INTRODUCTION:

Over the centuries we have been fighting through many infectious diseases like cholera, diphtheria, polio, tetanus, whooping cough, tuberculosis, swine flu, ebola & hanta, West Nile, SARS etc., which travels through hills, valleys and oceans spreading across. Starting in a tiny gene, these diseases hitting big and shaking nations. From the old sayings, prevention is better than cure, cure offers a symptomatic relief from that present disease but not a complete eradication. So foreseeing the actual damage of future prevention through vaccination always stands on high. Vaccination is a breakthrough in science when Edward Jenner in 1796 evaluated inoculation of cowpox virus in humans to prevent small pox in humans successfully.

This vaccination process stimulates and expresses the antibodies which will destroy the disease causing agents once they enter inside the human body. When the immune system detects a foreign particle (dead antigen) in a vaccine, it releases a group of antibodies which fights to eradicate the antigen, leaving behind a memory to the immune system. When the real pathogen makes its way inside the human body our immune system readily recognizes from the memory and defends effectively. From then this technique was only the gold way in eradicating smallpox and polio from nations. Recently India announced itself as a polio free nation.

In reality the core of vaccine is made of live/attenuation of (weakened in strength) pathogen (bacterial or viral), killed bacterial suspension, toxins produces by bacterial toxoids. Relying on only cure and prevention through vaccines alone helps only for the developed countries. Because the cost of production and the storage of these vaccines are the two main things to consider, not at all a problem for developed countries. Bending the sides in cost of production, storage and reaching out of under developed and poor nations is intolerable, while many of the vaccination techniques cannot reach to save the lives of millions peoples around the world. Then there comes help from a miracle mind with a vision of vaccinating entire world through food. In 1990’s, when world health organization gave a challenge of developing inexpensive production of oral vaccination techniques that needs no refrigeration. From the ages plant have been the free life saving magical content in the world.¹Charles J Arntzen, thought of genetically modified food which will produce vaccines in their plant parts, those are edible taken inside whenever they are needed.² These edible vaccines are antigenic proteins that are genetically engineered into a consumable crop. This food contains the proteins extracted from disease causing organisms. When the food was consumed it is digested and the proteins enters the blood stream, the immune response neutralizes the pathogenic protein and makes a memory mark of it. This visionary work is in its initial stages of development years ago. The products developed were under clinical trials. These vaccines exhibiting promising results in not only inducing immunization against polio, and also reducing the autoimmuity which is responsible for various diseases like rheumatoid arthritis, multiple sclerosis, type I diabetes.

The advantages of developing edible vaccines are more when compared with normal vaccine developing techniques. Because the selected plants can be grown locally using our normal farming techniques that are particular for a specific region and the cost of buying seeds for other season also decreases due to regenerative capacity of these genetically modified crops is more. These edible vaccines can be transported easily without any special storage techniques, because the stability of these proteins in food at normal temperature is best, that will hugely decrease the cost. These edible vaccines are the non invasive methods, don’t require any medical assistance, booster dose and contamination free. In the regard of safety, normally in classic vaccines the microorganisms comes back to life causing infection again, so the subunit vaccines are prepared which contains only the antigenic protein parts separated from pathogen, they themselves don’t have a capacity to spread infection but they too need to be in refrigerated condition. But these Food vaccines don’t contain antigen but only protein part which needs a gene copy of pathogen to form and needs no refrigeration. Thus from every side these edible vaccines are the safest thing ever invented.

Journey of edible vaccine through all these years:

Arntzen and his co-workers discovered that tomato and potato plants can synthesize antigens from Norwalk virus, enterotoxigenic E.Coli, vibrio cholera and hepatitis-B virus. Feeding these tubers to animals showed promising results of evoking immune response towards enterotoxigenic E.Coli, vibrio cholera. Additionally this vaccine provided protection against rabies virus, helicobacter pylori and mink enteric virus. In 1997 human trail in volunteers who took peeled and raw potatoes with antigenic protein of E. coli toxin evoked immune response. They also found out the immune reactivity in volunteers who took potato vaccine inoculated with Norwalk virus.³

Hilary Koprowski given transgenic lettuce inoculated with hepatitis B antigen to 3 volunteers exhibited favourable systemic response.^{1, 2}

Mason et al studied the immunogenic effect of genetically engineered potato tubers, tobacco leaves with Norwalk virus capsid protein in mice which exhibited successful results, and also successful immune evoking response was seen tomato propagated with rabies virus G- protein.^4,5

Wu et al noted a success in inoculating potatoes with rotavirus VP7, mice on consuming these tubers elicited a positive immunological response.⁷

There is a positive response evoked when young and aged mice were treated with corn derived antigen against E.coli heat labile enterotoxin.

Ma et al studied the activity hepatitis E virus partial gene in tomatoes.⁶

A positive result was obtained when partial gene of anthrax expressed in plant system. Thus there is a possibility of developing edible vaccine for anthrax is understudy.

Papilloma virus L1 protein showed positive result in expressing transgenic plant which paves the way of development of edible vaccine for cervical cancer.

A team of scientists from Singapore developed an edible vaccine for SARS virus, by vaccinating mice with genetically modified lactic acid bacteria.

Canadian scientists studied a transgenic tobacco plant which is made to produce interlukin 10 used against crohn’s disease.⁸

Process of developing an edible vaccine:

Pharmaceutical and therapeutic antibodies synthesized in plants can be produced in a variety of ways. Conventional methods use stable transformation and transient expression to introduce new genes into a host cell. Once DNA from the transformed host cell is isolated and purified, it can be injected into the embryo of a maturing plant. The plant can then propagate in an open field allowing for large-scale production of antibodies. However, purification of these proteins is generally long and tedious. Upon isolation of the antibody, several proteins, organic molecules, glycan and herbicides must also be isolated, leading to a complex purification process.⁹

Creating edible vaccines involves introduction of selected desired genes into plants and then inducing these altered plants to manufacture the encoded proteins. This process is known as "transformation," and the altered plants are called "transgenic plants." Like conventional subunit vaccines, edible vaccines are composed of antigenic proteins and are devoid of pathogenic genes. Thus, they have no way of establishing infection, assuring its safety, especially in immune compromised patients. Introduction of foreign DNA into plant's genome can either be done by bombarding embryonic suspension cell cultures using gene-gun or more commonly through Agrobacterium tumefaciens, a naturally occurring soil bacterium, which has the ability to get into plants through some kind of wound (scratch, etc.). It possesses a circular "Ti plasmid" (tumor inducing), which enables it to infect plant cells, integrate into their genome and produce a hollow tumor (crown gall tumor), where it can live. This ability can be exploited to insert foreign DNA into plant genome. But prior to this, the plasmid needs to be disarmed by deleting the genes for auxin and cytokinin synthesis, so that it does not produce tumor. Genes for antibiotic resistance are used to select out the transformed cells and whole plants, which contain the foreign gene; and for expressing the desired product, which can then be regenerated from them. The DNA integrates randomly into plant genome, resulting in a different antigen expression level for each independent line, so that 50-100 plants are transformed together at a time, from which one can choose the plant expressing the highest levels of antigen and least number of adverse effects. Production of transgenic plants is species dependent and takes 3-9 months. Reducing this time to 6-8 weeks is currently under investigation. Some antigens, like viral capsid proteins, have to self-assemble into VLPs (viruslike particles). VLPs mimic the virus without carrying DNA or RNA and therefore are not infectious. Each single antigen expressed in plants must be tested for its proper assembly and can be verified by animal studies, Western blot; and quantified by enzyme-linked immunosorbent assay (ELISA).¹⁰

Second generation edible vaccines:

Second generation edible vaccines are also called as multi component vaccines that provide protection against several pathogens. Successful expression of foreign genes in plant cells and/or its edible portions has given a potential to explore further and expand the possibility of developing plants expressing more than one antigenic protein. Multi component vaccines can be obtained by crossing two plant lines harboring different antigens. Adjuvants may also be co-expressed along with the antigen in the same plant. B subunit of Vibrio cholerae toxin (VC-B) tends to associate with copies of itself, forming a doughnut-shaped five-member ring with a hole in the middle. This feature can bring several different antigens to M cells at one time - for example, a trivalent edible vaccine against cholera, ETEC (Enterotoxigenic E. coli) and rotavirus could successfully elicit significant immune response to all three.^11,12

Mechanism of action:

The antigens in transgenic plants are delivered through bio-encapsulation, i.e., the tough outer wall of plant cells, which protects them from gastric secretions, and finally break up in the intestines. The antigens are released, taken up by M cells in the intestinal lining that overlie peyer’s patches and gut-associated lymphoid tissue (GALT), passed on to macrophages, other antigen-presenting cells; and local lymphocyte populations, generating serum IgG, IgE responses, local IgA response and memory cells, which would promptly neutralize the attack by the real infectious agent. Edible vaccines activate both mucosal and systemic immunity, as they come in contact with the digestive tract lining. This dual effect would provide first-line defense against pathogens invading through mucosa, like Mycobacterium tuberculosis and agents causing diarrhoea, pneumonia, STDs, HIV, Norwalk virus, Rotavirus, Vibrio cholera and enterotoxigenic E. coli (ETEC) etc., Administration of edible vaccines to mothers might be successful in immunizing the fetus-in-utero by transplacental transfer of maternal antibodies or the infant through breast milk.^11,13,14

Plants for vaccine development:

An important issue in producing edible vaccine is choosing a plant material which should express more levels of vaccine and should be extensively stored and should be used for oral delivery. Number of plants showed a reliable level of production of recombinant proteins in plants like leafy crops, cereals, legume seeds, oil seed, fruits, vegetables, higher plant tissue and cell cultures, hydroponic systems, algae, and halobios.

Vegetables:

Some vegetables are used as the receptor of plant-derived vaccines because many of them are appetizing, free of toxicant, full of nutrients and fresh edible. Potato (Solanum tuberosum), tomato (Lycopersicon esculentum), and carrot (Daucus carota subsp. Sativus) have already been reported to successfully express vaccine candidates. Potato is considered as an optimal model plant on research of plant oral vaccines, Tomato is a new expression system developed in recent years. Antigen genes encoding HBsAg, HIVgag, and Rabies capsid proteins have been successfully transformed to tomato. In particular, the latest discovery of seed-specific promoters converges a target protein in seeds, so high expression proteins are available. Carrot is one of the first plant for tissue culture, and transgenic technology for carrot is gradually mature. It was reported that high level of recombinant protein expressions was observed in proplastids of cultured carrot cells. Oral delivery of therapeutic proteins via edible carrots preserved the structural integrity of their target proteins, as no cooking is needed. In addition to these plants as noted above, more and more vegetable transgenic systems, such as lettuce (Lactuca sativa), celery cabbage (Brassica rapa var. pekinensis), cauliflower (B. oleracea var. botrytis) are now under the process of establishment, but low expression level is still a problem in these plant expression systems.¹⁵

Fruits

Banana (Musa acuminate) is one of the earliest fruits used for plant transgenic programs. A research showed that promoter MaExp1 could be an important tool for expressing foreign proteins (vaccine) in banana fruit at the time of ripening. Papaya (Carica papaya) is a widespread tropical and semi-tropical fresh edible fruit. Embryogenic callus of papaya was genetically transformed with different constructions including the reporter GUS gene by biobalistics. Papaya is considered as another ideal plant species for vaccines production. Sciutto E, reported that inserted transgenes were identified in papaya, and the expression of a novel synthetic vaccine SPvac, based on three synthetic peptides of 18, 12, and 8 amino acids shared by Taenia solium and T. crassiceps, was investigated to select the best candidates for production of homozygote seeds.¹⁶

Crops

Some spermatophytes are even more suitable for oral delivery vaccines production, because they have abundant soluble proteins and are easy to store. Alfalfa (Medicago sativa) is a perennial crop with strong regenerative capacity and propagation, which continuously produces large clonal populations by stem cuttings in a limited period of time for up to 5 years after establishment. The low levels of secondary metabolites and high protein content in alfalfa leaves make it a good bioreactor for production of recombinant proteins. Maize (Zea mays) was investigated as a commercial platform by several companies (e.g. ProdiGene Inc.) for producing a range of pharmaceutical and technical proteins, such as recombinant antibodies, vaccine candidates, and enzymes. Similar to maize, some proteins have been expressed in rice (Oryza sativa L.) at very high levels by using constitutive and endosperm-specific promoters. Cereal crops are also ideal plant species because the endosperms of such crops are full of soluble proteins and can be easily separated from the whole plant, which enhances the antigen concentration and reduces the oral doses.^17,18

Algae and other halobios:

Although the biotechnological processes based on transgenic microalgae are still in their infancy, researchers are considering the potential of microalgae as green cell-factories to produce value-added metabolites and heterologous proteins for pharmaceutical applications. As far as we know, five species have been transformed: Chlamydomonas reinhardtii, Phaeodactylum tricornutum, Amphidinium carterae, Symbiodinium microadriaticum and Cylindrotheca fusiformis. As a consequence, the research of plant derived recombinant proteins have been developed from the earth to oceans.^19,20 Using algae as a bioreactor to produce oral vaccines can solve many problems (e.g. low expression content, toxicity, incapable of fresh edible, and long-growth period) which are hardly to be overcome by higher organisms.²¹

Challenges for edible vaccines:

1) The first technical challenge for a potential edible vaccine is to keep the protein from being degraded upon ingestion long enough to have immunogenic activity. Current situation is that proteins taken orally will be completely degraded in the digestive tract, with insufficient time to elicit an immune response.

2) The next technical question beyond survivability in the digestive system is: can the protein produce an immune response?

3) The next question is whether this immune response would provide protection.

4) Is the gene stable inside the plant, can it produce required amount of antigenic proteins.

5) Palatibility is also a critical problem.

6) How to fix the dose for these edible vaccines. Low doses of the edible vaccine if consumed produces less number of antibodies and high doses causes immune tolerance.

7) Consistency of dosage form differs from plant to plant and generation to generation, protein content, patient’s age, weight, ripeness of the fruit and quantity of the food eaten in absence of availability of methods for standardization of plant material/product.

8) Some food cannot be eaten raw (e.g. potato) and needs cooking which will denature or weaken the protein present in it.

9) Variable conditions for edible vaccine are also a major problem. Potatoes containing vaccine to be stored at 4°C and could be stored for longer time while a tomato does not last long. Thus these vaccines need to be properly stored to avoid infection through microbial spoilage.

10) Another challenge regarding edible vaccine is how to distinguish characters to identify between ‘vaccine fruit’ and ‘normal fruit’ to avoid misadministration of vaccine which could lead to tolerance.

CONCLUSION:

Edible vaccine are the elements of a double cure where it can create immunization as well as in treating malnutrition, its place seems to be very worthful. Still some problems need to be overcome in production of edible vaccines. Now that the miracle compound in science edible vaccine was invented, as the time goes by no one ever believe what the science magic is upto.

REFERENCES:

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2. C. O. Tacket. Immunogenicity in Humans of a Recombinant Bacterial Antigen Delivered in a Transgenic Potato. Nature Medicine. 4 (5); 1998: 607–609.

3. Takeshi Arakawa, Jie Yu, D. K. Chong, John Hough, Paul C. Engen and William H. R. Langridge. A Plant-Based Cholera Toxin B Subunit-Insulin Fusion Protein Protects against the Development of Autoimmune Diabetes. Nature Biotechnology. 16 (10); 1998: 934–938.

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Received on 25.02.2015 Modified on 15.03.2015

Res. J. Pharm. Dosage Form. & Tech. 7(2): April-June, 2015; Page 161-165

DOI: 10.5958/0975-4377.2015.00024.5