IndianApril-June Journal 2007 of Medical Microbiology, (2007) 25 (2):93-102 93 Review Article

EDIBLE : CURRENT STATUS AND FUTURE P Lal, VG Ramachandran, *R Goyal, R Sharma Abstract

Edible vaccines hold great promise as a cost-effective, easy-to-administer, easy-to-store, fail-safe and socioculturally readily acceptable delivery system, especially for the poor developing countries. It involves introduction of selected desired genes into and then inducing these altered plants to manufacture the encoded proteins. Introduced as a concept about a decade ago, it has become a reality today. A variety of delivery systems have been developed. Initially thought to be useful only for preventing infectious diseases, it has also found application in prevention of autoimmune diseases, birth control, cancer therapy, etc. Edible vaccines are currently being developed for a number of human and animal diseases. There is growing acceptance of transgenic crops in both industrial and developing countries. Resistance to genetically modiÞ ed foods may affect the future of edible vaccines. They have passed the major hurdles in the path of an emerging vaccine technology. Various technical obstacles, regulatory and non-scientiÞ c challenges, though all seem surmountable, need to be overcome. This review attempts to discuss the current status and future of this new preventive modality.

Key words: Chimeric, edible vaccines, transgenic

Vaccines have been revolutionary for the prevention of desired genes into plants and then inducing these altered infectious diseases. Despite worldwide immunization of plants to manufacture the encoded proteins. This process is children against the six devastating diseases, 20% of infants known as “transformation,” and the altered plants are called are still left un-immunized; responsible for approximately “transgenic plants.” Like conventional subunit vaccines, two million unnecessary deaths every year, especially in the edible vaccines are composed of antigenic proteins and remote and impoverished parts of the globe.1 This is because are devoid of pathogenic genes. Thus, they have no way of the constraints on vaccine production, distribution and of establishing infection, assuring its safety, especially in delivery. One hundred percent coverage is desirable, because immunocompromised patients. Conventional subunit vaccines un-immunized populations in remote areas can spread are expensive and technology-intensive, need puriÞ cation, infections and epidemics in the immunized “safe” areas, require refrigeration and produce poor mucosal response. which have comparatively low herd immunity. For some In contrast, edible vaccines would enhance compliance, infectious diseases, immunizations either do not exist or they especially in children, and because of oral administration, are unreliable or very expensive. Immunization through DNA would eliminate the need for trained medical personnel. Their vaccines is an alternative but is an expensive approach, with production is highly efÞ cient and can be easily scaled up. disappointing immune response.2 Hence the search is on for For example, hepatitis-B required to vaccinate whole cost-effective, easy-to-administer, easy-to-store,(www.medknow.com). fail-safe of China annually, could be grown on a 40-acre plot and all and socioculturally readily acceptable vaccines and their babies in the world each year on just 200 acres of land!4 They delivery systems. As Hippocrates said, “Let thy food be thy are cheaper, sidestepping demands for puriÞ cation (single dose medicine,” scientists suggest that plants and viruses can of hepatitis-B vaccine would cost approximately 23 paise), be genetically engineeredThis to produce PDFa site vaccines is hosted available against diseases by for Medknowgrown free locally download usingPublications standard from methods and do not require such as dental caries; and life-threatening infections like capital-intensive pharmaceutical manufacturing facilities. diarrhea, AIDS, etc.3 This is the concept of edible vaccines. Mass-indeÞ nite production would also decrease dependence The following discussion will address issues relating to on foreign supply. They exhibit good genetic stability. They their commercial development, especially their usefulness in are heat-stable; do not require cold-chain maintenance; can preventing infectious diseases in developing countries. be stored near the site of use, eliminating long-distance transportation. Non-requirement of syringes and needles also Concept of Edible Vaccines decreases chances of infection.5 Fear of contamination with Creating edible vaccines involves introduction of selected animal viruses - like the mad cow disease, which is a threat in vaccines manufactured from cultured mammalian cells - is *Corresponding author (email: ) eliminated, because plant viruses do not infect humans. Departments of Microbiology (PL, VGR, RG) and Pediatrics (RS), University College of Medical Sciences, Guru Teg Bahadur Hospital, Edible vaccines activate both mucosal and systemic New Delhi - 110 095, India. immunity, as they come in contact with the digestive tract Received : 28-06-06 lining. This dual effect would provide Þ rst-line defense against Accepted : 14-10-06 pathogens invading through mucosa, like Mycobacterium

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93 CMYK 94 Indian Journal of Medical Microbiology vol. 25, No. 2 tuberculosis and agents causing diarrhea, pneumonia, STDs, dependent and takes 3-9 months. Reducing this time to 6-8 HIV, etc. Scientists place high priority on combating the weeks is currently under investigation. Some , like diarrheal agents – Norwalk virus, Rotavirus, Vibrio cholerae viral capsid proteins, have to self-assemble into VLPs (virus- and enterotoxigenic E. coli (ETEC) – responsible for about like particles). VLPs mimic the virus without carrying DNA three million infant deaths/year, mainly in developing or RNA and therefore are not infectious. Each single antigen countries.1 Administration of edible vaccines to mothers expressed in plants must be tested for its proper assembly and might be successful in immunizing the fetus-in-utero by can be veriÞ ed by animal studies, Western blot; and quantiÞ ed transplacental transfer of maternal antibodies or the infant by enzyme-linked immunosorbent assay (ELISA).11 through breast milk. Edible vaccines seroconvert even in the presence of maternal antibodies, thus having a potential role in “Second-Generation” Edible Vaccines protecting infants against diseases like group-B Streptococcus, Successful expression of foreign genes in plant cells and/or respiratory syncytial virus (RSV), etc., which are under its edible portions has given a potential to explore further and 6 investigation. Edible vaccines would also be suitable against expand the possibility of developing plants expressing more neglected/rare diseases like dengue, hookworm, rabies, etc. than one antigenic protein. Multicomponent vaccines can They may be integrated with other vaccine approaches, and be obtained by crossing two plant lines harboring different multiple antigens may also be delivered. Various foods under antigens. Adjuvants may also be co-expressed along with the study are banana, , , lettuce, rice, etc. Edible antigen in the same plant. B subunit of Vibrio cholerae toxin vaccines are currently being developed for a number of human (VC-B) tends to associate with copies of itself, forming a and animal diseases, including measles, cholera, foot and doughnut-shaped Þ ve-member ring with a hole in the middle.1 7 mouth disease and , C and E. This feature can bring several different antigens to M cells at one time - for example, a trivalent edible vaccine against Mechanism of Action cholera, ETEC (Enterotoxigenic E. coli) and rotavirus could The antigens in transgenic plants are delivered through successfully elicit signiÞ cant immune response to all three.12 bio-encapsulation, i.e., the tough outer wall of plant cells, Global alliance for vaccines and immunization (GAVI) which protects them from gastric secretions, and finally accords very high priority to such combination vaccines for break up in the intestines. The antigens are released, taken developing countries. up by M cells in the intestinal lining that overlie peyer’s Various Strategies patches and gut-associated lymphoid tissue (GALT), passed on to macrophages, other antigen-presenting cells; and local Approaches to mucosal vaccine formulation include (i) lymphocyte populations, generating serum IgG, IgE responses, gene fusion technology, creating non-toxic derivatives of local IgA response and memory cells, which would promptly mucosal adjuvants; (ii) genetically inactivating antigens by neutralize the attack by the real infectious agent.8 deleting an essential gene; (iii) co-expression of antigen and a cytokine, which modulates and controls mucosal immune Preparation of Edible Vaccines response; and (iv) genetic material itself, which allows DNA/ Introduction of foreign DNA into plant’s genome can either RNA uptake and its endogenous expression in the host cell. be done by bombarding embryonic suspension cell(www.medknow.com). cultures Various mucosal delivery systems include biodegradable using gene-gun or more commonly through Agrobacterium micro- and nanoparticles, liposomes, live bacterial/viral 13 tumefaciens, a naturally occurring soil bacterium, which has vectors and mucosal adjuvants. “Prime-boost” strategy the ability to get into plants through some kind of wound combines different routes of administration and vaccine types, 14 (scratch, etc.). It Thispossesses PDF aa circularsite is hosted“Ti available plasmid” by(tumor forMedknow especiallyfree download where Publications multiple antigens from or doses are required. For inducing), which enables it to infect plant cells, integrate example, a single parenteral dose of MV-H DNA (measles into their genome and produce a hollow tumor (crown gall virus haemagglutinin) followed by multiple oral MV-H tumor), where it can live. This ability can be exploited to boosters could induce greater quantities of MV-neutralizing 15 insert foreign DNA into plant genome. But prior to this, the antibodies than with either vaccine alone. plasmid needs to be disarmed by deleting the genes for auxin Chimeric Viruses and cytokinin synthesis, so that it does not produce tumor. Genes for antibiotic resistance are used to select out the Certain viruses can be redesigned to express fragments of transformed cells and whole plants, which contain the foreign antigenic proteins on their surface, such as CPMV (cowpea gene; and for expressing the desired product, which can then mosaic virus), alfalfa mosaic virus, TMV (tobacco mosaic be regenerated from them.9 The DNA integrates randomly virus), CaMV (cauliflower mosaic virus), potato virus X into plant genome, resulting in a different antigen expression and tomato bushy stunt virus. Technologies involved are level for each independent line,10 so that 50-100 plants are overcoat and epicoat technology.16 Overcoat technology transformed together at a time, from which one can choose the permits the plant to produce the entire protein, whereas epicoat plant expressing the highest levels of antigen and least number technology involves expression of only the foreign proteins. A of adverse effects. Production of transgenic plants is species plant-derived mink enteritis virus (MEV) injectable vaccine,

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94 CMYK April-June 2007 Lal et al – Edible Vaccines 95 expressed on chimeric CPMV, was shown to protect minks Table 1: Considerations in developing a plant-based against the clinical disease. Fragments of gp41 surface protein vaccine of HIV virus put into CPMV could evoke a strong neutralizing antibody response in mice.3 It may even be possible to present Antigen selection a cocktail of speciÞ c HIV epitopes on the surface of the plant • Is the antigen safe and non-pathogenic in all virus. CPMV is genetically and thermally stable. It can survive circumstances? acidity (pH 1) for one hour. Wide range of epitopes has been • Can the antigen induce a protective immune response? expressed in CPMV. They include HIV-1 (gp41), (gp120); • Is the antigen suitable for expression in plants? human rhinoviruses; foot and mouth disease virus; canine Effi cacy in model systems parvovirus; mammalian epitopes from hormones or from colon • Does the antigen accumulate in plants in sufficient cancer cells; fungal epitopes and protozoan epitopes from quantities? Plasmodium falciparum. Wide range of routes is available • Is the plant-derived antigen immunogenic? – parenteral and nasal (puriÞ ed particles), oral (formulated leaf • Do trial animals develop protective immune responses? extracts), whole and homogenized leaves, fruits or vegetable • Possible plant cell interference with antigen presentation tissues.16 In all these instances, plant viruses are engineered to • Possible induction of immune tolerance carry the desired genes and used to infect their natural hosts Choice of plant species for vaccine delivery such as the edible plants where the cloned genes are expressed • Best food plant? to varying degrees in different parts of the plant, including • Ability to be eaten raw and unprocessed? their edible portions. • Suitable for infants? • Widely and easily grown? Challenges • Easily stored? Resistant to spoiling? • Amenable to transformation and regeneration? There are many questions which need to be answered • Possible cost to plant of multiple transgenes before developing a plant-based vaccine (Table 1). Three Delivery and dosing issues successful human clinical trials have shown that adequate • Requirement of mucosal adjuvants for protective doses of antigen can be achieved with plant-based vaccines.17- 19 response? To determine the right dosage, one needs to consider the • Can a large enough dose be delivered by simply eating the person’s weight, age; fruit/plant’s size, ripeness and protein plant? content. The amount to be eaten is critical, especially in • Number of doses required? infants, who might spit it, eat a part or eat it all and throw it up Safety issues later. Too low a dose would fail to induce antibodies, and too • Allergenic and toxic (e.g., glycans, nicotine, etc.) potential high a dose would, instead, cause tolerance. Concentrating the of plant components vaccine into a teaspoon of baby food may be more practical • Potential for interference than administering it in a whole fruit. The transformed • Production of oral tolerance? plants can also be processed into pills, puddings, chips, etc. • Risk of atypical measles (in plants with cloned measles Regulatory concerns would include lot-to-lot consistency, virus genes)? uniformity of dosage and purity. (www.medknow.com).• Health and environmental risks of genetically modiÞ ed Foreign proteins in plants accumulate in low amounts organisms (0.01-2% of total protein) and are less immunogenic; therefore • Prevention of misuse/overuse the oral dose far exceeds the intranasal/parenteral dose. For Public perceptions and attitudes to genetic modifi cation example, oral hepatitis-BThis dose PDFa is 10-100site is timeshosted available the parenteral by for Medknow• free Will negative download Publications attitudes to geneticallyfrom modiÞ ed organisms dose; and 100 gm potato expressing B subunit of labile toxin inß uence vaccine acceptability? of ETEC (LT-B) is required in three different doses, to be • Legal and ethical considerations regarding products from immunogenic.19 Attempts at boosting the amount of antigens plants with status like tobacco often lead to stunted growth of plants and reduced tuber/fruit Quality control and licensing formation, as too much m-RNA from the transgene causes • Can antigen expression be consistent in crops? gene-silencing in plant genome.20 Some of the techniques • Who will control vaccine availability and production? to overcome these limitations are (i) optimization of coding ModiÞ ed from “Webster DE et al. Appetising solutions: An edible sequence of bacterial/viral genes for expression as plant vaccine for measles. MJA 2002; 176: 434-437. ©Copyright 2002. 5 nuclear genes, (ii) expression in plastids,21 (iii) plant viruses The Medical Journal of Australia” - reproduced with permission. 22 23 expressing foreign genes, (iv) coat-protein fusions, (v) on the genes more readily; or do so only at selected times 24 viral-assisted expression in transgenic plants and (vi) (after the plant is nearly fully grown); or only in its edible promoter elements of bean yellow dwarf virus with reporter regions. Exposure to some outside activator molecule may genes GUS (β-glucuronidase) and GFP (green ß uorescent also be tried.25 protein), substituted later with target antigen genes.20 Antigen genes may be linked with regulatory elements which switch To enhance immunogenicity, mucosal adjuvants, better

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95 CMYK 96 Indian Journal of Medical Microbiology vol. 25, No. 2 targeted to the immune system, may be used, like molecules most research, as edible vaccines are targeted to markets of that bind to M cells in the intestine lining and pass them developing nations. Large companies are more interested to immune cells. These include CT-B (Cholera toxin - B in livestock market than human application. Only few subunit), LT-B (ETEC), mammalian/viral immunomodulators international aid organizations and some national governments and plant-derived secondary metabolites.1,12 To decrease are rendering support, but the effort remains largely under- toxicity and allergic potential, mutant forms of E. coli-labile funded. Some of the companies funding edible vaccines toxin, like LT-K63 and LT-R72 and hinge cleavage mutant LT- research have failed to click due to lack of investors’ G192, are used.26 Another challenge would be in dealing with conÞ dence in returns on investments in genetically-modiÞ ed diseases caused by multiple serotypes (dengue) or by complex (GM) foods. There is also a lack of research and development parts from different life cycles of parasites (malaria) or by (R&D) personnel in the pharmaceutical companies. In rapidly mutating organisms (HIV, trypanosomes, inß uenza). addition, the recombinant (injectable) vaccines against Each plant possesses its set of advantages and disadvantages diphtheria, tetanus, etc., are so cheap now, that there would be (Table 2). little incentive to develop edible vaccines for them.

Nonscientifi c Challenges Regulatory Issues

Presently, small technology companies are undertaking It is still unclear whether the edible vaccines would be

Table 2: Advantages and disadvantages of different plants Plant/fruit Advantages Disadvantages Tobacco Good model for evaluating recombinant proteins Produces toxic compounds* Low-cost preserving system (numerous seeds, stored for long time) Easy puriÞ cation of antibodies stored in the seeds, at any location Large harvests, number of times/year Potato Dominated clinical trials Needs cooking, which can denature the Easily manipulated/transformed antigens and decrease immunogenicity** Easily propagated from its “eyes” Stored for long periods without refrigeration Banana Do not need cooking Trees take 2-3 years to mature Proteins not destroyed even if cooked Transformed trees take about 12 months to bear fruit Inexpensive Spoils rapidly after ripening Grown widely in developing countries Contains very little protein, so unlikely to produce large amounts of recombinant proteins Tomato Grow quickly Spoils readily Cultivated broadly High content of vitamin A may boost(www.medknow.com). immune response Overcome the spoilage problem by freeze-drying technology Heat-stable, antigen-containing powders***, made into capsules DifferentThis batches PDFa blendedsite is hosted toavailable give uniform by doses forMedknow of freeantigen download Publications from Rice Commonly used in baby food because of low allergenic potential Grows slowly High expression of proteins/ antigens Requires specialized glasshouse conditions Easy storage/transportation Expressed protein is heat-stable Lettuce Fast-growing Spoils readily Direct consumption Soybean Large harvests, number of times/year and Alfalfa Musk melon Fast growing (cantaloupe) Easily propagated by seed Easily transformed Others Carrots, peanuts, wheat, corn *Currently, therapeutic proteins in tobacco are being produced. **Some kinds of South American potatoes can be eaten raw. Although some studies show that cooking does not destroy full complement of antigen in potatoes.3 ***Freeze-dried tomato powder containing NV capsid and LT-B was found immunogenic. Same technology is also used for potatoes and carrots. www.ijmm.org

96 CMYK April-June 2007 Lal et al – Edible Vaccines 97 regulated under food, drugs or agricultural products and prove better for achieving its eradication. The success in mice what vaccine component would be licensed - antigen itself, has prompted similar experiments in primates. Transgenic genetically engineered fruit or transgenic seeds. They would rice, lettuce and baby food against measles are also being be subjected to a very close scrutiny by the regulatory bodies developed. When given with CT-B (adjuvant), 35-50 gm MV- in order to ensure that they never enter the food supply. H lettuce is enough; however, an increased dose would be This would include greenhouse segregation of medicinal required if given alone.7 plants from food crops to prevent out-crossing and would Hepatitis B necessitate separate storage and processing facilities. Although edible vaccines fall under “GM” plants, it is hoped that these For hepatitis B, parenteral VLPs could invoke speciÞ c vaccines will avoid serious controversy, because they are antibodies in mice.36 First human trials of a potato-based intended to save lives. vaccine against hepatitis B have reported encouraging results. The amount of HBsAg needed for one dose could be achieved Clinical Trials in a single potato. Levels of speciÞ c antibodies signiÞ cantly exceeded the protective level of 10 mIU/mL in humans. When Antigen expression in plants has been successfully shown cloned into CaMv (cauliß ower mosaic virus), plasmid HBsAg in the past, like LT-B (ETEC) in tobacco and potato, rabies subtype ayw showed higher expression in roots as compared to virus-G protein in tomato,27 HBsAg in tobacco28 and potato, leaf tissue of the transgenic potato. Further studies are required norwalk virus in tobacco and potato; CT-B (Vibrio cholerae) in to increase the production of antigen by using different potato.1 Ethical considerations usually preclude clinical trials promoters, like patatin promoter. The resulting plant material from directly assessing protection, except in a few cases.29 In proved superior to the yeast-derived antigen in both priming contrast, veterinary researchers can assess immune protection and boosting immunity in mice. Prime boost strategy in mice more directly. with a single sub-immunogenic parenteral dose of yeast- ETEC derived recombinant HBsAg and subsequent oral transgenic potatoes led to the development of antibodies that immediately Charles Arntzen at Boyce Thompson Institute, USA, peaked at >1,000 mIU/mL and were maintained at >200 accomplished the Þ rst published successful human trial in 1997.30 mIU/mL for Þ ve months. This could be a useful immunization Eleven volunteers were fed raw transgenic potatoes expressing strategy for developing countries.37 Tomatoes expressing LT-B. Ten (91%) of these individuals developed neutralizing hepatitis B are being grown in guarded greenhouses. Enough antibodies, and six (55%) developed a mucosal response. antigens for 4,000 vaccine doses were obtained from just 30 tomato plants.38 Transgenic lettuce is also being developed. Norwalk virus Rabies Nineteen (95%) out of 20 people fed with transgenic potato expressing norwalk virus antigen showed seroconversion.31 Tomato plants expressing rabies antigens could induce 39 40 Attempts are underway to engineer bananas and powdered antibodies in mice. Alternatively, TMV may also be used. tomatoes expressing norwalk virus. Transformed tomato plants using CaMV with the glycoprotein (G-protein) gene of rabies virus (ERA strain) was shown to be Cholera (www.medknow.com).immunogenic in animals.41 Transgenic potato with CT-B gene of Vibrio cholerae was HIV shown to be efÞ cacious in mice. Eating one potato a week for Initial success in splicing HIV protein into CPMV has 32 a month with periodicThis boosters PDF awas site said isto hostedprovide available immunity. by for Medknowbeen free achieved. download Publications39 Two HIV proteinfrom genes and CaMV as Co-expression of mutant cholera toxin subunit A (mCT-A) and promoter were successfully injected into tomatoes with LT-B in crop seed has been shown to be effective by nasal a needle, and the expressed protein was demonstrable by 33 administration and is extremely practical. polymerase chain reaction (PCR) in different parts of the Measles plant, including the ripe fruit, as well as in the second- generation plant.42 Recently, spinach has been successfully Mice fed with tobacco expressing MV-H (measles inoculated for Tat protein expression cloned into TMV. Each virus haemagglutinin from Edmonston strain) could attain gram of leaf tissue of spinach was shown to contain up to antibody titers Þ ve times the level considered protective for 300-500 µg of Tat antigen.43 Mice fed with this spinach humans, and they also demonstrated secretory IgA in their followed by DNA vaccinations resulted in higher antibody faeces.34 Prime boost strategy by combining parenteral and titers than the controls, with the levels peaking at four weeks subsequent oral MV-H boosters could induce titers 20 times post-vaccination. the human protective levels. These titers were signiÞ cantly STDs greater than with either vaccine administered alone.5,15 MV-H edible vaccine does not cause atypical measles, which may Human papilloma virus type-11 (HPV-11) recombinant be occasionally seen with the current vaccine.35 Thus it may VLPs produced in insect cells are immunogenic when given

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97 CMYK 98 Indian Journal of Medical Microbiology vol. 25, No. 2 orally to BALB/c mice. The response is dose-dependent, could induce high titers of IgG and mucosal IgA in mice.48 For conformationally-dependent and genotype-restricted. Thus, parasites like malaria, advances in vaccine have been hindered VLPs may be effective oral immunogens for the prevention by the complex multistage life cycle of the parasite, its of anogenital HPV disease.41 inaccessibility to study and by its large genome. Nevertheless, Anthrax chimeric coat proteins of CPMV expressing malarial and foot- and-mouth disease epitopes have been reported.39 Tobacco leaves bombarded with pag gene (anthrax Veterinary Sciences protective antigen - PA) using a gene gun could express a protein structurally identical to the major protein present in The first patented edible vaccine to demonstrate existing vaccine. Billions of units of anthrax antigen could efficacy in animal trials was against the transmissible be produced. In addition, this vaccine was devoid of edema gastroenteritis virus (TGEV) in pigs and was under planning factor and lethal factor, responsible for the toxic side effects. to be made commercially available.49 Vaccines against The same anthrax antigen is now being put in tomato plants.44 porcine reproductive and respiratory syndrome (PRRS) and Scientists are also trying to transform spinach by inoculating other diseases like parvovirus are being investigated. Various it with TMV-expressing PA, as spinach might be a safer transgenic animal feeds are currently undergoing clinical trials 45 vaccine. in pigs.50 Other candidates are diseases of pets, animals in swine and poultry industries, draft and wild animals. Different Others plant viral system patents exist (Table 3). Transgenic Arabidopsis thaliana plants are able to produce Passive Immunization a fusion protein consisting of LT-B and early secretory antigen ESAT-6 of Mycobacterium tuberculosis, demonstrating both Just as any antigenic protein can be expressed in plants, the antigens by ELISA.46 Respiratory syncytial virus (RSV) antibody molecules too can be expressed in plants. Protective pneumonia takes a heavy toll of life of children below two antibodies produced in plants are sometimes referred to as years of age, each year. RSV expressed in tomato and potato “plantibodies.” To date, only four antibodies produced in plants has shown encouraging results in mice. Further clinical plants have the potential to be used as therapeutic agents, and trials are being planned. ‘Apple juice’-based RSV vaccine is only IgG-IgA against Streptococcus mutans has been tested on also being developed.47 Programs for development of new humans. Attempts are being made to formulate these secretory vaccines against rotavirus and Streptococcus pneumoniae antibodies into toothpaste to protect against tooth decay. have been instituted by program for appropriate technology Human trials are also being planned. The other antibodies in health and GAVI, with work originating in developing have been tested on animals and also show great promise. countries. For rotavirus, transgenic potatoes expressing VP7 Producing complete secretory antibodies was quite difÞ cult

Table 3: Plant viral system patents Virus Target host Plant Mode of administration Enterotoxigenic E. coli (ETEC) (www.medknow.com). Humans Tobacco Oral ETEC Humans Potato Oral ETEC Humans Maize Oral Vibrio cholerae Humans Potato Oral Hepatitis B virus This PDFa site is hosted available by Humans forMedknow free download Publications Tobacco from Injection (extracted protein) Hepatitis B virus Humans Potato Oral Hepatitis B virus Humans Lettuce Oral Norwalk virus Humans Tobacco Oral Norwalk virus Humans Potato Oral (VLPs) Rabies virus Humans Tomato Intact glycoprotein CMV Cytomegalo virus Humans Tobacco Immunologically related protein Rabbit hemorrhagic disease virus Rabbits Potato Injection Foot-and-mouth disease Agricultural domestic animals Arabidopsis Injection Foot-and-mouth disease Agricultural domestic animals Alfalfa Injection or oral Transmissible gastroenteritis coronavirus (TGEV) Pigs Arabidopsis Injection TGEV Pigs Tobacco Injection TGEV Pigs Maize Oral Reproduced with permission41

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98 CMYK April-June 2007 Lal et al – Edible Vaccines 99 because apart from four protein chains (two heavy, two light greater quantities of transgenic protein. chains), they contain two additional proteins, one of which Role in autoimmune diseases is added in vivo during secretion. This was Þ nally achieved by crossing four separate lines of transgenic tobacco plants. For unknown reasons, oral delivery of auto-antigens can This process occurring in plants can be exploited to great suppress immune activity by switching on suppressor cells advantage. Complex and ‘high molecular weight’ antigens can of the immune system, producing immunological tolerance. be expressed in different lines of the same plant, optimally as For example, intake of collagen in arthritis patients resulted component parts, and cross-pollination between these lines in relief. This phenomenon is frequently seen in test animals of plants may yield a progeny producing the complex antigen and is time- and dose-dependent. To induce tolerance, in its native conÞ guration, obviating the need for any in vitro antigen is required to be given repeatedly or continuously in manipulation. This would also make enormous economic doses larger than the immunogenic dose. MisidentiÞ cation sense. Another advantage of IgA plantibodies is that major and misadministration of plants/fruits may unknowingly proportion (57%) exists in the dimerized form, in contrast induce tolerance. Hence it becomes essential to identify the to IgA produced in insect cells using Baculovirus, which transgenic plant/fruit and to determine safe, effective doses is mostly monomeric.51 Antibodies expressed in seeds are and feeding schedules, which would determine whether an preferred over antibodies in leaves, which risk early decay, antigen would stimulate or depress immunity. Among the thus requiring immediate extraction. In contrast, antibodies autoimmune disorders that might be prevented or eased are in seeds can be preserved for long at room temperature until type 1 diabetes, multiple sclerosis, rheumatoid arthritis, their extraction or consumption. This has been achieved transplant rejection, etc.1,53 Potatoes expressing insulin and a by linking antibody genes to a genetic “switch.” Injectable protein called GAD (glutamic acid decarboxylase), linked to materials are now being targeted. They are further associated CT-B subunit, were able to suppress immune attack in a mouse with challenges of puriÞ cation.3 HBV antibodies in tobacco strain that would become diabetic and could delay the onset plants are being investigated. A number of other plantibodies of high blood sugar.54 Another diabetes-related protein is also like anti-HIV antibodies are under development. A cream being investigated. Work on multiple sclerosis, rheumatoid containing anti-HIV antibodies, which might help reduce arthritis, lupus and transplant rejection is under progress. For the risk of HIV transmission during sexual intercourse, has autoimmune diseases, production of increased amounts of been proposed. Monoclonal antibodies (Mabs) against STDs self-antigen in plants is still required. like genital herpes have been expressed in soybean. When Recombinant drugs/proteins compared with humanized anti-HSV-2 Mab expressed in mammalian cell culture, they were found to be similar in Apart from vaccines and antibodies, plants are also being stability in human semen and cervical mucus over 24 hours, modiÞ ed by engineered virus inoculation to produce enzymes; with similar efÞ cacy in preventing vaginal HSV-2 infection drugs such as albumin, serum protease and interferon, which in mice.41 are otherwise difÞ cult or expensive to produce - for example, Other Potential Domains glucocerebrosidase (hGC) production in tobacco plants for treating Gaucher’s disease. This development would bring Cancer therapy down its cost thousand-fold.39 Transgenic tobacco plant (www.medknow.com).producing Interleukin-10 is being tested to treat Crohn’s Plants can make monoclonal antibodies for cancer disease. Industrial processes for the large-scale production therapy in sufÞ cient quantities. Soybean has been genetically of recombinant therapeutic proteins in plants have been engineered to make monoclonal antibody (BR-96) as a developed. Other novel compounds include an anti-viral vehicle for targetingThis doxorubicin PDFa site for breast,is hosted available ovarian, bycolon for Medknowprotein free that download inhibits Publications the HIV from virus in vitro, trichosanthin 3,39 and lung tumors. Non-Hodgkin’s lymphoma is also being (ribosome inactivator) and angiotensin-I (antihypertsensitive investigated. drug).39 Transgenic plant-derived biopharmaceutical hirudin Birth control (an antithrombin) is now being commercially produced. Food crops are being increasingly used to produce pharmaceuticals TMV has been designed to express protein found in mouse and drugs. zona pellucida (ZB3 protein).3,39 When injected, resulting antibodies were able to prevent fertilization of eggs in mice. The Future of Edible Vaccines The future of edible vaccines may be affected by resistance Chloroplast transformation to GM foods, which was reß ected when Zambia refused GM The chloroplast genome is maternally inherited; so maize in food aid from the United States despite the threat of following chloroplast transformation, the protein would not be famine.55 Transgenic contamination is unavoidable. Besides present in pollen, thereby reducing the risk of transmission of pollen, transgenes may spread horizontally by sucking insects, the transgene to neighboring crops or weed species by cross- transfer to soil microbes during plant wounding/breakdown pollination.52 It may also result in accumulation of signiÞ cantly of roots/rootlets and may pollute surface and ground water.56

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99 CMYK 100 Indian Journal of Medical Microbiology vol. 25, No. 2

Recently, a GM corn approved only for animal consumption species barriers, like between plant and animal kingdoms, and appeared in human foods. It was found growing from seeds transferring genes by overcoming their defense mechanisms left behind from the previous plantings. Quarantine done to which are physiologically operative against such genetic prevent any further spread caused monetary loss, penalties and assaults.58 Inadvertent birth of a Frankenstein would result in possible criminal charges for alleged violation of the permit unmitigated disaster. Naked DNA vaccines are perhaps the to grow the gene-altered crops.55 This incident shows failure riskiest, as these short pieces of DNA are readily taken up by at an elementary level. Overall, transgenic contamination cells of all species and may become integrated into the cell’s has cost the United States approximately $12 billion. Before genome material. Unlike chemical pollutants, these small endorsing such vaccines for human use, the WHO’s concerns DNA fragments can multiply and mutate indeÞ nitely. Feeding of quality assurance, efficacy and environmental impact GM products like maize to animals may also carry risks, not need to be addressed and GM crops in greenhouse facilities just for the animals but also for human beings consuming the should be rigidly controlled, and they need to be surrounded animal products. The ecological and environmental risks of by protective buffer crops. GM crops have not been able to edible vaccines need to be considered. It is still a very crude signiÞ cantly increase yield or reduce herbicide/pesticide use, science and has a long way to go before it will be ready for as proposed. In a few cases, the instability of transgenic lines large-scale testing in people for combating infectious diseases and transgene inactivation have led to major crop failures. and for autoimmunity. Dangerous/harmful gene products or potent immunogens/ Increase in global area utilized in cultivating transgenic allergens may also be incorporated. Some are reported to crops from 1.7 to 44.2 million hectares from 1996 to 2000 and produce side effects - for example, cytokines induce sickness the number of countries growing them from 6 to 13 reß ects and CNS toxicity; α-interferon causes dementia, neurotoxicity, the growing acceptance of transgenic crops in both industrial mood/cognitive changes, etc. Terminator crops with “suicide” and developing countries.59 At least 350 genetically engineered genes for male sterility, as a means of “containing” the spread pharmaceutical products are currently under development in of transgenes, actually spread both male sterile suicide genes as the United States and Canada. Edible vaccines offer major well as herbicide tolerance genes via pollen. Emerging multiple economic and technical beneÞ ts in the event of bioterrorism, herbicide-tolerant volunteers and weeds (super weeds) and as their production can be easily scaled up for millions of biotech-resistant pests demand the use of highly toxic herbicides doses within a limited period of time (smallpox, anthrax, (atrazine, glufosinate ammonium, glyphosate).57 plague, etc.).41 Few credible studies are there on the safety of GM foods. Conclusion An investigation on GM foods showing ‘growth factor’- like effects in the stomach and small intestine of young rats Edible plant-derived vaccine may lead to a future of safer was not followed up to see whether CaMV-35S promoter and more effective immunization. They would overcome was responsible for this. This promoter is present in most some of the difÞ culties associated with traditional vaccines, commercially grown GM crops today and is especially like production, distribution and delivery, and they can unstable and prone to horizontal gene transfer, recombination be incorporated into the immunisation plans. They have or mutations by random insertion. Random insertion of genes passed the major hurdles in the path of an emerging vaccine can destabilize the genomes of its plant and animal hosts, and technology. Before becoming a reality, the technical obstacles, the effects could ricochet through the neighboring(www.medknow.com). ecosystem. though all seem surmountable, need to be overcome. However, By facilitating horizontal gene transfer/recombination, genetic with limited access to essential health care in much of the engineering may contribute to emergence and re-emergence world and with the scientiÞ c community still struggling with of infectious, drug-resistantThis PDFa diseases, site is hosted riseavailable of autoimmune by forMedknow complexfree downloaddiseases Publications like HIV, malaria,from etc., a cost-effective, safe diseases, cancers and reactivation of dormant viruses. Bacteria and efÞ cacious delivery system in the form of edible vaccines may take up transgenic DNA in food in human gut. Antibiotic will become an essential component in our disease-prevention resistance marker genes can spread from transgenic food to arsenal. pathogenic bacteria, making infections very difÞ cult to treat. Minor genetic changes in pathogens can result in dramatic References changes in host spectrum and disease-causing potentials, 1. Landridge W. Edible vaccines. Scientifi c Am 2000;283:66-71. and inadvertently plants may become their unintentional reservoirs. There is also the risk of creating altogether new 2. Ramsay AJ, Kent SJ, Strugnell RA, Suhrbier A, Thomson SA, Ramshaw IA. Genetic vaccination strategies for enhanced strains of infectious agents, like super viruses. By DNA cellular, humoral and mucosal immunity. Immunol Rev shufß ing, geneticists can create in a matter of minutes in 1999;171:27-44. the laboratory, millions of recombinant viruses that have never existed in billions of years of evolution. This may be 3. Moffat AS. Exploring transgenic plants as a new vaccine source. misused for the intentional creation of bio-weapons. Genetic Science 1995;268:658,660. engineering is inherently hazardous because it involves 4. Available from: http://www.molecularfarming.com/plant- creating vectors/carriers speciÞ cally designed to cross wide derived-vaccines.html.

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5. Webster DE, Thomas MC, Strugnell RA, Dry IB, Wesselingh candidate against hepatitis C virus. Arch Virol 2000;145:2557- SL. Appetising solutions: An edible vaccine for measles. Med J 73. Aust 2002;176:434-7. 23. Modelska A, Dietzschold B, Sleysh N, Fu ZF, Steplewski K, 6. Available from: http://www.niaid.nih.gov/Þ nal/infds/la_infds. Hooper DC, et al. Immunization against rabies with plant- htm. derived antigen. Proc Natl Acad Sci USA 1998;95:2481-5.

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49. Available from: http://www.sciencedaily.com/ Source of Support: Nil, Confl ict of Interest: None declared. releases/1999/09/990913145730.htm.

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