PLANT DERIVED EDIBLE VACCINE

THROUGH TRANSGENICS

EDIBLE VACCINES Edible vaccines are produced through molecular

farming with the assistance of transgenic engineering.

The process of producing transgenic plants is known as transformation.

These vaccines are important for developing countries since they are inexpensive.

Assist in diseases such as diabetes, diarrhea, multiple sclerosis, rheumatoid arthritis.

Provide mucosal immunity responses in the body.

HOW EDIBLE VACCINES PROVIDE PROTECTION

http://www.quasar.ualberta.ca/edse456/apt/webquests/vaccines/article.html

HOW EDIBLE VACCINES PROVIDE PROTECTION

http://www.quasar.ualberta.ca/edse456/apt/webquests/vaccines/article.html

PRODUCTION OF EDIBLE VACCINES It is crucial to determine the surface antigens to

make edible vaccines against different pathogens.

Transformation is known as the process of insertion of a gene into the host plant genome and the manifestation of the desired gene.

Common method that used involves manipulation of bacteria in the genus Agrobacterium tumefaciens (which is a bacterium that can shift its DNA material to another species).

Desirable genes encoding the pathogen are transferred into the plant utilizing the Agrobacterium.

The methods of insertion consist of chemical treatment or electroporation of protoplasts with the use of a ``gene gun``.

Exposure of plants to chemical substances or to an electrical field that causes the cell membrane of the protoplast to become more porous.

STEP 2: Understanding the Genetics Behind Edible Vaccines, http://www.quasar.ualberta.ca/edse456/apt/webquests/vaccines/step2.html

GENE GUN

An analyst using the gene gun to inject DNA into a plant cell culture.

http://www.nature.com/nprot/journal/v1/n2/fig_tab/nprot.2006.145_F2.html, Nature Protocols

Microscopic particles containing DNA, are ``shot`` utilizing a gene gun into the plant tissues.

Transfer DNA from A. tumefaciens is necessary in order to integrate the DNA into the host plant genome.

A. tumefaciens is a promoter gene which assist in the transfer of the necessary genes into a plant`s genome.

Transformed plant cells are grown onto a callus and into complete plants which then produce transgenic fruit that can be eaten.

PRODUCTION OF EDIBLE VACCINES

STEP 2: Understanding the Genetics Behind Edible Vaccines, http://www.quasar.ualberta.ca/edse456/apt/webquests/vaccines/step2.html

CLONING INTO A PLASMID

Access excellence @ the national health museum, http://www.accessexcellence.org/RC/VL/GG/plasmid.php

Prior to DNA transfer from the bacterium to the plant, the plasmid needs to be disarmed by deleting the genes for auxin and cytokinin synthesis.

By disarming the two synthesis the bacterium will not produce a tumor.

Genes for antibiotic resistance are used to select out the transformed cells and whole plants, which contain the foreign gene.

AGROBACTERIUM TUMEFACIENCS

To get the antibiotic gene into the potato’s genome A.tumefaciens is used.

A. tumefaciens is a naturally occurring soil bacterium, which has the ability to get into plants through some kind of wound.

It possesses a circular "Ti plasmid" (tumor inducing), which enables it to infect plant cells with it own DNA.

Through the previous step the tumor inducing gene is removed, and integrates the new genome producing the edible vaccine.

CALLUSES Formed through the production of plant cells

with the Agrobacteria. Pieces of plant tissue are divided and developed

into a collection of cells in a liquid medium (suspension culture).

Certain conditions exist for this process to occur: A suitable plant tissue must be chosen High concentrations of auxin and cytokinin are required in the growth media Growth medium should consist of organic and inorganic compounds for the maintenance of cells Microorganisms must be prohibited from the medium with the aseptic conditions, of adding antibiotics during cell culture

The callus is the aim for DNA insertion into the plant tissue.

Sean Carrington http://www.cavehill.uwi.edu/FPAS/bcs/cape/capefl.html

PLANTS

Production of transgenic plants is species dependent and takes 3-9 months. Reducing this time to 6-8 weeks is currently under investigation.

The DNA integrates randomly into plant genome from the bacterium and therefore there is no consistency between plants’ antigen expression level.

EXPRESSION SYSTEMS (BIOREACTORS)

banana

s

(G. B. Sunil Kumar, 2007)

corn

rice

lettuce

PROCESS DIAGRAM

Production of plant derived vaccine (oral vaccine ) for hepatitis B. (G. B. Sunil Kumar, 2007)

HEPATITIS B VIRUS The Hepatitis B virus (HBV) infection is probably

the single most important cause of persistent viremia in humans.

The disease is characterized by acute and chronic hepatitis, which can also initiate hepatocellular carcinoma.

Currently, two forms of HBV vaccines are available, both of which are injectable and expensive.

The prevalence of this disease in developing countries justified initial efforts to express HBV candidate vaccines in plants.

PROOF OF TRANSGENIC EDIBLE VACCINES

Study focused on two main types of plants, which were the lupin callus (a wild perennial with violet flowers) and lettuce leaves.

Transgenic lupin callus was obtained to express the hepatitis B antigen, which was fed to mice.

Mice developed high levels of the antigen!!!!! Transgenic lettuce was fed to humans to analyze their

immune response. Volunteers experienced significant increases in their

immunoglobulin levels, approximately two weeks after the second ingestion.

Study proved that orally administered transgenic edible vaccines can protect against the hepatitis B virus.

PROOF OF TRANSGENIC EDIBLE VACCINES

(J. Kapusta, 1999) A Plant Derived Edible Vaccine Against Hepatitis B Virus. USA.

ADVANTAGES

Administration and safety are improved compared to injection.

Systemic and mucosal immunity. (First Line Defense)

Heat stability. Stable at room temperature. No need of refrigeration.

Mass production is possible.

Reduction in production costs-plants can be easily reproduced.

DISADVANTAGES

Edible plants are GM, and are therefore under ethical debate.

Must be eaten raw to avoid denaturation of the protein, significantly reduces possible plants.

Another problem is ensuring the vaccine has consistent dosage that survives the digestive processes of the gut.

Currently, most experimental vaccines do not provide adequate protection with just one dose.

Greenhouse segregation of medicinal plants from food crops to prevent out-crossing and would necessitate separate storage and processing facilities.

GLOSSARY

Antigen: substances that stimulate an immune response in the body resulting in the production of antibodies.

Pathogens: agents that generate diseases such as a virus or bacterium.

Epitope: proteins or peptides that can be selected to provoke anitbody responses

Plasmid: Plasmids are small circles of DNA found in bacteria cells, separate from the bacterial chromosome and smaller than it.

Viremia:when viruses enter the bloodstream and have access to the rest of the body

Auxin synthesis: synthesis of plant growth hormone Cytokinin synthesis: synthesis of plant growth hormone in

regards to promoting cell division

COMPANIES

SUMMARY

Although not in major production edible vaccines are turning out to be an advantageous choice of producing and sending vaccines to third world countries.

They provide mucosal immunity, which most vaccines do not offer.

It is accomplished through DNA exchange from A. tumefacien to the plant cells, after having undergone Ti plasmid cloning.

QUESTIONS

Assignment:

Mid-Term: