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Molecular Farming of Vaccines from Transgenic Plants

Ardian Coku

Infectious diseases kill millions world wide everyyear due to the lack of safe, effective, andaccessible vaccines. A novel method for vaccineproduction using transgenic plants has emergedthat has the potential to provide immunization ona global scale particularly if delivered orally.Numerous plant produced vaccines have beenresearched; such as, vaccines for rabies virusand anthrax, proving the effectiveness of thetechnology to provide immnunoprotection. Whilethe technology has several advantages overcurrent vaccine production methods, challengessuch as differences in glycosylation betweenmammals and plants have hampered thetechnologies effectiveness. Nevertheless, thetechnology is improving gradually and will one

day be utilized as a means of human vaccineproduction.

Biochemistry and Molecular Biology, MichiganState University, East Lansing, MICorressponding author: [email protected]

Abbreviations:CMP - Cytidine 5'monophosphoPA - Protective antigenLF - Lethal factorEF - Edema factorTSP - Total Soluble Protein

IntroductionThe need for new methods of vaccine pro-duction that are cheap, accessible, safe, and

effective is of the greatest importance, asinfectious disease account for millions of thedeaths globally. Current vaccine expressionsystems such as mammalian cell cultures areinsufficient to meet the growing need forvaccines due to a risk of pathogen contami-nation as well as the high production costsassociated with the technology [1]. While

MMG445 Basic Biotechnology eJournal, 2007

This review comes from a themed issue based oncurrent advances in the fields of applied microbiology,biotechnology, and pharmacology. It fulfills in part theassignment of the contributing author in MMG 445,Basic Biotechnology, Department of Microbiology andMolecular 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 produc-ing vaccines, their lack of a glycosylationmechanism severely limits their applicationin producing vaccines [1]. However, recentadvances in genetic modification have al-lowed for novel ways for plants to be of po-tential significance in producing a diverserange of pharmaceutically important prod-ucts including vaccines. This relatively newand diverse field is known as molecular

pharming, and it has the potential to make plants into vaccine factories [1]. Plantmade vaccines offer significant advantages

compared to current vaccine expression sys-tems as well as a few disadvantages (Table1). This paper will highlight the advances in

plant produced vaccines as well as evaluatethis new technologys viability as an alterna-tive method of vaccine production.

Comparison to other Vaccineproductions methodsIn evaluating transgenic plants as a novelmethod for the production of vaccines, costsand logistics must be taken into considera-

tion. Due to the fact that common agricul-tural techniques (eg. tiling, planting, irriga-tion, fertilizing, harvesting) can be used toscale up and sustain transgenic crops, trans-genic plants are significantly less expensivewhen compared to other means of vaccine

production such as mammalian cell cultures[1]. While the setup costs are low, 80% ofthe total production costs are caused by theneed to extract and purify the recombinant

proteins, which can be quite expensive [1].However, these costs can be reduced by ex-

pressing the protein in watery tissues (eg.

tomatoes) which are easier to extract fromthan dry tissue (eg. wheat) [1]. Nonethe-less transgenic cereal crops (eg. wheat) can

be stored at room temperature for more thana year without loss of recombinant proteinstability and function, thus eliminating theneed for refrigeration which has limited theuse of current vaccines in third world coun

Table 1. Comparison of currently used vectors for recombinant protein production Adapted from Balen et al.,2007, Food Technology and Biotechnology45:1-10.

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tries [1, 2]. Furthermore, though there aresignificant differences in the level of proteinexpression among different plant species,transgenic plants yield greater amounts of

protein than do mammalian cell cultures [1,3]. Plants also provide a greater degree ofsafety as there is no possibility of contami-nation by human pathogens that can occur in

mammalian cell cultures [3]. While bacteriaare able to produce large quantities of pro-teins at comparably low costs, they are notable to produce glycosylated proteins,whichare needed to produce vaccines for

pathogens such as rabies virus. Furthermore,using bacteria to produce recombinant pro-teins carries the risk of toxins being pro-duced and contaminating the protein prod-ucts, as in the case of anthrax vaccine pro-duction

Oral deliveryOne of the greatest advantages that plantvaccine production offers is its potential to

be used as edible vaccines. Edible vaccineswould make mass immunization possible atextremely low costs as no equipment such asneedles and personnel would be required[2]. While microorganisms could be used to

produce vaccines in such foods as yoghurtand cheese, this would require processingand thus would limit the accessibility of thevaccines as well as increase costs. Further-more, plants offer a far more nutritious vec-

tor for vaccine expression in comparison toany by-products of microorganisms, andthus would be far more suitable for con-sumption by malnourished populationswhich are often in the greatest need of vac-cinations. A concern with oral delivery ofantigens results from the fact that expressedantigens would enter the stomach and thusthe antigens could be broken down by prote-

ases. However, it was found that due to theantigens being encapsulated by the planttissue they resisted immediate break down[2]. Another concern is the possible sideeffects of accidental consumption of antigencontaining plants. It has been shown in re-cent years that GM crops not approved forhuman consumption have contaminated the

human food supply as a result of human er-ror in storage and transportation. However,one study determined that ingesting lowamounts of a highly immunogenic antigen(20 g), E. coli heat-labile toxin, in maizedid not cause any detectable harmful sideeffects in mice [4]. However, significantlymore research must be conducted to deter-mine the side effects of consuming a varietyof different antigens of varying antigenicityand at varying doses.

Several studies have been carried out to de-

termine the effectiveness of oral plant pro-duced vaccines. In one study it was shownthat transgenic rice could be used to provideoral immunoprotection for cholera toxineven after being stored at room temperaturefor 1.5 years [5]. In another study femalemice were fed transgenic alfalfa containingan antigen for a class of rotavirus. Mice bornto those females that had eaten the trans-genic alfalfa were found to have gained par-tial passive immunity to a strain of rotavirus[6]. In another recent study, a protein, Tat,

necessary for the production of HIV viruswas produced in transgenic tomato plantsand elicited the production of anti-Tat anti-

bodies in mice that ingested plant tissue thatcontained the antigen [7]. This shows theimmense potential for orally administered

plant made vaccines to combat even themost virulent and complex pathogens.

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VectorCost toProduce

Length ofTime toProduce

GlycosylationRisk of Pathogen

ContaminationCost toStore

Bacteria Low Short No Medium Moderate

MammalianCells

High Long Yes High Expensive

TransgenicPlants

Low-medium

LongYes (somedifferences)

Low Low

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The Glycosylation problemGlycosylation is an important post-translational modification in which sugarsare added onto proteins to aide in the activ-ity of the protein, folding, and guardingagainst denaturation [8]. Glycosylation in

plants and animals have many similarities;however, recombinant glycoproteins pro-duced in plants contain sugars which havethe potential to induce allergic reactions in a

portion of the population [9]. Added onto the N-glycan of mammalian glycoproteins aregalactose 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 andhumans, there is a real possibility that plantglycosylated recombinant protein wouldelicit severe allergic reactions. While the

majority of the population would not sufferany significant side effects, a small minorityof individuals may exhibit allergic reactionsas a result of being injected with plant gly-coproteins [9]. It has been found that 50% ofnonallergic human test subjects had detect-able levels of antibodies for either xylose or (1,3)-fucose [9]. While the presence ofthese antibodies is by itself not definitiveevidence that recombinant proteins producedin plants will elicit negative side effects in

patients, it may show that plant glycopro-teins would be quickly removed from the

body and thus inhibit the effectiveness of theproteins [9].

Nevertheless, much research is being con-ducted to produce plant glycoproteins withexactly the same residues as mammalianglycoproteins. One study demonstrated that

plants can be genetically modified to expresshuman enzymes essential for the addition ofsialic acid to proteins, CMP-sialic acid syn-thetase and CMP-sialic acid transporter[8]. CMP-sialic acid synthetase produces

the substrate needed by sialyltransferase toadd sialic acid to an N-Glyan, and CMP-sialic acid transporter sends CMP-sialic acidto the site in the cell where it is involved in

protein glycosylation [8]. The study didnot produce proteins with sialic acid addedto it, as sialyltransferase was not expressedin the plants. However, that missing portion

of the pathway has been expressed in plantsin previous studies [8]. Other research has

been conducted to eliminate the fucosyl andxylosyl residues from the N-glycans byknocking out the genes that encoded for theenzymes (fucosyltransferase and 1,2-

xylosyltransferase) necessary to add theresidues to the proteins [10]. These knock-out mutants also contained a gene encodingfor a human recombinant glycoproteinwhose production was not affected by theknocking out of the glycosylation enzymes[10].

Current plant made vaccinesbeing pursuedRabies virus vaccineRabies is a virus that is fatal in 100% ofcases when no treatment is given. Even to-day, more than 120 years after Louis Pas-teurs discovery of an effective rabies treat-ment, there are 60,000 deaths every yearworldwide from rabies [11]. A specific pro-tein of the rabies virus, known as G protein,is the major antigen that causes immunity;however, this protein must undergo glycosy-lation to provide immunoprotection [11].Thus, G protein cannot be produced in E.coli, as it has no glycosylation mechanism asanimals and plants do [11]. Before the genethat codes for the G protein could be ex-

pressed in tobacco plants, the gene was sig-nificantly modified to facilitate a higher ex-pression of the protein in plants, such as in-creasing the GC content of the gene and in-serting a promoter [11]. Agrobacterium tu-mefaciens was used to insert the gene thatencodes for the antigen as well as a genethat encodes for herbicide resistance, whichallows for the selection for plants that haveonly been transformed with the T-DNA [2].

ELISA was used to screen for tobacco plantsthat expressed G protein. Tobacco plants

found to express G protein were harvestedfor leaf tissue and the protein was purified[11]. A general overview of the productionof plant made vaccines can be found in Fig-ure 1. To determine if glycosylation of the

plant made G protein was comparable to thatof the rabies G protein Western blot was per-formed and found that the plant produced G

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

Molecular farming of vaccines 112

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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 conca-navalin A column, which is used to bindglycoproteins with specific sugars on theirglycan groups [11]. The effect to which the

plant vaccine was able to invoke an immuneresponse was performed by injecting onegroup of mice with the plant produced G

protein, injecting another group with acommercially available inactivated rabies

vaccine from rabbits, and injecting a controlgroup with a buffer [11]. The results dem-onstrated the plant made vaccine produced ahigher level of antibodies for the rabies virusthan the commercially available rabies vac-cine (Fig. 2). Mice from each group werethen injected with 10LD50 of rabies virus[11]. The tobacco produced vaccine and the

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

Anthrax vaccineBacillus anthracis is perhaps the most feared bacterial pathogen due its high fatality aswell as its ability to be used as a biologicalweapon [12]. The toxins PA, LF, and EF areresponsible for the pathogenicity of deadlystrains ofBacillus anthracis [12]. However,PA, encoded by the gene pagA, is the pro-tein that causes the immune system to pro-duce antibodies against anthrax and is thusthe primary component of current anthraxvaccines [12]. However, due to the natureof 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 weremeasured from samples that were taken from pre-immunization serum (PIS), control mice (CON), miceinjected with the plant derived protein (PDP), and mice injected with commercially available rabies vaccine(V). From Ashraf et al., 2005, Journal of Biotechnology119:1-14.

they also contain minute amounts of LF andEF, which can cause severe side effects[12].

The pagA was chosen to be expressed in Nicotiana tabacum through chloroplasttransformation. Unlike nuclear transforma-tions, chloroplast transformations are per-formed by bombarding particles, covered inDNA at plant embryos. Producing proteinsin chloroplasts has several advantages, suchas there being more copies of the gene ofinterest as there are roughly 10,000 copies ofchloroplast DNA in every cell [12]. Thisresults in 1025 times higher protein expression through chloroplast transformationwhen compared to nuclear transformation

[13]. Furthermore, the translation of T-DNAis significantly higher in transgenic chloroplasts in comparison to nuclear transgenicplants due to a lack of gene silencing [12].In addition, transforming only the chloro-

plast DNA greatly reduces the risk of atransgene proliferating throughout the envi-ronment due to the fact that chloroplastDNA is not transmitted by pollen [12].

A plasmid containing the pagA gene wascoated onto gold particles which were then

bombarded into Nicotiana tabacum leavesand after a short incubation period, theleaves were cut into pieces and then placedonto a medium containing growth hormones[12]. To determine the amount of PA being

produced, ELISA was performed and it wasfound that up to 14% of the TSP in leaveswas PA [12]. Remarkably, this correlates to

being able to extract 1.8 mg of PA for everygram of leaf tissue, and thus, 1 acre of landcan generate enough leaf tissue to be able to

produce 360 million doses of anthrax vac-cine [12].

In order to determine the effectiveness of the

plant produced PA as a vaccine, sevengroups of mice were injected with chloro-plast PA (with and without adjuvant), crudechloroplast PA with adjuvant, Bacillus an-thracis PA (with and without adjuvant), andcrude wild type with adjuvant and a salinesolution to serve as controls [12]. Eachmouse was then injected with 1.5LD100 ofLT. The results of the challenge were that allthe mice immunized with PA fromNicotiana



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tabacum with adjuvant survived the chal-lenge, and the mice immunized with unpuri-fied extract of plant PA had a survival rate of80% [12]. However, none of the mice im-munized with the plant PA without adjuvantsurvived [12]. The results of this study

demonstrate the high potential plants have tomass produce effective vaccines againstdeadly pathogens.

Market readinessOne of the main challenges that plant madevaccines will face in becoming commer-cially available, is gaining approval fromregulatory agencies [1]. Unlike, vaccines

produced by conventional means, plantmade vaccines must go through lengthy andrigorous approval processes from both theFDA and USDA [1]. Nevertheless, much

research has gone on in both academia andthe industrial sector, resulting in 45 antigens

being expressed in plants from 1992-2003[1]. Furthermore, a small number of plantmade vaccines, such as vaccines for Nor-walk virus, Hepatitis B, and rabies are cur-rently in Phase I clinical trials [2]. How-ever, major developments still need to bemade to make these vaccines more cheaply

produced and easy to distribute [2]. None-theless, plant made vaccines have proven to

be effective and with further intensive re-search the technology will undoubtedly en-ter the market within the next 20 years.

ConclusionPlant made vaccines have demonstrated thatthey offer a safe, cheap, effective, andhighly accessible alternative to currentmethods of vaccine production. Intensiveresearch has been done to show the vast po-tential that the technology has in savinghundreds of thousands or perhaps millionsof lives every year. The technology has alsoshown how robust and versatile it is as an

array of pathogens, plant species, and trans-formation technology can be used to pro-duce vaccines in plants. The technology hasshown to have numerous advantages overcurrent methods of vaccine production aswell as a few disadvantages. While the chal-lenges facing plant made vaccines have in-hibited efforts to make the technology viablefor human use, these challenges are gradu-

ally being overcome. Thus, the technology ishighly promising and will in all likelihood

be a major contender in vaccine productionfor the 21st century.

Acknowledgements

I would like to thank Dr. Garrity and Dr. Marsh forgiving me the opportunity to research a topic in

biotechnology of great personal interest. I would alsolike to thank my classmates for helping me to improvemy manuscript by critiquing it.

References and recommendedreadingPapers of special significance that have beenpublished within the period of review arehighlighted as follows: of significance of special significance

1. Schillberg S, Twyman RM, Fischer R:

Opportunities for recombinant antigenand antibody expression in transgenicplants - technology assessment. Vaccine2005, 23:1764-1769.

This was an excellent review that provided detailsof the logistics and costs of the technology aswell as provide an introduction of the field ofmolecular farming. It also provided informationabout the federal regulation of the technology.

2. Streatfield, SJ: Mucosal immunizationusing recombinant plant-based oralvaccines. Methods 2006, 38:150-157.

This review article provided crucial informationabout the different aspects of oral delivery ofplant made vaccines. It also provided informationabout the current plant made vaccines currentlybeing researched for human use.

3. Balen B, Krsnik-Rasol M, Lt: N-glycosylation of recombinant therapeuticglycoproteins in plant systems. FoodTechnology and Biotechnology2007,45:1-10.

This review article provided importantcomparisons between plant made recombinantproteins and current systems of recombinantprotein production. This information was crucial toprovide an evaluation of the technology as aalternative method of vaccine production.

4. Beyer AJ, Wang K, Umble AN, Wolt JD,Cunnick JE: Low-dose exposure andimmunogenicity of transgenic maizeexpressing the Escherichia coliheat-labile toxin B subunit.EnvironmentalHealth Perspectives 2007, 115: 354-360.

5. Nochi T, Takagi H, Yuki Y, Yang LJ,Masumura T, Mejima M, Nakanishi U,Matsumura A, Uozumi A, Hiroi T, et al.: Rice-based mucosal vaccine as a globalstrategy for cold-chain- and needle-free

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vaccination. Proceedings of the NationalAcademy of Sciences of the United States ofAmerica 2007, 104:10986-10991.

6. Dong JL, Liang BG, Jin YS, Zhang WJ,Wang T: Oral immunization with pBsVP6-transgenic alfalfa protects mice againstrotavirus infection. Virology2005,

339:153-163.

7. Ramirez YJP, Tasciotti E, Gutierrez-OrtegaA, Torres AJD, Flores MTO, Giacca M, LimMAG, Hk: Fruit-specific expression of thehuman immunodeficiency virus type 1 Tatgene in tomato plants and itsimmunogenic potential in mice. Clinicaland Vaccine Immunology2007, 14:685-692.

8 Misaki R, Fujiyama K, Seki T: Expressionof human CMP-N-acetylneuraminic acidsynthetase and CMP-sialic acidtransporter in tobacco suspension-cultured cell.Biochemical and BiophysicalResearch Communications 2006,

339:1184-1189.This article provided research in solving animportant problem facing plant-made vaccines. Itshowed that the challenges with the technologycan be overcome and thus has the potential to beused for human vaccines.

9. Bardor M, Faveeuw C, Fitchette AC, GilbertD, Galas L, Trottein F, Faye L, Lerouge P:Immunoreactivity in mammals of twotypical plant glyco-epitopes, core alpha(1,3)-fucose and core xylose. Glycobiology2003, 13:427-434.

10. Koprivova A, Stemmer C, Altmann F,

Hoffmann A, Kopriva S, Gorr G, Reski R,Decker EL: Targeted knockouts ofPhyscomitrella lacking plantspecificimmunogenic N-glycans. PlantBiotechnology Journal2004, 2:416-423.

11. Ashraf S, Singh PK, Yadav DK, ShahnawazM, Mishra S, Sawant SV, Tuli R: High level

expression of surface glycoprotein ofrabies virus in tobacco leaves and itsimmunoprotective activity in mice. Journal

of Biotechnology2005, 119:1-14.

12 Koya V, Moayeri M, Leppla SH, Daniell H:Plant-based vaccine: Mice immunized withchloroplast-derived anthrax protectiveantigen survive anthrax lethal toxinchallenge.Infection and Immunity2005,73:8266-8274.

This research article showed the immensepotential plant made vaccines have in creatingvast quantities of safe and effective vaccinesagainst deadly pathogens. Furthermore, it

provided information about the important use ofchloroplast transformations in enhancing proteinyields and making transgenic plants safe for theenvironment.

13. Soria-Guerra RE, Rosales-Mendoza S,Marquez-Mercado C, Lopez-Revilla R,Castillo-Collazo R, Alpuche-Solis NG, Eh:Transgenic tomatoes express anantigenic polypeptide containingepitopes of the diphtheria, pertussis andtetanus exotoxins, encoded by asynthetic gene.Plant Cell Reports 2007,26:961-968.



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