The concept of edible vaccines stem from Charles Arntzen of Texas University, USA, when he saw a mother offering a piece of banana to a crying baby. With the advent of introducing gene into the plants, he then mooted the idea that food could be genetically altered to produce vaccine in their edible parts, which could then be eaten to combat disease.
Active vaccines have been developed for over 70 infectious diseases that strike people at every stage of life. Recent advances in vaccinology have created an array of novel vaccines, living and non-living antigen and delivery systems. These are administered basically via painful injection process.
When mass immunization was undertaken in developing countries, it was imperative to produce bulk of vaccines. In the entire exercise, World Health Organization (WHO) programmes for vaccines and immunology has declared war on unsafe injection process using unsterilise needles, syringes, which in turn inadvartantly transmits risk of HIV and other pathogen from one infant to other.
When WHO called for the production of inexpensive oral vaccines for scientific community that needed no refrigeration, the concept on the development of safe plant based vaccines caught greater attention. Although, Hiatt and co-workers attempted to produce plantibodies plants in 1989, however, in the early 1990, first report on the production of edible vaccines appeared in the form of a patent application.
The idea of edible vaccine production in plants is to simply eat edible portion of the plants to be immunizied against infectious disease. Nearly decades of studies carried out in animals hope that edible vaccine can work. The experimental animals fed or injected with transgenic plant derived vaccines shows considerable promise.
Production of vaccines in plants as biofarming was initially carried out in non-edible plant tobacco as model system. Later it was then produced in potato to feed experimental animals. The techniques of vaccine production in edible portion have been successfully produced.
Bananas are particularly appealing as vaccine because they can be cultivated in wide range of areas of developing world and can be eaten raw so that they can avoid degradation during cooking. Further, vaccination process will become easier and children liked this vaccine fruits. In addition, tomato, watermelon and cucumber plants are generally a choice for vaccine production.
This new development in biotechnology opens up a hope that through molecular farming or biofarming of plants may provide an inexpensive source of edible vaccines and antibodies to help in the fight against diseases. Such a development which involves a combined effort of medical and plant biotechnology will have significant implications in developing countries including India.
It is however, several questions need to be addressed in the whole exercise on food vaccine production. They are (a) how vaccines for infection diseases are delivered (b) how do vaccines present in the fruits. Would the antigen be degraded in the stomach before being induced immune response?
It has been shown that vaccines present in the food have shown to induce immune response. Once food vaccines are given, presences of foreign organisms in the form of biomolecules are immediately detected and immune system behaves as if the body were under attack by pathogens. The immune machinery swiftly mobilizes its faces to eliminate invader.
The immune response later leaves behind memory cells that remain alert, ready to strike the offender, a real pathogen appears in the body. One of the modes of immune response triggered by edible vaccines known as mucosal immunity. There are associated in the mucosal membrane lining the airways, the digestive tract and the reproduction tract.
When this system is effective, it generates molecules like secretary antibodies that neutralize any pathogens in the pathways. The injected vaccines, however, bypass mucous membrane and typically are the poor candidate in inducing mucosal response. The edible vaccines can however, trigger mucosal response when they come into contact with the lining of the digestive tract (Fig. 16.1).
Thus, one can presume theoretically that they can induce both humorus and nucosal immunity. It is amazing that antigen delivered in plant food survive in the stomach and reach the target to activate immune response.
The rigid plant cell wall apparently serve as armor for the antigen, rescued them from gastric secretions. Thus, food can act as perfect bioencapsulated system and helps in the safe delivery to the target for better immune response.
Edible Vaccines for Human Diseases (hepatitis, cholera, enteric diarrhoea and rabies):
The first published report on the production of edible vaccines in plants is the cell surface adhesion protein, known as SpaA or streptococcus antigen from streptococcus mutans. This is a main causative-agent in dental carries, colonising adult teeth. Transgenic tobacco expressed introduced SpaA gene and its level reached upto 0.02% total protein. It is a large protein of M.W. 185,000.
Although hepatitis B surface antigen (HBsAg) was the first recombinant protein product commercially produced in yeast, its cost of production, however, through recombination method was a serious concern among anterpreuners from developing countries. In addition, the expense of immunization properties for large segment of population is prohibited.
Moreover, this virus cannot be cultured. Fortunately with the advent of food vaccine technology, Arnitzens group has successfully developed tobacco plants producing a vaccine against hepatitis B with the hope of developing a less expensive production system.
Tobacco plants were genetically transformed with the gene encoding hepatitis B surface antigen (HBsAg) linked to a constitutive 35S promoter. Maximum level of HB Ag in transgenic leaf represents 0.01% of the soluble leaf protein.
The expressed HBsAg assembled into virus like particle (VLPs), resembles the yeast and serum derived HBsAg. Further studies established plant derived hepatitis B surface antigen was able to trigger immune response in mice. The anti-hepatitis B response to the tobacco derived HBsAg was qualitatively similar to that obtained by immunizing mice with yeast derived commercially producing recombinant HBsAg.
Presence of both B and T-cell epitopes in plant derived vaccines elicites HBsAg specific B and T cell response. It was also demonstrated that after priming with tobacco derived HBsAg, mice developed T-cells in responses to the yeast recombinant HBsAg. The primed and also by synthetic peptide that represents part of the protective determinant in S region of HBsAg.
Further support for the integrity of the T-cell epitops of the tobacco-derived rHBsAg was obtained by testing ability of the primed T cell to proliferate (in vitro) after stimulate with a monoclonal antibody, that mimics the same determinant. Since purification of HBsAg for tobacco pose serious hurdles due to the existance of several secondary metabolites a problem was averted by producing the antigen in transgenic potatoes.
After initial success, to target the diarrheal disease, Dr. Arnitzen group has also developed transgenic tobacco and potato plants containing a highly active immunogen of E. coli heat labile enterotoxin (LT-B). The enterotoxigenic Echerichi a coli (ETEc), vibrio cholorae causes acute and water diarrhea by colonizing small intestine and producing one or more enterotoxins including heat labile enterotoxin (ETEC).
The pentameric structure of the enterotoxin from E. coli (LT) and cholera toxin (CT) shares common features. The LTA has a one 27 KD A subunit and a pentamers of B subunit 11.6 KD (LT-B). The subunit LT-B is a non-toxin, which can bind to GM, ganglioside toxin LT-A subunits into the cells.
The conventional methods of tobacco and potato plants were engineered with the transgene coding LT-B or a LT-B fusion protein with a microsomal retension signal. The plants expressed the protein shows partial pentamerization. In order to test immunogenic, raw potato containing vaccines were fed to the experimental mice and obtained both humoral and mucosal immune response.
The extended work of clinical trials on human being has also been established with the help of health volunteers, who ate raw potato. The health individuals were screened for gut derived antibody secretion cells, which were detetable 7-8 days after immunization. Since cholera toxin was similar to E. coli enterotoxin LT, it has been expressed in transgenic tobacco and potato plants in pentameric form.
The cholera CT molecule consists of one A subunit and five B subunits. A subunit is 28-kDa polypeptide composed of two major domains (A1, and A2). Both cholera enterotoxins CT-A or CT-B were expressed in tobacco plants in which CT-A produced was not cleaved to A1 and A2 subunits.
Abundant form of toxin B was expressed as processed pentameric form and anti-genetically they are similar to bacterial protein. Even after binding, nearly 50% of the CT-B was found to exist in the pentameric GM, ganglioside binding form.
The cholera toxin B subunit (CTB) was shown to function as a effective carrier molecule for fused foreign protein including mucosal vaccine antigen and autoantigens. Transgenic potatoes were engineered to synthesise a cholera toxin B subunit (CTB) with affinity for GM, ganglioside and both serum and intestinal CTB-specific antibody were induced in orally immunized mice.
Similarly, Mc Garvey, along with Hilesy Koprowsk (1995), developer of oral polio rabies vaccines at Thomas Jefferson University in philadelphia has produced tomato plants that express the rabies vaccines. Rabies is one of the potential fatal diseases caused by Rabies virus, transmitted from animals to man.
Since mass immunization of animals is practically impossible current strategy employ aircraft to drop baits loaded with oral recombinant vaccines in the forest in developed countries. This strategy has proved very effective in North America but the practice is however, cost effective.
These plants would thus provide an inexpensive and effective alternation to immunization of both wild animals and street dogs. The expression of rabies virus glycoprotein in tomato was driven under strong 35S cauliflower mosaic virus promoter. Both leaf and fruit tissues were found to have accumulation of glycoprotein.
The glycoprotein in transgenic tomato expressed in two forms with a molecular weight of 62 kDa. Though antigenic proteins of edible rabies glycoprotein was encouraging, their value as vaccine is still requires strategic important. Further demonstration of immunogenecity of plant derived recombinant proteins was reported.
Transgenic tobacco and potato plants were produced that express the capsid protein of Norwalk virus. This pathogenic virus causes epidemic acute gastroenteritis in humans. Transgenic tobacco and potato plants were created that express capsid protein of Norwalk.
The capsid protein could be extracted from tobacco leaves in the form of 38 nm Norwalk virus like particles. The capsid protein expressed in bacculo virus self-assembles in to VLPs that are immunogenic. Plant derived recombinant capsid proteins also assembled into VLPs. Plant expressed rNV was orally immunogenic in mice.
Immunization with plant derived antigen resulted in serum and mucosal antibody response i.e., mice developed both IgG and secretary IgA specific to rNV. Further results shows that when potato tuber expressed rNv fed directly to mice, they developed seven IgG specific for rNv. Production of vaccines for food and mouth disease has also been reported.
Foot and mouth disease virus (FMDV) is the major causative agent affecting meat and milk producing domestic animals. Current vaccines are based on the usage of inactivated virus, but their production includes risk of contamination from vaccine factories. Thus, it is important to develop alternative approach for producing edible vaccines.
The genes encoding antigens of bacterial and viral pathogens have been expressed in plant in which they retain native immunogenic properties. The expression of structural protein VP1 for foot and mouth was shown in transgenic Arabidopsis thaliana and mice were fed with leaf extracts elicites specific antibody response.
Mobilizing vaccine to desirable target sites can be accomplished by using many carrier molecules. For example, cholera toxin B subunit (CTB) was effective carrier molecule for fused foreign proteins including vaccine antigens and autoantigen (Insulin).
The CTB pentamer bind selectively to sugar lipid GM ganglioside molecule embedded in the membrane of intestinal epithelial micro-fold (M) cells. Similarly, utilising CTB for triggering of mucosal immunal response is the expression of rotavirus NSP4 fusion protein oligomers in transgenic potato in the production of auto-antigen insulin in transgenic potato.
Rotavirus is the single most important cause of virus-based severe diarrhea in infants and young children. This life-threatening virus account for 18 million cases and 900,000 deaths each year. Synthesis and oligomer assembly of the CTB-rotavirus enterotoxin NSP is a 22 aminoacid immunodominal epitopic fusion protein i.e., CTB-NSPH22 was expressed in transgenic potato plant. Further, NSP422 epitope was able to generate antibody in orally immunized mice.