Edible Vaccines

Prepared by: Rubielyn Narra

Plants as Bioreactors
Transgenic non-food GM plant pharming; biopharming; molecular farming; or simply, pharming

MOLECULAR FARMING
Molecular farming is a method used to integrate a foreign gene into plants. Molecular farming is the term for new use plants only (not animals) and is different in that this does not affect and has nothing to do with food.

Plants genetically engineered to make products that are not of plant origin Products: therapeutics, vaccines, antibodies, industrial proteins, bioplastics Pros: large amounts, no bacterial or viral contamination, low production cost Cons: different sugar residues There are two types of Molecular Farming: Medical and non-Medical.

Medical Molecular Farming

The first synthesis of a pharmaceuticallyrelevant protein, human growth hormone, was described in transgenic tobacco plants in 1986. Now, molecular farming has become commercially interesting as a method for the production of recombinant pharmaceutical proteins, in particular antibodies.

Non-Medical Molecular Farming

Non-Medical Molecular Farming includes Industrial Enzymes and Polymers. Industrial enzymes: for example laccase in transgenic maize, Technical proteins for research purposes: for example avidin, which is also produced in maize, Milk proteins such as human beta casein, which is produced in transgenic tomatoes, Protein polymers: collagens, which are used for medical as well as industrial purposes.

 Potentially the biggest development in this field could be the development of plants growing biodegradable plastics.
Other uses could be as Industrial oils such as hydraulic oil or high yielding biodiesels, new solid Biofuels, new Fibers and Papers, and as agents for Bioremediation and Phytoremediation, environmentally cleaning up contamination.

Molecular Farming

Key processes
Transform Express Patenting

WHY PLANTS?
Plants are also very flexible and can produce a wide variety of proteins. Crop plants can synthesize a wide variety of proteins that are free of mammalian toxins and pathogens. Crop plants produce large amounts of biomass at low cost and require limited facilities. Crops are therefore well suited for the production of safe low-cost therapeutic proteins.

Edible Vaccines
Biotech Plants Serving Human Health Needs
Works like any vaccine A transgenic plant with a pathogen protein gene is developed Potato, banana, and tomato are targets Humans eat the plant The body produces antibodies against pathogen protein Humans are immunized against the pathogen Examples: Rabies Norwalk virus (cold virus) Anthrax

Rabies Virus Vaccine

Rabies is fatal in 100% of cases when no treatment is given resulting in 60,000 deaths every year worldwide. A specific protein of rabies virus, known as G protein, is the major antigen that causes immunity.

Gene that codes for the G protein was significantly modified to facilitate a higher expression of the protein in plants.
Agrobacterium tumefaciens was used to insert the gene that encodes for the antigen as well as a gene that encodes for herbicide resistance.

Rabies Virus Vaccine

The effect to which the plant vaccine was able to invoke an immune response was performed by injecting mice with the protein derived from tobacco. The plant made vaccine produced a higher level of antibodies for the rabies virus than the commercially available rabies vaccine. Mice from each group were then challenged with 10LD50 of rabies virus.
(Adapted from Ashraf S, Singh PK, Yadav DK, Shahnawaz M, Mishra S, Sawant SV, Tuli R: High level expression of surface glycoprotein of rabies virus in tobacco leaves and its immunoprotective activity in mice. Journal of Biotechnology 2005, 119:1-14.)

RABIES VIRUS G PROTEIN IN TOMATO

Gene introduced to tomato plants by Agrobacteriummediated transformation Expression of recombinant glycoprotein in leaves and fruits Protein localized in golgi bodies, vesicles and plasmalemma

Norwalk virus (cold virus) capsid protein in potato and tobacco

Causative agent for acute epidemic gastroenteritis Transformation by Agrobacterium Expression level:

0.37% in potato tubers

0.23% in tobacco leaves

Anthrax Vaccine
The toxins PA, LF, and EF are responsible for the pathogenicity of deadly strains of Bacillus anthracis. The PA gene was expressed in Nicotiana tabacum through chloroplast transformation
~14% in leaves was PA, 1 acre of land=360 million doses of anthrax vaccine.

Producing proteins in chloroplasts has several advantages: ~10,000 copies of chloroplast DNA in every cell, 1025 times higher protein levels lack of gene silencing. reduces the risk of a transgene proliferating throughout the environment

Anthrax Vaccine in Transgenic Tomatoes are in early stage of development.

Tomato Callus Differentiating On Selection Medium

Putative Transgenic Tomato Plants at Bottle Stage

Putative Tomato Transgenic Plants Transferred To Pots

Comparison to other Vaccine Productions Methods

Transgenic plants are significantly less expensive than other means of vaccine production. ~80% of the total production costs are attributed to extraction and purification of the recombinant proteins Costs can be reduced by expressing the protein in watery tissues. Transgenic cereal crops can be stored at room temperature. Plants also provide a greater degree of safety.

Comparison to other Vaccine Productions Methods Table 1

Comparison of currently used vectors for recombinant protein production

(Adapted from Balen B, Krsnik-Rasol M, Lt: N-glycosylation of recombinant therapeutic glycoproteins in plant systems. Food Technology and Biotechnology 2007, 45:1-10.)

Oral delivery
Edible vaccines would make mass immunization possible at extremely low costs.

Concern that the expressed antigens could be broken down by proteases upon entering the stomach.
Possible side effects of accidental consumption of antigen containing plants.

Oral delivery
Transgenic rice has been shown to orally immunize against cholera toxin even after prolonged storage. Mice born to females that had eaten transgenic alfalfa containing an antigen for a class of rotavirus gained partial passive immunity. A protein necessary for the production of HIV virus was produced in transgenic tomato plants and elicited the production of antibodies in mice.

Conclusion
Plant made vaccines are safe, cheap, effective, and highly accessible alternative to current methods of vaccine production. An array of pathogens, plant species, and transformation technology can be used to produce vaccines in plants.

The technology has shown to have numerous advantages over current methods of vaccine production as well as a few disadvantages.

Thank you.