Corn as a production and delivery system for oral vaccines

1. Corn as a production and delivery system for oral vaccines Kan Wang January 10, 2003

2. Louis Pasteur Chemist 1822-1895 Pasteur and invention of vaccine • Attenuation of virulent microorganisms • Chicken cholera • Anthrax • Swine erysipelas • Prevention of infectious diseases • Therapeutic vaccines • Anti-tumor (cancer) • Anti-HIV

3. How vaccines work? A. Vaccine contain antigens (weakened or dead virus, bacteria that cause disease). When enter the body, antigens stimulate the immune system (B cells to produce antibodies with help of T cells). B. The antibodies are produced to fight the weakened or dead viruses in the vaccine. C. The antibodies “practice” on the weakened viruses, preparing the immune system to destroy real and stronger viruses in the future. D. When new virulent strain enter the body, white blood cells called macrophages engulf them, recognize the antigens, and send it to the T-cells so that the immune system response can be mobilized. Illustration by Electronic Illustrators Group

4. Vaccination: benefits and risks • Conventional vaccines: • Three types: • Killed whole organisms • Live but severely weakened (attenuated) organisms • Inactive specific parts or products of infectious organisms • Not always effective • Can cause allergic reactions or even death • Modern vaccines: • DNA vaccines • Subunit vaccines

5. Why oral vaccine? • First line of defense • Effective and economical in inducing systemic immunity • Vertical transmission • Safer compare to injectable vaccines

6. Current limitations in subunit vaccine production • Chemical synthesis • Size • Cost • Microbial fermentations • Protein processing • Downstream purification • Animal tissue cultures • Cost • Possible pathogen contamination

7. SV40 contamination in polio vaccine in 1960’s Farm-aceuticals: why use plant as bioreactor ? • Safer • Plants carries no common mammalian pathogenes • Animal viruses – Bovine viral diseases • Microbes – E. coli 0157, Salmonella • Prions – Mad cow diseases

8. Farm-aceuticals: why use plant as bioreactor ? • Safer • High-volume production • Seeds allow long-term protein storage and stability • 11 million children each year die from • Pneumonia, Diarrhea, Measles, Malaria, Malnutrition Source: WHO Fact Sheet No 178, 1998

9. Plant process vs other process: Modified from Fischer et al., 1999.

10. Which plant ? Which tissue ?

11. Why corn ? • Major staple food and feed worldwide • Most tolerated plant for both humans and animals • Can be fed raw • Yield • Seeds allow long-term protein storage and stability • Established infrastructure for production and protein extraction • Possible low-cost production and administration of proteins • Genetic transformation is routine in ISU

12. Transformed plant cells divide rapidly and form plantlets Agrobacterium delivers the bacterial gene into plant cells and integrated into plant’s own chromosome Transgenic Maize plants are grown in greenhouse to maturity How vaccine corn is made? Altered bacterial gene is spliced into Agrobacterium

13. Ubiquitin promoter 27 kD  -zein promoter Title: Development of Corn-based Edible Vaccine for Livestock USDA-NRI Award ID # 99-35504-7799 Objective Long term goal:to produce effective vaccines in corn to protect livestock against viral pathogens. Specific objective: to produce high levels of vaccines against 1) human diarrhea disease and 2) porcine transmissible gastroenteritis (TGE) in transgenic maize plants.

14. Mouse feeding trial Dry corn pellet BALB/c mice LT-B corn

15. Spiked Functional analyses of maize generated LT-B – Mice Feeding Anti-LT-B antibody analysis in orally immunized mice Serum IgG Fecal IgA Serum IgA Negative Immunization dates: days 0, 3, 7 and 21 Sampling dates: days –1, 6, 13, 20, 27 Transgenic

16. Patent mouse assay for toxin challenge of mice Mouse gavaged with 25 mg of LT or PBS at day 28 and gut/carcass ratio determined 3 h after euthanasia.

17. Comparing level and stability of seed expression of LT-B LT-B expression increased by 53-fold over 2 generations

18. I am wondering if it is a transgenic apple?

19. Plant-Made Pharmaceuticals - points to consider: • Scientific considerations: • Proof of concept • Genetic stability • Regulatory considerations: • Product comparability • Purity • Safety considerations: • Environmental impact • Product protection • Gene flow • Commercialization considerations: • Legal issues

20. Product comparability Potential problems: • Different glycosylation pattern What is Glycosylation: - Adding sugar moieties to protein Why is it bad? • Can affect activity of products • Can affect optimal dose • Can cause allergic reactions in some patients What should we do? • Structure, function, bio-activity tests

21. Purity Potential problems: Contamination of • plant alkaloids • plant macromolecules (DNA, polysaccharides, lipids etc) • pesticides, herbicides • bacterial and fungi endotoxin • other protein products

22. Purity (cont’n) Why is it bad? • direct toxic effects on the recipient • effects on product stability and biodistribution • allergic reactions What should we do? • effective purification process • rigorous testing and/or validation protocols • identity preservation

23. Solid Phase Immunoassay Commonly Allergenic Less commonly Allergenic Assessment of the allergenic potential of GM food (decision tree) Source of Gene (Allergenic) Yes No Sequence similarity Yes No No No Stability to Digestion/processing Skin Prick Yes Yes No Yes Consult with Reg. Agency DBPCFC (IRB) Yes Label as to source No No Market

24. Environmental impact Potential problems: • horizontal gene transfer to soil microorganisms • recombinant toxin may contaminate soil or affect wildlife eating the plant What should we do? • Containment or restricted field release • Risk assessment

25. Containment and restricted field release • Physical isolation:one mile (radius) away from any corn field • Temporary isolation:three weeks delayed planting • Biological isolation:male sterile (not this project) Summer, 2002

26. 60 inch 30 inch 120 inch Demonstration of corn pollen flow Courtesy Dr. P. Peterson (Department of Agronomy, ISU)

27. Louis Pasteur Chemist 1822-1895 Molecular Pharming “Do not put forward anything that you cannot prove by experimentation.” “Chance favors only the prepared mind.”