MMG 445 Basic Biotechnology eJournal

Molecular Farming of Vaccines from Transgenic Plants

Ardian Coku
Infectious diseases kill millions world wide every year due to the lack of safe, effective, and accessible vaccines. A novel method for vaccine production using transgenic plants has emerged that has the potential to provide immunization on a global scale particularly if delivered orally. Numerous plant produced vaccines have been researched; such as, vaccines for rabies virus and anthrax, proving the effectiveness of the technology to provide immnunoprotection. While the technology has several advantages over current vaccine production methods, challenges such as differences in glycosylation between mammals and plants have hampered the technologies effectiveness. Nevertheless, the technology is improving gradually and will one day be utilized as a means of human vaccine production. Biochemistry and Molecular Biology, Michigan State University, East Lansing, MI Corressponding author: cokuardi@msu.edu MMG445 Basic Biotechnology eJournal, 2007
This review comes from a themed issue based on current advances in the fields of applied microbiology, biotechnology, and pharmacology. It fulfills in part the assignment of the contributing author in MMG 445, Basic Biotechnology, Department of Microbiology and Molecular Genetics, Fall semester, 2007. Edit by George M. Garrity and Terry L. Marsh Board of Trustees, Michigan State University. All rights reserved.

bacteria offer a low cost solution to producing vaccines, their lack of a glycosylation mechanism severely limits their application in producing vaccines [1]. However, recent advances in genetic modification have allowed for novel ways for plants to be of potential significance in producing a diverse range of pharmaceutically important products including vaccines. This relatively new and diverse field is known as molecular pharming, and it has the potential to make plants into vaccine factories [1]. Plant made vaccines offer significant advantages compared to current vaccine expression systems as well as a few disadvantages (Table 1). This paper will highlight the advances in plant produced vaccines as well as evaluate this new technologys viability as an alternative method of vaccine production.

Comparison to other Vaccine productions methods

In evaluating transgenic plants as a novel method for the production of vaccines, costs and logistics must be taken into consideration. Due to the fact that common agricultural techniques (eg. tiling, planting, irrigation, fertilizing, harvesting) can be used to scale up and sustain transgenic crops, transgenic plants are significantly less expensive when compared to other means of vaccine production such as mammalian cell cultures [1]. While the setup costs are low, 80% of the total production costs are caused by the need to extract and purify the recombinant proteins, which can be quite expensive [1]. However, these costs can be reduced by expressing the protein in watery tissues (eg. tomatoes) which are easier to extract from than dry tissue (eg. wheat) [1]. Nonetheless transgenic cereal crops (eg. wheat) can be stored at room temperature for more than a year without loss of recombinant protein stability and function, thus eliminating the need for refrigeration which has limited the use of current vaccines in third world coun

Abbreviations: CMP - Cytidine 5'monophospho PA - Protective antigen LF - Lethal factor EF - Edema factor TSP - Total Soluble Protein

Introduction
The need for new methods of vaccine production that are cheap, accessible, safe, and effective is of the greatest importance, as infectious disease account for millions of the deaths globally. Current vaccine expression systems such as mammalian cell cultures are insufficient to meet the growing need for vaccines due to a risk of pathogen contamination as well as the high production costs associated with the technology [1]. While

Table 1. Comparison of currently used vectors for recombinant protein production Adapted from Balen et al., 2007, Food Technology and Biotechnology 45:1-10.

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Vector Bacteria Mammalian Cells Transgenic Plants

Cost to Produce Low High Lowmedium

Length of Time to Produce Short Long Long

Glycosylation No Yes Yes (some differences)

Risk of Pathogen Contamination Medium High Low

Cost to Store Moderate Expensive Low

tries [1, 2]. Furthermore, though there are significant differences in the level of protein expression among different plant species, transgenic plants yield greater amounts of protein than do mammalian cell cultures [1, 3]. Plants also provide a greater degree of safety as there is no possibility of contamination by human pathogens that can occur in mammalian cell cultures [3]. While bacteria are able to produce large quantities of proteins at comparably low costs, they are not able to produce glycosylated proteins, whichare needed to produce vaccines for pathogens such as rabies virus. Furthermore, using bacteria to produce recombinant proteins carries the risk of toxins being produced and contaminating the protein products, as in the case of anthrax vaccine production

ases. However, it was found that due to the antigens being encapsulated by the plant tissue they resisted immediate break down [2]. Another concern is the possible side effects of accidental consumption of antigen containing plants. It has been shown in recent years that GM crops not approved for human consumption have contaminated the human food supply as a result of human error in storage and transportation. However, one study determined that ingesting low amounts of a highly immunogenic antigen (20 g), E. coli heat-labile toxin, in maize did not cause any detectable harmful side effects in mice [4]. However, significantly more research must be conducted to determine the side effects of consuming a variety of different antigens of varying antigenicity and at varying doses. Several studies have been carried out to determine the effectiveness of oral plant produced vaccines. In one study it was shown that transgenic rice could be used to provide oral immunoprotection for cholera toxin even after being stored at room temperature for 1.5 years [5]. In another study female mice were fed transgenic alfalfa containing an antigen for a class of rotavirus. Mice born to those females that had eaten the transgenic alfalfa were found to have gained partial passive immunity to a strain of rotavirus [6]. In another recent study, a protein, Tat, necessary for the production of HIV virus was produced in transgenic tomato plants and elicited the production of anti-Tat antibodies in mice that ingested plant tissue that contained the antigen [7]. This shows the immense potential for orally administered plant made vaccines to combat even the most virulent and complex pathogens.

Oral delivery
One of the greatest advantages that plant vaccine production offers is its potential to be used as edible vaccines. Edible vaccines would make mass immunization possible at extremely low costs as no equipment such as needles and personnel would be required [2]. While microorganisms could be used to produce vaccines in such foods as yoghurt and cheese, this would require processing and thus would limit the accessibility of the vaccines as well as increase costs. Furthermore, plants offer a far more nutritious vector for vaccine expression in comparison to any by-products of microorganisms, and thus would be far more suitable for consumption by malnourished populations which are often in the greatest need of vaccinations. A concern with oral delivery of antigens results from the fact that expressed antigens would enter the stomach and thus the antigens could be broken down by prote-

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The Glycosylation problem

Glycosylation is an important posttranslational modification in which sugars are added onto proteins to aide in the activity of the protein, folding, and guarding against denaturation [8]. Glycosylation in plants and animals have many similarities; however, recombinant glycoproteins produced in plants contain sugars which have the potential to induce allergic reactions in a portion of the population [9]. Added onto the N-glycan of mammalian glycoproteins are galactose residues with sialic acids, while plants on the other hand have xylose and/or (1,3)-fucose added onto the N-glycan [8]. As these sugars are not found in animals and humans, there is a real possibility that plant glycosylated recombinant protein would elicit severe allergic reactions. While the majority of the population would not suffer any significant side effects, a small minority of individuals may exhibit allergic reactions as a result of being injected with plant glycoproteins [9]. It has been found that 50% of nonallergic human test subjects had detectable levels of antibodies for either xylose or (1,3)-fucose [9]. While the presence of these antibodies is by itself not definitive evidence that recombinant proteins produced in plants will elicit negative side effects in patients, it may show that plant glycoproteins would be quickly removed from the body and thus inhibit the effectiveness of the proteins [9]. Nevertheless, much research is being conducted to produce plant glycoproteins with exactly the same residues as mammalian glycoproteins. One study demonstrated that plants can be genetically modified to express human enzymes essential for the addition of sialic acid to proteins, CMP-sialic acid synthetase and CMP-sialic acid transporter [8]. CMP-sialic acid synthetase produces the substrate needed by sialyltransferase to add sialic acid to an N-Glyan, and CMPsialic acid transporter sends CMP-sialic acid to the site in the cell where it is involved in protein glycosylation [8]. The study did not produce proteins with sialic acid added to it, as sialyltransferase was not expressed in the plants. However, that missing portion

of the pathway has been expressed in plants in previous studies [8]. Other research has been conducted to eliminate the fucosyl and xylosyl residues from the N-glycans by knocking out the genes that encoded for the enzymes (fucosyltransferase and 1,2xylosyltransferase) necessary to add the residues to the proteins [10]. These knockout mutants also contained a gene encoding for a human recombinant glycoprotein whose production was not affected by the knocking out of the glycosylation enzymes [10].

Current plant made vaccines being pursued

Rabies virus vaccine Rabies is a virus that is fatal in 100% of cases when no treatment is given. Even today, more than 120 years after Louis Pasteurs discovery of an effective rabies treatment, there are 60,000 deaths every year worldwide from rabies [11]. A specific protein of the rabies virus, known as G protein, is the major antigen that causes immunity; however, this protein must undergo glycosylation to provide immunoprotection [11]. Thus, G protein cannot be produced in E. coli, as it has no glycosylation mechanism as animals and plants do [11]. Before the gene that codes for the G protein could be expressed in tobacco plants, the gene was significantly modified to facilitate a higher expression of the protein in plants, such as increasing the GC content of the gene and inserting a promoter [11]. Agrobacterium tumefaciens was used to insert the gene that encodes for the antigen as well as a gene that encodes for herbicide resistance, which allows for the selection for plants that have only been transformed with the T-DNA [2]. ELISA was used to screen for tobacco plants that expressed G protein. Tobacco plants found to express G protein were harvested for leaf tissue and the protein was purified [11]. A general overview of the production of plant made vaccines can be found in Figure 1. To determine if glycosylation of the plant made G protein was comparable to that of the rabies G protein Western blot was performed and found that the plant produced G

Figure 1. Flow diagram of the general procedures used to produce plant made vaccines.

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protein cross reacted with anti-G antibodies [11]. Furthermore, the G protein produced by plants had the ability to bind to a concanavalin A column, which is used to bind glycoproteins with specific sugars on their glycan groups [11]. The effect to which the plant vaccine was able to invoke an immune response was performed by injecting one group of mice with the plant produced G protein, injecting another group with a commercially available inactivated rabies vaccine from rabbits, and injecting a control group with a buffer [11]. The results demonstrated the plant made vaccine produced a higher level of antibodies for the rabies virus than the commercially available rabies vaccine (Fig. 2). Mice from each group were then injected with 10LD50 of rabies virus [11]. The tobacco produced vaccine and the

commercial vaccine both immunized the mice completely and so the mice in those groups had a 100% survival rate, while all the mice in the control group died within 14 days of being injected with the virus.

Anthrax vaccine
Bacillus anthracis is perhaps the most feared bacterial pathogen due its high fatality as well as its ability to be used as a biological weapon [12]. The toxins PA, LF, and EF are responsible for the pathogenicity of deadly strains of Bacillus anthracis [12]. However, PA, encoded by the gene pagA, is the protein that causes the immune system to produce antibodies against anthrax and is thus the primary component of current anthrax vaccines [12]. However, due to the nature of the production of current anthrax vaccines

Figure 2. Level of antibodies measured in mice injected with a protein derived from rabies virus. Antibody

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levels were measured after the second ( ) and third ( ) round of injections. Antibody levels were measured from samples that were taken from pre-immunization serum (PIS), control mice (CON), mice injected with the plant derived protein (PDP), and mice injected with commercially available rabies vaccine (V). From Ashraf et al., 2005, Journal of Biotechnology 119:1-14.

they also contain minute amounts of LF and EF, which can cause severe side effects [12]. The pagA was chosen to be expressed in Nicotiana tabacum through chloroplast transformation. Unlike nuclear transformations, chloroplast transformations are performed by bombarding particles, covered in DNA at plant embryos. Producing proteins in chloroplasts has several advantages, such as there being more copies of the gene of interest as there are roughly 10,000 copies of chloroplast DNA in every cell [12]. This results in 1025 times higher protein expres sion through chloroplast transformation when compared to nuclear transformation [13]. Furthermore, the translation of T-DNA is significantly higher in transgenic chloroplasts in comparison to nuclear transgenic plants due to a lack of gene silencing [12]. In addition, transforming only the chloroplast DNA greatly reduces the risk of a transgene proliferating throughout the environment due to the fact that chloroplast DNA is not transmitted by pollen [12].

A plasmid containing the pagA gene was coated onto gold particles which were then bombarded into Nicotiana tabacum leaves and after a short incubation period, the leaves were cut into pieces and then placed onto a medium containing growth hormones [12]. To determine the amount of PA being produced, ELISA was performed and it was found that up to 14% of the TSP in leaves was PA [12]. Remarkably, this correlates to being able to extract 1.8 mg of PA for every gram of leaf tissue, and thus, 1 acre of land can generate enough leaf tissue to be able to produce 360 million doses of anthrax vaccine [12]. In order to determine the effectiveness of the plant produced PA as a vaccine, seven groups of mice were injected with chloroplast PA (with and without adjuvant), crude chloroplast PA with adjuvant, Bacillus anthracis PA (with and without adjuvant), and crude wild type with adjuvant and a saline solution to serve as controls [12]. Each mouse was then injected with 1.5LD100 of LT. The results of the challenge were that all the mice immunized with PA from Nicotiana
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tabacum with adjuvant survived the challenge, and the mice immunized with unpurified extract of plant PA had a survival rate of 80% [12]. However, none of the mice immunized with the plant PA without adjuvant survived [12]. The results of this study demonstrate the high potential plants have to mass produce effective vaccines against deadly pathogens.

ally being overcome. Thus, the technology is highly promising and will in all likelihood be a major contender in vaccine production for the 21st century.

Acknowledgements
I would like to thank Dr. Garrity and Dr. Marsh for giving me the opportunity to research a topic in biotechnology of great personal interest. I would also like to thank my classmates for helping me to improve my manuscript by critiquing it.

Market readiness
One of the main challenges that plant made vaccines will face in becoming commercially available, is gaining approval from regulatory agencies [1]. Unlike, vaccines produced by conventional means, plant made vaccines must go through lengthy and rigorous approval processes from both the FDA and USDA [1]. Nevertheless, much research has gone on in both academia and the industrial sector, resulting in 45 antigens being expressed in plants from 1992-2003 [1]. Furthermore, a small number of plant made vaccines, such as vaccines for Norwalk virus, Hepatitis B, and rabies are currently in Phase I clinical trials [2]. However, major developments still need to be made to make these vaccines more cheaply produced and easy to distribute [2]. Nonetheless, plant made vaccines have proven to be effective and with further intensive research the technology will undoubtedly enter the market within the next 20 years.

References and recommended reading

Papers of special significance that have been published within the period of review are highlighted as follows: of significance of special significance 1. Schillberg S, Twyman RM, Fischer R: Opportunities for recombinant antigen and antibody expression in transgenic plants - technology assessment. Vaccine 2005, 23:1764-1769. This was an excellent review that provided details of the logistics and costs of the technology as well as provide an introduction of the field of molecular farming. It also provided information about the federal regulation of the technology. 2. Streatfield, SJ: Mucosal immunization using recombinant plant-based oral vaccines. Methods 2006, 38:150-157. This review article provided crucial information about the different aspects of oral delivery of plant made vaccines. It also provided information about the current plant made vaccines currently being researched for human use. 3. Balen B, Krsnik-Rasol M, Lt: Nglycosylation of recombinant therapeutic glycoproteins in plant systems. Food Technology and Biotechnology 2007, 45:1-10. This review article provided important comparisons between plant made recombinant proteins and current systems of recombinant protein production. This information was crucial to provide an evaluation of the technology as a alternative method of vaccine production. 4. Beyer AJ, Wang K, Umble AN, Wolt JD, Cunnick JE: Low-dose exposure and immunogenicity of transgenic maize expressing the Escherichia coli heatlabile toxin B subunit. Environmental Health Perspectives 2007, 115: 354-360. Nochi T, Takagi H, Yuki Y, Yang LJ, Masumura T, Mejima M, Nakanishi U, Matsumura A, Uozumi A, Hiroi T, et al.: Ricebased mucosal vaccine as a global strategy for cold-chain- and needle-free

Conclusion
Plant made vaccines have demonstrated that they offer a safe, cheap, effective, and highly accessible alternative to current methods of vaccine production. Intensive research has been done to show the vast potential that the technology has in saving hundreds of thousands or perhaps millions of lives every year. The technology has also shown how robust and versatile it is as an array of pathogens, plant species, and transformation technology can be used to produce vaccines in plants. The technology has shown to have numerous advantages over current methods of vaccine production as well as a few disadvantages. While the challenges facing plant made vaccines have inhibited efforts to make the technology viable for human use, these challenges are gradu-

5.

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vaccination. Proceedings of the National Academy of Sciences of the United States of America 2007, 104:10986-10991. 6. Dong JL, Liang BG, Jin YS, Zhang WJ, Wang T: Oral immunization with pBsVP6transgenic alfalfa protects mice against rotavirus infection. Virology 2005, 339:153-163. Ramirez YJP, Tasciotti E, Gutierrez-Ortega A, Torres AJD, Flores MTO, Giacca M, Lim MAG, Hk: Fruit-specific expression of the human immunodeficiency virus type 1 Tat gene in tomato plants and its immunogenic potential in mice. Clinical and Vaccine Immunology 2007, 14:685-692.

Hoffmann A, Kopriva S, Gorr G, Reski R, Decker EL: Targeted knockouts of Physcomitrella lacking plantspecific immunogenic N-glycans. Plant Biotechnology Journal 2004, 2:416-423.

11. Ashraf S, Singh PK, Yadav DK, Shahnawaz

7.

M, Mishra S, Sawant SV, Tuli R: High level expression of surface glycoprotein of rabies virus in tobacco leaves and its immunoprotective activity in mice. Journal of Biotechnology 2005, 119:1-14.

Misaki R, Fujiyama K, Seki T: Expression of human CMP-N-acetylneuraminic acid synthetase and CMP-sialic acid transporter in tobacco suspensioncultured cell. Biochemical and Biophysical Research Communications 2006, 339:1184-1189. This article provided research in solving an important problem facing plant-made vaccines. It showed that the challenges with the technology can be overcome and thus has the potential to be used for human vaccines. 9. Bardor M, Faveeuw C, Fitchette AC, Gilbert D, Galas L, Trottein F, Faye L, Lerouge P: Immunoreactivity in mammals of two typical plant glyco-epitopes, core alpha (1,3)-fucose and core xylose. Glycobiology 2003, 13:427-434.

12 Koya V, Moayeri M, Leppla SH, Daniell H: Plant-based vaccine: Mice immunized with chloroplast-derived anthrax protective antigen survive anthrax lethal toxin challenge. Infection and Immunity 2005, 73:8266-8274. This research article showed the immense potential plant made vaccines have in creating vast quantities of safe and effective vaccines against deadly pathogens. Furthermore, it provided information about the important use of chloroplast transformations in enhancing protein yields and making transgenic plants safe for the environment. 13. Soria-Guerra RE, Rosales-Mendoza S, Marquez-Mercado C, Lopez-Revilla R, Castillo-Collazo R, Alpuche-Solis NG, Eh: Transgenic tomatoes express an antigenic polypeptide containing epitopes of the diphtheria, pertussis and tetanus exotoxins, encoded by a synthetic gene. Plant Cell Reports 2007, 26:961-968.

10. Koprivova A, Stemmer C, Altmann F,

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