FORMULATION: Vaccines

Taking the Bite Out of Injections

Edible Vaccines Offer Alternatives to Conventional Immunization

ABSTRACT

Vaccines are primary tools in programs for health intervention for both humans and animals, and vaccines would perhaps be more widely used, especially in developing countries, if cost of production could be reduced and distribution could be accomplished without refrigeration.

Research underway is dedicated to solving these limitations by finding ways to produce edible (oral) vaccines in transgenic plants. A variety of plants have now been used as hosts for vaccines, still at the experimental stage, including tobacco, potatoes, carrots, rice and tomatoes. The main goal of an oral vaccine is the induction of a mucosal immune response and a subsequent systemic immune response. By stimulating a strong mucosal immune response, edible vaccines may provide effective immunity against pathogens that invade both mucosal and systemic sites.

Immunization studies with plant-derived, sub-unit vaccines have demonstrated that plants are able to produce recombinant proteins that retain antigenic and immunogenic properties. Although many issues and concerns still remain regarding the safety and efficacy of edible vaccines in humans, current data indicate that edible vaccines are indeed a promising option for the future.

Introduction

Vaccination is one of the most important and cost-effective methods of preventing infectious diseases of animals. A vaccine is an immunological substance designed to confer specific protection against a disease. Edible vaccines are sub-unit vaccines where the selected genes are introduced into the plants and the transgenic plant is then induced to manufacture the encoded protein. It is mucosal-targeted vaccine where stimulation of both systematic and mucosal immune network takes place. It stimulates the immune system both humoral and cell mediated to generate specific protection against an infectious agent. Vaccine may be prepared from live modified organisms, inactivated or killed organisms, toxoids or combinations thereof.

There are two types of immunity, active immunity and passive immunity, and vaccines produce active immunity. A concern with oral vaccines is the degradation of protein components in the stomach (due to lowpH and gastric enzymes) and gut before they can elicit immune responses, but the rigid plant cell walls could provide protection from intestinal degradation. The degradation can be compensated by repeating the exposure of the antigen until immunological tolerance is accomplished. The value of new or replacement vaccines produced in plant cells and delivered orally must be considered alongside the probability and severity of potential risks in their production and use, and the cost of not deploying this technology-the risk of continuing with the status quo alternative. The process of using DNA vaccines to prevent or slow down the spread of disease is known as polynucleotide immunization. DNA that is injected into the subject undergoes the transcription and translations that yield protein, making the specific T and B cells to differentiate and proliferate. With vaccination, therefore, the immune system is ready to combat invading pathogens very quickly, greatly minimizing the chance of spreading throughout the body while causing discomfort to the host. Food vaccines are like sub-unit preparations in that they are engineered to contain antigens but bear no genes that would enable whole pathogens to form. These vaccines basically work in the same way as the injected DNA vaccine, since a peptide sequence similar to an infectious part of a pathogen is synthesized, by itself, and is used to prime T and B cells in the body.

ADVANTAGES OF EDIBLE VACCINES

• Plant derived antigens assemble spontaneously into oligomers and into virus like particles.

• Multiple vaccinations per plant are possible.

• Stimulates mucosal immunity.

• More effective against pathogens that invade mucosal surfaces.

• Administration is safe and painless.

• Cost could be very low.

• Therapeutic proteins are free of pathogens and toxins.

• Requires no special facilities for storage and transportation.

DISADVANTAGES OF EDIBLE VACCINES

• Certain foods like potato are not eaten raw, and cooking the food might weaken the medicine incorporated into it.

• Stability of the vaccines in the fruits is not known.

• Survival in the gut is difficult to predict.

• Evaluating dosage requirement is tedious.

• Selection of the best food plant is difficult.

• Cost of plant multiple transgenesis is yet to be known.

Developing a Sub-Unit Plant Vaccine

Initially, sub-unit vaccines were produced by purifying the specific antigens from cultures of the pathogenic bacteria or viruses. However, this required large-scale production facilities and costly downstream processing procedures. While the sub-unit antigen is free of toxins and immunosuppressive components associated with the pathogen, there is a significant risk during production, especially with Level III organisms, due to the potential of accidental release or escape to the external environment and subsequent transmission. It is for these reasons that native-organism, purified sub-unit vaccines are not often economically viable for use in veterinary vaccines except as crude preparations.

Molecular biology and genetic engineering have had an enormous impact on vaccine development by providing the tools and techniques to produce a single protein in a prokaryotic or eukaryotic system. Furthermore, if the protein is produced in prokaryotic systems, it can be tailored in such a way that the protein of interest is expressed on the surface of the bacteria, in the periplasm, as insoluble inclusion bodies or secreted in the media.

The recombinant approach to sub-unit vaccines is to clone the gene that encodes the protective-antigen into a secondary, preferably non-pathogenic organism that is capable of expressing the immunogen in its native form or with minimal alteration. This protein can then be expressed and harvested using traditional bacterial antigen production methods, or delivered by a live non-pathogenic vector. Recombinant subunit vaccines eliminate the risks associated with handling a pathogenic organism, and the risks associated with live or killed products reverting to a pathogenic state due to incomplete inactivation or attenuation. Plant-derived antigenic proteins have delayed or prevented the onset of disease in animals and have proven to be safe and efficacious in human clinical trials.

Administration of a Plant-Based Vaccine

The original concept of an "edible" vaccine was, for the most part, naive. The vaccine product that is envisioned now is a powdered formulation that is pressed into a paste for oral administration. Powdered formulation can be produced from seeds or freeze-dried fruits and leaves using standard inexpensive food industry by milling processing technique. Large-scale batches of powered plant material can be tested to ensure consistency of vaccine dose and adequate quality control. This material may also be supplemented with mucosal adjuvant, vitamins and other vaccines thereby maximizing the use of infrastructure and reducing the number of contacts necessary with remote communities. For countries that do not wish to grow genetically modified crops, this provides the ability to supply the vaccine as an acceptable compromise.

Therapeutic Applications for Edible Vaccines: Enterotoxigenic E. coli and vibrio cholerae vaccine

Toxins produced by E. coli and V. cholerae can cause acute diarrhea. Infection by these microorganisms is common in places where there is poor sanitation and untreated water supplies. If allowed to go untreated, there are fatal consequences of exposure to these microorganisms, especially in children. The synthetic genes of E.coli heat-labile enterotoxin B sub-unit (LTB) and cholera toxin B sub-unit (CTB) are related proteins with similar structure, function and immunochemistry to the actual toxins found in each species. The production of these recombinant proteins in yeast or bacteria is expensive compared to the production of a recombinant potato that produces modified LTB. It has been shown that mice which eat the transgenic potato raise antibodies in response to the potato LTB that were effective in inhibiting LT activity on mammalian cells. Synthetic LTB coding sequence (sLTB) was modified for cloning in plants. The potato plant cells were transformed by leaf disc co cultivation. The transformants were selected and are regenerated as plantlets on a selective media. Tubers from a mature plant were used as seed for the next production cycle. Later, the mice were fed with transformed potato and developed a resistance to the pathogen. The transgenic potato is useful as a vaccine component or as a booster vaccine.

Hepatitis B Vaccine

It is estimated that more than 400 million people around the globe are infected with the Hepatitis B virus, making it one of the most common human pathogens. Since immunization is the only known method to prevent the disease of the Hepatitis B virus, any attempt to reduce its infection requires the availability of large quantities of vaccine, Hepatitis B surface antigen (HbsAg). The HbsAg subtype ayw was cloned into CaMv plasmid and the regenerated plants from the transformed cells were shown to produce HbsAg. Furthermore, expression of the antigen was found to be higher in roots of the transgenic potato than in leaf tissues. However the expression of HbsAg in transgenic potatoes is not sufficient for using as oral vaccine. Further studies are underway to increase the level of the HbsAg by using different promoters such as the patatin promoter, and different transcription regulating elements.

Measles Vaccine

Measles is a highly contagious viral disease caused by airborne Paramyxovirus and includes such symptoms as high fever, skin rash and spots and it can lead to many different complications, which can be even more severe than the disease itself. Each year, almost one million children die from the measles and many of the survivors are weakened by pneumonia or encephalitis and suffer such permanent disabilities as deafness.

Recent studies report expression of the Paramyxovirus surface protein hemagglutinin in tobacco, potato, rice and lettuce with satisfying results. Serum samples from healthy experimental animals, fed with transgenic banana, were analyzed for the presence of anti-hemagglutinin-specific antibodies. The results are highly significant and demonstrate that the banana plant can produce the antigenic hemagglutinin protein of the measles virus and elicit immune responses in experimental animals.

Cholera Vaccine

Cholera is one of the leading causes of death among children in developing countries. Clean water and hygienic conditions have effectively controlled this in the industrialized countries, but many poor countries have a long way to go before their sensitization infrastructure can be improved. Since Cholera is effectively controlled through oral immunization, edible vaccine may provide exactly such a tool. Food plant-based vaccines integrate vaccine production and delivery systems into one product and they are safe, nutritious and easy to administration.

Malaria Vaccine

Malaria remains one of the most significant causes of human morbidity and mortality worldwide, with 300 to 500 million new cases of infection annually resulting in 1.5 to 2.7 million deaths. The world malaria situation has become significantly worse in recent years as the main forms of malaria control, spraying programs and chemotherapy, become less effective due to the development of vector and parasite resistance. The capacity of transgenic plants to produce one or several antigens at low cost, and the ease of oral delivery makes a plant-based malaria vaccine particularly attractive. Three antigens are currently being investigated for the development of a plant-based malaria vaccine, merozoite surface protein (MSP) 4 and MSP5 from Plasmodium falciparum, and MSP4/5 from P. yoelii. Studies have shown that oral vaccination with 25mg of recombinant (E. coli) MSP4/5 can protect mice against lethal challenge. Preliminary results indicate that tobacco plants can make MSP4/5, however expression levels were low.

Edible Vaccine for Diabetes

There are 16 million people with diabetes in the U.S. alone, and more than 100 million afflicted worldwide. Plants can be bio-engineered to fight this silent killer. Insulin dependent diabetes mellitus (IDDM) affects children and young adults. It is an auto-immunize disease, for the pancreatic beta cells that produce insulin are destroyed by the immune system. As a result, glucose builds up in the blood, it overflows into urine and, thus, the body loses its main source of fuel. Research shows that diabetes can be prevented in mice by feeding them with plants engineered to produce diabetes related protein. The pancreatic protein, glutamic acid decarboxylase (GAD67), is linked to the onset of IDDM, and when injected into mice it is known to prevent diabetes. The researchers developed transgenic potato and tobacco plants with a gene for GAD67, which is easy and inexpensive to produce. Plants can be fed directly, without cooking, to non-obese diabetes mice, which develop insulin dependent diabetes spontaneously. As a result, mice showed increased levels of IgG1, an antibody associated with cytokines that suppress harmful immune response and prevent diabetes in an animal model. The vaccine may even lead to a cure for diseases like multiple sclerosis and rheumatoid arthritis.

Food Poisoning Vaccine

Researchers say they're making strides toward developing a number of vaccines that may offer protection against some of the most common causes of foodborne illness. Preliminary tests of two new experimental vaccines show they're effective in protecting against infection with several types of dangerous bacteria found in food, including:

• Salmonella: A bacteria often found in raw poultry and eggs;

• Listeria: A bacteria often found in improperly pasteurized milk or cheese;

• E. coli: A potentially deadly bacteria

spread through contact with human or animal feces or contaminated water or food. Most illness has been associated with eating undercooked, contaminated ground beef.

Allergy Vaccine

An Australian microbiologist may have found a new way to vaccinate against asthma, and there will be no needles, just open and swallow. Dr. Simon Hogan from the ANU in Canberra is part of the brave new world of genetically-modified plants being designed not for food but as plants that can deliver a vaccine. His remarkable finding in experiments with asthmatic mice reveal that it's possible to introduce an allergen to their stomach, which creates an immune reaction, activating cells against the allergen. When their lungs were exposed to the same allergen, the previous immune response cells in the stomach rushed to the lungs via the bloodstream, protecting the mice from sensitization, thus preventing asthma. One day, these plants could provide a cheap, sustainable and easily accessible vaccination not only for asthma but many of the diseases, like plague, in the developing world.

Stopping Autoimmunity

In the past 15 years, investigators have identified several beta cell proteins that can elicit autoimmunity in people predisposed to Type I diabetes. The development of plant based diabetes vaccines in potatoes and tobacco containing insulin linked to the innocuous B sub-unit of the V. cholerae toxin (to enhance uptake of the antigens by M cells) was attempted. The developed transgenic potato and tobacco plants when fed to non-obese diabetic mice showed increased levels of IgG1, an antibody associated with cytokines that suppress harmful immune responses. "Molecular Pharming" to produce auto antigens in plants targeting other autoimmune diseases such as multiple sclerosis, rheumatoid arthritis, systemic lupus and even transplant rejection is underway.

Limitations of Edible Vaccines

Even though edible vaccines are stable and easily accessible there are some limitations, which restrict its development. For example, one could develop immunotolerance to the vaccine peptide or protein. Little research has been done on this. Another concern with whole fruit or vegetable vaccines is the consistency of dosage from fruit-to-fruit, plant-to-plant and generation-to-generation. Another limitation is storage of edible vaccines. Potatoes containing vaccine proteins seem to store well at 40^o C, but tomatoes will not last very long. Using potatoes or bananas may require some processing such as smashing and a liquoting as in baby food jars of bananas. Since these fruits are being used as vectors for the vaccines in question, they have to be properly stored to avoid infection or disease through spoilage. Other concerns are transgene escape and identification of "vaccine" fruit against a normal fruit. Fruit vaccines should be easily identifiable to avoid the misadministration of the vaccine, which may lead to complications such as immunotolerance.

Safety of an Edible Vaccine

Conventional sub-unit vaccines have not been associated with oral tolerance. However, repeated exposure to an oral antigen has the potential to produce immunological tolerance. The induction of oral tolerance is both time-dependent and dose-dependent. The antigen dose necessary to induce protection is generally smaller than required to produce tolerance. In addition, repeated or continuous exposure is usually necessary to induce tolerance. Expression of vaccines in commonly consumed foods does not mean that they will become a component of regular diets. As medicines, edible vaccines should be administered appropriately. In this setting, we believe it is unlikely that an edible vaccine would lead to oral tolerance. However, this remains an area of vaccine development that needs to be closely monitored.

The Future of Edible Vaccines

Vaccines have been one of the most far-reaching and important public health initiatives. Advancing technology, such as oral DNA vaccines, intranasal delivery and edible plant-derived vaccines, may lead to a future of safer and more effective immunization. Edible vaccines, in particular, might overcome some of the difficulties of production, distribution and delivery associated with traditional vaccines. Significant challenges are still to be overcome before vaccine crops can become a reality. However, while access to essential healthcare remains limited in much of the world and the scientific community is struggling with complex diseases such as HIV and malaria, plant derived vaccines represent an appetizing prospect.

Edible vaccines are superior means of antigen administration as compared to synthetic carriers from a technical, economical and safety point of view. Oral administration produces a more convenient way to induce systemic and mucosal immune response and this would help in eradication of infectious diseases. �

For references to this article go to www.pharmaquality.com and click on current issue.

Swamy Krishna Tripurani, N. S. Reddy and K. R. S. Sambasiva Rao, of the Centre for Biotechnology, Nagarjuna University (Nagarjuna Nagar, Guntur-522510, A.P., India), can be reached through K. R. S. Sambasiva Rao at 91-863-2293400