Ph02 edible vaccines

DETAILED PRESENTATION OF EDIBLE VACCINES

1. E D I B L E V A C C I N E S LET THY FOOD BE THY MEDICINE Presentation by, Mr. Srinivas R. Bhairy M.Pharmacy (F.Y) Roll No. PH02 Dept. of Pharmaceutics VES College of pharmacy, chembur Guidance Dr. Rajashree S. Hirlekar M.Pharm , PhD Dept of Pharmaceutics VES College of pharmacy, chembur

2. FLOW OF CONTENTS Introduction to vaccines Introduction to edible vaccines Steps in the production of plant-derived vaccine antigens Methods for transformation of DNA/gene into plants Candidates for edible vaccines Consideration factors/ factors affecting efficacy of edible vaccines Regulatory aspects / issues Applications of edible vaccines Some patents of edible vaccines Future aspects Case study Conclusion References

3. INTRODUCTION TO VACCINES

4. VACCINES A vaccine is a biological preparation that improves immunity to a particular disease. It contains an agent that resembles a disease-causing microorganism and is often made from weakened or killed forms of the microbe, its toxins or one of its surface proteins. The process of distributing and administrating vaccines is referred to as vaccination. Vaccination is a form of immunization.

5. Continued….. Routes of administration, including oral, nasal, intramuscular (IM), subcutaneous (SC), and intradermal (ID). Immunization science of prophylaxis. Jenner in 1796 studied that inoculation of cowpox virus prevents small pox in human. VACCINES PROPHYLACTIC (e.g. to prevent the effects of a future infection by any natural or "wild" pathogen) THERAPEUTIC (e.g. vaccines against cancer )

6. EDIBLE VACCINES

7. CONCEPT OF EDIBLE VACCINE Developed by Arntzen in the 1990s. Introduce genes of interest into plants (Transformation) Genes expressed in the plant tissues edible parts (Transgenic plants) Genes encode putatively protective vaccine antigens from viral, bacterial, and parasitic pathogens that cause disease in humans and animals Ingestion of the edible part of the transgenic plant (Oral delivery of vaccine)

8. IDEAL PROPERTIES EDIBLE VACCINES Nontoxic or Nonpathogenic very low levels of side effects Not cause problems in individuals with impaired immune system Long lasting humoral and cellular immunities Vaccination should be Simple Not contaminate the Environment Should be effective in affordable

9. DIFFICULTIES IN TRADITIONAL VACCINES Dependence on cold chain system , store and transport the vaccine under strict controlled conditions . Risk of adverse reactions Restricted production Painful needle procedure

10. Edible Vaccines Cheap Mass-production Can be ingested The need to process and purify does not arise Extensive storage Trigger the immunity at the mucosal surfaces , Which is the body’s first line of defense The difficulty in providing a standard dose Contaminate the food supply with antigens or weedy relatives Ideal plant with expression of stable gene is difficult task ADVANTAGES DISADVANTAGES

11. MECHANISM OF ACTION

12. S T E P S

13. METHODS FOR TRANSFORMATION OF GENES INTO PLANTS

14. PLASMID/VECTOR CARRIER SYSTEM: AGROBACTERIUM TUMIFACIENS METHOD

15. MICRO PROJECTILE BOMBARDMENT (BIOLISTICS)/ GENE GUN METHOD Selected DNA sequences are precipitated onto metal (e.g. Gold, tungsten) micro-particles Bombarded against the vegetable tissue with a particle gun at an accelerated speed Micro-particles penetrate the walls and release the exogenous DNA inside the cell where it will be integrated in the nuclear genome Produce large number of genetically identical crop

16. CHIMERIC VIRUSES METHOD CPMV (Cowpea mosaic virus), alfalfa mosaic virus, TMV (Tobacco mosaic virus), CaMV (Cauliflower mosaic virus), potato virus and tomato bushy stunt virus .

17. ELECTROPORATION METHOD Introduction of DNA into cells by exposing them for brief period to high voltage electrical pulse which is thought to induce transient pores in the plasma lemma. The cell wall presents an effective barrier to DNA. Therefore, it has to be weakened by mild enzymatic treatment so as to allow the entry of DNA into cell cytoplasm.

18. Candidate plants

19. Easily transformation Stored for long period without refrigeration No Cooking 2-3 years to mature&12 months to bear fruit Spoils rapidly after ripening Contains very little protein Grow quickly High content of vitamin A may boost immune response Heat-stable Do not need special facilities for storage and transportation. They taste good. Spoils easily BANANA TOMATO

20. Commonly used in baby food low allergenic potential High expression of proteins Vaccine does not dissolve when exposed to stomach acids. Less risk of contaminating than normal crop Grows slowly specialized glasshouse conditions cheaper not need to be refrigerated need cooking to use take a time to reach RICE MAIZE

21. Dominated clinical trials Easily transformed Stored for long period without refrigeration Cooking of the potatoes does not always destroy the full complement of an antigen Need cooking Good model for evaluating recombinant proteins. Easy purification of antibodies stored in the seeds Large harvests, number of times/year Produces high level of toxic alkaloids POTATO TOBACCO

22. Lettuce Fast-growing But, Spoils readily Wheat Large number of seeds help in increased harvest. but, Need cooking Carrot Rich in β carotein , production of Insulin

23. FACTORS AFFECTING EDIBLE VACCINES Antigen selection (Safe, suitable, Stable) Efficacy in model systems (small qty) Choice of plant species (Suitable, easy grown, storage, cost) Delivery and dosing issues Safety issues (allergic & toxic potential) Public perceptions and attitudes to genetic modification Quality control and licensing (consistent) FACTORS AFFECTING EFFICACY OF EDIBLE VACCINES

24. REGULATORY ASPECTS It has to be decided whether edible vaccines would be regulated under food, drugs or agricultural products Undecided that licensing shall be required for the antigen or genetically engineered fruit or transgenic seeds . Transgenes may spread by pollen, sucking insects, transfer to soil microbes during plant wounding or breakdown of roots and may pollute surface and ground water. Ethical considerations usually restrict clinical trials from directly assessing protection in humans.

25. APPLICATIONS

26. MALARIA Three antigens are currently being investigated for the development of a plant-based malaria vaccine , merozoite surface protein (MSP) 4 and MSP 5 from Plasmodium falciparum , and MSP 4/5 from P. yoelli . Wang et al have demonstrated that oral immunization of mice with recombinant MSP 4, MSP 4/5 and MSP1, co-administered with CTB as a mucosal adjuvant, induced antibody responses effective against blood-stage parasite. 2. MEASLES Mice fed with tobacco expressing MV-H (measles virus haemagglutinin from Edmonston strain) could attain antibody titers five times the level considered protective for humans and they also demonstrated secretory IgA in their faeces . Carrot, banana and rice are the potential candidates

27. HEPATITIS B potato-based vaccine against hepatitis B have reported The amount of HBsAg needed for one dose could be achieved in a single potato. Levels of specific antibodies significantly exceeded the protective level of 10 mIU / mL in humans.. 4. STOPPING AUTOIMMUNITY The transgenic potato and tobacco plants when fed to nonobese diabetic mice showed increased levels of IgG , an antibody associated with cytokines that suppress harmful immune response.

28. 5. CHOLERA plants were transformed with the gene encoding B subunit of the E. coli heat liable enterotoxin (LT-B). Transgenic potatoes expressing LT-B were found to induce both serum and secretory antibodies when fed to mice; these antibodies were protective in bacterial toxin assay in vitro . This is the first “proof of concept” for the edible vaccine. ETEC 11 volunteers were fed raw transgenic potatoes expressing LT-B. Ten (91%) of these individuals developed neutralizing antibodies and six (55%) developed a mucosal response .( Tacket et al., 1998).

29. 7. NORWALK VIRUS   Nineteen (95%) out of 20 people Fed with transgenic potato expressing norwalk virus antigen showed seroconversionn ( tacket et al., 1998). Other applications of edible vaccines under research are:- HIV STDs Anthrax Bovine pneumonia pasteurellosis

30. S. No. Patent holder Claim 01 Prodigene Recombinant antigen production and transfer to plants cells using plasmid vector system; Vaccine produced in genetically engineered plants for hepatitis and transmissible gastroenteritis virus 02 Found Advan Mil Med (USA) Antibacterial vaccine expressed in plant cells, particularly useful against shigellosis 03 Ribozyme-Pharm Nucleic acid vaccine used to treat or prevent viral infections in plants, animals or bacteria 04 Rubicon-Lab Retrovirus expressed in animal or plant cells useful as virus and cancer vaccine 05 Applied Phytologics Gene constructs for disease resistance, vaccine production in rice, barley, wheat, corn 06 Biosource (now Large Scale Biology) Plant viral vector with potential as anti-AIDS vaccine; recombinant proteins for use in vaccines to protect against parasitic infection, eg malaria 07 University of Yale Vaccine against invertebrates (insects, arachnids, helminthes, etc) 08 University of Texas Hepatitis B virus core antigen recombinant vaccine 09 Biocem; Rhone-Merieux Rabies vaccine in transgenic plants 10 Institute Pasteur Attenuated E coli vaccine for use in gene therapy Some patents OF EDIBLE VACCINES

31. Future aspects The future of edible vaccines depends on following factors: Socio-cultural acceptability of genetically modified plants Genetic stability of transgenic varieties Proper segregation of transgenic plants and prevention of environment contaminatio n and prevention of potent side effects of transgenes as production of allergens.

32. CASE STUDY

33. AIM: Transgenic Rice Expressing Amyloid β-peptide for Oral Immunization RESULTS AND DISCUSSION Various vaccine therapies for Alzheimer’s disease (AD) have been investigated. Here, transgenic rice expressing amyloid β-peptide ( Aβ ). The Aβ42 gene fused with a green fluorescent protein gene was introduced into rice using the Agrobacterium method. When transgenic brown rice expressing Aβ was orally administered to mice, serum anti- Aβ antibody titers were elevated. The same results were observed when mice were fed boiled, transgenic brown rice.

34. Western blotting was used to investigate the accumulation of the Aβ -GFP fusion protein in Aβ transgenic rice. The signal intensity of the band was compared against the signal intensity of Aβ42 as a control, and differences were observed among lines. The highest concentration, 8 μg of Aβ in a single grain of brown rice (400 μg /g brown rice) was found in samples Immunogenicity of Aβ rice was assessed by feeding brown Aβ rice to C57BL/6J mice , from 8 to 11 weeks of age, and assessing serum anti- Aβ antibody titer by ELISA. At 12 weeks age, we observed a significant increase in serum anti- Aβ antibody titer in mice fed boiled Aβ rice; the increase was not signifi -cant in mice fed uncooked Aβ rice.

35. Edible vaccine creating inexpensive vaccines that might be particularly useful in immunizing people in developing countries, where high cost, transportation and the need for certain vaccines to be refrigerated, can hamper effective vaccination programs. Edible vaccine might be solution to get rid of various ailments as it has more advantages compared to traditional vaccine. Edible plant-derived vaccine may lead to a future of safer and more effective immunization. CONCLUSION

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