Imagine about eating a ripe appetizing banana fruit and getting rid of all your worries about catching hepatitis B at the same time. Is it a scientist's dream or just a step away from reality?
Across the globe, scientists are developing vaccines which will be produced by edible plants, and then be consumed by human beings in need of such vaccine. The plants are genetically engineered to carry genes from disease-causing microbes. Within those microbes, the genes control production of antigens which provoke immune responses in people infected with the microbes. When inside a plant's DNA, the genes force the plant to produce the desired vaccinating antigens. Such edible plant vaccines hold great potential, especially in Third World countries where transportation costs, poor refrigeration and needle use complicate vaccine administration. Plants ideally suited to be vaccine producers and vehicles are tasty, cheap, and can be eaten raw, since heat would inactivate protein antigens; for instance, it has recently been shown that foreign genes can be inserted into banana DNA.

The day will come when bananas, potatoes, peas and other common fruits or vegetables may save lives and lower the cost of protecting people and animals from common killer diseases due to a specific advance that enables them to carry a vaccine.The use of plants to produce vaccines is a concept first proposed in the 1980s. Substantial progress has been achieved in the laboratory, and there have been a small number of promising clinical trials, but the promise of plant-derived vaccines remains unfulfilled.

Over the last decade, there has been a significant increase in sophistication about the production and distribution of plant-derived vaccines (PDV). In the early years of research, investigators proposed that antigen bearing fruits or vegetables could be consumed directly. While "immunization-by-eating" is a fascinating prospect, detailed considerations have led to a more refined view of the use of plant-derived vaccines. Regulatory concerns will call for lot-to-lot consistency, uniformity of dosage, and purity, none of which are achievable through immunization-by-eating strategies. Thus the future of plant-derived vaccines will be the development of either orally administered or injected vaccines. The orally administered vaccines may consist of purified antigen-bearing plant tissue, with or without excipients, delivered in a capsule.

There are many important characteristics of plant-derived vaccines that could make them particularly attractive for addressing infectious disease control in developing countries.
*Plant derived vaccines could be produced on a very large scale and thus promise to be very low in cost.
*The vaccines could be orally active thus facilitating needle free administration.
*The vaccines stimulate the immune response at the mucosal level and thus would be especially effective against diseases - TB, pneumonia, diarrhoeal diseases, STDs, HIV, etc., that infect the mucosal system facilitating needle free administration.
*It might be possible to make multi-antigen vaccines either by multiple gene splicing or by mixing various plant-derived vaccines. For example, it could theoretically be possible to make a plant producing antigen to stimulate effective immune response to cholera, ETEC, rotavirus, etc.

The current research-based international vaccine companies are not interested in plant-derived vaccines because of concerns about return on investment, lack of regulatory framework, lack of precedent, and lack of skilled plant biologists in their existing research corps. For plant-derived vaccine development to move forward, the public sector will have to take the lead. However, if plant-derived vaccines are to become a major component of international immunization efforts, the private sector will eventually need to embrace this technology. The private sector will put certain questions such as
1.What is the unmet medical need, i.e. why would these products be used?
2.What is the product(s) that will emerge? If they are new versions of existing products, what are the advantages and will these advantages be important in a decade or so when the products emerge from development?
3. Can the product be made at the right scale and cost, i.e. manufacturing feasibility?
4. Are there world class people who can lead and support the effort?
The first challenge with respect to plant-derived vaccines is to demonstrate that plants represent a viable and attractive means to produce vaccines. There is a large array of methods for producing vaccines including attenuation of live pathogens, e.g., the Sabin polio vaccine; inactivation of live pathogens, e.g., rabies vaccine; isolation of pathogen subunits, e.g., hepatitis B vaccine from human plasma; and production of subunit vaccines using recombinant DNA technologies. Each of these methods has been used successfully on a commercial scale.

A preferred strategy to validate plant-derived vaccines, therefore, should be to produce a vaccine that is already made successfully by some other method. The work should be conducted in such a way as to make possible to compare the two methods of production to demonstrate conclusively that plants represent a significant improvement, by one or more measures, over proven methods for making vaccines. It is essential, then, to define clearly the criteria by which a plant-derived vaccine would be considered to be superior to a vaccine produced by conventional means. With success achieved in this exercise, there would be a firm basis for seeking to develop other vaccines in plants.

Why Plants?
Plants are among the most efficient bioreactors as they allow production of recombinant proteins in large quantities and at relatively low costs. This production step replaces the traditional production step of fermentation. The benefits of plant-based production include: The ability to increase production at low cost by planting more acres, rather than building fermentation capacity; lower capital and operating costs compared to traditional production facilities; simplified downstream processing compared to traditional technology. From a technology perspective plants have an advantage over microbes in that they can accommodate the production of a wide range of protein types.So, with sunlight and soil based nutrients as inputs, plants produce large quantities of material, plant derived vaccines being one of those.

Further elaborating the above discussion, there are some more advantages of plant system in the development of oral vaccines:
*Edible plants are very effective as a delivery vehicle for inducing oral immunization
*Adjuvant for immune response is not necessary
*Easy for separation and purification of vaccines from plant materials
*Effective prevention of pathogenic contamination from animal cells
*Effective maintenance of vaccine activity by controlling the temperature in plant cultivation
*Easy for mass production system by breeding compared to an animal system
*Reduced dependence on foreign supply
*Storage near the site of use
*Antigen protection through bioencapsulation
*Subunit vaccine (not attenuated pathogens) means improved safety
*Seroconversion in the presence of maternal antibodies
*Generation ofsystemic and mucosal immunity
*Enhanced compliance (especially in children)
*Integration with other vaccine approaches

Which plant is PDV friendly?
There are some criteria on which plants are evaluated for their efficiency to produce vaccines. A plant-derived vaccine would be deemed superior to other methods of production if it met with the following criteria and were not inferior in any significant way.

*Its unit cost of production at a commercial scale was < 50% of the cost of production by the currently used method.
*It could be formulated and delivered in a rea-dily acceptable orally administered form that achieved acceptable correlates of protection.
*It was demonstrated to be safe and, in particular, did not cause immune tolerance.
*It was shown to be heat stable, e.g., had a shelf-life of at least 12 months at 27 degrees Celsius.Eat Your Veggie Vaccines
*It provided equal or enhanced levels of mucosal effectiveness compared to the existing vaccine.
*It could be produced readily in developing countries.

Edible rice-based vaccine may combat hay fever
Eating a bowl of rice will solve the problem of seasonal misery of pollen allergies.
In experiments with mice, Japanese scientists found that an edible vaccine produced in genetically modified rice was able to prevent the immune response that triggers allergies. Mice that were fed the vaccine showed a dampened immune reaction to pollen, and they sneezed far less often than their non-vaccinated brethren. Takaiwa, a researcher at Japan's National Institute of Agrobiological Sciences, said he and his colleagues have already developed a human version of the rice vaccine that should be ready for safety testing in a few years.

Hepatitis B antigen expressed in potato root
Hairy roots of potato are an attractive system for the production of recombinant proteins due to their genetic stability, fast growth, and ability to grow in hormone-free media. G.B. Sunil Kumara and colleagues of the Bhabha Atomic Research Center and Shantha Biotechnics Limited, India work on the "Expression of hepatitis B surface antigen in potato hairy roots." The results of their research are reported in a recent issue of the journal Plant Science.

In the study, the scientists used Agrobacterium to introduce the hepatitis B surface antigen (HBsAg) gene to Bahar, an Indian potato cultivar. After inducing hairy-root growth, they verified the presence of the transgene and its product by PCR and ELISA. Scientists noted that HBsAg was expressed in potato plants, microtubers, and hairy roots. Plants regenerated from hairy roots also exhibited similar levels of HBsAg expression to that of transgenic plants.

Eating transgenic tobacco prevents cervical cancer
Human Papillomaviruses (HPV) are the causative agent for cervical cancer, being also involved in skin, head and neck tumours. The most commonly found HPVs in cervical carcinomas are HPV 16 and 18. The cancer causing factors are the virus proteins E6 and E7, which are known as onco-proteins.

Some E7-based HPV vaccines are currently being explored and the first promising results have been disclosed, but they still need further improvement to ensure protection. One method to produce high amounts of E7 protein in a relatively short time is by genetically engineering plants or plant viruses. "An advantage of the plant-derived systems is that they generally lack human pathogens, oncogenic DNA sequences and endotoxins", explained Dr. Rosella Franconi from ENEA (Ente per le nuove Tecnologie, Energia e Ambiente). "This minimizes health risks and lowers the production costs."

In 2002, Dr. Franconi, in collaboration with Dr. A. Venuti from IRE (the Cancer Institute "Regina Elena") and C. Giorgi, from the ISS (Istituto Superiore di Sanita) in Rome, produced the HPV 16 E7 protein in the cytoplasm of tobacco (Nicotiana benthamiana) plants. To do so, they used the potato virus X (PVX). PVX is a safe tool for genetic engineering, because it does not infect animals, but does effectively transfer genetic material to a variety of plant species.

Hepatitis B vaccination by eating a banana
A team from India succeeded in producing a Hepatitis B antigen in bananas. The antigen can then be used to make a vaccine against Hepatitis B.

Bananas are an ideal host since most of the edible bananas do not set seeds and fruits develop parthenocarpically, which means directly from the maternal tissue of the flower. This prevents the imported gene from being transferred to other vegetation.

Equally important, the banana is an ideal meal for infants, and is available year-round in the tropics and subtropics - precisely where economical vaccines are required most urgently. The researchers working on the banana vaccine are from the Bhabha Atomic Research Centre in Mumbai, India, and the Shantha Biotechnics Ltd., Hyderabad, India.

Dr. Bapat's team is working out strategies to enhance the amount of Hepatitis B surface antigen a given banana can produce, and feel that the use of certain promoters, codon optimization and the use of banana UTRs (un-translated regions) may increase the level of antigen produced in future bananas. All these methods support the gene expression in one way or the other.

New plant-made vaccine wins first federal approval
Dow AgroSciences has won the first federal approval of a plant-made vaccine, the product of a laboratory process that avoids the controversial use of pharmaceutical field crops.

The chicken vaccine will not be commercialized, but official with Dow AgroSciences said that winning approval from the U.S. Agricultural Department's Center for Veterinary Biologics in Ames, Ia., showed the promise for making pharmaceuticals from plant cells, rather than animal products or whole plants.Dow AgroSciences, an Indianapolis-based unit of Dow Chemical Co., developed its vaccine by fermenting bioengineered tobacco cells in steel tanks.

Vaccines are typically made from chicken eggs or in mammalian cells, which can carry diseases. The Dow process also uses fragments of the virus, rather than the entire pathogen, in making the vaccine. Dow has several commercial products in development, all intended for animals. The first product is not expected to reach the market before 2009 or 2010. There are already several Newcastle vaccines on the market.

The Dow AgroSciences Concert Plant-Cell-Produced System represents a new category of plant-made vaccines. This leading edge technology utilizes plant cells instead of whole plants in a secure, bio-contained environment to produce vaccines. Because of this bio-contained production system, concerns and challenges associated with making vaccines in whole plants or food crops are eliminated. The Concert Plant-Cell-Produced System uses only the necessary parts of the disease causing agent to stimulate immunity in a manufacturing process that is totally free of animal components:
"Being the first company to ever register a plant-made vaccine is another example of Dow AgroSciences' cutting edge approach to bringing highly novel and differentiated solutions to the market," said Jerome Peribere, Dow AgroSciences' president and CEO.

Vaccination by needle soon to be a history?
Though it is still very early to say anything, the experimental know-how and results strongly suggest that plant-derived edible vaccines are likely to become a reality in the next few years. Future research will demonstrate if these vaccines meet the standards of quality defined for vaccines by the World Health Organization.

After the demonstration that plants can be engineered as to produce edible vaccines that trigger an immune response in mice and humans, we are now confronted with the successive problems of clinical trials, process development, registration and marketing. Clinical trials with populations at risk are already under way in some laboratories. The definition of the overall immune response to plant-derived edible vaccines is of the utmost importance. With the growing availability of plant-derived vaccines, this will soon be verified.

Process development primarily concerns achieving sufficiently high levels of expression of the recombinant antigen, and defining the optimal way of antigen administration. Solutions to the first point are well under way, as described above, while approaches to the second will be manifold. While the initial concept was to induce an immune response by directly feeding a crude edible plant portion (fruit, leaf, tuber), it is now felt that this may not be the ideal solution as it would be difficult to standardise antigen concentration in different harvests of the same crop.

Furthermore, fresh products may have short shelf life. Dried products, for instance banana slices, may offer a partial solution, but the best solution (as for shelf-life, stability and title standardisation) would be delivery in the form of a dry powder. This can be achieved by using low cost food processing technology. A dried tomato powder has been stored for one year in C. Arntzen's laboratory without loss of antigen activity.
(The author is with Accure Labs Pvt. Ltd., New Delhi)