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LET THY FOOD BE THY MEDICINE

Presentation by,Mr. Srinivas R. Bhairy

M.Pharmacy (F.Y)Roll No. PH02

Dept. of PharmaceuticsVES College of pharmacy,

chembur

GuidanceDr. Rajashree S. Hirlekar

M.Pharm, PhDDept of Pharmaceutics

VES College of pharmacy, chembur

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

A vaccine is a biological preparation that improvesimmunity to a particular disease.

It contains an agent that resembles a disease-causing microorganism and is often made fromweakened or killed forms of the microbe, its toxinsor one of its surface proteins.

The process of distributing and administratingvaccines is referred to as vaccination. Vaccination isa form of immunization.

Continued…..

Routes of administration, including oral, nasal,intramuscular (IM), subcutaneous (SC), and intradermal(ID).

Immunization science of prophylaxis.

in studied that inoculation ofcowpox virus prevents small pox in human.

VACCINES

PROPHYLACTIC(e.g. to prevent the effects ofa future infection by anynatural or "wild" pathogen)

THERAPEUTIC(e.g. vaccines against

cancer )

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)

EDIBLE VACCINES

Nontoxic orNonpathogenic

very low levels ofside effects

Not causeproblems inindividuals withimpairedimmune systemLong lasting humoral

and cellular immunities

Vaccinationshould beSimple

NotcontaminatetheEnvironment

Should be effective in affordable

Dependence on cold chain system, store and transport the vaccine under strict controlled

conditions.

Risk of adverse reactions

Restricted production

Painful needle procedure

Cheap

Mass-production

Can be ingestedThe need to processand purify does notarise

Extensive storage

Trigger theimmunity at themucosal surfaces ,Which is the body’sfirst line of defense

The difficulty inproviding astandard dose

Contaminate thefood supply withantigens or weedyrelatives

Ideal plant withexpression of stablegene is difficult task

ADVANTAGESDISADVANTAGES

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

CPMV (Cowpea mosaic virus), alfalfa mosaic virus, TMV (Tobacco mosaicvirus), CaMV (Cauliflower mosaic virus), potato virus and tomato bushystunt virus.

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.

Easily transformationStored for long period without

refrigerationNo Cooking

2-3 years to mature&12 months to bear fruit

Spoils rapidly after ripeningContains very little protein

Grow quicklyHigh content of vitamin A may boost

immune responseHeat-stable

Do not need special facilities for storageand transportation.They taste good.

Spoils easilyTOMATO

Commonly used in baby food low allergenic potential

High expression of proteinsVaccine does not dissolve when exposed to

stomach acids.Less risk of contaminating than normal crop

Grows slowlyspecialized glasshouse conditions

cheaper not need to be refrigerated

need cooking to use take a time to reach

RICE

MAIZE

Dominated clinical trialsEasily transformedStored for long period without

refrigerationCooking of the potatoes does not

always destroy the fullcomplement of an antigen

Need cooking

Good model for evaluatingrecombinant proteins.

Easy purification of antibodiesstored in the seedsLarge harvests, number of

times/year

Produces high level of toxic alkaloids

POTATO

TOBACCO

LettuceFast-growing But, Spoils readily

WheatLarge number of seeds help in increased

harvest. but, Need cooking

CarrotRich in β carotein, production of Insulin

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)

It has to be decided whether edible vaccines would be

shall be required for theor or .

Transgenes may spread by pollen, sucking insects, transferto soil microbes during plant wounding or breakdown ofroots and may

usually restrict clinical trials fromdirectly assessing protection in humans.

1. MALARIAThree antigens are currently being investigatedfor the development of a plant-based malariavaccine

Wang et al have demonstratedthat oral immunization of mice withrecombinant MSP 4, MSP 4/5 and MSP1, co-administered with CTB as a mucosal adjuvant,induced antibody responses effective againstblood-stage parasite.

2. MEASLESMice fed with tobacco expressing MV-H (measlesvirus haemagglutinin from Edmonston strain) couldattain antibody titers five times the level consideredprotective for humans and they also demonstratedsecretory IgA in their faeces.

Carrot, banana and rice are the potential candidates

3. HEPATITIS B

potato-based vaccine against hepatitis B have reportedThe amount of HBsAg needed for one dose could beachieved in a single potato.

Levels of specific antibodies significantly exceeded theprotective level of 10 mIU/mL in humans..

4. STOPPING AUTOIMMUNITYThe transgenic potato and tobacco plantswhen fed to nonobese diabetic miceshowed increased levels of IgG, an antibodyassociated with cytokines that suppressharmful immune response.

5. CHOLERAplants were transformed with the geneencoding B subunit of the E. coli heatliable enterotoxin (LT-B). Transgenicpotatoes expressing LT-B were found toinduce both serum and secretoryantibodies when fed to mice; theseantibodies were protective in bacterialtoxin assay in vitro. This is the first“proof of concept” for the edible vaccine.

6. ETEC11 volunteers were fed raw transgenic potatoes expressing LT-

B. Ten (91%) of these individualsand six (55%) developed a

.(Tacket et al., 1998).

7. NORWALK VIRUS

people withshowed seroconversionn

(tacket et al., 1998).

Other applications of edible vaccines under research are:-

8. HIV9. STDs10.Anthrax11.Bovine pneumonia pasteurellosis

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

The future of edible vaccines depends on following factors:

of genetically modified plants

of transgenic varieties

Proper segregation of transgenic plants and n and

of transgenes as production of allergens.

Transgenic Rice Expressing Amyloid β-peptide for Oral Immunization

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 were observed

when mice were

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.

creating vaccines that might beparticularly useful in

where high cost, transportation and the needfor certain vaccines to be refrigerated, can hampereffective vaccination programs.

Edible vaccine might be solution to get rid of variousailments as it has

Edible plant-derived vaccine may lead to a future of

1. Charmi P et al, A Better Way For Immunization, Int J Curr Pharm Res,3(1):53-562. Hafiz Esmael et al, Review on Edible Vaccine, Acad. J. Nutri, 4 (1): 40-49, 2015.3. Vyas et al, Edible Vaccines: A New Approach to Oral Immunization Ind.J. Biotech.,(7): 283-294, 2008. 4. Sambasiva Rao et al, Green Revolution Vaccines, Edible Vaccines, Afri. J.Biotech. 2(12), 679-683, 2003.5. Swarnali Dasa et al, advances In Vaccination: A Review, Int.J. App.Pharm, 1(1), 1-21 2009.6. Akhilesh e al, Edible Vaccines: Let Thy Food Be Thy Medicine, Int.J. Pharmacolo Screen. 4(2): 105-108, 2014.7. Waghulkar e al, Fruit Derived Edible Vaccines: Natural Way for the Vaccination, Int. J.Pharmtech Res.2 (3):2010, 2124-2127.8. Madhumita Naithani et al, Edible Vaccines-A Review, Inter. J. Of Pharmacotherapy, 4(1): 2014, 58-61.9. Chaitanya et al, Edible Vaccines, J. Med.1 (1):2006, 33-34.10. Jacob et al, Edible-Vaccines-Against-Veterinary-Parasitic-Diseases—Current-Status-And-Future-Prospects, vaccines, 31:2013,

1879_188511. Swapna et al, Edible Vaccines: A New Approach For Immunization In Plant Biotechnology Sch. Acad. J. Pharm., 2(3): 2013; 227-23212. William H R, Edible Vaccines Scientific American 2000 ,66-7113. Aswathi et al, Plant Based Edible Vaccines against Poultry Diseases: A Review, Adv. Animal & Veterinary Sci, 2 (5): 305 – 311.14. Hire Rajendra et al, A Review On Edible Vaccines, IJPRBS, 2012:1 (3):133- 144.15. Dosh et al, Edible Vaccines From Gm Crops, J. Of Pharm&Sci Innova, 2(3):2013:1-616. Kumar et al, Edible Vaccine: A Prospective Substitute For Better Immunization In Future, Int J Pharm Bio Sci ,3(3):2012, (B) 948 –

955.17. Siddharth et al, plants As Bioreactors For The Production Of Vaccine Antigens, Biotech. Adva. 27 (2009): 449–467.18. Goyal et al, Edible Vaccines: Current Status And Future, Ind.J. Med. Microbio., 25 (2):2007, 93-10219. Vinod Kumar et al, Transgenic Plants As Green Factories For Vaccine Production, Afr. J. Biotechnol. 12(43): 2013, 6147-6158.20. Pant et al, Edible Vaccines: A Boon To Medical Science, Int. J.Curr.Agri. Res.3(5):2014,076-080.21. Lee Rw et al, Towards Development Of An Edible Vaccine Against Bovine Pneumonic Pasteurellosis Using Transgenic White Clover

Expressing A Mannheimia Haemolytica A1 Leukotoxin 50 Fusion Protein. Infect Immun 2001, 69:5786-5793.22. Smith et al, Factors Important in the Extraction, Stability and in Vitro Assembly of the Hepatitis B Surface Antigen Derived from

Recombinant Plant Systems. Biotechnol Prog 2002, 18:538-550.23. Castan et al, The Effect Of The Promoter On Expression Of Vp60 Gene From Rabbit Hemorrhagic Disease Virus In Potato Plants. Plant

Sci 2002, 162:87-95.24. Amanda et al, Plant Cell Factories And Mucosal Vaccines, Curr.Opinion Biotech, 14:2003, 145–150.25. ajaz Malik et al, “Edible” Vaccine-Vegetables As Alternative To Needles, Int.J.Current Res.33(5):2011,018-026.26. Taiji Yoshida et al, Transgenic Rice Expressing Amyloid Β-Peptide For Oral Immunization, Int.J. Bio. Sci. 2011; 7(3):301-307.27. Renuga Ga et al, Transgenic Banana Callus Derived Recombinant Cholera Toxin B Subunit-As Potential Vaccines, Int J Curr Sci 2014,

10: E 61-68.