EDIBLE VACCINES

THEORY BEHIND EDIBLE VACCINES
One of the major challenges of biotechnology is to reduce clinical innovations to economically viable practices. Plant derived edible vaccines were first conceived and are continuing to be developed with this prime directive in mind: merging innovations in medical science and plant biology for the creation of efficacious and affordable pharmaceuticals. Since the emergence of the original idea about 10 years ago, it was embraced by a growing number of laboratories in academia and industry.
Despite notable success, traditional vaccine technology has its limitations. Almost all vaccines now commercially available consist of either inactivated or attenuated strains of pathogens which are almost always delivered by injection (the oral polio vaccine is an exception). In contrast, many of the currant vaccine development efforts focus on subunit vaccines, and these are being considered for either mucosal or parental delivery.
A subunit vaccine refers to a pathogen derived protein (or even just an immunogenic domain of a protein, i.e., an “epitope”) that cannot cause disease but can elicit a protective immune response against the pathogen. Very often the subunit vaccine candidate is a recombinant protein made in transgenic production–hosts (such as cultured yeast cells), then purified, and injected into vaccines to immunize against a specific disease. Subunit vaccines are generally considered safer to produce (eliminating the need to culture pathogenic organisms) and more importantly, to use.
However, immunization by injection (parental delivery) rarely results in specific protective immune responses at the mucosal surfaces of the respiratory, gastrointestinal and genito -urinal tracts. Mucosal immune responses represent a first line of defense against most pathogens. In contrast, mucosally targeted vaccines achieve stimulation of both the systemic as well as the mucosal immune networks. In addition, mucosal vaccines delivered orally increase safety and compliance by eliminating the need for needles. While subunit vaccines are effective, they currently depend on capital-intensive fermentation-based technology and a “ cold chain ” (refrigeration) for delivery. Both of these factors create constraints in use in the developing world, where vaccines are needed the most. Combining a cost effective production system with a safe and efficacious delivery system, plant edible vaccines, provide a compelling new opportunity.

THEORY PUT TO CLINICAL TRIALS
In 1992 the expression of Hepatitis-B surface antigen (HbsAg) in tobacco plants was described. To prove that plant derived HbsAg can stimulate mucosal immune responses via the oral route, potato tubers were used as an expression system and were optimized to increase accumulation of the protein in the plant tubers. The resulting plant material proved superior to the yeast-derived antigen in both priming and boosting of immune responses to oral immunogen in mice. In parallel with evaluation of plant derived HbsAg, Mason and Arntzen explored plant expression of other vaccine candidates including the ‘labile toxin B subunit (LT-B) of entertotoxigenic Escherichia coli (ETEC) and the capsid protein of Norwalk virus(NVCP). The plant derived proteins correctly assembled into functional oligomers that could elicit the expected immune responses when given orally to animals.
Success in mouse experiments provided motivation for conducting Phase І / ІІ human clinical trials to test the safety and immunogenicity of plant produced LT-B, NVCP and HBsAg. In the three cases tested, humans who consumed raw potato tubers containing tens of microgram amounts of the antigens developed specific serum and more importantly mucosal immune responses. Significantly the three antigens in these studies come from three very different pathogens. Taken together these results provide the basis for wider-scale clinical trials with these antigens.
Although mucosal and systemic antibody titers were evaluated in vaccines who received the plant-based oral vaccines, we do not yet have evidence of protection against pathogen challenge. Ethical considerations usually preclude (prevent) clinical trials from directly assaying protection except in few cases. In contrast working with veterinary vaccines provides researchers an opportunity to asses the degree of immune protection more directly.

SECOND GENERATION EDIBLE VACCINES
Multi component vaccines that provide protection against several pathogens are very desirable. An elegant approach to achieve this goal, based on epitope fusion to both subunits of the cholera toxin (CT), was demonstrated by Yu and Langridge. CT provides a scaffold for presentation of protective epitopes of rotavirus and ETEC, acts as a vaccine candidate by its own right and as a mucosal adjuvant devoid of toxicity. The trivalent edible vaccine elicited significant humoral responses, as well as immune memory B cells and T-helper cell responses, important hallmarks of successful immunization. Commonly, foreign proteins in plants accumulate to relatively low levels (0.01% to 2% of total soluble protein). In the clinical trials described above, 100gm of raw potato tubers expressing LT-B of ETEC in three doses had to be consumed in order to overcome digestive losses of the antigen and to elicit a significant immune response. Less immunogenic proteins would require even larger doses to be effective. Even with more palatable alternatives to potatoes (e.g. bananas), these accumulation levels may limit the practicality of edible vaccines.
Two solutions to overcome this limitation are being explored. First, techniques to enhance antigen accumulation in plant tissues are being explored. These include, optimization of the coding sequence of bacterial or viral genes for expression as plant nuclear genes, and defining the sub cellular compartment in which to accumulate the product for optimal for optimal quality and quantity. Several laboratories are also developing alternative expression systems to improve accumulation. For example the expression in plastids is advocated by some. Other systems involve plant viruses for expression of foreign genes or coat-protiens fusions and even viral assisted expression in transgenic plants.
The second approach is to enhance the immunogenicity of the orally delivered antigens by using mucosal adjuvants. One such approach is making use of bacterial enterotoxins such as CT or LT (e.g. Yu and Langridge, 2001), mammalian and viral immune-modulators as well as plant derived secondary metabolites.At door step of the 21st century, the fear of a surge in naturally occurring epidemics is heightened by the threat of bio-terrorism. This new reality makes disease prevention through vaccination a necessity in our ever more interconnected world. Any tools we can master and all tools we can afford will have to be employed. Technical problems and skeptics aside, edible vaccines have passed the major hurdles of an emerging vaccine technology.