The concept of edible vaccine got impetus after Arntzen and coworkers expressed HBsAg in tobacco. The first edible vaccine was produced in tobacco in 1990 in which 0.02 % recombinant protein (a surface protein from Streptococcus) of the total soluble leaf proteins was found. It appeared in the form of a patent application published under the International Patent Cooperation Treaty. Transgenic tobacco is successfully engineered for the production of edible vaccines against hepatitis B antigen using ‘s’ gene of hepatitis B virus (HBV). The optimum level of recombinant protein was obtained in leaves and seeds. Since acute watery diarrhea is caused by enterotoxigenic E. coli and Vibrio cholerae that colonize the small intestine and produce one or more enterotoxin, an attempt was made toward the production of edible vaccine by expressing heat-labile enterotoxin (LT-B) in tobacco. Besides, antibodies against dental caries, expressed in tobacco, are already in preclinical human trials. Italian researchers have now developed an immunologically active, cost-efficient vaccine against human papilloma viruses (HPV). HPV are the causative agents for cervical cancer, and are also involved in skin, head, and neck tumors. Cervical cancer is one of the main causes of cancer-related deaths.
12.3.2 Potato
Genetically modified potatoes are also a viable option and seem to be the desired vector. Many of the first edible vaccines were synthesized in potato plants. The transgenic potatoes were developed and grown by Arntzen and Mason and their research group at the Boyce Thompson Institute for Plant Research, Cornell University. Previously, NIAID supported in vitro and preclinical studies by John Clements and colleagues at Tulane University School of Medicine, in which 14 volunteers ate bite-sized pieces of raw potato that had been genetically engineered to produce part of the toxin secreted by E. coli causing diarrhea. The investigators periodically collected blood and stool samples from the volunteers to evaluate the vaccine’s ability to stimulate both systemic and intestinal immune responses. Ten of the 11 volunteers (91 %) who ingested the transgenic potatoes had fourfold rise in serum antibodies at some point after immunization, and 6 of the 11 (55 %) also showed fourfold mount in intestinal antibodies. The potatoes were well tolerated and no one experienced serious adverse side effects. Vaccine development has successfully tested a potato-based vaccine to combat the Norwalk Virus, which is spread by contaminated food and water. The virus causes severe abdominal pain and diarrhea.A research team led by William Langridge of the Loma Linda University in California has reported that transgenic potatoes engineered with a cholera antigen, CTB can effectively immunize mice. Mice fed transgenic potatoes produce cholera-specific antibodies in their serum and intestine; IgA and IgG antibodies reach their highest levels after the fourth feeding. In yet another experiment genetically engineered potatoes containing a hepatitis B vaccine have successfully boosted immunity in their first human trials.
Attempts have also been made to boil the potatoes as raw potatoes are not very appetizing but unfortunately the cooking process breaks down about 50 % of the proteins in the vaccine. While some proteins are more tolerant to heat, for most proteins it will be necessary to amplify the amount of protein in the engineered foods if they are to be cooked before consumption.
12.3.3 Tomato
Tomatoes are an excellent candidate because they are easy to manipulate genetically and new crops can be grown quickly. Moreover, they are palatable and can be eaten raw. While tomatoes do not grow well in the regions in which the edible vaccines are most needed, the engineered tomatoes can be dried or made into a paste to facilitate their delivery.The anti-malaria edible vaccines in different transgenic tomato plants expressing antigenic type(s) have been proposed by Chowdhury and Bagasara in 2007. They hypothesized that immunizing individuals against 2–3 antigens and against each stage of the life cycle of the multistage parasites would be an efficient, inexpensive and safe way of vaccination. Tomatoes with varying sizes, shapes, and colors carrying different antigens would make the vaccines easily identifiable by lay individuals.
Tomatoes serve as an ideal candidate for the HIV antigen because they unlike other transgenic plants that carry the protein, are edible and immune to any thermal process, which help to retain their healing capabilities. Scientists have claimed that tomatoes could be used as a vaccine against Alzheimer’s disease. The work is in progress to genetically modify the fruit to create an edible vaccine that fires up the immune system to tackle the disease by attacking the toxic beta-amyloid protein that destroys vital connections between brain cells, causing Alzheimer’s.
Researchers have engineered tomato plants (Lycopersicon esculentum Mill var. UC82b) to express a gene for the glycoprotein (G-protein), that coats the outer surface of the rabies virus. The recombinant constructs contained the G-protein gene from the ERA strain of rabies virus, including the signal peptide, under the control of the 35S promoter of cauliflower mosaic virus (CaMV).
12.3.4 Banana
A common fruit—the banana—is currently being considered as a potential vehicle for vaccines against serious as well as too common diseases. The advantage of bananas is that they can be eaten raw as compared to potatoes or rice that need to be cooked and can also be consumed in a pure form. Furthermore, children tend to like banana and the plants grow well in the tropical areas in which the vaccines are needed the most. Hence, the research is leaning toward the use of banana as the vector since a large number of third-world countries, who would benefit the most from edible vaccines have tropical climates. On the negative side, a new crop of banana plants takes about 12 months to bear fruit. After fruiting, the plants are cut down and a new crop of vaccine-bearing plants must be planted.Researchers have also developed bananas that deliver a vaccine for HBV. The banana vaccine is expected to cost just 2 cents a dose, as compared to the $125 for the currently available injectable vaccine.
12.3.5 Maize
Maize has also been used as a vector for various edible vaccines. Egyptian scientists have genetically engineered the maize plants to produce a protein known as HbsAg which elicits an immune response against the hepatitis B virus and could be used as a vaccine. If human trials are successful more than 2 billion people infected with hepatitis B, and about 350 million of these at high risk of serious illness and death from liver damage and liver cancer would be benefited.Researches are in offing at Iowa State University with the aim to allow pigs and humans to get a flu vaccination simply by eating corn or corn products. It is quite likely that corn vaccine would work in humans when they eat corn or even corn flakes, corn chips, tortillas, or anything that contains corn.
Genetically modified maize could provide protection to chickens against a highly contagious and fatal viral disease affecting most species of birds. Mexican researcher Octavio Guerrero-Andrade and his colleagues at the Centre for Research and Advanced Studies in Guanajuato, Central Mexico, genetically modified maize to create an edible vaccine against Newcastle disease virus (NDV). They inserted a gene from the NDV, a major killer of poultry in developing countries, into the maize DNA and found antibodies against the virus in chickens that ate the genetically modified maize. One pig vaccine has also been produced in corn successfully.
Efforts are being made by US company ProdiGene to genetically modify maize to contain a key protein found on the surface of the monkey form of HIV. According to US National Institute of Health this development brings an edible, more effective, HIV vaccine for people a step closer.
Transgenic maize expressing the rabies virus glycoprotein (G) of the Vnukovo strain has also been produced using ubiquitin maize promoter fused to the whole coding region of the rabies virus G gene, and a constitutive promoter from CaMV. Maize embryogenic callus were transformed with the above construct by biolistics. Regenerated maize plants were recovered and grown in a greenhouse. The amount of G-protein detected in the grains was approximately 1 % of the total soluble plant protein.
12.3.6 Rice
Rice is another potential crop which has been used for developing vaccines. It offers several advantages over traditional vaccines; it does not require refrigeration. In fact, the rice proved just as potent after 18 months of storage at room temperature and the vaccine did not dissolve when exposed to stomach acids. In an attempt, predominant T cell epitope peptides, which were derived from Japanese cedar pollen allergens, were specifically expressed in rice seeds and delivered to the mucosal immune system (MIS); the development of an allergic immune response of the allergen-specific Th2 cell was suppressed. Furthermore, not only the specific IgE production and release of histamine from mast cells were suppressed, but the inflammatory symptoms of pollinosis, such as sneezing, were also suppressed. These results suggest the feasibility of using an oral immunotherapy agent derived from transgenic plants that accumulate T cell epitope peptides of allergens for allergy treatment.The transfer of genetic material from the microbe responsible for producing cholera toxin into a rice plant has been achieved. The plants produced the toxin and when the rice grains were fed to mice they provoked immunity from the diarrhea-causing bacterium.
12.3.7 Spinach
Genetically modified spinach has also been considered for the development of edible vaccine. Spinach is being investigated as a plant-derived, edible vehicle for anthrax vaccine, as well as a vehicle for the HIV-1 Tat protein (a prospective vaccine candidate). In an experiment a fragment of protective antigen (PA) that represents most of the receptor-binding domain was expressed as a translational fusion with a capsid protein on the outer surface of tobacco mosaic virus, and spinach was inoculated with the recombinant virus. The plant-expressed PA is highly immunogenic in laboratory animals.Among other food crops with potential to be developed as edible vaccine; sweet potato, peanuts, lettuce, watermelon, and carrots are on the top priority. The development of plant-based vaccines to protect against many other diseases, such as HIV-1, hepatitis B, rabies, and non-Hodgkin’s lymphoma are ongoing throughout the globe using one of these edible plants.
The advantages and disadvantages of various plant host systems are given in Table 12.1.
Table 12.1
Features of different plant host systems
Plant | Advantages | Disadvantages |
|---|---|---|
Tobacco | Facile and efficient transformation system; abundant material for protein characterization | Toxic alkaloids incompatible with oral delivery; potential for outcrossing in field |
Banana | Cultivated widely in developing countries where vaccines are needed; eaten raw by infants and adults; clonally propagated; low potential for outcrossing in field; once established, plentiful and inexpensive fruits are available on a 10–12 month cycle | Inefficient transformation system; little data available on gene expression, especially for fruit specific promoters; high cultivation space requirement; very expensive in greenhouse |
Potato | Facile and efficient transformation system; tuber is edible raw though not palatable; tuber-specific promoters available; microtuber production for quick assay; clonally propagated, low potential for outcrossing in field; Industrial tuber processing well established | Relatively low tuber protein content; unpalatable in raw form; cooking might cause denaturation and poor immunogenicity of vaccine |
Tomato | Relatively efficient transformation system; fruit is edible raw; fruit specific promoters available; crossing possible to stack antigen genes; industrial greenhouse culture and industrial fruit processing well established | Relatively low fruit protein content; acidic fruit may be incompatible with some antigens or for delivery to infants; no in vitro system to test fruit expression |
Legumes | Production technology widely established; high protein content in seeds; stable protein in stored seeds; well suited for animal vaccines; industrial seed processing well established | Inefficient transformation systems; heating or cooking for human use might cause denaturation and poor immunogenicity of vaccine; potential for outcrossing in field for some species |
Alfalfa | Relatively efficient transformation system; high protein content in leaves; leaves edible uncooked | Potential for outcrossing in field; deep root system problematic for cleaning field |
12.4 Advantages
Conventional subunit vaccines are expensive and technology-intensive, need purification, require refrigeration, and produce poor mucosal response. In contrast, edible vaccines would enhance compliance, especially in children, and because of oral administration would eliminate the need for trained medical personnel. Their production is highly efficient and can be easily scaled up. For example, hepatitis B antigen required to vaccinate whole of China annually, could be grown on a 40-acre plot and all babies in the world each year on just 200 acres of land. They are cheaper, sidestepping demands for purification (single dose of hepatitis B vaccine would cost approximately 0.43 cents), grown locally using standard methods and do not require capital-intensive pharmaceutical manufacturing facilities. Mass-indefinite production would also decrease dependence on foreign supply. Fear of contamination with animal viruses—like the mad cow disease, which is a threat in vaccines manufactured from cultured mammalian cells, is eliminated as plant viruses do not infect humans.
Edible vaccines activate both mucosal and systemic immunity, as they come in contact with the digestive tract lining which is not possible with subunit vaccines which provide poor mucosal response. This dual effect of edible vaccines provides first-line defense against pathogens invading through mucosa, such as Mycobacterium tuberculosis and agents causing diarrhea, pneumonia, STDs, HIV, etc.
The specific advantages are stated below:
1.
Edible means of administration.
2.
No need of medical personnel and syringes.
3.
Sterile injection conditions are no more required.
4.
Economical in mass production by breeding compared to an animal system.
5.
Easy for administration and transportation.
6.
Effective maintenance of vaccine activity by controlling the temperature in plant cultivation.
7.
Therapeutic proteins are free of pathogens and toxins.
8.
Storage near the site of use.
9.
Heat stable, thus eliminating the need of refrigeration.
10.
Antigen protection through bioencapsulation.
11.
Subunit vaccine (not attenuated vaccine) means improved safety.
12.
Seroconversion in the presence of maternal antibodies.
13.
Generation of systemic and mucosal immunity.
14.
Enhanced compliance (especially in children).
15.
Delivery of multiple antigens.
16.
Integration with other vaccine approaches.
17.
Plant-derived antigens assemble spontaneously into oligomers and into virus like particles.
18.
No serious side effect problems have been noticed until now.
19.
Reduced risk of anaphylactic side effects from edible vaccine over injection system is one benefit reported by the Bio-Medicine.org. They reported that the edible vaccine carries only part of the allergen compared to injection methods which reduce anaphylactic risk.
20.
Administration of edible vaccines to mothers to immunize the fetus-in utero by trans-placental transfer of maternal antibodies or the infant through breast milk. Edible vaccines have a potential role in protecting infants against diseases like group-B Streptococcus, respiratory syncytial virus (RSV), etc., which is under investigation.
21.
Edible vaccines would also be suitable against neglected/less common diseases like dengue, hookworm, rabies, etc. They may be integrated with other vaccine approaches and multiple antigens may also be delivered.
12.5 Limitations and Challenges
With advancement come many hurdles and problems, so is true for edible vaccines. Like, one could develop immunotolerance to the vaccine peptide or protein, though a little research has been done on it. One of the key goals of the edible-vaccine pioneers was to reduce immunization costs but later many limitations were reported as given below:
1.
Consistency of dosage from fruit to fruit, plant to plant, lot to lot, and generation to generation is not similar.
2.
Stability of vaccine in fruit is not known.
3.
Evaluation of dosage requirement is tedious.
4.
Selection of best plant is difficult.
5.
Certain foods like potatoes are generally not eaten raw and cooking the food might weaken the medicine present in it.
6.
Not convenient for infants as they might spit it, eat a part or eat it all, and throw it up later. Concentrating the vaccine into a teaspoon of baby food may be more practical than administering it in a whole fruit.
7.
There is always possibility of sideeffects due to the interaction between the vaccine and the vehicle.
8.
People could ingest too much of the vaccine, which could be toxic, or too little, which could lead to disease outbreaks among populations believed to be immune.
9.
A concern with oral vaccines is the degradation of protein components in the stomach due to low pH and gastric enzymes. However, the degradation can be compensated by repeating the exposure of the antigen until immunological tolerance is accomplished (Mason et al. 2002).
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