Edible Vaccines:A novel approach

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Parag A.Kulkarni

Parag A. Kulkarni

Edible vaccines play a vital role in immunization. Antigens or antibodies expressed in plants (transgenic plant) can be administered orally through edible parts of the plant.

Edible vaccines are the best option and they are more convenient over traditional vaccines. In future a great ability for the immunization throughout the glob with the use of edible vaccine can be possible with its economic production will lower down the cost  of immunization, separation and purification is easy and pathogenic contamination can be avoided therefore safe, no constricted criteria for its storage so can be stored near the site of use, aseptic condition dose not require for oral immunization as it is given through oral route Many plants like tobacco, potato, tomato, banana, rice, maize are used for the production of edible vaccines. These vaccines can be utilized for the treatment of various diseases in human or animals such as measles, cholera, Hepatitis-B.


Vaccines are primary tools in programs for health intervention for both humans and animals. They would be more widely used especially in developing countries if their cost of production could be reduced and if they could be distributed without refrigeration. Research underway is dedicated to solving these limitations by finding ways to produce oral (edible) vaccines in transgenic plants.

With the advent of modern molecular biology techniques in the 1980s, new strategies were developed for the production of subunit vaccines. These are vaccines comprised of proteins derived from pathogenic viruses, bacteria or parasites; in general the proteins are produced not by the pathogens themselves, but by expression of the gene encoding the protein in a “surrogate organism. In the last decade we have learned that green plants also be used as the “surrogate production organism” to produce antigens of human pathogens (including HBsAg), and that these proteins can elicit priming and boosting immune response in humans when given orally. In addition, unlike almost all other cell lines used for production of vaccines, components of plant cells have always been an important part of the normal human diet. Plants, therefore, offer significant new opportunities for making safe and effective oral vaccines 1.

Transgenic plants for immunotherapy

Since 1984, when transformation of tobacco – the first plant to be transformed with a foreign gene was reported4, great effort has been put into the developing of efficient methods for genetic transformation of plants, and optimizing expression of foreign genes in plants. The techniques used to introduce foreign genes into plants have been extended to major crops, including vegetables, as well as into ornamental, medicinal, fruit, tree and pasture plants5. Various foreign proteins including serum albumin, human α-interferon, human erythropoetin, and murine IgG and IgA immunoglobulins have been successfully expressed in plants6. In recent years, several attempts have been made to produce various antigens and antibodies in plants2,7. Antigens or antibodies expressed in plants can be administered orally as any edible part of the plant, or by parenteral route (such as intramuscular or intravenous injection) after isolation and purification from the plant tissue. The edible part of the plant to be used as a vaccine is fed raw to experimental animals or humans to prevent possible denaturation during cooking, and avoid cumbersome purification protocols.

While Agrobacterium-mediated transformation still remains the method of choice for dicots, a general method, the biolistics method, of transformation of plants, including monocots, has come into existence 5,8. Various strategies for expression of foreign genes in high amounts in plants include use of strong and organ-specific plant promoters, targeting of the protein into endoplasmic reticulum (ER) by incorporating ER-targeting and ER-retention signals, creation of optimized translation start site context as well as alteration of codons to suit the expression of prokaryotic genes in a plant9, 10. For production of edible vaccines or antibodies, it is desirable to select a plant whose products are consumed raw to avoid degradation during cooking. Thus, plants like tomato, banana and cucumbers are generally the plants of choice. While expression of a gene that is stably integrated into the genome allows maintenance of the material in the form of seeds, some virus-based vectors can also be used to express the gene transiently to develop the products in a short period (Figure 1). This may have the additional advantage of allowing expression of the product at very high level; not always attainable in transgenic systems.

Figure 1. Strategies for expression of antigens in plants 2

Strategies for expression of antigens in plants

Table no.1 Antigens produced in transgenic plants2.




Hepatitis-B surface Antigen


3, 11

Rabbis virus Glycoprotein



Norwalk virus capside protein



E. coli Heat labile enterotoxin β-subunit



Cholera toxin β-subunit

Potato, Tobacco

16 – 18

Mouse glutamate decarboxylase



VPI protein of foot and mouth disease virus






Glycoprotein’s of swins-transmissible gastroenteritis corona virus



One of the alternative strategies of producing a plant-based vaccine is to infect the plants with recombinant viruses carrying the desired antigen that is fused to viral coat protein. The infected plants have been reported to produce the desired fusion protein in large amounts in a short time. The technique involved either placing the gene downstream a subgenomic promoter, or fusing the gene with capsid protein that coats the virus (Table no.2, Figure 1). The latter strategy is perhaps the strategy of choice since fusion proteins in particulate form are highly immunogenic. It should, however, be kept in view that recombinant viruses need to be highly purified for parenteral administration or partially purified for oral administration. Modelska et al.29 have shown that immunization of mice intraperitoneally or orally by gastric incubation or by feeding of plants infected with the recombinant alfalfa mosaic virus (AIMV) carrying rabies peptide CPDrg 24 mounted local as well as systemic immune response. Oral administration could stimulate both serum IgG as well as IgA synthesis. After immunization, 40% of the mice were protected against the challenge with a lethal dose of the virus.

Table no. 2 Transient production of antigens in plants after infection with plant virus expressing recombinant gene.





Influenza antigen




Murine zona pellucida antigen




Rabies antigen




HIV- 1 antigen




Mink Enteritis virus antigen

Black eyed bean



Colon cancer antigen




TMV Tobacco Mosaic virus, AlMV Alfalfa Mosaic virus, CPMV Cowpea mosaic virus.

Recently, scientists at Axis Genetics, Cambridge , have shown that injecting mink with extracts of plants infected with a cowpea mosaic virus, that expresses a mink enteritis antigen gene, protects the animal against subsequent virus challen26. While much remains to be done, indications are that plant-based vaccines can compete with vaccines produced by other approaches, particularly keeping in view the low cost and ease of production/distribution.

Though the first report on production of edible vaccine appeared in 1990 in the form of a patent application2 the concept of edible vaccine got impetus after Arntzen and co-workers3 expressed Hepatitis-B surface antigen in tobacco in 1992 to produce immunologically active edible vaccine.


· Edible plants are very effective as a delivery vehicle for inducing oral immunization

· Adjuvant for immune response is not necessary

· Excellent , feasibility of oral administration compared to injection

· Easy for separation and purification of vaccines from plant materials

· Effective prevention of pathogenic contamination from animal cells

· Convenience and safety in storing and transporting vaccines

· Effective maintenance of vaccine activity by controlling the temperature in plant cultivation

· Easy for mass production system by breeding compared to an animal system

· Possible production of vaccines with low costs

· Reduced need for medical personnel and sterile injection conditions

· Economical to mass produce and transport

· Reduced dependence on foreign supply

· Storage near the site of use

· Heat stable, eliminating the need for refrigeration

· Antigen protection through bioencapsulation

· Subunit vaccine (not attenuated pathogens) means improved safety


· Dosage of vaccines would be variable.

· Not convenient for infants.

Scientific background on plant-based vaccines1

Scientific advancements in plant-derived vaccines have occurred over the last decade; they include four major milestones.

·  First, insertion of genes encoding antigenic proteins of human pathogens resulted in successful expression and assembly of multi-component structures within plant cells. These structures, which mimic the native immunogens, include "virus-like particles" (VLPs) for the Hepatitis-B surface antigen (HBsAg), Norwalk virus capsid protein (NV capsid), and oligomeric β-subunit of the heat labile enterotoxin of E. coli (LT-B) either by itself or in association with the enzymatically active α-subunit to form a holotoxin (LT). (Similar studies have also been completed for cholera toxin - CT.) Other than introduction of the genes encoding the antigens with an appropriate DNA vector modified to optimize gene expression, no further cellular engineering of the plant cells were required to obtain immunogens resembling the native pathogen proteins. Subsequent studies, which are continuing in other laboratories around the world, are verifying these findings for other antigenic proteins from human and animal pathogens.

· Second, oral immunogenicity of HBsAg, LT-B, and NV capsid was demonstrated by feeding plant material expressing these antigens directly to animals as feed. While two of these are from enteric pathogens, which might be anticipated to contain mucosally-active immunogens, Hepatitis-B is not an enteric pathogen and is usually not thought to invade the body via the gut. The emerging results portend success with different types of antigens through oral immunization, albeit with very significantly higher levels of immunogen than would be required for injection.

· Third, in Phase 1 human clinical trials, LT-B and NV capsid were found to stimulate both humoral and mucosal immune responses (as evidenced by serum and mucosal antibody responses) and HBsAg gave a strong boosting response in volunteers who had previously received the yeast-derived, injected commercial vaccine. Although the immune responses to NV capsid was modest in amplitude, it was achieved with unprocessed plant tissues (raw potato) with no adjuvants, buffers or additives; in all human clinical trials, the immunogens were active simply when the plant sample was eaten.

· Fourth, in unpublished studies, we have found that standard food industry freeze-drying technology can be used for multiple plant tissues (including tomato, potato and carrot) to yield heat-stable, antigen-containing powders. Freeze-dried tomato powder containing NV capsid and LT-B has been found to be immunogenic in pre-clinical trials, and studies of other antigens are underway. Different batch samples can be blended to give uniform doses of antigen and can be stored at room temperature without antigen loss.

Table No. 3 Proteins with applications for human vaccines and expressed by transgenic plants 31




Enterotoxigenic E.coli



Enterotoxigenic E.coli



Enterotoxigenic E.coli



Vibrio cholerae



Hepatitis-B virus



Hepatitis-B virus



Hepatitis-B virus



Norwalk virus



Norwalk virus



Rabies virus



Human cytomegalovirus



1) Tobacco: -


The first report of the production of edible vaccine (a surface protein from Streptococcus) in tobacco, at 0.02% of total leaf protein level, appeared in 1990 in the form of a patent application published under the International Patent Cooperation Treaty2.Subsequently, a number of attempts were made to express various antigens in plants (Table 1). Since acute watery diarrhoea is caused by enteroxigenic Escherichia coli and Vibrio cholerae that colonize the small intestine and produce one or more enterotoxin, an attempt was made towards the production of edible vaccine by expressing heat-labile enterotoxin (LT-B) in tobacco14.

2) Potato: -


The transgenic potatoes were created and grown by Charles Arntzen, and Hugh S. Mason, and their colleagues at the Boyce Thompson Institute for Plant Research, an affiliate of Cornell University . Previously, National Institute of Allergy and Infectious Diseases (NIAID) supported in vitro and preclinical studies by John Clements, and colleagues at Tulane University School of Medicine showed that transgenic potatoes containing this segment of the toxin stimulated strong immune responses in animals.An edible vaccine could stimulate an immune response in humans. Volunteers ate bite-sized pieces of raw potato that had been genetically engineered to produce part of the toxin secreted by the Escherichia coli bacterium, which causes diarrhea.The trial enrolled 14 healthy adults; 11 were chosen at random to receive the genetically engineered potatoes and three received pieces of ordinary potatoes. 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 rises in serum antibodies at some point after immunization, and six of the 11 (55٪) developed fourfold rises in intestinal antibodies. The potatoes were well tolerated and no one experienced serious adverse side effects38.

3) Tomato: -


Tomatoes serve as an ideal candidate for this HIV antigen because unlike other transgenic plants that carry the protein, tomatoes are edible and immune to any thermal process, which help retain its healing capabilities. Even more importantly, tomatoes were found to grow at a high rate of success in Russia , compared to bananas30.

Introducing the Protein to the Tomato

Getting the artificial protein gene into the tomato germs was accomplished with the help of a needle. The next step was to cultivate the germs on a special nutrient medium. From there, the plants that grew roots were planted into the soil and grown in the hothouse until they reached maturity and produced fruit. The plants were then tested for the protein, which could be found in the leaves and the tomato plant itself30.

 4) Banana: -


Vaccines or subunit vaccinations, bananas seem to be the desired vector.  The advantage of bananas is that they can be eaten raw as compared to potatoes or rice that need to be cooked and bananas can also be consumed in a pure form. Research is leaning towards the use of bananas as the vector since most third-world countries, who would benefit most from edible vaccines, are in tropical climates that are suitable for growing bananas.

5) Maize: -


Egyptian scientists have genetically engineered maize plants to produce a protein used to make the Hepatitis-B virus vaccine. More than 2 billion people are infected with Hepatitis-B, and about 350 million of these are at high risk of serious illness and death from liver damage and liver cancer. A team of researchers led by Hania El-itriby, director of Cairo's Agricultural Genetic Engineering Research Institute, developed genetically modified (GM) maize plants that produce the protein known as HbsAg, which elicits an immune response against the Hepatitis-B virus and could be used as a vaccine34.  

6) Rice: -


When predominant T cell epitope peptides, which were derived from Japanese cedar pollen allergens, were specifically expressed in rice seed and delivered to the mucosal immune system, the development of an allergic immune response of the allergen-specific Th2 cell was suppressed. Furthermore, not only were specific IgE production and release of histamine from mast cells 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 treatment37.

First characterized the seed promoters suitable for the expression of foreign genes in appropriate sites of the rice seed. After demonstrating that the individual promoters of the storage proteins glutelin, globulin and prolamin directed high levels of expression at different sites in the endosperm, they decided to use primarily the major seed storage protein glutelin GluB-1 promoter for expression of foreign genes. In addition, they demonstrated that the accumulation levels of the foreign protein were enhanced when expressed within the genetic background of a low storage protein character.When the seed expression system is used as a platform for foreign protein production, substantial amounts of recombinant proteins can be accumulated, because the seed is a natural storage organ for accumulating the starch, protein, and oil required for seedling growth. Also, artificial peptides or proteins accumulate in seed, which is in remarkable contrast with other tissues37.

Table No.4 Advantages and disadvantages of different plants39

Plant / Fruit





Good model for evaluating recombinant proteins.

Low cost preserving system.

Easy purification of antibodies stored in the seeds, at an location.

Large harvests, number of times/year

Produces toxic compounds*





Dominated clinical trials

Easily manipulated/transformed

Easily propagated from its “eyes”

Stored for long periods without refrigeration

Needs cooking which can denature antigen and decrease immunogenicity**




Do not need cooking

Proteins not destroyed even if cooked


Grown widely in developing countries


Trees take 2-3 years to mature

Transformed trees take about 12 months to bear fruit

Spoils rapidly after ripening

Contains very little protein, so unlikely to produce large amounts of recombinant proteins



Grow quickly

Cultivated broadly

High content of vitamin A may boost immune response

Overcome the spoilage problem by freeze-drying technology

Heat-stable, antigen-containing powders***, made into capsules

Different batches blended to give uniform doses of antigen

Spoils readily




Commonly used in baby food because of low allergenic potential

High expression of proteins/ antigens

Easy storage/transportation

Expressed protein is heat-stable

Grows slowly

Requires specialized glasshouse conditions





Direct consumption

Spoils readily


and Alfalfa

Large harvests, number of times/year


*Currently, therapeutic proteins in tobacco are being produced. **Some kinds of South American potatoes can be eaten raw. Although some studies show that cooking does not destroy full complement of antigen in potatoes. ***Freeze-dried tomato powder containing NV capsid and LT-B was found immunogenic.

Current development

Currently researchers are seeking to develop genetically altered plants that could provide immunity to infectious diseases. Studies have already shown that genetically engineered plants can act as a vaccine. Plants acting as vaccines would offer the advantage of inexpensive to produce, and thus they could more easily be made available to developing countries. In addition, contamination with animal viruses would be eliminated, since cultured cells would not be used in the production process. Many of the quality control tests that require animals also could be eliminated. 

Edible vaccines are currently being developed for a number of human and animal diseases, including measles, cholera, foot and mouth disease, and Hepatitis-B and C. Many of these diseases are likely to require booster vaccinations or multiple antigens to induce and maintain protective immunity. Plants have the capacity to express more than one transgene, allowing delivery of multiple antigens for repeated inoculations.

Recently the glare of the media spotlight has fallen on genetically engineered food crops bred to resist herbicides and insects. Meanwhile, plants engineered with human proteins to produce drugs and vaccines for human consumption have escaped notice. Well, take note: At least 350 genetically engineered pharmaceutical products are currently in clinical development in the United States and Canada . Scientists believe that potent drugs and vaccines will soon be harvested just like wheat and corn.

In Canada , a genetically engineered tobacco plant made to produce Interleukin 10 will be tested to treat Crohn's disease, an intestinal disorder.

Scientists have also discovered that fruits with high water content could result in proteolysis.  Experimentation with freeze-dried food to create pellets or powder is now being investigated to help avoid proteolysis and overall efficacy.

Arntzen and his colleagues plan to develop banana-based vaccines. They're also targeting papilloma virus, which can lead to cervical cancer, as well as Hepatitis-B virus. A reliable injectable vaccine already exists for Hepatitis-B, but a less expensive, edible vaccine would be better, particularly for use in the developing world.

Current measles vaccines are made from the actual virus and work by priming the immune system to attack if it becomes exposed to a full assault of the measles virus. In contrast, plant-based vaccines rely on the measles virus gene for the H protein being genetically cloned into the plant.31

Future Perspectives

Although still at an early stage of development, the experimental know-how and results strongly suggest that plant-derived edible vaccines are likely to become a reality, in the next few years. Future research will demonstrate if these vaccines meet the standards of quality (purity, potency, safety and efficacy) defined for vaccines by the World Health Organization. 

In future a great ability for the immunization throughout the glob with the use of edible vaccine can be possible with its economic production will lower down the cost  of immunization, separation and purification is easy and pathogenic contamination can be avoided therefore safe, no constricted criteria for its storage so can be stored near the site of use, aseptic condition dose not require for oral immunization as it is given through oral route.GM-plant (Genetically modified plants) may be grown in field and clinical trials are required to define the risk/ benefit ratio of a GM-plant before registration is granted.

In most countries of the world, plants engineered to produce vaccines fall under the very restrictive rules set up to control GM-crop plants. The present concern, especially in Europe , over the use of biotechnology for the genetic improvement of crop plants also negatively affects the acceptance of GM-plants for medicinal use. As a consequence, while the demonstration that plant-derived vaccines are effective on populations at risk is expected to arrive within 1-2 years, a further quarantine of 2-3 years will be required in order to fulfil requirements for registration and marketing. It is hoped that simpler rules will be set up for GM-plants producing vaccines and that they are seen as clearly and legally distinct from GM-plants grown for nutrition purposes.


Edible plant vaccines offer significant new opportunities for making safe and effective oral vaccines would be more widely used especially in developing countries. Due to some positive aspects like they are economical, safe, easy to administered and can be stored without refrigeration so that these vaccines are prominent over typical traditional vaccines. Plant-based vaccines are obtained from specific transition plants. This technique can be utilized for the treatment of Hepatitis-B, measles, cholera, foot and mouth disease but still diseases like cancer, malaria, HIV, asthma, etc. are major health problems in developing countries. By means of edible vaccines these problems can be solved.


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About Authors

Parag A.Kulkarni

Parag A. Kulkarni (M. Pharm).
Lecturer of Pharmaceutics, Padmashree Dr. D. Y. Patil College of Pharmacy, Akurdi. Pune-411044
For correspondence

Dr.Pramod V.Kasture

Dr. Pramod V. Kasture (Pharm. PhD.)
Principal and Professor of Pharmaceutics, Padmashree Dr. D. Y. Patil College of Pharmacy, Akurdi. Pune-411044

Mr.Santosh S.Bhujbal

Mr.Santosh S.Bhujbal
Asst. Professor of Pharmacognosy, Padmashree Dr. D.Y. Patil College of Pharmacy, Akurdi, Pune (MS) 411044.

Mrs.Sugandha G.Chaudhari

Mrs.Sugandha G.Chaudhari (M. Pharm.)
Lecturer of Pharmacology, Hydrabad (Sind) National Collegiate Board’s College of Pharmacy, Ulhasnagar.421003

Ms.Ujwala R.Suralkar

Ms.Ujwala R.Suralkar (B. Pharm.)
Padmashree Dr. D.Y. Patil College of Pharmacy, Akurdi. Pune-411044

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