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MOLECULAR FARMING OF EDIBLE VACCINES. {1998-2001}

{ with special thanks to Charles J. Arntzen, the global expert on edible vaccines }

1998 - FIRST HUMAN TRIAL SHOWS THAT AN EDIBLE VACCINE IS FEASIBLE

Baltimore, Maryland, April 28, 1998 Opening a new era in vaccine delivery, researchers supported by the National Institute of Allergy and Infectious Diseases (NIAID) have shown for the first time that an edible vaccine can safely trigger significant immune responses in people.

The report, by collaborators from the University of Maryland in Baltimore, the Boyce Thompson Institute for Plant Research in Ithaca, N.Y., and Tulane University in New Orleans, appears in the May issue of Nature Medicine. "Edible vaccines offer exciting possibilities for significantly reducing the burden of diseases like hepatitis and diarrhea, particularly in the developing world where storing and administering vaccines are often major problems," says Anthony S. Fauci, M.D., director of NIAID.

The Phase 1 proof-of-concept trial began last fall at the University of Maryland School of Medicine's Center for Vaccine Development under the direction of Carol O. Tacket, M.D., professor of medicine. The goal of the study was to demonstrate that 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. Previously, NIAID-supported in vitro and preclinical studies by John Clements, Ph.D., and colleagues at Tulane University School of Medicine showed that transgenic potatoes containing this segment of the toxin stimulated strong immune responses in animals. The transgenic potatoes were created and grown by Charles Arntzen, Ph.D., and Hugh S. Mason, Ph.D., and their colleagues at the Boyce Thompson Institute for Plant Research, an affiliate of Cornell University. The trial enrolled 14 healthy adults; 11 were chosen at random to receive the genetically engineered potatoes and three received pieces of ordinary potatoes.

Encouraged by the results of this study, NIAID-supported scientists are exploring the use of this technique for administering other antigens. Edible vaccines for other intestinal pathogens are already in the pipeline--for example, potatoes and bananas that might protect against Norwalk virus, a common cause of diarrhea, and potatoes and tomatoes that might protect against hepatitis B. Regina Rabinovich, M.D., oversees NIAID's Vaccine and Treatment Evaluation Program, of which the University of Maryland's vaccine center is a part. "This first trial is a milestone on the road to creating inexpensive vaccines that might be particularly useful in immunizing people in developing countries, where high cost and logistical issues, such as transportation and the need for certain vaccines to be refrigerated, can thwart effective vaccination programs," she comments. "The hope is that edible vaccines could be grown in many of the developing countries where they would actually be used."

Details of the Study

The study nurse at the University of Maryland peeled the potatoes just before they were eaten, because potato skin sometimes contains a compound that imparts a bitter taste and can cause nausea and stomach upset. The potatoes were then cut into small, uniform pieces and weighed into 50- gram and 100-gram doses. Each person received three doses of either 50 grams or 100 grams of potato over a three-week period, at 0, 7 and 21 days. The dosage size varied in order to evaluate any side effects from eating raw potatoes. NIAID is a component of the National Institutes of Health (NIH). NIAID conducts and supports research to prevent, diagnose and treat illnesses such as AIDS and other sexually transmitted diseases, malaria, tuberculosis, asthma and allergies. NIH is an agency of the U.S. Department of Health and Human Services.

References: Arntzen CJ. Pharmaceutical foodstuffs-oral immunization with transgenic plants. Nature Medicine (vaccine supplement) 1998;4(5):502-03.

Haq TA, Mason HS, Clements JD, and Arntzen CJ. Oral immunization with a recombinant bacterial antigen produced in transgenic plants. Science 1995;268:714-16.

Mason HS, Haq TA, Clements JD, and Arntzen CJ. Edible vaccine protects mice against E. coli heat-labile enterotoxin (LT): potatoes expressing a synthetic LT-B gene. Vaccine, In Press.

Tacket CO, Mason HS, Losonsky G, Clements JD, Levine MM and Arntzen CJ. Immunogenicity in humans of a recombinant bacterial antigen delivered in a transgenic potato. Nature Medicine 1998;4(5):607-09.

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PLANTS AND HUMAN HEALTH: DELIVERY OF VACCINES VIA TRANSGENIC PLANTS

Tsafrir S. Mor and Charles J. Arntzen © 2002
Arizona Biomedical Institute and the Plant Biology Department, PO Box 1601, Arizona State University, Tempe, AZ 85287-1601 (emails: tsafrir.mor@asu.edu, charles.arntzen@asu.edu)

Keywords mucosal immunity, infectious diseases, subunit oral vaccines

1. THE THEORY BEHIND EDIBLE VACCINES

One of major challenges of biotechnology is to reduce clinical innovations to economically viable practices. Plant-derived edible vaccines were first conceived and are continuing to be developed with this prime directive in mind: merging innovations in medical science and plant biology for the creation of efficacious and affordable pharmaceuticals. Since the emergence of the original idea about 10 years ago, it was embraced by a growing number of laboratories in academia and industry. Recent reviews provide detail about progress acheived (Daniell, et al., 2001; Mor, et al., 1998; Tacket and Mason, 1999).
Despite notable successes, traditional vaccine technology has its limitations. Almost all vaccines now commercially available consist of either inactivated or attenuated strains of pathogens which are almost always delivered by injection. (The oral polio vaccine is an exception.) In contrast, many of the currant vaccine development efforts focus on subunit vaccines, and these are being considered for either mucosal or parenteral delivery.
A "subunit vaccine" refers to a pathogen-derived protein (or even just an immunogenic domain of a protein, ie. "an epitope") that cannot cause disease but can elicit a protective immune response against the pathogen. Very often the subunit vaccine candidate is a recombinant protein made in transgenic production-hosts (such as cultured yeast cells), then purified, and injected into vaccinees to immunize against a specific disease. Subunit vaccines are generally considered safer to produce (eliminating the need to culture pathogenic organisms) and more importantly, to use.
However, immunization by injection (parenteral delivery) rarely results in specific protective immune responses at the mucosal surfaces of the respiratory, gastrointestinal and genito-urinary tracts. Mucosal immune responses represent a first line of defense against most pathogens. In contrast, mucosally targeted vaccines achieve stimulation of both the systemic as well as the mucosal immune networks. In addition, mucosal vaccines delivered orally increase safety and compliance by eliminating the need for needles. While subunit vaccines are effective, they currently depend on capital-intensive fermentaion-based technology and a "cold chain" (refrigeration) for delivery. Both of these factors create constraints in use in the developing world, where vaccines are needed the most. Combining a cost-effective production system with a safe and efficacious delivery system, plant edible vaccines, provide a compelling new opportunity.

2. THE THEORY IS PUT TO CLINICAL TRIALS

In 1992 our research team described the expression of hepatitis B surface antigen (HBsAg) in tobacco plants (Mason, et al., 1992). A subsequent succession of papers characterizing the recombinant product which assembled into virus like particles (VLPs, Mason, et al., 1992), and could invoke specific immune responses in mice upon parenteral delivery (Thanavala, et al., 1995). To prove that plant-derived HBsAg can stimulate mucosal immune responses via the oral route, our group switched to potato tubers as an expression system and optimized it to increase accumulation of the protein in the plant tubers (Richter, et al., 2000). The resulting plant material proved superior to the yeast-derived antigen in both priming and boosting of immune responses to oral immunogen in mice (Kong, et al., 2001; Richter, et al., 2000). In parallel with evaluation of plant-derived Hepatitis B surface antigen, Mason and Arntzen explored plant expression of other vaccine candidates including the labile toxin B subunit (LT-B) of entertotoxigenic Escherichia coli (ETEC) and the capsid protein of Norwalk virus (NVCP). The plant derived proteins correctly assembled into functional oligomers that could elicit the expected immune responses when given orally to animals (Haq, et al., 1995; Mason, et al., 1996; Mason, et al., 1998).
Success in mouse experiments provided motivation for conducting Phase I/II human clinical trials to test the safety and immunogenicity of plant-produced LT-B, NVCP and HBsAg (Tacket, et al., 1998; Tacket, et al., 2000, and Thanavala, Mason and Arntzen, unpublished). In the three cases tested, humans who consumed raw potato tubers containing tens of microgram amounts of the antigens developed specific serum and more importantly mucosal immune responses. Significantly, the three antigens in these studies come from three very different pathogens including viral (NV and HBV) and bacterial (E. coli) pathogens, and enteric (NV and E. coli) as well as non-enteric (HBV) disease (Tacket, et al., 1998; Tacket, et al., 2000). Taken together these results provide the basis for wider-scale clinical trials with these antigens which we plan to conduct with the aid of international agencies.
Although mucosal and systemic antibody titers were elevated in vaccinees who received the plant-based oral vaccines, we do not yet have evidence of protection against pathogen challenge. Ethical considerations usually preclude clinical trials from directly assaying protection except in a few cases (e.g. Mason, et al., 1998). In contrast, working with veterinary vaccines provides researchers an opportunity to assess the degree of immune protection more directly. An excellent example of this approach is represented by a series of papers originating from the group of Borca (Carrillo, et al., 2001 and references therein).

3. "SECOND GENERATION" EDIBLE VACCINES

Multicomponent vaccines that provide protection against several pathogens are very desirable. An elegant approach to achieve this goal, based on epitope fusion to both subunits of the cholera toxin (CT), was recently demonstrated by Yu and Langridge (2001). CT provides a scaffold for presentation of protective epitopes of rotavirus and ETEC, acts as as a vaccine candidate by its own right and as a mucosal adjuvant devoid of toxicity. The trivalent edible vaccine elicited significant humoral responses, as well as immune memory B cells and T-helper cell responses, important hallmarks of successful immunization (Yu and Langridge, 2001)..
Commonly, foreign proteins in plants accumulate to relatively low levels (0.01-2% of total soluble protein). In the clinical trials described above, 100 g of raw potato tubers expressing LT-B of ETEC in three doses had to be consumed in order to overcome digestive losses of the antigen and to elicit a significant immune response (Tacket, et al., 1998). Less immunogenic proteins would require even larger doses to be effective. Even with more palatable alternatives to potatoes (e.g. bananas), these accumulation levels may limit the practicality of edible vaccines
Two solutions to overcome this limitation are being explored. First, techniques to enhance antigen accumulation in plant tissues are being explored. These include, optimization of the coding sequence of bacterial or viral genes for expression as plant nuclear genes, and defining the subcellular compartment in which to accumulate the product for optimal quantity and quality. Several laboratories are also developing alternative expression systems to improve accumulation. For example the expression in plastids is advocated by some (Daniell, et al., 2001; Ruf, et al., 2001). Other systems involve plant viruses for expression of foreign genes (e.g. Nemchinov, et al., 2000) or coat-protein fusions (e.g. Modelska, et al., 1998) and even viral assisted expression in transgenic plants (Mor, et al., 2002).
The second approach is to enhance the immunogenicity of the orally delivered antigens by using mucosal adjuvants. One such approach is making use of bacterial entertoxins such as CT or LT (e.g. Yu and Langridge, 2001), mammalian and viral immunomodulators (Matoba, Soreq, Arntzen and Mor unpublished) as well as plant-derived secondary metabolites (Joshi and Arntzen, unpublished).
At doorstep of the 21st century, the fear of a surge in naturally occurring epidemics is heightened by the threat of bio-terrorism. This new reality makes disease prevention through vaccination a necessity in our ever more interconnected world. Any tools we can master and all the tools we can afford will have to be employed. Technical problems and skeptics aside, edible-vaccines have passed the major hurdles of an emerging vaccine technology. We believe production of vaccines in transgenic plants will become an essential component in our disease prevention arsenal.

4. REFERENCES

Carrillo C., A. Wigdorovitz, K. Trono, M.J. Dus Santos, S. Castanon, A.M. Sadir, R. Ordas, J.M. Escribano and M.V. Borca. 2001. Induction of a virus-specific antibody response to foot and mouth disease virus using the structural protein VP1 expressed in transgenic potato plants. Viral Immunol. 14:49-57.
Daniell H., S.J. Streatfield and K. Wycoff. 2001. Medical molecular farming: production of antibodies, biopharmaceuticals and edible vaccines in plants. Trends Plant Sci. 6:219-226.
Haq T.A., H.S. Mason, J.D. Clements and C.J. Arntzen. 1995. Oral Immunization with a recombinant Bacterial antigen produced in transgenic plants. Science 268:714-716.
Kong Q., L. Richter, Y.F. Yang, C.J. Arntzen, H.S. Mason and Y. Thanavala. 2001. Oral immunization with hepatitis B surface antigen expressed in transgenic plants. Proc. Natl. Acad. Sci. U.S.A. 98:11539-11544.
Mason H.S., D.M.K. Lam and C.J. Arntzen. 1992. Expression of hepatitis B surface antigen in transgenic plants. Proc. Natl. Acad. Sci. U.S.A. 89:11745-11749.
Mason H.S., J.M. Ball, J.-J. Shi, X. Jiang, M.K. Estes and C.J. Arntzen. 1996. Expression of Norwalk virus capsid protein in transgenic tobacco and protein and its oral immunogenicity in mice. Proc. Natl. Acad. Sci. U.S.A. 93:5335-5340.
Mason H.S., T.A. Haq, J.D. Clements and C.J. Arntzen. 1998. Edible Vaccine Protects Mice Against E. coli Heat-labile Enterotoxin (LT): Potatoes Expressing a Synthetic LT-B Gene. Vaccine 16:1336-1343.
Modelska A., B. Dietzschold, N. Sleysh, F.Z. Fu, K. Steplewski, D.C. Hooper, H. Koprowski and V. Yusibov. 1998. Immunization against rabies with plant-derived antigen. Proc. Natl. Acad. Sci. U.S.A. 95:2481-2485.
Mor T.S., M.A. Gómez-Lim and K.E. Palmer. 1998. Edible vaccines: a concept comes of age. Trends Microbiol. 6:449-453.
Mor T.S., Y.-S. Moon, K.E. Palmer and H.S. Mason. 2002. Geminivirus Vectors for High Level Expression of Foreign Proteins in Plant Cells. Biotechnol. Bioeng. in press.
Nemchinov L.G., T.J. Liang, M.M. Rifaat, H.M. Mazyad, A. Hadidi and J.M. Keith. 2000. Development of a plant-derived subunit vaccine candidate against hepatitis C virus. Arch. Virol. 145:2557-2573.
Richter L.J., Y. Thanavala, C.J. Arntzen and H.S. Mason. 2000. Production of hepatitis B surface antigen in transgenic plants for oral immunization. Nat. Biotechnol. 18:1167-1171.
Ruf S., M. Hermann, I.J. Berger, H. Carrer and R. Bock. 2001. Stable genetic transformation of tomato plastids and expression of a foreign protein in fruit. Nat. Biotechnol. 19:870-875.
Tacket C.O., H.S. Mason, G. Losonsky, J.D. Clements, S.S. Wasserman, M.M. Levine and C.J. Arntzen. 1998. Immunogenicity in humans of a recombinant bacterial-antigen delivered in transgenic potato. Nat. Med. 4:607-609.
Tacket C.O. and H.S. Mason. 1999. A review of oral vaccination with transgenic vegetables. Microbes Infect. 1:777-783.
Tacket C.O., H.S. Mason, G. Losonsky, M.K. Estes, M.M. Levine and C.J. Arntzen. 2000. Human immune responses to a novel Norwalk virus vaccine delivered in transgenic potatoes. J. Infect. Dis. 182:302-305.
Thanavala Y., Y.-F. Yang, P. Lyons, H.S. Mason and C.J. Arntzen. 1995. Immunogenicity of transgenic plant-derived hepatitis B surface antigen. Proc. Natl. Acad. Sci. U.S.A. 92:3358-3361.
Yu J. and W.H. Langridge. 2001. A plant-based multicomponent vaccine protects mice from enteric diseases. Nat. Biotechnol. 19:548-552.

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2000 --- { AN ARTICLE FROM E.N.N. }

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. Welcome to the new world of molecular farming.

In Canada, a genetically engineered tobacco plant made to produce Interleukin 10 will be tested to treat Crohn's disease, an intestinal disorder. Molecular farming uses the science of genetic engineering to turn ordinary plants into factories for the production of inexpensive drugs and vaccines. Researchers at the London Health Sciences Center in London, Ontario, Canada, are growing potatoes that have been genetically altered to produce a special diabetes-related protein. When the potatoes are fed to diabetic mice, scientists find that most don't develop Type I diabetes, also known as juvenile-onset diabetes. Scientists believe that the low-cost production of this protein may help the 100 million people worldwide affected by diabetes. In the lab, the new transgenic potatoes produce large amounts of a human protein that suppresses the destructive immune response and prevents diabetes from developing.

Molecular biologist Shengwu Ma of the London Health Sciences Center says his team's research has similar potential to combate other autoimmune diseases such as multiple sclerosis, rheumatoid arthritis, lupus and even transplant rejection. "Plants are ideal because they can synthesize and assemble proteins to provide huge quantities of soluble proteins at relatively low cost," says Ma. Many traditional drugs are difficult to make and hence, costly. However, once this technology is perfected, growing transgenic potatoes will cost very little, he notes.

Edible vaccines were first tested on humans in 1997, when scientists asked volunteers to eat anti-diarrheal transgenic potatoes produced by the Boyce Thompson Institute at Cornell University. After consuming the potatoes, almost all the volunteers produced antigens in their bodies just as if they had received a traditional anti-diarrheal vaccination. And they experienced no adverse side effects. Volunteers are also testing raw potatoes engineered to produce a Hepatitis B antigen at the Roswell Park Cancer Institute in Buffalo, New York. Results are expected this summer. Hugh Mason, an associate research scientist in edible vaccines at the Boyce Thompson Institute, hopes to develop "methods to increase production of foreign protein in plant cells and to engineer protein antigens that will enhance their potential as human and animal vaccines." This fall Mason hopes to do human tests on Hepatitis B antigens grown in transgenic tomatoes if the FDA approves. "This technology will be a big plus for the developing world," he says.

copyright for this article - Enviromental News Network 2000 Article by Stephen Leahy
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2001 AUSTRALIAN DEVELOPMENTS
Edible vaccines the key to better immunisation

Monash University scientists have successfully grown a genetically engineered plant containing a measles vaccine in a technique that may eventually lead to simpler and cheaper immunisation programs for a range of viral diseases, including HIV and malaria.

Led by Professor Steve Wesselingh, the research team successfully produced a tobacco leaf containing a viral protein found in the measles virus. When the plant was processed and fed to mice, their immune system responded by producing protective antibodies. Testing has now begun on primates.

The research team is now developing the protein in a range of foods including rice and lettuce and have recognised the potential for the protein to be incorporated into baby food.

"There is no real reason why we couldn't be working with any type of food, but we believe that rice flour, when mixed with breast milk as baby food, is a simple and cheap option even for poor or remote communities," said Professor Wesselingh.

Although measles can be effectively prevented by a 'live' measles vaccine injection, it still causes up to one million deaths each year, mostly among young children in developing countries. In these countries, injectable vaccines are inhibited by many factors, including the need to provide a stable and cold environment during storage and transportation and a lack of trained medical staff to administer the vaccine.

The quest for new and better ways to immunise people against infectious diseases has led to a variety of alternatives to injections, with the food-based vaccine research providing the greatest potential.

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.

The H protein sits on the outside of the virus and has a role in provoking the immune response in the body. The edible vaccines, therefore, do not contain the complete 'live' virus - only the key protein to trigger the immune response.

The Monash researchers are working closely with scientists at the CSIRO Plant Industry and at the University of Melbourne

2001 - from TRENDS IN PLANT SCIENCE . Vol.6 No.5 {page 222} [by permission of Elsevier Science]

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

KEYS - DISEASE TARGET {Source of the protein} and target species for the VACCINES - PLANT EXPRESSION SYSTEM - Notes and PROTECTIVE CAPACITY of the VACCINES

Enterotoxigenic E. COLI (humans)-TOBACCO - Immunogenic when administered orally
Enterotoxigenic E. COLI {humans}- POTATO - Immunogenic and protective when administered orally
Enterotoxigenic E. COLI {humans}- MAIZE - Immunogenic and protective when administered orally

Vibrio cholerae [CHOLERA] (humans) - POTATO- Immunogenic and protective when administered orally

Hepatitis B virus {humans}- TOBACCO -Extracted protein is immunogenic when administered by injection
Hepatitis B virus {humans} - POTATO - Immunogenic when administered orally
Hepatitis B virus {humans}- LUPIN - Immunogenic when administered orally
Hepatitis B virus {humans}- LETTUCE - Immunogenic when administered orally

Norwalk virus (humans) - TOBACCO - Immunogenic when administered orally
Norwalk virus (humans) - POTATO - Virus-like particles form and immunogenic when administered orally

RABIES virus (humans) -TOMATO - Intact Glycoprotein

Human cytomegalovirus {humans} - TOBACCO - Immunologically related protein

Rabbit hemorrhagic disease virus {rabbits} - POTATO - Immunogenic and protective when administered by injection
FOOT-AND-MOUTH disease {agricultural domestic animals} - ARABIDOPSIS - Immunogenic and protective when administered by injection

FOOT-AND-MOUTH disease {agricultural domestic animals}- ALFALFA - Immunogenic and protective when administered by injection or orally

Transmissible gastroenteritis coronavirus (pigs) - ARIBIDOPSIS - Immunogenic when administered by injection
Transmissible gastroenteritis coronavirus (pigs) - TOBACCO - Intact protein and immunogenic when administered by injection
Transmissible gastroenteritis coronavirus (pigs) - MAIZE - Protective when administered orally

More on edible vaccine from the University of Wisconsin HERE