.[5]. Comparing the production of HPV19 L1 in cytoplasm or chloroplast of Nicotiana benthamiana showed that the vaccine was produced most effectively in the chloroplasts. Adjustments in codon preferences showed that the human codon preference was most effective in enhancing production of the vaccine. The optimally engineered gene configuration produced up to 11 percent of the plant’s soluble protein as L1 vaccine protein [6]. The HPV 16 L1 protein produced in N. benthamiana proved very immunogenic following injection in mice [7].  Another N. benthamiana  chloroplast transformation produced up to 1.5 percent total leaf protein as HPV L1 whose half life in the leaf was at least 8 hours [8].

The plant chloroplast system not only produces satisfactorily high levels of HPV L1 protein but avoided the spread of the transgene in pollen for the most part. But the spread of L1 protein in plant debris polluting surface and ground water and in dust to the respiratory tracts of humans and other animals cannot be avoided unless the transgenic plants are carefully confined in a secure greenhouse facility

Transgenic microbes as oral Vaccines

The transgenic yeast, Schizosaccharomyces pombe, modified to produce HPV 16 L1 [1].  Currently, a lyophilized preparation of S. pombe containing HPV 16 L1 as an oral vaccine was the subject of a patent application [9].  S. pombe is a native of Africa and has been used there to make beer. The potential pollution of the African environment with transgenic pombe yeasts requires fuller consideration.

A bacterial system has been developed for both a prophylactic and a therapeutic treatment for cervical cancer.  Bacterial expression vectors are designed to produce coat protein ( L1) or tumour associated proteins of HPV. These proteins are displayed on the surface of the modified bacterium.  The bacteria-based vaccine is potentially capable of preventing viral infection and of targeting cancer cells. Gram positive bacteria such as Lactobacilli, or gram negative bacteria such as Salmonella, may both serve as display vectors [10].

Vaccine production from viruses

A rabbit papilloma virus similar to the human virus served as a model for producing vaccine using tobacco mosaic virus (TMV).  The DNA codes for epitopes (protein amino acid sequences that are recognized by elicited antibodies) were identified and used to modify coat proteins from TMV. The modified TMV proteins were capable of eliciting antibodies that were active against the rabbit papilloma virus. The modified TMV coat proteins served as a vaccine to prevent rabbit paillomavirus infection. The modified vaccine was produced rapidly and in quantity by infecting N. benthamiana with modified TMV [11]. Using modified TMV to produce recombinant vaccines is convenient, but inherently hazardous, as the recombinant virus may give rise to new pathogens.

A potyvirus (potato virus A) coat protein gene was modified by fusing an epitope from the HPV L2 minor protein to its N terminus, and an epitope from E7 oncooprotein (cancer gene) to its C terminus.  That construct was cloned into a potato virus X vector, and used to transform N. benthamiana and the food crop Brassica rapa variety Rapa (turnip tops). Both transformed crops produced edible vaccine believed to be capable of both preventing and treating HPV cancers [12]. The purified HPV vaccine was most stable as freeze dried material stored at minus 20 degrees C [13].  N. benthamiana is not a food crop nor is it used to produce tobacco. B.  rapa is both a food crop and a weed known to spread transgenic pollen great distances, and is almost certain to cross pollinate Brassica food crops. No transgenic crops producing vaccines and drugs should be allowed in open fields for reasons stated earlier.

The Cervarix vaccine available commercially [2] is produced by GlaxoSmithKline using a baculovirus vector propagated in an insect cell line. A number of other vaccines are also being produced using baculovirus vectors. Baculoviruses are soil inhabiting viruses that infect insects. Baculovirus expression vectors propagated in insect cells were originally hampered by the appearance of many  interfering baculovirusese with chromosomal deletions, which arise as an intrinsic property of the native baculovirus [14,15]. The intrinsic deletions in the viral chromosome may provide a source of diversity as the virus faces environmental challenges.  Such instability is undesirable in producing vaccines. Some progress has been achieved in making more stable baculovirus expression vector lines [16].  Nevertheless, regulators and the vaccine producer have not made public comment about the genetic stability of the baculovirus lines producing Cervarix vaccine, nor the fact that baculovirus is capable of  infecting mammalian cells and tissues. If the GM baculovirus infects mammalian cells and tissues in vivo, they would also transfer transgenes to those infected cells as gene therapy experiments have demonstrated since 2001 [17]. Baculovirus can also serve as a gene delivery vector for stem cell and bone tissue engineering [18].

The use of GM viruses to produce HPV vaccines in yeast, insect cells, crop plants and bacteria has proceeded without much warning. And the pharmaceutical corporations commercializing such products appear to have scant regard over the safety of their products.

Hazards of horizontal transfer of transgenes

A safety issue that has been persistently ignored by regulators is horizontal transfer of transgenes to unrelated species. GM microbes and viruses have the strongest potential to transfer transgenes horizontally and contribute to creating new pathogenic bacteria and viral strains  Recent evidence confirms that transgenic DNA does jump species to bacteria and even plants and animals [19] (Horizontal Gene Transfer from GMOs Does Happen, SiS 39), as some of us had predicted. The widespread use of eukaryotic cell cultures and crops plants to produce vaccines in conjunction with viruses creates abundant opportunities for horizontal gene transfer and recombination to generate potentially more deadly viruses than the vaccines are meant to protect against.     


1. Cummins J. Recombinant cervical cancer vaccines  Science in Society 29, 20-21, 2006.

2. Cummins J and Ho MW. The HPV vaccine controversy. Science in Society 41 (to appear).

3. Cummins J.  Pharm crops for vaccines and therapeutic antibodies.  Science in Society 24, 22-23,  2004.

4. Santia L, Huanga Z, Mason H. Virus-like particles production in green plants. Particle-based Vaccines Methods 2006, 40, 66-76.

5. Warzecha H, Mason HS, Lane C, Tryggvesson A, Rybicki E, Williamson AL, Clements JD, Rose RC.Oral immunogenicity of human papillomavirus-like particles expressed in potato. J Virol. 2003; 77(16),:8702-11.

6.   Maclean J, Koekemoer M, Olivier AJ, Stewart D, Hitzeroth II, Rademacher T, Fischer R, Williamson AL, Rybicki EP.Optimization of human papillomavirus type 16 (HPV-16) L1 expression in plants: comparison of the suitability of different HPV-16 L1 gene variants and different cell-compartment localization. J Gen Virol. 2007; 88(Pt 5), 1460-9.

7. Fernández-San Millán A, Ortigosa SM, Hervás-Stubbs S, Corral-Martínez P, Seguí-Simarro JM, Gaétan J, Coursaget P, Veramendi J.Human papillomavirus L1 protein expressed in tobacco chloroplasts self-assembles into virus-like particles that are highly immunogenic. Plant Biotechnol J. 2008; 6(5), 427-41.

8.  Lenzi P, Scotti N, Alagna F, Tornesello ML, Pompa A, Vitale A, De Stradis A, Monti L, Grillo S, Buonaguro FM, Maliga P, Cardi T.Translational fusion of chloroplast-expressed human papillomavirus type 16 L1 capsid protein enhances antigen accumulation in transplastomic tobacco. Transgenic Res. 2008, 17(6), 1091-102.

9.  Sasagawa T, Tohda H, Hama Y. Edible vaccine  United States Patent Application 2007 20070154491

10. Sung M, Poo H, Lee J, Jung C, Hong S, Kim C, Park S, Pyo H.  United States Patent 2009, 7,425,438

11.  Palmer KE, Benko A, Doucette SA, Cameron TI, Foster T, Hanley KM, McCormick AA, McCulloch M, Pogue GP, Smith ML, Christensen ND. Protection of rabbits against cutaneous papillomavirus infection using recombinant tobacco mosaic virus containing L2 capsid epitopes. Vaccine 2006, 24(26), :5516-25 

12.  Hoffmeisterová H, Čeřovská N, Moravec T,  Plchová H, Folwarczna J, Velemínský J. Transient expression of fusion gene coding for the HPV-16 epitopes fused to the sequence of potyvirus coat protein using different means of inoculation of Nicotiana benthamiana  and Brassica rapa , cv. Rapa plants. Plant Cell, Tissue and Organ Culture 2008, 94, 261-7.

13.  Čeřovská N, Hoffmeisterová H, Moravec T, Plchová H, Folwarczna J, Hadámková R. Optimum storage conditions for product of transiently expressed epitopes of Human papillomavirus using Potato virus X-based vector. Biologia Plantarum 2008, 52, 184-6.

14. Pijlman GP, van den Born E, Martens DE, Vlak JM. Autographa californica baculoviruses with large genomic deletions are rapidly generated in infected insect cells. Virology. 2001, 283(1),132-8.

15. Pijlman GP, van Schijndel JE, Vlak JM. Spontaneous excision of BAC vector sequences from bacmid-derived baculovirus expression vectors upon passage in insect cells.J Gen Virol. 2003; 84(Pt 10), 2669-78.

16. Pijlman GP, de Vrij J, van den End FJ, Vlak JM, Martens DE.Evaluation of baculovirus expression vectors with enhanced stability in continuous cascaded insect-cell bioreactors. Biotechnol Bioeng. 2004; 87(6),743-53.

17. Pieroni L, Maione D, La Monica N. In vivo gene transfer in mouse skeletal muscle mediated by baculovirus  vectors. Hum Gene Ther. 2001,12(8), 871-81.

18. Chuang CK, Sung LY, Hwang SM, Lo WH, Chen HC, Hu YC. Baculovirus as a new gene delivery vector for stem cell engineering and bone tissue engineering. Gene Ther. 2007t;14(19), 1417-24.

19. Ho MW and Cummins J. Horizontal gene transfer from GMOs does happen. Science in Society 39, 22-24, 2008.

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