It is estimated that one-third of all prescription drugs on markets around the world are originally derived from plants, although most are now synthetic analogues of chemicals found in plants. With the advent of genetic engineering, plants may again stage a comeback but now as a novel source of preventive drugs. Transgenic plants producing antigens against cholera, hepatitis B and rabies have been developed in US laboratories.

Recombinant DNA technology has already radically altered the field of vaccines. We now understand better how our body interacts with microbes at the molecular level. Molecular biology also facilitates the development, production and delivery of safe and effective vaccines. For example, antigen genes introduced into yeast and baculovirus have proved to be efficient means of producing ‘subunit’ vaccines against rabies and hepatitis B. This approach provides purified protein antigens. Recombinant vaccinia virus is being used as a carrier virus for a vaccine against rabies but has met resistance because of occasional reports of adverse reactions. However, current vaccines produced by the use of pathogenic organisms contain risks of reversion or contamination.
Edible vaccines produced in plants, according to Dr. Charles Arntzen of the Boyce Thompson Institute for Plant Research, USA, obviate many hurdles associated with the cell-culture systems currently used in the large-scale vaccine production. These hurdles, which are relatively more difficult to overcome in developing countries, include the need for fermentation technology, strict purification protocols, refrigeration during shipment including ‘cold chain’, risk and pain associated with parenteral delivery and high costs. Additionally, the concept is appealing since it would not carry risks associated with the use of live pathogens, and sterility requirements of injected vaccines. The production of antigens in genetically-engineered plants could provide an inexpensive source of edible vaccines and antibodies to help in the fight against infectious diseases such as rabies, cholera, hepatitis B, malaria, and AIDS. The idea is that by simply eating these plants the consumer will be immunized against these diseases.

Active immunization
Dr. Arntzen’s group has successfully developed tobacco plants producing a vaccine against hepatitis B. It found that the vaccine produced in plants is similar in form and function to that from human serum or recombinant yeast and provoked a strong immune response when injected into mice, while B and T-cell epitopes were preserved. An estimated 300 million people carry the hepatitis B virus, which affects the liver.
Another target is formed by diarrhoeal diseases. The same group has also developed transgenic tobacco and potato plants containing an highly active immunogen of E. coli heat labile enterotoxin (LT-B) which is structurally similar to cholera toxin. Mice orally immunized with transgenic extracts exhibited a strong immune response. Human clinical trials with volunteers will be conducted soon with this plant-derived vaccine against diarrhoea using fresh potatoes.
Researchers at the Thomas Jefferson University in Philadelphia, USA, have produced tomato plants that express rabies antigens. Rabies is a fatal viral disease transmitted to man by bites of animals such as dogs, foxes, bats and raccoons.

Passive immunization
In contrast to the above examples of ‘active immunization’, the concept of ‘passive immunization’ (the application of antibodies) is also being explored by producing these protective antibodies in plants. Researchers at Guy’s Hospital, London and at Salk Institute, USA have developed transgenic plants producing antibodies against Streptococcus mutans, a common tooth-decay bacteria. These scientists hope that one day their plant-produced antibodies will be incorporated into toothpaste for protection against dental diseases. However, to produce complete antibodies, it was necessary to develop individual transgenic plants producing a single chain and then sexually hybridize them to develop a plant producing complete antibodies consisting of heavy and light chains.
The private biotechnology company Agracetus, Wisconsin, USA, has developed transgenetic soya beans that produce a tumor-reactive monoclonal antibody called BR 96 which can be used as a drug carrier to treat breast, colon, ovarian and lung cancers. These soya beans are now being grown in Puerto Rico, and Agracetus plans to start clinical trials using antibodies isolated from these crops.
Plants are also being modified to produce other drugs such as albumin, serum protease and interferon which are otherwise difficult or expensive to produce. A dramatic example of this research involves the recent development of tobacco plants by Crop Tech Development Corp in Virginia, USA, which produce glucocerebrosidase (hGC), an expensive human enzyme for treating Gaucher’s disease. Currently, this enzyme is derived from human placentae. It takes 2,000 to 8,000 placentae to produce a single dose of this drug, raising the cost of a single treatment to US$ 300,000 per year. It is expected that this development should bring down the cost of this drug by a thousand fold.

Plant viruses as vaccine producers
Genetically-engineered plant viruses are also being employed to produce vaccines and other medicinal compounds in infected plants. At the Scripps Research Institute, USA, tobacco mosaic virus (TMV) has been engineered genetically to contain mouse zonna pellucida ZB3 protein which is an immuno-contraceptive as it covers the unfertilized eggs preventing fertilization. In the long run, the results of this research may serve as a cheap source of oral contraceptives. Researchers at John Innes Institute have engineered cowpea mosaic virus (CPMV) to contain a surface protein of human immuno-deficiency virus (HIV), the virus responsible for AIDS. Chimeric coat proteins of CPMV that express the malarial and foot-and-mouth disease epitopes have also been produced. Other novel compounds including an anti-viral protein that inhibits the HIV virus in vitro, trichosanthin (ribosome inactivator) and angiotensin-I (an antihypersensitive drug) have also been expressed in infected plants through engineered virus inoculation.

Finding suitable plants
A plant will be suitable for oral vaccine production if it:

• produces edible parts that can be consumed uncooked since vaccine antigens are heat sensitive;
• is rich in protein because vaccine protein will only be a small percentage of the total protein content of a plant;
• can be transformed by genetic engineering;
• can be grown widely;
• is fast growing;
• does not produce toxic products.

At least a decade to go
Transgenic plants that produce medicinal compounds such as subunit oral vaccines and antibodies have already been developed, but experts concede that application of this technology is at least a decade away. There are several technical and logistical problems which need to be addressed before edible vaccines through plants become a reality in practice.
Firstly, most inserted genes are expressed in very low levels in plants. To enhance expression, focus is on the development of efficient promoters especially to target the production of proteins into edible parts of the plants, and on factors such as enhancers, signal sequences and optimized codon usage. The group of Arntzen recently reported that a synthetic cholera vaccine gene that was more ‘plant’ like in its sequence, is four times more productive than the original gene.
Secondly, the stability of vaccine proteins when transgenic fruits or leaves are stored at ambient conditions is another concern. Antibodies in leaves have to be extracted immediately, or they will decay with the leaves themselves. German researchers reported that by linking antibody genes to a genetic "switch", antibodies were produced in the seeds instead of in the leaves. These antibodies in seeds did not deteriorate significantly after a year of storage at room temperature. This could imply, that antibodies would be preserved until extraction or consumption of the seed at a later stage.
Thirdly, there is also concern about oral tolerance in case increased levels of vaccines become ineffective when consumed orally  by suppressing systemic immunity. Further research may identify useful adjuvants that enhance oral immunogenicity.
Fourthly, the dosage is a major problem as vaccine content in plants may vary depending on where and when they are grown. Therefore, Arntzen proposes that a delivery scheme should be developed to ensure the required dosage level, and that edible vaccine producing plants are taken as a routine food source. Only further collaborative research between plant and medical scientists may resolve these and other issues.
In the near-term, the edible-vaccine technology might be better targeted at animals. In fact, such an approach may benefit agriculture as billions of dollars are spent presently on vaccinating farm animals and poultry. Transgenic plants supplying feedstock containing edible vaccines may represent the first commercial application of this intriguing technology.
C.S. Prakash

Center for Plant Biotechnology Research, Tuskegee University, Milbank Hall, Tuskegee, AL 36088, USA. Fax (+1) 334 727 8552; E-mail prakash@acd.tusk.edu

Sources
Andy Coghlan (1995), "Pick-your-own Antibodies." New Scientist. 28 October 1995, p. 23.

T.A. Haq, H.S. Mason, J.D. Clements and C.J. Arntzen (1995), "Oral Immunization with a Recombinant Bacterial Antigen Produced in Transgenic Plants." Science. No. 268, pp. 714-716.

H. Mason and C.J. Arntzen (1995), "Transgenic Plants as Vaccine Production Systems." Trends in Biotech, Vol 13, pp. 388-392.

P.B. McGarvey et al (1995), "Expression of the Rabies Virus Glycoprotein in Transgenic Tomatoes." Bio/Technology Vol. 13, No. 13, pp. 1484-1487.

A.S. Moffat (1995). "Exploring Transgenic Plants as a New Vaccine Source." Science. No. 268, pp. 658-660.

Y. Thanavala, Y-F. Yang, P. Lyons, H.S. Mason and C.J. Arntzen (1995), "Immunogenicity of Transgenic Plant-derived Hepatitis B Surface Antigen." Proc. Natl. Acad. Sci. USA. Vol. 92, pp. 3358-3361.

Personal communication with Charles Arntzen, Peter McGarvey and Hilary Koprowski.