Potato Vaccine

against

Hepatitis B

PLANT & MAMMALIAN CELL TECHNOLOGY

BSB316313TH DEC 2011

BY:

KALAISELVI MOHANRAJ SB09031RUBINI DEVI SELVARAJOO SB09005SUMITHALAKSMY GUNASEGARAN SB09063

BACKGROUND

Hepatitis B is a viral infection that attacks

the liver and can cause both acute and

chronic disease.

The virus is transmitted through contact

with the blood or other body fluids of an

infected person - not through casual

contact.

(Fact sheet N°204 Revised August 2008)

Over two billion people worldwide are infected with hepatitis B, a serious liver infection that can result in

Jaundice

Cirrhosis and

Liver cancer.

(ISB News Report, February 2001)

The current hepatitis vaccine extracted from yeast (injectable vaccine) requires chemical modification to become active, increasing the cost of the vaccine, which also must be stored under refrigeration.

This has severely limited its utility to more than one-third of the world's population, especially in third world regions where the disease in rampant. The situation is further complicated by the need for three separate injections of the vaccine at 0, 1, and 6 months of age.

(ISB News Report, February 2001)

Dr. Charles Arntzen, of Arizona State University, has generated and forward the idea of a stable, plant-based vaccine as an attractive alternative.

Dr. Charles Arntzen and his colleagues studied various

ways to increase plant production of the Hepatitis B antigen, HbsAg, in potato.

(ISB News Report, February 2001)

The transgenic potatoes were

created and grown by Dr. Charles

J. Arntzen and Hugh S. Mason and

their colleagues at the Boyce

Thompson Institute for Plant

Research, an affiliate of Cornell

University, USA.(ISB News Report, February 2001)

Dr. Charles J. Arntzen and his colleagues have took a gene out of the hepatitis B virus and incorporated it in the potato plant, which responded by producing the Hepatitis B virus antigen.

Once ingested, this antigen protein creates an immune response in the human body that acts as a booster shot against the Hepatitis B virus.

(Science News, 2000)

•Risks to the environment include:

- gene transfer- exposure to antigens or

selectable marker proteins - detrimental effects to the

environment.

Identification of risk and possible problems

posted by risk

•Risks to human health include:

- oral tolerance,- allergenicity,- inconsistent dosage-worker exposure- unintended exposure to antigens

or selectable marker proteins in the food chain.

•Another concern is if the transgenic plants are mass produced, they may have an inconsistent expression caused by the small interfering RNAS.

Possible Research Solution For The Risk Identified

•mutant forms of E. coli -labile toxin can be used to reduce the risk of allergic reactions and also toxicity.

•According to studies, inconsistent expression can be resolved by selecting and reselecting high expression lines, but it requires a monitoring for high expression lines.

•To enhance immunogenicity, mucosal adjuvants, better targeted to the immune system, may be used, like molecules that bind to M cells in the intestine lining and pass them to immune cells.

Advantages Of

Edible Vaccine

Dominated clinical trialsEasily manipulated/transformedEasily propagated from its “eyes”Stored for long periods without refrigeration• Easy for mass production system by breeding compared to an animal system• Possible production of vaccines with low costs• Reduced need for medical personnel and sterile injection conditions• Economical to mass produce and transport• Heat stable, eliminating the need for refrigeration• Antigen protection through bioencapsulation• Subunit vaccine (not attenuated pathogens) means improved safety

•Dosage of vaccines would be variable.

•Not convenient for infants.

Needs cooking which can denature antigen and decrease immunogenicity*Some kinds of South American potatoes can be eaten raw. Although some studies show that cooking does not destroy full complement of antigen in potatoes.

Disadvantages of Edible Vaccine

Edible vaccines are

thought to be

possible and

promising yet there

are still some issues!

• Long term effect of those who

consume transgenic plants are not

known

• Some say “playing God” by making

organisms produce things they

normally would not produce is

unnatural and wrong

Ethical Issues

Safety Issues

• Plant/crops (food) contamination through

cross

pollination and of vaccine itself in plant debris

spreading dust and other pollutant in surfaces

and ground waters.

• The vaccine antigen may affect browsing

animals and humans living in the area drinking

vaccine polluted water or breathing vaccine

polluted

dust.

• The cultivation and production of

pharmaceutical crops should be

limited to control the production

facilities like greenhouse, or in plant

tissue culture, that prevent the

environmental release of

biopharmaceuticals

• a dosage problem might occur, as the production of

antigens is likely to vary from plant to plant.

• A possible way to overcome this would involve

processing the transgenic plants into concentrated

forms, so that dosing could be uniform.

• More studies need to be performed in order to

determine the safety of edible vaccines, especially

since horizontal gene transfer may occur, thus

increasing the risk of creating new strains of

viruses.

COMPANY• Wisconsin by American Ag- Tec International,

Ltd., a Delavan, Wisconsin based pharmaceutical agricultural technology company in NY.

• Axis Genetics Cambridge, England, a plant pharmaceutical company now holds an exclusive License for this medical technology.

• Boyce Thompson Institute for Plant Research in Ithaca, New York develop Edible Plant Vaccines for the prevention of Hepatitis B.

(SeedQuest 13thNov 1998)

Chemicals

/ Reagents

Distilled water

FeSO4.7H2O

Na2EDTA

NaOH

MS macronutrient

MS micronutrient

Gamborg’s B5 vitaminSucrose

Agar

Buffer solutions (pH 4, pH 7 and pH 10)

2,4-Dichlorophenoxyacetic acid (2,4-D)

Benzo(a)pyrene (BAP)NH4NO3

CaCl2.2H2O

MgSO4.7H2O

KH2PO4

H3BO3

MnSO4.H2O

ZnSO4.7H2O

KI

Na2MoO4.2H2O

CuSO4.5H2O

CoCl2.6H2O

Glycine

Myo-inositol

Nicotinic acid

Pyridoxine

Thiamine HCl

0.1 M NaOH

Commercial ethanol

Apparatus /

Equipment

Beakers 250 mL

Beakers 500 mL

Electronic Balance

Spatula

Magnetic stirrer

Measuring cylinder, 100 mLMeasuring cylinder, 500 mLMeasuring cylinder, 1000 mLMicrowave

Sterile jars (X10)

Autoclave

Reagent bottles (1000 mL and 100 mL)Blue cap Scott bottle 500 mLReagent bottles

Pipettes

Refrigerator

Volumetric Flask

Culture plates

Potato explant

Knife

Forceps

Bunsen Burner

Aluminium FoilPara film tape

METHOD(S)Extract virus strain from HBV and inactivate it into HBsAg

Clone HBsAg into pROK2S shuttle plasmid

Electroporate the plasmid into Agrobacterium tumefaciens

Plate the bacterium and make colonies

Co-cultivate the leaf fragment to induce callus using gel medium

Transfer explant grown into soil

Select a potato explant and introduce the Agrobacterium into the explant

(JARED SCHNEIDMAN DESIGN)

REFERENCES

Kapusta.J, Modelska. A, et.al, A Plant Derived Edible Vaccine Against Hepatitis B Virus, The Faseb Journal, 13: 1796-1798, Oct 1999.

Kirk D.D, McIntosh.K, Walmsley A, et.al, Risk Analysis for Plant Made Vaccines, Springer Transgenic Research, 14:449–462, 2005.

ISB News Report, February 2001(online)http://www.biotechinfo.net/hepatitus_vaccine.html• American Ag-Tec International Ltd, 2006(online)

http://www.ag-tec.com/potato.htm