Since prehistory, mankind has used plants for medicinal purpose. Thousands of plants have been employed for the treatment and therapies of human and animals. Still today, the search for new phytochemicals is still going on. In recent years, the development of genetically engineered plants has paved the way for using plants to produce vaccines and other pharmaceuticals. This technique is relatively new and its gaining momentum. The traditional method of producing proteins and vaccine using fermentation technique is limited due to high production cost, sophisticated purification technique and construction, and long validation period of manufacturing products (Mei et al, 2006). However, modern plant biotechnology has used plants in providing an alternative way of producing vaccine which has the potential to overcome the constraints faced by traditional method, ie using fermentation technique. Modern method of vaccine production is the use of principles of genetic engineering to express foreign gene in plants. Plants can be genetically transformed to express any recombinant protein in them from which they can be purified or can be consumed directly. Moreover, when these recombinant proteins are expressed in the edible plant tissue like seeds, fruits, there is no need of purification, cold chain delivery of vaccine, and injection for the administration of vaccine.
Plants are used as a biofactories for manufacturing large quanitities of recombinant protein in short period of time. Plant made vaccine and therapeutics refer to protein products with clinical applications for both humans and animals. There are numerous benefits of employing plants for the production of recombinant proteins. Plants are inexpensive to grow as they have very simplistic requirement of sunlight, soil water and cheap fertilizers and when they are engineered to express recombinant protein in edible tissues, overall production cost will significantly get lowered. Also, the need of expensive fermentation, purification, cold storage, transportation and sterile delivery is eliminated (Daniell et al, 2009). The products that are currently being produced in plants include bioactive peptides, vaccine antigens, antibodies, diagnostic proteins, nutritional supplements, enzymes and biodegradable plastic (Sharma and Sharma, 2009). These products can be expressed in edible plant tissue like fruits, seeds or leaves and easily processed or consumed directly (Warzecha and Mason, 2003). Therefore the edible plant vaccine is a cost effective means of delivering vaccines. Edible vaccines are like subunit preparations in which the antigens are expressed in them using genetic engineering, but they bear no genes that would enable whole pathogens to form. Also, the risk of contamination of plant vaccine with pathogens is eliminated. Subunit vaccines are just as efficacious as whole pathogen vaccine. In addition to that, they are safer means of vaccination (Streatfield et al, 2001). Plant derived antigenic proteins (vaccine) have been proved as an efficient means of delaying or preventing the onset of disease in animals and have also been proven to be safe and functional in human clinical trials (Walmsley and Arntzen, 2000).
Steps in production of plant vaccines
WHO has defined vaccine as any preparation which is able to stimulate the production of antibody conferring immunity against a particular disease. Vaccines may include killed or attenuated microorganisms or purified products derived from them. Traditionally, vaccines have been produced using variety of transgenic systems, including cultured mammalian cells, microorganism like bacteria, viruses and fungi. The use of transgenic plants in the production of vaccine and pharmaceutics is much more recent and promising technique. In transgenic plants, bacterial and viral antigens can be faithfully expressed to form immunogenic proteins. When these proteins are expressed in edible plant tissue, they form edible vaccine.
Sharma and Sharma (2009) has mentioned the following steps for the manufacture of plant vaccine:
- selection of host plant system,
- Selection of vector and promoter,
- Integration of gene constructs into the plant genome and regeneration of plants expressing the desired protein, and
- Identification and purification of recombinant proteins.
1. Selection of host plant system
Selection of plant system is the key issue in producing plant vaccine. Selection of host depends upon the type of recombinant product to be obtained. Different plants have different ability to express recombinant proteins. Also the life cycle of host, biomass yield, containment and scale up costs are the deciding factors for efficient production of recombinant product. Plant or plant parts should express high level of recombinant proteins and should be suitable for extensive storage and oral delivery. These days, tobacco tomato, banana, rice, maize, wheat, carrot, soybean, pea, potato, lettuce and alfalfa are used in production of plant vaccine (Rybicki, 2009).
2. Selection of Vector and promoter
Figure 1. Showing two different approaches of engineering plants for the expression of recombinant protein in them.
The factor involved in controlling the expression of high level antigen coding gene is the selection of suitable promoter. Selection of promoters is desirable to achieve high level of transcription which is needed for optimising expression level of recombinant protein. Promoters can be constitutive, tissue specific or inducible (Sharma and Sharma, 2009). High level expression of protein is essential to develop economically competitive transgenic plant within confined field with controlled envirionmental and contained biosafety conditions (Tiwari et al, 2009). Also, expression of proteins other than the target organ leads to heavy loss.
3. Production and Purification
Transgenic plant vaccine can be obtained or used in two ways: one is plentiful production of antigen proteins and then converting into vaccine by separation and purification; and the other is direct consumption of edible plant tissue (Mei et al, 2006). Purification of recombinant proteins accounts for high cost of pharmaceutical products. Purification of recombinant plant proteins has many problems like influence of polyphenols, alkaloids, quinonoid compounds, and other secondary metabolites (Mei et al, 2006). Therefore, the better approach will be the expression of recombinant proteins in the edible tissues of plant which can be consumed without processing. This eliminates the purification expenses. Also, proteins expressed in edible parts are stable as they can be stored for a longer period and transportation is easier (Tiwari et al, 2009). The most important aspect of recombinant protein expressed in edible tissue is the reduced production cost. Oral delivery of recombinant protein reduced about 90 percent of the production cost.
Applications of Plant derived vaccine
Vaccination is one of the most effective means of preventing infectious diseases. Since the discovery of vaccine in 1796 by Edward Jenner, many vaccines have been developed and helped in saving life of humans and animals. The traditional method of vaccine production includes the use of cultured mammalian cells, or recombinant microbes. These production methods are limited due to high cost, sophisticated technique, risks of human pathogenic contamination, etc.. Also microbes are not ideal for synthesizing many mammalian proteins because of the differences in metabolic pathway, protein processing, codon usage and the formation of inclusion bodies (Giddings et al, 2000). Also, lack of post translational modification can be taken as a main draw back in bacterial fermentation (Peeters et al, 2001).
Production of vaccine in plants has overcomed this problem and has been proved to be an efficient and inexpensive way of producing and delivering vaccines. Following are some of the major advantages of plant based vaccine:
ü Low production cost
ü Rapid and easy scale up
ü Convenient storage
ü Need of cold chain transport is eliminated
ü Reduced use of needles and syringes
ü Contamination of vaccine with human pathogens is eliminated
ü The need of trained health professionals for the administration of vaccine is minimized
ü The fear of vaccination via injection, particularly in children is eliminated.
The main advantage of plant vaccine is the reduced manufacturing cost. Sala et al (2003) has mentioned that the production cost will be reduced by 100-1000 times as compared with that of traditional vaccine. The capital investment is low as the existing infrastructures can be used for cultivation, harvesting, storage and processing of transgenic crops (Giddings et al, 2000). The plants can be grown in green houses which eliminate the expensive cost of using fermenters and bioreactors. Plant derived vaccine are stable so that they can be conveniently stored. Also the need of cold chain method of storing and transporting vaccine is eliminated. All these reduce the overall cost of vaccine production in plant.
Plant vaccine has the advantage to scale up the vaccine manufacturing time. Gleba et al (2005) has mentioned that milligrams and grams quantities of recombinant protein are available in 3-4 weeks, and the within less than one year, the scale up to 100 kg is possible. Therefore, plant vaccine can be employed for combating mutating viruses like flu virus and norovirus. Bernstein (2009) has repoted that scientists have already discovered the first vaccine against norovirus also called cruise ship virus in tobacco plants. Viruses like noroviruses are always mutating, making it difficult for developing vaccines in short period of time. However, plant biotechnology has the potential of quicker manufacturing technique suitable to combat mutating viruses like norovirus and the flu. It is mentioned that with plant based vaccine has the advantage to scale up vaccine manufacture within weeks instead of months (Bernstein, 2009).
One of the main advantages of plant derived vaccine is the protein folding mechanism in the endomembrane system of plant that is homologous to those in mammalian cells. This allows the expression of monoclonal antibodies, other types of immunoglobin molecules and multimeric complexes (Ma et al, 2004). Also when recombinant proteins are expressed in plants, there is minimized risk of contamination with human or animal pathogens.
The other advantage of plant vaccine is oral delivery. Plant derived oral vaccines are not digested in stomach as the antigens are bioencapsulated by plant cell wall. When plant derived vaccines are given orally, they are also capable of inducing both mucosal and humoral immunity (Arntzen et al, 2005). Streatfeild et al (2001) has mentioned that vaccines against enterotoxigenic strains of E.Coli and swine transmissible gastroenteritis virus (TGEV) have been expressed in corn seeds and these antigens were able to elicit protective immune responses. Walmsley and Arntzen (2000) has cited the expression of hepatitis antigen in tobacco and lettuce, rabies antigen in tomato, and cholera antigen in tobacco and potatoes; human cytomegalovirus in tobacco. Also, when vaccines are delivered orally, there is no need of syringes and needles. Therefore, it eliminates the fear of vaccination via injection, particularly in children. Also, the need of trained health professionals for the delivery of vaccine is minimized.
One of the greatest breakthroughs of plant vaccine will be for the people in developing countries where people don’t have access to medicines and vaccines vaccine because of their high cost and complicated delivery system. However, plant derived vaccine has the potential to overcome these problems as it is inexpensive and the need of cold chain storage or delivery is eliminated. So, plant derived vaccines can be accessed by poor and people at remote areas. Therefore vaccines and medicines derived from plants will be a great boon of modern plant biotechnology to these people.
Plant vaccine is regarded as the safer means of vaccine production. Vaccines that are produced in plants are less likely to get contaminated with human or animal pathogens because plant doesn’t act as a host for those infectious agents (Giddings et al, 2000). Also, plants have the ability to carry out post translational modification that is similar to higher eukaryotes (Streatfield and Howard, 2003). These features are beneficial for the safe consumption of plant derived vaccines.
Limitations and potentials of plant derived vaccine
Plant biotechnology is a rapidly emerging technology and is extending from the boundaries of crop improvement to the production of vaccines. Plant derived vaccine holds considerable potential in the vaccination of humans and animals as it is the inexpensive and efficient way of delivering vaccines. Despite having numerous benefits, there is significant public debate regarding the use of genetically modified crops for vaccine production. Doran (2000) has mentioned some of the pitfalls of plant vaccine as follows:
- There is a possible fear of containment of genetically modified plants in the environment;
- Recombinant proteins might cause allergic reactions to plant protein glycans and plant antigens;
- There is a possibility of contamination of recombinant product by mycotoxins, pesticides, herbicides, and endogenous plant secondary metabolites;
- There is a regulatory uncertainty, particularly for protein requiring approval for using plant vaccine as a human drug.
The major concern against using genetically engineered crops for vaccine production is the cross contamination of wild type plants with genetically altered crops. Amos, J. (2004) has reported that the transgenic plants should be grown pharmaceutical production units on dedicated land, isolated from food crops with regulatory oversights. Also, these plants should be genetically isolated, i.e. sterility in male so that they don’t produce pollen. The crops should be harvested and stored using dedicated equipments. Also, these contained conditions should be properly regulated.
Also, Mei et al (2006) has mentioned that repeated exposure to an oral antigen has the potential to produce immunological tolerance or unresponsiveness. However, the antigen dose to induce protection is generally smaller than the dose for unresponsiveness. Also, all vaccines should go extensive testing to define the correct dosage and the appropriate schedule for boosting. Therefore, tolerance from prescribed doses is highly unlikely (Streatfield and Howard, 2003).
The production of plant vaccine is an economical and efficient means of vaccinating animals. When large herds of farmed animals are to be vaccinated, they require huge amount of vaccines. Therefore, plant derived vaccine can be sucessfully implemented. Also, plant derived vaccine can be used in immunization of wild animals, which may act as a reservoir of disease, like rabies. Giddings et al (2000) reported that feeding pigs with edible maize vaccine protects them from the transmissible gastroenteritis virus.
One concern regarding plant based vaccine is the extent and nature of glcosylation, which is sometimes different from that found in animals. Giddings et al (2000) has acknowledged that some carbohydrates are unique to plants and when they are administered regularly, may present an antigenic challenge to the immune system. Therefore, sufficient knowledge and awareness should be provided to the people about the consumption of plant derived vaccine. It is mentioned that the specific problem of transgene expression in plants like gene silencing, low expression level and altered glycolysation pattern must be overcomed for the efficiency of plant derived vaccines (Warcheza and Mason, 2003).
Discussion
Plant based vaccine holds considerable promise in future vaccination programmes. Plants have been proven as a virtually unlimited source of inexpensive vaccines for human and animal therapeutics. Plants are proved as ideal bioreactors for the production of vaccines and pharmaceuticals. Plant vaccines are far more advantageous and economical than fermentation technique. The production of vaccine in plants has been taken as a breakthrough for the delivery of vaccine to the people in developing and poor countries. Plant derived vaccine doesn’t need the cold chain process for its delivery and it can be administered without the need of trained health professionals. These vaccines and pharmaceuticals can be stored and distributed as seeds, tubers, or fruits. This makes vaccination cheaper and easier to administer. Therefore, using plant derived vaccine, diseases can be prevented or cured through consumption of food. Another major scope of plant derived vaccine is the vaccination of farmed and wild animals. Since, plant derived vaccines are cheap, edible and with a rapid scale up, it has the potential of protecting large number of animals from infectious diseases. Plant vaccine is a new technology and various challenges are still to be overcomed. So there is a need of adequate regulation, constant monitoring and research for the safe production of vaccines in plants. The use of genetically modified plants should be given utmost emphasis and also developed countries should be supportive to the developing countries for vaccine production in plants by providing advice, education, and monetary support.
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