FUTURE PROSPECTS

A.    Humanized  Monoclonal Antibody Produced  In Transgenic Plants For Immuno-Protection Of The Vagina Against Genital   Herpes 

The ability to produce monoclonal antibodies (Mabs) in plants offers the opportunity for the development of an inexpensive method of mucosol immuno protection against sexually transmitted diseases. To investigate the suitability of plant expressed Mabs for vaginal preventive applications, workers had compared a humanised anti herpes simplex virus 2 (HSV-2) Mab expressed in mammalian cell culture with the same antibody expressed in soyabean. They found these Mabs to be similar in their stability in human semen and cervical mucus over 24 hr., and their efficacy for prevention of vaginal HSV-2 infection in the mouse. 

B.     Immunogenicity In Humans Of A  Recombinant Bacterial Antigen Delivered In A Transgenic Potato  

Compared with vaccine delivery by injection, oral vaccines offer the hope of more convenient immunization strategies and a more practical means of implementing universal vaccination programs throughout the world. Oral vaccines act by stimulating the immune system at effector sites (lymphoid tissue) located in the gut. Genetic engineering has been used with variable success to design living and non-living systems as a means to deliver antigens to these sites and to stimulate a desired immune response. More recently, plant biotechnology techniques have been used to create plants which contain a gene derived from a human pathogen; the resultant plant tissues will accumulate an antigenic protein encoded by the foreign DNA. In pre-clinical trials, workers found that antigenic proteins produced in transgenic plants retained immunogenic properties when purified; if injected into mice the antigen caused production of protein-specific antibodies. Moreover, in some experiments, if the plant tissues were simply fed to mice, a mucosal immune response occurred. The present study was conducted as a proof of principle to determine if humans would also develop a serum and/or mucosal immune response to an antigen delivered in an uncooked foodstuff. 

C.     Efficacy Of A Food Plant-Based Oral Cholera  Toxin B Subunit Vaccine 

Transgenic potatoes were engineered to synthesize a cholera toxin B subunit (CTB) pentamer with affinity for G sub(M1)- ganglioside. Both serum and intenstinal CTB-specific antibodies were induced in orally immunized mice. Mucosal antibody titers declined gradually after the last immunization but were restored following an oral booster of transgenic potato. The cytopathic effect of cholera holotoxin (CT) on Vero cells was neutralized by serum from mice immunized with transgenic potato tissues. Following intraileal injection with CT, the plant-immunized mice showed up to a 60% reduction in diarrheal fluid accumulation in the small intestine. Protection against CT was based on inhibition of enterotoxin binding to the cell-surface receptor G sub(M1) - ganglioside. These results demonstrate the ability of transgenic food plants to generate protective immunity in mice against a bacterial enterotoxin.

D.   Expression Of The Rabies Virus Glycoprotein In Transgenic Tomatoes 

Researchers have engineered tomato plants (Lycopersicon esculentum Mill var. UC82b) to express a gene for the glycoprotein (G-protein), which coats the outer surface 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. Plants were transformed by Agrobacterium tumefaciens-medicated transformation of cotyledons and tissue culture on selective media. PCR confirmed the presence of the G-protein gene in plants surviving selection. Northern blot analysis indicated that RNA of the appropriate molecular weight was produced in both leaves and fruit of the transgenic plants. The recombinant G-protein was immunoprecipitated and detected by Western blot from leaves and fruit using different antisera. The G-protein expressed in tomato appeared as two distinct bands with apparent molecular mass of 62 and 60 kDa as compared to the 66 kDa observed for G-protein from virus grown in BHK cells. Electron microscopy of leaf tissue using immunogold-labeling and antisera specific for rabies G-protein showed localization of the G-protein to the Golgi bodies, vesicles, plasmalemma and cell walls of vascular parenchyma cells. In light of  previous demonstration that orally administered rabies G-protein from the same ERA strain elicits protective immunity in animals, these transgenic plants should provide a valuable tool for the development of edible oral vaccines. 

E.     Oral Immunisation Of Native And Primed Animals  With Transgenic Potato Tubers Expressing Lt-B 

The efficacy of edible vaccines produced in potato tubers was examined in mice. Transgenic plants were developed by Agrobacterium tumefaciens-medicated transformation. The antigen selected was the non-toxic B subunit of the Escherichia coli enterotoxin (recLT-B). A synthetic gene coding for recLT-B was made and optimised for expression in potato tubers and accumulation in the endoplasmic reticulum. 

Introduction of this gene under control of the tuber-specific patatin promotor in potato plants resulted in the production of functional, i.e. Gm1-binding, recLT-B pentamers in tubers. Selected tubers containing about 13 µg of recLT-B per gram fresh weight were used for immunisation. Subcutaneous immunisation with an extract of recLT-B tubers yielded high antibody titres in serum that were similar to those obtained with bacterial recLT-B. The efficacy of oral administration of recLT-B tubers was determined by measuring mucosal and systemic immune responses in naive and primed mice. Animals were primed by subcutaneous injection of an extract of recLT-B tuber plus adjuvant. Naive and primed mice were fed 5 g of tubers ( ~ 65 µg of recLT-B) or were intubated intragastrically with 0.4 ml of tuber extract (~2 µg of recLT-B). In naive mice, feeding recLT-B tubers or intubation of tuber extract did not induce detectable anti-LT antibody titres. In primed animals, however, oral immunisation resulted in significant anti-LT lgA antibody responses in serum and faeces. Intragastric intubation of tuber extract revealed higher responses than feeding of tubers. 

F.    Genomic Tools For Gene And Protein Discovery In Malaria : Toward New Vaccines

Advances in malaria vaccine and drug development have been hindered in part by the complex multistage life cycle of the parasite, much of which is inaccessible to study, and by a large genome encoding over 5000 genes. Two human models of immunity to malaria, however, suggest that the development of an effective vaccine is within reach. Scientists have outlined a strategy to identify the expression of hundreds to thousands of potential vaccine targets employing recently developed technologies for gene and protein expression. Combined with the exciting developments of malaria DNA vaccine technologies, these approaches form the basis for malaria subunit vaccines that may mimic the protective efficacy of our human model systems and provide the foundation for novel approaches to vaccine development for a range of pathogens. 

G.    Plants Expressing Human Papillomavirus Proteins For Use As Edible/Oral Vaccines 

Most edible vaccines are for gut pathogens. The question was raised as to whether the HPV VLPs would be stable enough to induce an immune response if inoculated via intragastric gavage. The following experiment was done in collaboration with Dr Bob Rose (Rochester University, NY), to investigate this possibility. Human papillomavirus type 11 (HPV-11) recombinant virus-like particles (VLPs) were produced in insect cells and evaluated for oral immunogenicity in BALB/c mice. When tested in an enzyme-linked immunosorbent assay (ELISA), sera from immunized animals demonstrated HPV-11 VLP-specific immunoglobulin G (IgG) and IgA responses that were dose-dependent, conformationally dependent, and genotype-restricted. Moreover, when tested in a newly developed VLP binding inhibition (VBI) enzyme-linked immunosorbent assay (ELISA), orally induced VLP antibodies were found to efficiently inhibit VLP binding by rabbit HPV-11 virus-neutralizing polyclonal antibodies. These results suggest that VLPs may be effective oral immunogens for the prevention of anogenital HPV disease. 

H.    A Candidate Vaccine for Anthrax 

Dr. Henry Daniell a UCF professor and biomolecular researcher, has created a potential vaccine for anthrax using a tobacco plant. Daniell explains, “The genetic makeup of the tobacco plant makes it ideal to produce many kinds of proteins needed to make medicine.” 

Daniell’s team uses a gene gun to bombard tobacco leaves with the gene for anthrax protective antigen, called pag, obtained from the National Institutes of Health. Pag is eventually integrated into the tobacco genome. “The material produced is completely free of the lethal factor found in the commercial vaccine,” Daniell said. 

The anthrax toxin is made up of three components: a break-in protein called protective antigen (so called because it seeks to “break in” to cells), the edema factor and the lethal factor. Either the edema factor (PA-EF) or the lethal factor (PA-LF) binds with the protective antigen. PA-EF causes swelling and PA-LF causes the hyper-inflammatory reaction that can lead to shock and death. 

The problems with the current vaccine lie in the nature of the anthrax bacterium, Daniell said. The current vaccine filters the actual Bacillus anthracis bacterium to obtain the protective antigen. Some of the edema and lethal factors can get through and this is what can cause toxic side effects. This vaccine, designed in the 1950s and reformulated in the late 1960s, is now made by BioPort Corp., of Lansing, Michigan, the only company licensed by the U.S. Food and Drug Administration to make an anthrax vaccine. “Our protein is exactly the same structurally as the major protein in the existing vaccine,” Daniell said. “We have sent samples to NIH so they can determine if our protein is functionally the same as well.” 

Daniell’s tobacco plant process can produce billions of units of the anthrax antigen. Daniell said, “My team and I have created a safe candidature anthrax vaccine that can be produced in large quantities.” Daniell is also working on a more user-friendly anthrax vaccine, putting the same protective antigen in tomato plants. “This would be an edible vaccine that would allow a person to eat the tomato fruit instead of having to receive an injection,” he said.  

Proteins with applications for human or animal vaccines and expressed by transgenic plants: 

I.    Spinach Makes A Safer Anthrax Vaccine 

Spinach could provide an answer to the quest for a safer, purer anthrax vaccine, suggest US researchers. The plant could readily be used as a vehicle for the production of an edible vaccine against the infection, say immunologists at Thomas Jefferson University in Philadelphia. 

Efforts to develop a new vaccine have been stepped up since the current anthrax vaccine, which was licensed for human use in 1970, was recently deemed sub-optimal. 

Anthrax is caused by the spore-forming bacterium Bacillus anthracis, which exists as spores in the soil and, therefore, commonly affects grazing animals such as cattle and sheep. Human infection, although rare, occurs following direct skin contact with infected animals or their wool, hides or tissues, by ingestion of contaminated meat, or via inhalation of the spores. Left untreated, inhalation anthrax is almost always fatal and early intervention with antibiotics, such as ciprofloxacin, is essential. 

Vaccination is recommended for persons at risk of exposure to anthrax spores. The current vaccine is based on cell-free culture supernatants of an attenuated strain of B. anthracis adsorbed on aluminium hydroxide (in the USA) or precipitated with aluminium phosphate (in the UK) - aluminium acts as an adjuvant. It is incompletely characterized and difficult to standardize and, therefore, exhibits inconsistency between lots. It is also relatively reactogenic, with side effects including a possible link to Gulf War syndrome (whose symptoms include chronic fatigue, depression, skin rashes and gastrointestinal disorders), and requires a lengthy dosing schedule, all of which suggest the need for an improved, alternative vaccine. 

The main immunogenic component of the current vaccine has been determined to be the protective antigen (PA), and vaccination with PA alone can include protective immunity to anthrax. PA binds mammalian cell surface receptors, where it is proteolytically cleaved and activated to form a heptameric pore-like structure that binds either edema factor (EF) or lethal factor (LF) to form edema toxin and lethal toxin complex, PA facilitates the passage of the toxins into the host cell cytoplasm where they disrupt normal signaling pathways leading to cell lysis, toxic shock and, ultimately, death. However, "if you can block the very first stage - binding of the PA to the receptor - you block the mechanism - of-action of the toxin and essentially block the disease," said Alexander Karasev, assistant professor of microbiology and immunology at Thomas Jefferson University. 

"The new vaccines will be based on recombinant PA and will be much purer than the current vaccine," said  Meryl Nass, a Diplomat on the American Board of Internal Medicine, but "whether a pure PA vaccine will be safer is a big question." 

Stephen Leppla, senior investigator in the Microbial Pathoenesis Section at the National Institute of Allergy and Infectious Diseases, (NIH) concurs. "The concerns about the existing vaccine may well apply to protective antigen - based vaccines regardless of whether they are made in plants, bacteria, yeast or other systems. There is no a priori reason to suggest that plant-derived vaccines will produce fewer side effects." 

Nevertheless, many scientists believe that it is unlikely that  the PA protein itself is associated with adverse reactions. Furthermore, plant-based vaccines are appealing. "One of the beauties of the plant system is that there are no pathogens that infect both plants and animals," said Karasev, and without having to continuously screen the production medium for contaminants, screening costs are significantly reduced. 

Mohammed Azhar Aziz, senior research fellow at the Centre For Biotechnology, Jawaharlal Nehru University, India adds that the "production of subunit vaccines in plants offers the additional unique advantage of delivery in commonly consumed foodstuff, which may enhance the availability and ease of delivering immunizations." Aziz is part of a group of scientists that successfully integrated the PA gene into the nuclear genome of tobacco plants last year. 

Because there is some public opposition to the genetic modification of plants, and tobacco is considered an experimental plant, the scientists at Thomas Jefferson University designed a system to transiently express PA within a normal spinach plant, moving yet another step closer to an edible anthrax vaccine. 

Specifically, a fragment of PA that represents most of the receptor - binding domain was expressed as translational fusion with a capsid protein on the outer surface of tobacco mosaic virus, and spinach was inoculated with the recombinant virus particles. "One of the rationales for using just a fragment of the protein is that basically you don't need the whole protein to elicit a protective immune response," said Karasev, whose data were presented last month at the American Society for Microbiology Biodefense Research Meeting, in Baltimore, USA. 

The plant-expressed PA is highly immunogenic in laboratory animals, but producing antibodies that are specific to PA is not sufficient. "The key question is whether or not the antibodies are protective," remarked Saanford Kimmel, professor of family medicine at the Medical College of Ohio. 

Karasev's group plans to first test whether these antibodies inactivate the anthrax toxin in vitro and then determine whether they protect laboratory animals against anthrax. Karasev also admits that evoking a good systemic immune response after the vaccine is delivered through the digestive system is "probably the biggest challenge today." The PA fragment was extracted and purified from the spinach to test it as an Immunogen "but immune response you can probably just eat it as a vegetable salad," said Karasev. 

The development of plant-based vaccines to protect against many other disease, such as HIV-1, hepatitis B, rabies and non-Hodgkin's lymphoma, are ongoing.

THE FUTURE OF EDIBLE VACCINES

Vaccines have been one of the most far-reaching and important public health initiatives of the 20th century. Advancing technology, such as oral DNA vaccines, intranasal delivery and edible plant-derived vaccines, may lead to a future of safer and more effective immunisation. 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 remain 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 appetising prospect.  

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. (Milstien J.) 

When is this expected to happen ? A realistic appraisal of the state of the art should consider that after the ongoing event of discovery (i.e., the demonstration that plants can be engineered as to produce edible vaccines that trigger an immune response in mice and humans), we are now confronted with the successive problems of clinical trials, process development, registration and marketing. Clinical trials with populations at risk are already under way in some laboratories. The definition of the overall immune response to plant-derived edible vaccines is of the utmost importance. With the growing availability of plant-derived vaccines, this will soon be verified. Process development primarily concerns achieving sufficiently high levels of expression of the recombinant antigen, and defining the optimal way of antigen administration. Solutions to the first point are well under way, as described above, while approaches to the second will be manifold. While the initial concept was to induce an immune response by directly feeding a crude edible plant portion (fruit, leaf, tuber), it is now felt that this may not be the ideal solution as it would be difficult to standardise antigen concentration in different harvests of the same crop. Furthermore, fresh products may have short shelf life. Dried products, for instance banana slices, may offer a partial solution, but the best solution (as for shelf-life, stability and title standardisation) would be delivery in the form of a dry powder. This can be achieved by using low cost food processing technology. A dried tomato powder has been stored for one year in C. Arntzen's laboratory without loss of antigen activity. In cases in which effectiveness is much more relevant than cost, for example with cancer antigens, administration may be through injection of appropriately purified antigens. 

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. 

Important social questions still exist. Who will benefit from this development ? Who will be able to perform research, produce and control plant- derived edible vaccines ? Will the resultant vaccines be affordable to developing countries ? Definitely, the answer is that there is no danger of monopoly in the hands of powerful economic groups. Many countries in the world are already greatly involved in research on plant vaccines; these include the USA, the European Community, China, Japan, India, Korea and others. The reason for this is that the applications are based on established gene cloning and plant transformation technology and that development requires relatively limited investment. 

Opportunity Against The Threat Of Bio-Weapons  

A number of infectious diseases, including smallpox, anthrax and plague have recently raised concern for their possible use in actions of bio-terrorism. Nations at risk are now faced  with the need to be ready to vaccinate part or all of their population within limited periods of time. This means that millions of vaccine doses have to be prepared, stored and renewed at intervals of time. The economic and technical benefits offered by plant - derived vaccines propose these vaccines as ideal substitutes for traditional vaccines. Research on plants that produce antigen against major pathogens feared in case of bio-terrorism is already under way.