ISIS Press Release 31/08/04
Pharm Crops for Vaccines and Therapeutic Antibodies
Prof. Joe Cummins warns of special health impacts of vaccine and antibodies in pharm crops
Reckless disregard of known risks
The European Union (EU) recently announced a major program to produce plant-based vaccines and therapeutic antibodies [1], despite the risks that came to public attention two years ago [2]. The crops plants currently used to produce vaccines include tobacco, maize, potato, tomato, rice and alfalfa. In spite of the threat to the food supply, maize is a favorite crop for vaccine production because the transgenic protein can be concentrated in the kernels. In general, field-test releases of crop plants modified for vaccine production have been undertaken with little regard for the health and environmental consequences of contaminating food crop with the vaccine genes.
Risks of vaccine proteins and antibodies
Vaccines are made using antigen proteins from disease organisms such as viruses or bacteria to elicit production of antibodies following injection into the blood stream or ingestion with food. Plant-based vaccines are mainly produced from synthetic transgenes whose DNA code words have been altered for maximal activity in a crop plant [3]. Apart from vaccines, antibodies are also produced in plants for treating both animal and plant diseases. These antibodies are effective, but plagued by the powerful immune response to the antibodies themselves following repeated exposure.
Plant-based vaccines are mainly geared towards mucosal immunization following oral intake. Oral vaccines may elicit oral tolerance on repetitive exposure. Oral tolerance is the animal’s defence against antigens in food. Thus, after repeated exposure to an oral antigen, the mucosal immune system ceases to view the antigen as such, leaving the animal susceptible to the pathogen for which the vaccine is supposed to protect against [4]. The problem of oral tolerance has been mentioned in at least one review of plant-based vaccines [5]. Oral tolerance has been used to treat autoimmune disease such as diabetes by feeding patients with plants producing an antigen eliciting the autoimmune response [6]. Oral tolerance to pathogens is one main threat from the contamination of our food supply with vaccine genes, whereas therapeutic antibodies threaten a direct immune response; these two impacts are seldom discussed by promoters of plant genetic modification or by science journals reporting the studies.
Risks from synthetic genes and viral vectors
Edible plant-based vaccines have been produced with synthetic nuclear genes, synthetic chloroplast genes or plant viruses modified with synthetic genes. These synthetic genes are completely unknown and untested for toxicities. The nuclear transgenes frequently failed to produce sufficient protein to evoke an oral immune response, while chloroplast transgenes tended to provide adequate protein levels. (Chloroplasts allow insertion of multiple transgene copies, with less problem of gene-silencing than nuclear transgene insertions). Chloroplast transformations produced antigens at high levels, up to 25% of total soluble protein while nuclear inserts generally produced less than 1% total soluble protein. The endosperm localization of nuclear gene products can boost antigen levels to 10% of protein in maize kernels [7].
Numerous plant viruses modified with vaccine antigens have been released in field tests. Such viruses can produce vaccine antigen up to10% total soluble protein in the infected plant but 1% is most frequent [8]. Little consideration has been given to containment of these GM viruses in field tests. They can be spread by sucking insects, plant wounding or by wind-blown plant debris. A recent study shows that plant viruses may be spread by wind, either in water droplets from the plant surface or by abrasive contact between plant leaves [9].
Box 1 provides a list of 30 human and animal diseases for which plant-based vaccines have been created. It is worth mentioning that about half of the transgenic vaccines on the list were produced using plant viruses as vectors, including tobacco mosaic virus, cowpea mosaic virus, alfalfa mosaic virus, potato virus X, plum pox poty virus and tomato bushy stunt virus. The virus constructions are productive but pose special long-term risks associated with the release of the virus to the environment and predictable viral recombination to produce novel disease agents. Little effort has been made to monitor these hazardous experiments.
Box 1
Plant-based vaccines [8]
Disease agents Species protected
1. Enterotoxigenic strains of E. coli humans & farmed animals
2. Vibrio cholerae/ Cholera toxin B subunit humans
3. Enteropathogenic E. coli/ Pilus structural subunit A humans
4. Vibrio cholerae/ Cholera toxin B subunit, rotavirus humans
5. Enterotoxigenic strains of E. coli humans
6. Hepatitis B virus/ Surface antigen humans
7. Hepatitis C virus/ Hypervariable region 1
of envelope protein 2 fused to cholera toxin humans
8. Norwalk virus &Rotavirus humans
9. Measles/ Haemagglutinin protein humans
10. HIV-1/ Peptide of gp41 protein humans
11. HIV-1/ V3 loop of gp120 protein humans
12. HIV-1/ Peptide of transmembrane protein gp41 humans
13. HIV-1/ Nucleocapsid protein p24 humans
14. Cytomegalovirus/ Glycoprotein B humans
15. Rhinovirus type 14/ Peptide of VP1 protein humans
16. Respiratory syncytial virus/ Peptides of G protein humans
17. Staphylococcus aureus/ D2 peptide of
bronectin-binding protein FnBP humans
18. Pseudomonas aeruginosa/ Peptides of outer-membrane humans
19. Protein F Plasmodium falciparum (malaria) &
Peptides of circumsporozoite protein humans
20. Human papillomavirus type 16/ E7 oncoprotein humans
21. Bacillus anthracis/ Protective antigen humans
22. Rabies virus/ Glycoprotein humans, domestic & wild animals
23. Foot-and-mouth disease virus/ Structural protein VP1 farmed animals
24. Transmissible gastroenteritis virus/ Glycoprotein pigs
25. Bovine group A rotavirus/ Major capsid protein VP6 cattle
26. Mannheimia haemolytica (bovine pneumonia
teurellosis)/ Leukotoxin fused to green fluorescent protein cattle
27. Mink enteritis virus/ Peptide of capsid protein VP2 mink, dogs & cats
28. Rabbit haemorrhagic disease virus/ Structural
protein VP60 rabbits
29. Rabbit haemorrhagic disease virus rabbits
30. Canine arvovirus/ Peptide of capsid protein VP2 dogs
Numerous plant based therapeutic antibodies for treating human, animal and plant diseases have been created and released in field tests. The antibodies are made from synthetic antibody genes and they are also greatly influenced by the pattern of glycosylation (sugar modification of protein) produced in the plant [10]. Further examples of plant-based antibodies include mice monoclonal antibodies that confer resistance to a herbicide by binding to it, thus inactivating the herbicide [11]. The antibody-bound herbicide was inactivated but not destroyed, and its ultimate fate is unknown; presumably it would be consumed with the transgenic crop. Kholer and Milstein discovered a method for preparing monoclonal antibodies in 1975 [12]. That discovery has made an exceptional contribution to the development of clinical analytical technology and to therapy, but that application has not fulfilled the expectation of a “magic bullet” for treating disease because the antibodies provoked a strong immune response if applied repeatedly.
Risks from cancer and HIV vaccines
In the reviews mentioned previously, numerous plant-based vaccines for treating infectious diseases have been described [7,8]. I shall now focus on cancer vaccines and vaccines against human immunodeficiency virus (HIV). A vaccine against a colorectal cancer was produced in tobacco plants [13], as was a vaccine for treating non-Hodgkins lymphoma [14]. A vaccine against the papilloma virus oncogene product causing human cervical cancer was produced using a potato virus-X vector carrying an antigen of the viral oncogene-encoded protein [15]. These cancer vaccines are an important effort to control cancer, but careless environmental release of the vaccines in crop plants could greatly increase people’s susceptibility to specific cancers through the development of oral tolerance.
The Gag gene from Simian Immunodeficiency virus (SIV) a surrogate for HIV, was used to transform potato [16]. In that experiment, the native SIV gene was used rather than a plant enhanced synthetic copy. Failure to alter the genetic code to the form most active in plants may explain the relatively low production of Gag protein. In another experiment, the coat protein of alfalfa mosaic virus was modified to express antigenic peptides for rabies virus and HIV. Antibodies against rabies and HIV were expressed in mice immunized with the antigenic peptides [17]. Simian-human immunodeficiency virus (SHIV) tat gene was fused to the cholera toxin subunit gene and the combination was used to transform potato and the fusion protein was found suitable for mucosal immunization [18]. In none of the above publications was the potential danger of the horizontal spread and recombination of the virus genes discussed.
A number of technical enhancements have been attempted to enhance the vaccine antigen production in plants. Codon usage enhancement has been mentioned [3]. Various combinations of promoters and enhancers were used to boost expression of a gene from rabbit hemorrhagic virus in potato [19]. The potato patatin promoter proved more effective than the CaMV or the ubiquitin promoter. Ricin B, a lectin sub-unit of the deadly poison ricin, has been proposed as a delivery adjuvant for mucosal vaccines [20]. At least as far as the published information is concerned, plant-based vaccines and antibodies are far from ready for major commercial production. Production of plant-based vaccines in primary food crops such as maize and rice is extremely unwise on environmental and health grounds, but a recent publication indicates that maize, at least, is still promoted by crop plant vaccine promoters [21].
Regulators must put the brakes on firmly now
In conclusion, there has been extensive creation and field tests of plant-based vaccines and therapeutic antibodies, with little care given to the environmental and health consequences of the field releases. The major accidental exposures of the public that have come to light have done little to dampen the accelerating pace of development and testing, most of which are taking place in secret away from public scrutiny.
We are heading towards a monumental poisoning of our primary food supply, unless the regulators put the brakes on firmly now.
References
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2. Ho, M. Pharmageddon. Science in Society 2003, 17, 23.
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5. Bonetta L. Edible vaccines: not quite ready for prime time. Nature Medicine 2002, 8, 94-7.
6. Ma S, Huang Y, Yin Z, Menassa R, Brandle J and Jevnikar A. Induction of oral tolerance to prevent diabetes with transgenic plants requires glutamic acid decarboxylase (GAD) Proceeding of the National Academy of Sciences USA 2004, 101, 5680-5.
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17. Yusibov V, Modelska A, Steplewski K, Agadjanyan M, Weiner D, Hooper D and Koprowski H. Antigens produced in plants by infection with chimeric plant viruses immunize against rabies virus and HIV-1 Proceedings of the National Academy of Science USA 1997, 94, 5784-8.
18. Kim T, Ruprecht R and Langridgea W. Synthesis and assembly of a cholera toxin B subunit SHIV 89.6p Tat fusion protein in transgenic potato. Protein Expression and Purfication 2004,3 5, 313-9.
19. Castanon S, Martın-Alonso J, Marın M, Boga J, Alonso P, Parra F and Ordas R. The effect of the promoter on expression of VP 60 gene from rabbit hemorrhagic disease virus in potato plants. Plant Science 2002, 162, 87-95.
20. Medina-Bolivar F, Wright R, Funka V, Sentz D, Barroso L, Wilkins T, Petri W Jr. and Cramer C. A non-toxic lectin for antigen delivery of plant-based mucosal vaccines. Vaccine 2003, 21, 997-1005.
21. Lamphear B, Jilka J, Kesl L, Welter M, Howard J and Streatfield S. A corn-based delivery system for animal vaccines: an oral transmissible gastroenteritis virus vaccine boosts lactogenic immunity in swine. Vaccine 2004, 22, 2420-4.