TRANSGENIC PLANTS AS EDIBLE VACCINES

Vaccinations are among the more cost - effective health care procedures. In the United States and Europe, a majority of newborn children are vaccinated against ten diseases (Recommended childhood immunization schedule from Centre for Disease Control, USA). Recently in the United States hepatitis B was added to the recommended vaccinations for infants. Of these different immunizations, only one is an oral vaccine with the others requiring injections. In addition, approximately thirty new vaccines are currently under development in the United States. In contrast to Western countries, vaccine are in limited use in many developing countries, with children receiving the "Expended Program on Immunization" (EPI) vaccine against six diseases.  

These are recommended, and in many countries financed, by the World Health Organization. The cost of vaccines is one factor preventing further use of vaccination, leaving hundreds of thousands of children susceptible to preventable diseases. The principle costs of most commercial vaccines are production, packaging and delivery. Injectable vaccines incur further expenses related to the use and disposal of needles and syringes, trained personnel to administer injections, and refrigeration required during shipping and storage.  

These same economic factors prevent widespread vaccination of livestock, poultry and swine against preventable diseases. The Children's Vaccine Initiative called for new technologies to make vaccines more widely available. This includes low cost production systems and to further develop oral vaccines. Oral vaccines are desirable due to their ease of administration and patients acceptance of non-injected vaccinations. Another advantage is that oral vaccine may stimulate production of mucosal antibodies more effectively then injected vaccines. This is important as the mucosal immune system is a first line of defence against many disease organisms.  

Vaccines are designed to elicit an immune response without causing disease. Typical vaccines are composed of killed or attenuated disease-causing organisms. Recombinant subunit vaccines are desirable as an alternative with potentially fewer side effects than delivering the whole organism. Recombinant subunit vaccines do not contain an infectious agent, and thus are safer to administer and prepare, and doses are more uniform. Advances in molecular biology of diseases have identified many candidate proteins or peptides that may function as effective subunit vaccines.  

Recombinant vaccines have potential for being highly effective in preventing disease, both in humans and animals, but are rather costly to produce, and therefore are in limited use worldwide. The choice of which system to use to produce a recombinant vaccine must take into consideration their advantages and disadvantage, costs of production, and the amount of product required on a global scale. For some vaccine antigens, transgenic plants may provide an ideal expression system, in which transgenic plant material can be fed directly to subject as their oral dose of recombinant vaccine.

Transgenic plants that express foreign proteins with industrial or pharmaceutical value represent an economical alternative to fermentation-based production systems. Specific vaccines have been produced in plants as a result of the transient or stable expression of foreign genes. It has recently been shown that genes encoding antigens of bacterial and viral pathogens can be expressed in plants in a form in which they retain native immunogenic properties. Transgenic potato tubers expressing a bacterial antigen stimulated humoral and mucosal immune responses when they were provided as food. These results provide ‘proof of concept’ for the use of plants as a vehicle to produce vaccines. 

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, causing an immunological response in mice that have eaten these plants. 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. 

Oral Vaccine Using A Plant System

Most vaccine-developing technologies of remixing genes use revealed systems of animal cells and a great deal of expenses to produce the vaccine are required according to certain conditions of the cultivation of animal cells. Also, a disease virus is various and there are many variants so we cannot develop vaccine without paying a great deal of expenses to research and develop such vaccine. But the workers had already kept producing technology of vaccine including transgenic plant, transgenic vector production, and control of genetic revelation, separation and refinement of remixing protein analysis of transgenic plants, producing an antibody and separating proteins, and examination technology of the cause of an animal immunity and others. We can produce a great deal of safe vaccine from vegetable cells at a low price. The technology of producing vaccine using vegetable systems has many good points and has attracted public attention as the technology of developing effective vaccine a (covering the introduction of producing oral vaccine from plants). 

But there have not nearly been the products using the technology of producing oral vaccine from plants until now and the vaccine is likely to commercialize this field without limit in the future. 

If our technology could secure immunity induced by oral medication, the prevention and cure for a disease will be simple without any inoculation and will be developed into effective programme of vaccine. Besides, the workers are carrying a study about the development of plants producing a compound vaccine against different situations between diseases.

Advantages Of Plant System In The Development Of Oral Vaccines  

• Edible plants are very effective as a delivery vehicle for inducing oral immunization
• Adjuvant for immune response is not necessary
• Excellent safety and economic 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
• Edible means of administration
• 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
• Seroconversion in the presence of maternal antibodies
• Generation of  systemic and mucosal immunity
• Enhanced compliance (especially in children)
• Delivery of multiple antigens
• Integration with other vaccine approaches

Protocol Of Vaccine Production In Plants

• Appropriate plant viruses are genetically engineered to express the desired peptides or proteins. Plant viruses currently being utilized are cowpea mosaic virus, potato virus X, tomato bushy stunt virus, and tobacco mosaic virus.
• Typically, two leaves of a plant are inoculated with the engineered virus.
• The plant grows new leaves.
• The new leaves are harvested and virus particles are extracted in a process that includes grinding the leaves, centrifuging the raw extract, and precipitation with polyethylene glycol.
• The virus particles are used to infect new plants.
• Large numbers of new plants are grown.
• Chimeric virions are extracted and purified.

The newest technique being developed to permit plant production of entire proteins, is termed “overcoat” technology. 

This still-experimental method uses the rod-shaped potato virus X PVX. PVX has an RNA core surrounded by a single coat protein. 

“The 25 kDa coat protein of PVX was fused, through a special linker peptide of 16-18 amino acids, to a high-molecular-weight foreign reporter protein - the green fluorescent protein (GFP) from a jellyfish, Aequorea victoria.” 

“Serendipitously, the 55 kDa fusion protein could still assemble to form PVX particles which were not only longer (because of the added 900 nucleotides of GFP - coding RNA) but also twice as wide as normal PVX.” 

Other foreign proteins - including large polypeptide epitopes from pathogens - can be fused to PVX coat proteins in similar overcoats. 

Further developed is “epicoat” technology for the expression of foreign peptides on the surfaces of icosahedral plant viruses. 

This technique uses the cowpea mosaic virus (CPMV). This RNA virus has a 28-30 nm diameter and consists of two RNA strands, one coding for regulatory proteins and one coding for structural proteins. 

By linking foreign genes to a specific site in the sequence, 60 copies of the desired peptides are expressed on peaks dotting the surface of the virus particle. 

“The largest polypeptide which has been expressed so far using the epicoat is 36 amino acids in length,” Rodgers noted. 

By weight, the foreign peptide represents 3 percent of the chimeric CPMV particle. 

To test the immunogenicity of antigen-expressing CPMV, Below is described an experiment in which CPMV was used to express an epitope from the HIV-1 [IIIB] gp41 transmembrane glycoprotein. 

Mice inoculated with 100 (micro)g of chimeric CPMV (containing 3 (micro)g of the HIV epitope) with alum adjuvant developed a strong neutralizing antibody response. These  antibodies also recognized heterologous gp41 from the RF (70 percent neutralization) and SF2 (60 percent neutralization) strains of HIV-1. 

In another experiment, the CPMV system was used to express the 17-amino-acid MEV epitope of canine parvovirus (CPV). 

Mink were inoculated with either 100 (micro)g (low dose) or 1 mg (high dose) of chimeric CPMV. While all six untreated control animals died within seven days of oronasal challenge with virulent CPV, none of the 12 vaccinated animals developed symptoms. 

Four of the six animals in the low-dose group became infected as indicated by viral shedding, but none developed symptoms. Only transient viral shedding was seen in two of the six animals in the high-dose group. 

Wide Range Of Epitopes Have Been Expressed In Cpmv Chimeric Virus Particles. These Include:

• Viral epitopes from HIV-1 gp41, HIV-1 gp120, human rhinovirus, foot and mouth disease virus, and canine parvovirus.
• Bacterial epitopes from Staphylococcus aureus up to 33 amino acids in length.
• Mammalian epitopes from hormones and from colon-cancer cells.
• Fungal epitopes.
• Protozoan epitopes from Plasmodium falciparum.

Further recommending the chimeric virus particles (CVPs) are their stability : 

• Genetic stability, as demonstrated after 20 passages in culture of CVP-HIV-1.
• Thermal stability : the thermal inactivation point of wild-type CPMV is 65 (degree)C.
• Acid stability, as demonstrated with CVP-HIV-1 culture at pH 1 for one hour at 37 (degree)C.
• Protease stability, as demonstrated with CVP-HIV-1 culture with pepsin at pH2 for one hour at 37ºC.

“The carrier particles are non-infectious to mammals, and contamination by adventitious agents is not a major concern for a plant-based production process. A wide range of vaccine administration routes is available: parenteral and nasal (purified particles), oral (formulated crude leaf extracts), and edible (whole or homogenized) leaves, fruits, or vegetable tissues.” 

Epicoat and overcoat techniques represent a platform technology for his firm.

   Dr Charles Arntzen

The Fruit Delivery System: A New Way To Vaccinate  

Vaccines produced in raw foods such as bananas may be a cost-effective alternative for controlling important diseases in developing countries, said a prominent plant sciences researcher speaking recently at the Arizona National University. 

“Big companies haven't invested the money that’s necessary to develop vaccines where the primary market is the third world, as the third world doesn’t have the resources to pay for what would be (by traditional techniques) very costly vaccines,” said Dr Charles Arntzen, President of the Boyce Thompson Institute for Plant Research in New York. 

“We're developing a very simple system of using plants as a manufacturing system for vaccines.” 

Dr Arntzen described the Institute's innovative research at a recent ANU 50th Anniversary Lecture sponsored by the ANU's Research School of Biological Sciences (RSBS). The lecture was also a plenary lecture for the combined conference of the Australian Society for Biochemistry and Molecular Biology and the Australian Society of Plant Physiologists, held at the National Convention Centre. 

The Boyce Thompson Institute, a not-for-profit foundation affiliated with Cornell University, is funded by a private endowment and hence free to promote the study of problems which might not otherwise be a priority. 

Five years ago work commenced on a project to determine whether plants were suitable subunit vaccine factories. Normally to make a subunit vaccine, the gene for one of the proteins of infectious agents, such as a bacteria or viruses, is produced in another system. Complicated and costly technology is needed to produce the protein in large quantities and purify it for use as a vaccine. 

Dr. Arntzen's overall plan is to put the gene into plant cells and use plant tissue to produce the protein. Then, instead of purification, the plant tissue, such as the fruit of a banana, would be given to the infant or adult orally. 

The proteins targeted for use in subunit vaccines are antigens, which may be thought of as the molecular signature of the pathogen. In viruses, antigens are usually proteins which appear on the surface of the virus and are well recognised by the immune system. If humans are exposed to an antigen, they recognise it as being a foreign molecule and develop an immune response against it, so, if exposed to the real bacteria or virus, their system can mount an effective and protective defence. 

The researchers are working on antigens from the enterotoxic. E. coli bacterium and the Norwalk virus, both of which are important causes of diarrhoea in developing countries.

“We chose proteins for which there is a lot of previous research showing their potential value as vaccines,” said Dr. Arntzen. 

The first step was to introduce the genes for the antigens into a test system, and the system of choice was the potato. It is easy to introduce foreign genes into potatoes and generate a lot of plant material in a comparatively short time. After finding that the antigens were produced in the tissue of the potato, the researchers turned to the banana.  

“We chose bananas for the three reasons,” he said,  

1. It's a crop that's grown throughout the developing world, in sub-tropics and even temperate areas in Asia, India, Africa and Latin America.
2. It's a food that's eaten uncooked, which is very important so that the protein we put in is not destroyed by cooking.
3. It's eaten by infants and children it's often the first food that mothers give to an infant.

In collaboration with scientists at the Queensland University of Technology, a method for transferring genes into the banana plant was developed from scratch. The efficiency of this system is currently being optimised.  

Another important area is to identify DNA signals in the banana which control when and where a gene is switched on, called promoters. For the vaccine antigens to be produced in the fruit of the banana at the right stage in the growth cycle, they must be linked up with suitable promoters.

“We now have our first plants back, little seedlings about three metres tall that do contain the gene for our first bacterial diarrhoea vaccine,” he said.  

The plant material now must be subdivided then grown to maturity in the greenhouse, taking a minimum of two years before fruit is produced. If an adequate amount of the protein is found in the fruit, testing can begin in animal followed by clinical trials in humans. 

The researchers have been encouraged by the finding that in test systems, mice develop an immune response to antigens given to them orally in potato tissue. 

“It shows up as serum antibodies in the blood and secretory antibodies which are excreted into the intestine.  

“An important part of this work is to try and establish a network so that we can transfer this technology to the developing world. I think we've seen a lot of cases where the science is taken to some endpoint then we just try and give it to a developing country and it's either not accepted or slowly accepted. Our strategy is to get individuals from the developing world involved very early on.” 

A scientist from Mexico, where diarrhoeal disease is the primary cause of infant mortality, has been funded by the Rockefeller Foundation to learn the technology for turning bananas into vaccine factories and take it back to Mexico. 

Dr Arntzen expects the project to expand in other areas and is looking for collaborators in Australia to move the work into South East Asia. 

The plant system of producing vaccines may be suitable for a number of other important diseases, such as hepatitis B, C and E and even AIDS. 

“There're over a billion dollars being spent each year on HIV research in the States and a lot of it focused on vaccines. When that finally pays off and someone gets an appropriate antigen, they're going to need a very inexpensive delivery system if they're going to provide that vaccine throughout central Africa and South East Asia. Our delivery system could be very useful for that,” said Dr Arntzen. 

Other candidates for use of edible plant vaccination are diseases of pets, production animals and wild animals. Rabies is already controlled in Europe by doping food bait with a rabies vaccine, so this would be a prime target for a low cost plant vaccine . 

Cost is also a key factor in the swine and poultry industries, where livestock have to be individually handled for vaccination against various respiratory and enteric diseases. A food-based vaccine would reduce stress to the animals and handling costs. 

“Every vaccine that is developed will need testing to make sure that the antigen will be appropriately recognised by the immune system, but results so far show great promise,” he said.