Plant-based vaccines

International Journal for Parasitology 33 (2003) 479–493 www.parasitology-online.com

Invited review

Plant-based vaccines Stephen J. Streatfield*, John A. Howard1 ProdiGene, Inc., 101 Gateway Boulevard, College Station, TX 77845, USA Received 19 July 2002; received in revised form 3 February 2003; accepted 3 February 2003

Abstract Plant systems are reviewed with regard to their ability to express and produce subunit vaccines. Examples of different types of expression systems producing a variety of vaccine candidates are illustrated. Many of these subunit vaccines have been purified and shown to elicit an immune response when injected into animal models. This review also includes vaccines that have been administered orally in a non-purified form as a food or feed product. Cases are highlighted which demonstrate that orally delivered plant-based vaccines can elicit immune responses and in some case studies, confer protection. Examples are used to illustrate some of the inherent advantages of a plant-based system, such as cost, ease of scale-up and convenience of delivery. Also, some of the key steps are identified that will be necessary to bring these new vaccines to the market. q 2003 Australian Society for Parasitology Inc. Published by Elsevier Science Ltd. All rights reserved. Keywords: Plant expression; Plant virus; Protective immunity; Subunit vaccine; Transgenic plant; Vaccine production

1. Introduction Vaccines have been used very effectively over decades to prevent the spread and in some countries, even to eradicate diseases. While their use has great potential, it has been limited severely by a number of practical impediments. These include identifying suitable vaccines, producing them in a cost-effective manner and providing a convenient way to deliver them. The use of oral vaccines can alleviate some of the practical barriers regarding delivery. Oral vaccines can remove the dependence on injections, which require specialised medical assistance, as well as the need for highly purified material. Having an oral vaccine can make irrelevant the fright most children (and adults) have of needles and can improve accessibility in many developing countries due to abolishing the need for a cold chain. These convenience factors could then lead to better compliance for patients, both in developing and developed countries. The development of a polio vaccine is an example of an oral vaccine programme, which has demonstrated wide acceptance. However, despite of the overwhelming * Corresponding author. Tel.: þ 1-979-690-8537; fax: þ1-979-690-9527. E-mail addresses: [email protected] (S.J. Streatfield), [email protected] (J.A. Howard). 1 Tel.: þ1-979-690-8537; fax: þ 1-979-690-9527.

advantages of oral delivery, the vast majority of vaccines being developed today are injectable. The reason is that solutions to the technical challenges for oral delivery have eluded researchers. Some of these challenges are (1) being able to identify subunit or modified virus vaccines which will provide protection; (2) being able to produce the vaccines in a suitable host which is cost-effective and (3) making large quantities of the vaccine. Since oral vaccines must survive the digestive tract, much larger quantities are needed than for injectables. Therefore, a 100 kg injectable vaccine product that costs $1 per dose for the raw material may now cost $1,000 per dose and require 100,000 kg as an oral vaccine. This illustrates the technical problems in obtaining both low cost and large volumes. These limitations are magnified when recombinant DNA technology is used to produce subunit vaccines. While subunit vaccines have the advantage of being inherently safe, it can be more difficult to obtain expression of the proteins in the absence of the virus or native organism. Although microorganisms are a cost-effective system for protein production, many of the proteins that are used in vaccines are glycosylated. Bacteria do not glycosylate, yeasts may hyper-glycosylate and both systems may express the protein poorly (Marino, 1991; Harashima, 1994; Archer, 1993). Mammalian cell cultures are the preferred choice with respect to post-translational modification; however,

0020-7519/03/$30.00 q 2003 Australian Society for Parasitology Inc. Published by Elsevier Science Ltd. All rights reserved. doi:10.1016/S0020-7519(03)00052-3

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the costs are approximately 100 times that of microbial fermentation, making them impractical production systems for the large quantities of subunit vaccines required for oral delivery. Transgenic animals are another possibility, with subunit vaccine candidates produced in milk. However, to date, the practical problems of commercialising such systems have not been solved. Recently, plants have been used as recombinant biofactories to express a number of proteins including pharmaceuticals and potential vaccines (Kusnadi et al., 1997; Jilka et al., 1999; Daniell et al., 2001a; Hood and Howard, 2002; Carter and Langridge, 2002; Korban, 2002). Plants offer some distinct advantages over other systems. These include: (1) the ability to carry out post-translational modifications similar to other higher eukaryotes; (2) the ability to rapidly scale-up and produce extremely large quantities; (3) the potential for greatly reduced costs of raw material and (4) the reduced fear of using plants, which do not harbour human pathogens. Food-based crops offer additional advantages if the vaccine can be delivered orally, by eliminating purification steps and delivering the product in a safe and palatable formulation. If a grain crop is used, the potential to store the vaccine at ambient temperature without adverse effects may remove the need for a cold chain (Kusnadi et al., 1998). It has also been shown that plants can express multiple transgenes at one time (Howard, unpublished). Therefore, the potential for using one formulation to deliver several vaccines is very practical in plants. The advantages of using this type of plant system for oral delivery compared to traditional vaccines are summarised in Table 1. Assuming plants can overcome previous limitations for subunit vaccines, there is still the challenge of developing a new regulatory path and social acceptance. The critical features that are needed in product development and production for orally delivered vaccines produced in plants to become established are outlined in this review.

Table 1 Potential advantages of plant-based oral vaccines Lower cost of raw material Rapid scale-up Multiple vaccines may be produced together Convenient storage of raw material Reduced need for a cold chain during transport Convenience of delivery in food or feed type products Reduced concerns over human pathogen contamination in vaccine preparations Eliminate cost of syringes and needles Reduced need of medical assistance in administration Eliminate fear of vaccination via injection, particularly for children Eliminate concern over blood borne diseases through needle reuse

2. Proof of concept 2.1. Antigen expression Since subunit vaccines consist of one or more defined proteins that are generally purified and then delivered at a set dose, they constitute a relatively simple and uniform material for administration. Antigens constituting subunit vaccines are generally prepared from recombinant sources. For example, many hepatitis B subunit vaccines are prepared from a recombinant cell surface antigen purified from yeast. The first critical point in the development of vaccines in plants is, can plants express the relevant proteins? There are now many examples demonstrating the successful expression of subunit vaccine candidates in transgenic plants (Table 2). These include antigens from bacterial and viral sources that infect humans, domestic or wild animals. The antigens represent several classes of protein, including secreted toxins and cell or viral coat surface antigens that may include membrane embedded regions. Levels of expression vary greatly depending on the protein expressed and the species of plant used to achieve expression (Table 2). Also, the specifics of the expression system deployed have been shown to strongly influence the level of antigen produced. Factors such as the organellar genome (nuclear vs. plastid) used for expression (Daniell et al., 2001b), strength and tissue specificity of the promoter (Chikwamba et al., 2002a), choice of untranslated leader and message processing sequences (Richter et al., 2000) and targeting of the expressed protein to a particular organelle within the cell (Richter et al., 2000; Streatfield et al., 2003), all affect the yield. The related technology of expressing selected antigens from plant viral genomes and infecting the target plant with these recombinant viruses has also been successful in producing subunit vaccine candidates in plant tissues (Table 2). Approximately 1 mg of recombinant virus can be harvested per gram of leaf tissue and typically about 2 – 4% of this corresponds to the antigenic peptide chosen for expression (Dalsgaard et al., 1997; Brennan et al., 1999a,b). In general, selected approaches taken from the above listed strategies have resulted in very high levels of expression of several antigens. However, comparisons between systems are difficult since, one of the most important factors is the stability of the particular antigen in the chosen plant system and specific antigens have rarely been tested in multiple systems. Exceptions include the structurally similar receptor-binding (B) subunits of the heat labile toxin of enterotoxigenic strains of Escherichia coli and of cholera toxin, which have been expressed in multiple plant systems. Using a nuclear expression system and either targeting the protein for secretion or retention in the endoplasmic reticulum, the B subunit of the heat labile toxin has been expressed at approximately 0.2% of total soluble protein in potato tubers (Mason et al., 1998; Lauterslager

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et al., 2001) and at up to approximately 4 or 12% of total soluble protein in corn seed depending on the regulatory sequences used (Chikwamba et al., 2002a; Streatfield et al., 2003). By comparison, the B subunit of cholera toxin has been expressed at approximately 0.3% of total soluble protein in potato tubers when targeted for retention in the endoplasmic reticulum (Arakawa et al., 1997) and at approximately 4% of total soluble protein in tobacco leaves using a chloroplast expression system (Daniell et al., 2001b). High expression levels of antigens in plants allow for economical methods to extract and purify the vaccines for formulation, similar to other production systems. Alternatively, in cases where the plant tissue is edible, either directly or in a processed form, a defined antigen dose capable of inducing protection can be administered in an easily manageable amount of edible plant material. In the case of the B subunit of the heat labile toxin expressed in corn, germ tissue fractionated from a bulk grain harvest contained a sufficiently high concentration of the antigen such that a 1 mg dose, anticipated to be the desired oral dosing, corresponded to approximately 2 g of edible tissue (Lamphear et al., 2002). Typically, membrane proteins are more difficult to express in transgenic systems than soluble proteins and the high expression levels attained in plants with soluble antigens such as the B subunit of the heat labile toxin have not so far been matched with membrane proteins (Table 2). However, even here, by optimising the expression system, levels of expression have been achieved that are consistent with administering a defined antigen dose in a manageable amount of edible plant material for early phase clinical trials. For example, the level of the surface antigen of hepatitis B in transgenic potato has been increased from approximately 1 mg per gram of tuber to approximately 16 mg per gram by applying various expression tools (Richter et al., 2000). This latter level of expression requires approximately 100 g of plant material to be fed to constitute a single dose of surface antigen in the targeted 1– 2 mg range. This assumes that to induce protective immune responses about 100-fold more antigen would need to be administered orally than is delivered with the current vaccines by injection. Further improvements in expression, and thus a reduction in the amount of material to be consumed, are anticipated as a wider array of expression technologies are applied to producing hepatitis B surface antigen and other candidate membrane proteins in plants. Such technologies will be particularly important for membrane proteins that do not form virus-like particles. Such particles have been observed for hepatitis B surface antigen expressed in tobacco and potato and may serve to stabilise this antigen (Mason et al., 1992; Kong et al., 2001). 2.2. Antigen integrity The structural integrity and functional activity of

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expressed antigens have been assessed for several subunit vaccine candidates. In most cases, this comprises using an antibody raised to the native protein to detect a plantexpressed protein of predicted mobility following sodium dodecyl sulfate (SDS)-PAGE or alternatively to detect the protein using an ELISA. Size separation on gels indicates that, in general, antigens of the predicted size are expressed, protein subunits can assemble into quaternary structures (Arakawa et al., 1997; O’Brien et al., 2000; Daniell et al., 2001b; Yu and Langridge, 2001; Streatfield et al., 2002; Chikwamba et al., 2002b) and proteins that are natively glycosylated can be glycosylated in plants, although some differences in the glycosylation pattern are likely (McGarvey et al., 1995). In some cases, a more detailed analysis of the expressed antigen produced in a plant or plant viral system has been undertaken. For example, the B subunits of the heat labile toxin and cholera toxin have GM1 receptor-binding activity (Haq et al., 1995; Arakawa et al., 1997; Mason et al., 1998; Lauterslager et al., 2001; Streatfield et al., 2002; Chikwamba et al., 2002a) and hepatitis B surface antigen and Norwalk virus capsid protein form virus-like particles (Mason et al., 1992, 1996). The formation of virus-like particles is considered advantageous for inducing immune responses since the antigens are likely to be further protected from degradation in the gut and antigen presentation is more akin to the situation that prevails during infection. Plant viral expression technology, in which selected antigenic epitopes are fused to plant viral coat proteins, artificially follows this strategy (Dalsgaard et al., 1997), although in these cases, the viral particles take the geometry of the selected carrier, not that of the pathogen, which is typically only contributing an immunogenic peptide. However, plant viruses can also be applied to express whole proteins, in which case, the viral particle geometry reflects the chosen protein (O’Brien et al., 2000). 2.3. Antigen delivery Focusing on oral delivery, the major technical hurdles for subunit vaccines are surviving the digestive processes of the gut, inducing an immune response and conferring protection. A variety of approaches have been taken to protect antigens from digestive enzymes. These can be divided into two broad categories, the use of attenuated bacterial strains such as those of Salmonella and Vibrio cholerae as delivery vehicles (Van De Verg et al., 1990; Ryan et al., 1997) and the encapsulation of the antigen in a protective coat. Attenuated strains have the advantage that they tend to direct the antigen to the surface of the gut where it will be available for sampling by the mucosal immune system. However, this approach has the same potential safety drawback of any vaccine based on an attenuated strain. Encapsulation is an inherently safer means of antigen protection and can be achieved using biodegradable polymers (Eldridge et al., 1991), liposomes (Jackson et al., 1990), proteosomes (Mallett et al., 1995) or a product

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Table 2 Subunit vaccine candidates expressed by transgenic plants or plant viruses Antigen

Production system

Expressiona

Efficacy

References

Enterotoxigenic strains of E. coli/humans and farmed animals Enterotoxigenic strains of E. coli/humans and farmed animals Enterotoxigenic strains of E. coli/humans and farmed animals

Heat-labile toxin B subunit

Tobacco leaf

0.001% TSP/0.0002% FW

Immunogenic by oral delivery to mice

Haq et al., 1995

Heat-labile toxin B subunit

Potato tuber

0.2% TSP/0.001% FW

Heat-labile toxin B subunit

Maize seed

10% TSP/0.1% FW

Immunogenic and protective by oral delivery to mice and humans Immunogenic and protective by oral delivery to mice

V. cholerae/humans

Cholera toxin B subunit

Potato tuber

0.3% TSP/0.002% FW

Haq et al., 1995; Mason et al., 1998; Tacket et al., 1998; Lauterslager et al., 2001 Streatfield et al., 2001, 2002; Chikwamba et al., 2002a,b; Lamphear et al., 2002; Streatfield et al., 2003 Arakawa et al., 1997, 1998

Enteropathogenic E. coli/humans

Bundle-forming pilus structural subunit A Cholera toxin B subunit Cholera toxin B subunit

Tobacco leaf

8% TSP/1% FW

Tobacco leaf Tomato leaf and fruit Potato tuber

4% TSP/0.5% FW 0.04% TSP/0.005% FW

V. cholerae/humans V. cholerae/humans V. cholerae, rotavirus, enterotoxigenic strains of E. coli/humans

Immunogenic and protective by oral delivery to mice Immunogenic by oral delivery to mice No published data No published data

da Silva et al., 2002 Daniell et al., 2001b Jani et al., 2002

0.0003% FW

Immunogenic and protective (passive immunity) by oral delivery to mice

Yu and Langridge, 2001

Hepatitis B virus/humans

Cholera toxin B subunit fused to rotavirus enterotoxin NSP4 and cholera toxin A subunit fused to enterotoxigenic E. coli fimbrial colonisation factor CFA/1 Surface antigen Tobacco leaf

0.007% TSP/0.0008% FW

Hepatitis B virus/humans

Surface antigen

Potato tuber

0.002% FW

Hepatitis B virus/humans

Surface antigen

Lupin callus

0.00002% FW

Mason et al., 1992; Thanavala et al., 1995 Richter et al., 2000; Kong et al., 2001 Kapusta et al., 1999

Hepatitis B virus/humans

Surface antigen

Lettuce leaf

0.0000006% FW

Hepatitis B virus/humans Hepatitis B virus/humans

Surface antigen Surface antigen

0.006% TSP/0.00004% FW 0.007% FW

Hepatitis B virus/humans

Surface antigen

0.0008% FW

No published data

Smith et al., 2002a,b

Hepatitis B virus/humans Hepatitis C virus/humans

0.001% TSP/0.000009% FW 0.04% TSP/0.005% FW

No published data Immunogenic by nasal delivery to mice

Ehsani et al., 1997 Nemchinov et al., 2000

Norwalk virus/humans

Middle protein Hypervariable region 1 of envelope protein 2 fused to cholera toxin B subunit Capsid protein

Potato tuber Soybean cell culture Tobacco cell culture Potato tuber Tobacco mosaic virus (tobacco leaf) Tobacco leaf

Immunogenic by intraperitoneal delivery to mice Immunogenic by oral delivery to mice Immunogenic by oral delivery to mice Immunogenic by oral delivery to humans No published data No published data

0.2% TSP/0.03% FW

Mason et al., 1996

Norwalk virus/humans

Capsid protein

Potato tuber

0.4% TSP/0.003% FW

Rotavirus/humans

Inner capsid protein VP6

Potato virus X (tobacco leaf)

0.005% FW

Immunogenic by oral delivery to mice Immunogenic by oral delivery to mice and humans No published data

Kapusta et al., 1999 Ehsani et al., 1997 Smith et al., 2002a,b

Mason et al., 1996; Tacket et al., 2000 O’Brien et al., 2000

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Pathogen/host

Table 2 (continued) Antigen

Production system

Expressiona

Efficacy

References

Measles/humans

Haemagglutinin protein

Tobacco leaf

No published data

Human immunodeficiency virus type 1/humans

Peptide of gp41 protein

No published data

Huang et al., 2001; Webster et al., 2002 Porta et al., 1994

Human immunodeficiency virus type 1/humans

V3 loop of gp120 protein

No published data

Immunogenic by intraperitoneal delivery to mice

Yusibov et al., 1997

Human immunodeficiency virus type 1/humans

Peptide of V3 loop of gp120 protein

0.03% FW

Immunogenic by subcutaneous delivery to mice

Joelson et al., 1997

Human immunodeficiency virus type 1/humans

Peptide of transmembrane protein gp41

2% VPW/0.002% FW

Immunogenic by subcutaneous, nasal or oral delivery to mice

McLain et al., 1996; Durrani et al., 1998; McInerney et al., 1999

Human immunodeficiency virus type 1/humans Human immunodeficiency virus type 1/humans

Peptide of transmembrane protein gp41 Nucleocapsid protein p24

No published data

Immunogenic by intraperitoneal or nasal delivery to mice No published data

Marusic et al., 2001

Human cytomegalovirus/humans Human rhinovirus type 14/humans

Glycoprotein B Peptide of VP1 protein

0.01% TSP/0.00007% FW No published data

No published data Immunogenic by intramuscular or subcutaneous delivery to rabbits

Tackaberry et al., 1999 Porta et al., 1994

Respiratory syncytial virus/humans

Peptides of G protein

0.006% FW

Immunogenic and protective by intraperitoneal delivery to mice

Belanger et al., 2000

Respiratory syncytial virus/humans

Fusion protein

Cowpea mosaic virus (cowpea leaf) Alfalfa mosaic virus (tobacco leaf) Tomato bushy stunt virus (tobacco leaf) Cowpea mosaic virus (cowpea leaf) Potato virus X (tobacco leaf) Tomato bushy stunt virus (tobacco leaf) Tobacco seed Cowpea mosaic virus (cowpea leaf) Alfalfa mosaic virus (tobacco leaf) Tomato fruit

Immunogenic by intraperitoneal or oral delivery to mice No published data

0.003% FW expression level

Sandhu et al., 2000

Staphylococcus aureus/humans

D2 peptide of fibronectin-binding protein FnBP

Immunogenic by oral delivery to mice Immunogenic by subcutaneous, nasal or oral delivery to mice or rats Immunogenic by subcutaneous delivery to mice Immunogenic and protective by subcutaneous delivery to mice Immunogenic and protective by intramuscular or subcutaneous delivery to mice No published data

Staczek et al., 2000

Immunogenic and protective by subcutaneous delivery to mice

Franconi et al., 2002

S. aureus/humans Pseudomonas aeruginosa/humans

P. aeruginosa/humans

Plasmodium falciparum (malaria)/humans Human papillomavirus type 16/humans

Cowpea mosaic virus (cowpea leaf) D2 peptide of fibronectin-binding Potato virus X protein FnBP (tobacco leaf) Peptides of outer-membrane Cowpea mosaic protein F virus (cowpea leaf) Peptide of outer-membrane protein Tobacco mosaic F virus (tobacco leaf) Peptides of circumsporozoite Tobacco mosaic protein virus (tobacco leaf) E7 oncoprotein Potato virus X (tobacco leaf)

0.4% TSP/0.05% FW

4% VPW/0.005% FW

2% VPW/0.0003% FW 4% VPW/0.005% FW

No published data

0.003% FW

0.0004% FW

Zhang et al., 2000, 2002

Brennan et al., 1999a,c

Brennan et al., 1999a Brennan et al., 1999b,d; Gilleland et al., 2000

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Pathogen/host

Turpen et al., 1995

(continued on next page) 483

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Table 2 (continued) Antigen

Production system

Expressiona

Efficacy

References

B cell lymphoma/humans

Single chain Fv fragment of immunoglobulin

0.003% FW

Immunogenic and protective by subcutaneous delivery to mice

McCormick et al., 1999

No published data 1% TSP/0.1% FW

No published data No published data

Aziz et al., 2002 McGarvey et al., 1995

10% VPW/0.0005% FW

Immunogenic and protective by intraperitoneal or oral delivery to mice and immunogenic by oral delivery to humans

Yusibov et al., 1997; Modelska et al., 1998; Yusibov et al., 2002

No published data

Immunogenic and protective by intraperitoneal delivery to mice Immunogenic and protective by intraperitoneal or oral delivery to mice Immunogenic and protective by intraperitoneal delivery to mice No published data

Carrillo et al., 1998

Foot-and-mouth disease virus/farmed animals Foot-and-mouth disease virus/farmed animals

Structural protein VP1

Tobacco mosaic virus (tobacco leaf) Tobacco leaf Tomato leaf and fruit Alfalfa mosaic virus and tobacco mosaic virus (tobacco and spinach leaf) Arabidopsis leaf

Structural protein VP1

Alfalfa leaf

No published data

Foot-and-mouth disease virus/farmed animals Foot-and-mouth disease virus/farmed animals

Peptide of structural protein VP1 fused to b-glucuronidases Peptide of structural protein VP1

Alfalfa leaf

0.004% TSP/0.0005% FW No published data

Foot-and-mouth disease virus/farmed animals

Structural protein VP1

0.02% FW

Immunogenic and protective by intraperitoneal delivery to mice

Wigdorovitz et al., 1999a

Foot-and-mouth disease virus/farmed animals Transmissible gastroenteritis virus/pigs Transmissible gastroenteritis virus/pigs Transmissible gastroenteritis virus/pigs Transmissible gastroenteritis virus/pigs Bovine group A rotavirus/cattle

Structural protein VP1

Cowpea mosaic virus (cowpea leaf) Tobacco mosaic virus (tobacco leaf) Potato leaf

0.01% TSP/0.001% FW

Carrillo et al., 2001

Glycoprotein S

Arabidopsis leaf

0.06% TSP/0.008% FW

Glycoprotein S

Potato tuber

0.07% TSP/0.0005% FW

Glycoprotein S

Tobacco leaf

0.2% TSP/0.03% FW

Glycoprotein S

Maize seed

2% TSP/0.02% FW

Major capsid protein VP6

Potato tuber

0.002% TSP/0.00001% FW

Mannheimia haemolytica (bovine pneumonia pasteurellosis)/cattle Mink enteritis virus/mink, dogs, cats

Leukotoxin fused to green fluorescent protein Peptide of capsid protein VP2

White clover leaf

0.5% TSP/0.0009% FW 2% VPW/0.002% FW

Rabbit haemorrhagic disease virus/rabbits

Structural protein VP60

Cowpea mosaic virus (cowpea leaf) Potato leaf

Immunogenic and protective by intraperitoneal delivery to mice Immunogenic by intramuscular delivery to mice Immunogenic by intraperitoneal or oral delivery to mice Immunogenic by intraperitoneal delivery to pigs Immunogenic and protective by oral delivery to pigs Immunogenic by intraperitoneal delivery to mice Immunogenic by intramuscular delivery to rabbits Immunogenic and protective by subcutaneous delivery to mink

Bacillus anthracis Protective antigen Rabies virus/humans, domestic and Glycoprotein wild animals Rabies virus/humans, domestic and Peptides of glycoprotein and wild animals nucleoprotein

0.3% TSP/0.04% FW

Immunogenic and protective by subcutaneous followed by intramuscular delivery to rabbits

Wigdorovitz et al., 1999b

Dus Santos et al., 2002 Usha et al., 1993

Gomez et al., 1998 Gomez et al., 2000 Tuboly et al., 2000 Streatfield et al., 2001; Lamphear et al., 2002 Matsumura et al., 2002 Lee et al., 2001 Dalsgaard et al., 1997

Castanon et al., 1999

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Pathogen/host

Pathogen/host

Antigen

Production system

Expressiona

Efficacy

References

Rabbit haemorrhagic disease virus/rabbits

Structural protein VP60

Plum pox potyvirus (tobacco leaf)

No published data

Fernandez-Fernandez et al., 1998, 2001

Canine parvovirus/dogs

Peptide of capsid protein VP2

No published data

Canine parvovirus/dogs Canine parvovirus/dogs

Peptide of capsid protein VP2 fused to b-glucuronidase Peptide of capsid protein VP2

Plum pox potyvirus (tobacco leaf) Arabidopsis leaf

Murine hepatitis virus/mice

Peptide of glycoprotein S

Immunogenic by intraperitoneal delivery to mice and immunogenic and protective by intramuscular and subcutaneous delivery to rabbits Immunogenic by intraperitoneal delivery to mice or intramuscular delivery to rabbits Immunogenic by intraperitoneal or oral delivery to mice Immunogenic by subcutaneous or intranasal delivery to mice and immunogenic and protective by subcutaneous delivery to dogs Immunogenic and protective by subcutaneous or nasal delivery to mice

0.1% TSP/0.0003% FW

Cowpea mosaic virus (cowpea leaf)

2% VPW/0.002% FW

Tobacco mosaic virus (tobacco leaf)

No published data

Fernandez-Fernandez et al., 1998

Gil et al., 2001 Langeveld et al., 2001; Nicholas et al., 2002

Koo et al., 1999)

TSP, total soluble protein; FW, fresh weight; VPW, viral particle weight. Expression levels are as reported in the literature, given to one significant figure and if necessary, also converted to give approximate FW. In each case, the values given are those reported as the highest levels of expression achieved in unprocessed plant material. Levels refer to the relevant protein of peptide and not to whole fusion proteins. a

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Table 2 (continued)

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derived from transgenic plants expressing the antigen. We have shown that feeding the protein avidin expressed in transgenic corn products to mice serves to protect the protein from degradation in the gut (Bailey, 2000). Avidin fed in corn products can be detected in the faeces, whereas a corresponding dose of purified avidin is completely degraded, demonstrating the potential of plant material to protect selected antigens. Similar results have been observed with the B subunit of the heat labile toxin (Streatfield et al., unpublished). Particularly in seeds, plants can provide a carbohydrate polymer matrix environment for subunit vaccines that is rich in protease inhibitors. Providing protection for these antigens through bioencapsulation in seeds is at no extra cost, in contrast to other means of antigen encapsulation, which require additional processing steps. 2.4. Immunogenicity Demonstrating the induction of immune responses is a key step in developing new vaccines. Subunit vaccine candidates produced by plants or plant viruses have been administered in trials to laboratory animals, target domestic animals and humans. Immune responses have been recorded with several of these vaccine candidates to antigens enriched from plants and then administered intraperitoneally, subcutaneously, intramuscularly, intranasally or orally and also to antigens delivered orally in the raw or processed plant tissues used for expression (Table 2). Levels of serum IgG and intestinal mucosal IgA are generally assessed to gauge immunogenicity. Responses at other mucosal sites have also been monitored. Consider examples of responses to purified antigens or plant extracts containing such antigens. Following the intranasal or subcutaneous administration of an immunogenic peptide from a Staphylococcus aureus fibronectinbinding protein expressed on the surface of cowpea mosaic virus, strong IgG responses were observed with the IgG2a and IgG2b isotypes dominating (Brennan et al., 1999a,c). This bias towards a Th1 response is independent of the route of administration or the presence of adjuvant, and was also observed when an immunogenic peptide from human immunodeficiency virus type 1 (HIV-1) gp41 was expressed on the surface of cowpea mosaic virus (Durrani et al., 1998). Thus, the Th1 emphasis may result from the nature of the viral carrier. By contrast, as an example of an antigen partially purified from a stable transgenic plant expression system, and thus produced without using plant viral components, hepatitis B surface antigen has been isolated from transgenic tobacco leaves and delivered to mice intraperitoneally. Although the magnitude of the total IgG response was reduced about twofold for mice injected with hepatitis B surface antigen isolated from plant tissue compared to mice injected with a commercially available recombinant yeastderived vaccine, a broader range of IgG subclasses together

with an increased level of IgM was observed with the tobacco material (Thanavala et al., 1995). Significantly, both B and T cell responses to the surface antigen expressed in tobacco were observed. Focusing on the oral delivery of antigens expressed in plants without viral particle isolation or antigen purification, many studies have been completed using various feeding strategies. Early studies centred on feeding mice highly immunogenic molecules such as the B subunits of the heat labile toxin and cholera toxin expressed in plant tissues. These are optimal antigens for oral delivery since the relevant pathogens infect via intestinal tissue and the toxin molecules themselves bind to receptors on the gut surface, so initiating host responses. Serum and intestinal mucosal IgG and IgA responses were recorded (Haq et al., 1995; Arakawa et al., 1998; Mason et al., 1998; Streatfield et al., 2001; Lamphear et al., 2002; Chikwamba et al., 2002b). In a separate clinical study with the B subunit of the heat labile toxin, IgA antibody secreting cells were detected, indicating that the intestinal mucosal immune system had been primed (Tacket et al., 1998). Hepatitis B surface antigen and Norwalk virus capsid protein have also been shown to induce serum Ig responses when fed to mice or humans (Mason et al., 1996; Kapusta et al., 1999; Richter et al., 2000; Tacket et al., 2000; Kong et al., 2001). Furthermore, in the clinical study with Norwalk virus capsid protein, mucosal IgG and IgA antibody secreting cells were detected (Tacket et al., 2000). For most antigens expressed in plants and fed in preclinical or clinical trials, there are not yet reports analysing the immune responses in detail or addressing cell mediated immunity. However, in a study with transgenic potatoes, where selected antigens from rotavirus and enterotoxigenic E. coli were fused to the B and A2 subunits of cholera toxin, respectively, immune responses to the antigens were shown to have a Th1 bias (Yu and Langridge, 2001). Interleukin 2 and interferon g were induced and increased levels of CD4þ lymphocytes were recorded. These Th1 biased responses could result from the choice of antigens or carrier. Delivery of a subunit vaccine with an adjuvant potentially increases the magnitude of the immune response and can bias the nature of the response. The co-expression of protein adjuvants such as attenuated versions of cholera toxin and the heat labile toxin in plants along with the antigen of interest may be beneficial to induce protective immune responses against poorly immunogenic antigens delivered orally. The adjuvants could either be expressed directly in the same plant tissue as the antigen through co-transformation or crossing during plant breeding or could be expressed in separate transgenic lines with material being pooled at the time of processing prior to delivery. The latter approach has the advantage of allowing the relative doses of antigen and adjuvant to be reliably set. In some cases, it may not be necessary for an orally delivered plant-expressed antigen to be able to induce a primary immune response, for there may be an ease of

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administration issue or an economic advantage in using the oral plant vaccine only as a primer or only as a booster. An example in the area of animal health is transmissible gastroenteritis virus, which causes diarrhoea in piglets that impacts weight gain and is often fatal. Currently, sows receive a combination of intramuscular injections and oral administrations with an attenuated virus with the aim of passing immunity to their piglets through the colostrum. The initial injection is delivered as a part of a combination vaccine and, therefore, an oral delivery of a transmissible gastroenteritis virus vaccine would not eliminate the need for injection of the other components. However, substitution of any of the booster injections with a plant vaccine would reduce the inconvenience and cost of immunising large herds. In a sow feeding study, the replacement of two intramuscular booster injections of attenuated virus with two oral booster administrations of the S glycoprotein of the virus expressed in corn seed resulted in an increase in the level of neutralising antibodies in the colostrum (Lamphear et al., unpublished). Furthermore, in a separate study, piglets fed the S glycoprotein corn vaccine had their immune systems primed as demonstrated by greatly elevated serum neutralisation titres following a subsequent subclinical viral challenge (Lamphear et al., 2002). As a human health example, though using a mouse model, consider feeding studies in which the cell surface antigen of hepatitis B expressed in potato tubers was administered orally. When used for the initial delivery, the oral plant material induces only a small serum immune response. However, this acts to prime a large response to a boost with a normally subimmunogenic dose of a commercial vaccine delivered by intraperitoneal injection (Richter et al., 2000; Kong et al., 2001). Conversely, the injection of a subimmunogenic dose of the commercial vaccine can act as a primer, facilitating a large boost response following the oral delivery of the transgenic potato tubers (Kong et al., 2001).

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the VP1 protein of foot and mouth disease virus using a tobacco mosaic virus system provides an example of an entire immunogenic protein being expressed by a plant virus (Wigdorovitz et al., 1999a). Viral particles administered intraperitoneally protected mice from challenge with foot and mouth disease virus. Consider next, antigens extracted from plant expression systems that are not based on viral infection. The VP1 structural protein of foot and mouth disease virus has also been expressed in Arabidopsis thaliana and alfalfa and leaf extracts containing VP1 have been administered by intraperitoneal injection to mice, so conferring protection against the virus (Carrillo et al., 1998; Wigdorovitz et al., 1999b). Similarly, the VP60 structural protein of rabbit haemorrhagic disease virus expressed in potatoes and delivered by subcutaneous and intramuscular injections to rabbits protected the animals from subsequent challenge with the virus (Castanon et al., 1999). To achieve the full potential of antigens expressed in plants, the candidate subunit vaccines should confer protection when administered orally without purification from the plant material. Significant progress towards this goal has been achieved with a few vaccine candidates. The B subunit of the heat labile toxin expressed in potato tubers or corn seed and the B subunit of cholera toxin expressed in potatoes have been fed to mice and offer protection against diarrhoea when the mice are challenged with the relevant native bacterial toxin by gavage (Arakawa et al., 1998; Mason et al., 1998; Streatfield et al., 2001; Chikwamba et al., 2002b). Furthermore, a multi-component vaccine candidate, that includes a rotavirus antigen fused to the B subunit of cholera toxin, has been expressed in potato tubers and fed to female mice. These mice conferred passive immunity to their pups demonstrated by protection to rotavirus challenge (Yu and Langridge, 2001). Finally, in a target animal study with an oral vaccine, the S glycoprotein of transmissible gastroenteritis virus delivered in corn seed is protective to piglets (Streatfield et al., 2001; Lamphear et al., 2002).

2.5. Protective efficacy Protection, particularly in the target species, is, of course, the principal goal during vaccine development and several plant-based vaccines have demonstrated efficacy in experimental or target species (Table 2). In some such cases, plant viral expression systems present immunogenic peptides of key pathogen proteins. For example, an epitope from the VP2 capsid protein of mink enteritis virus displayed by cowpea mosaic virus conferred protection when administered to mink by subcutaneous injection (Dalsgaard et al., 1997) and an epitope of the rabies glycoprotein displayed by alfalfa mosaic virus and administered either intraperitoneally or orally reduced symptoms in mice subsequently challenged with the rabies virus (Modelska et al., 1998). In the latter case, oral administration was protective whether viral particles were isolated prior to delivery or plant leaves harbouring viral particles were fed directly. Production of

3. Product development and what is needed Since the first report of using plants to produce subunit vaccines a decade ago (Mason et al., 1992), there has been considerable progress in raising the expression levels of antigens, demonstrating immune responses and protection in model animal studies and more recently, in completing application trials for target animals for veterinary vaccines and early phase clinical trials for human vaccines (Tacket et al., 1998; Kapusta et al., 1999; Tacket et al., 2000; Streatfield et al., 2001; Lamphear et al., 2002). However, there are several issues that need to be addressed before such vaccine candidates can be considered practical alternatives and additions to currently established programmes of vaccination. The critical issues differ depending on whether the vaccine candidate is to be purified from plant tissues prior to

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formulation and delivery by the chosen route or whether it is to be administered orally as recombinant plant material. In the former case, facilities for purifying the antigen from plant material will need to be operated under conditions of good manufacturing practises and issues such as lot-to-lot variability of the purified antigen will need to be monitored. This is similar to the current situation with the production of antigens produced in bacteria, yeasts or animal cell cultures. Characterisation would include biochemical and biophysical analysis of the antigen to ensure it meets defined quality standards for the product. A detailed analysis of proteins currently produced commercially in corn has demonstrated that the recombinant corn proteins are very similar to the corresponding native source proteins, the only differences being minor changes in glycosylation (Hood et al., 1997; Howard et al., unpublished). By contrast, when delivering a recombinant antigen in a processed plant product as an oral vaccine, the means of production will be based on food processing technology rather than protein purification schemes. However, good manufacturing practises and a detailed characterisation of the vaccine, including defining the dose, will still apply. Whichever production route is pursued, one yielding pure protein or an oral product, the highest priority for most plant vaccine candidates is to raise the level of antigen expression. This is necessary to make protein purification practical and economically viable when the vaccine candidate is a pure protein and to make the concentration of the antigen sufficiently high to allow feasible administration if an oral vaccine is favoured. Expression levels for certain proteins are already sufficiently high for economic production (Hood et al., 1997; Witcher et al., 1998; Zhong et al., 1999) and this should extend to vaccine candidates. Indeed, with some antigens, expression levels are already adequate to allow the delivery of the desired dose in a small mass of plant material (Lamphear et al., 2002). However, with most antigens, further improvements in expression are necessary before the vaccines can be considered practical, especially since oral delivery will likely require greatly increased doses of up to a 1,000-fold over those used for injections. Such expression targets may be achieved more easily with vaccine candidates for animals, since relatively large amounts of plant material can be used to deliver each dose if the plant material is also part of the normal feed (e.g. vaccines in corn fed to pigs). Consistency of product is important for oral vaccines as well as for purified antigens. This will only be achieved for oral vaccines if the final product is homogeneous. Thus, rather than administering fruits, vegetables or grains directly, it may be desirable to process the plant material into a uniform state. This requirement for homogeneous material necessitates that the antigen be stable to the food processing technology deployed. For many standard crops, processing technologies are well established and selected antigens can readily be tested for stability. For example, corn grain can be dry milled to separate out the germ

fraction, which can be ground to an even consistency and administered orally without further processing. Alternatively, corn grain can be subjected to extrusion processing to generate edible snacks. Both processes have been assessed using recombinant corn expressing the B subunit of the heat labile toxin, and the antigen has been shown to be stable to milling and modified extrusion conditions and to be evenly distributed in the products (Streatfield et al., 2002; Lamphear et al., 2002; Streatfield et al., 2003). Since the products are to be consumed orally, the means of processing must meet regulatory agency requirements for food grade material, as well as, for the reproducible quality and concentration of the antigen candidates. Homogeneous products of this kind lend themselves to easy blending with non-transgenic material to attain reproducible desired antigen concentrations for even dosing. Transgenic material expressing different antigens, or even protein adjuvants, could also be mixed together into a final combination vaccine product. Stability of antigens over time in processed food products stored at different temperatures will also need to be assessed. In the case of corn, the B subunit of the heat labile toxin and the S glycoprotein of transmissible gastroenteritis virus have been shown to be stable for at least a year, even when stored at ambient temperatures (Lamphear et al., 2002). This indicates the potential of removing the need for a cold chain during storage and distribution. The regulatory process will drive many of the steps and timelines needed for product development of plant-based vaccines. The Master Seed Bank and Working Seed Bank definitions used for microbial and mammalian cell lines can be adapted for plant cell material with similar meaning. It will be necessary to perform toxicity testing and clinical trials demonstrating the subunit vaccines are safe and efficacious. If the plant is a food product, then it should already have ‘generally recognised as safe’ status. Therefore, an oral vaccine regulatory package will have strict guidelines for the processing and formulation of the plant material. This will be similar to guidelines for food product manufacturing with the critical addition of validating the dose requirements for the vaccine. Good manufacturing practises will need to include the processing and formulation of the plant material. The timeline for product development in plants is comparable to animal cell culture systems. During the first two years, the research and the pre-clinical studies can be performed with limited amounts of material. Subsequently, there should be enough material to enter clinical trials. Scale-up of plant material only requires growing more acreage, not an increase in capital facilities. Since for most plants, this would represent a very small portion of the total acreage (, 0.1%), this would not impose undue stress on existing infrastructures. If a purified protein is required, capital investments will be necessary, albeit reduced compared to other production systems, since capital equipment will not be needed to generate the source material.

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Alternatively, if the vaccine were to be orally delivered, there would be minimal infrastructure requirements for the manufacturing facility. The rate-limiting step will not be the production of raw material, but the validation of the manufacturing site.

4. Opportunities Oral vaccines are potentially applicable to any vaccine formulation based on or including a subunit component. The oral delivery of a subunit vaccine is particularly suited to protect against pathogens that infect via the intestinal surface, such as diarrhoea causing agents. In addition, because of the linked nature of the mucosal immune system, these vaccines are very relevant to combating pathogens that infect other mucosal surfaces, prominent examples being hepatitis B and HIV. Early studies indicate that oral vaccines may also be a viable option to combat pathogens that typically invade via the circulatory system, such as rabies (Modelska et al., 1998). Plants allow for the rapid bulk up of large supplies of subunit vaccines, for example a 40,000-fold increase per year is possible using corn. Therefore, oral vaccines are particularly applicable to combating diseases that affect very large populations. Furthermore, oral plant-based vaccines are stable during storage at ambient temperatures (Lamphear et al., 2002) and do not require syringes, needles and trained personnel for administration. These features also favour the use of oral vaccines for large-scale immunisation programmes, particularly in developing countries with limited resources to provide a cold chain and the equipment and personnel needed for injections. This ability to stockpile plant-based vaccines without expensive refrigerated storage and administer them without injections also favours the development of such vaccines to protect against sudden outbreaks of disease in developed countries as a result of, for example, terrorist actions. Perhaps, most significantly, the low cost of plant-based vaccines make them ideal for large-scale programmes in developing countries. Inexpensive raw material and processing costs together with the absence of a cold chain and reduced administration costs should serve to make oral plant-based vaccines accessible worldwide. The advantages listed above also favour the use of oral vaccines in veterinary medicine to combat pathogens of domestic animals, particularly large herds of farmed animals. The development of an oral vaccine against transmissible gastroenteritis virus for swine is one such example (Streatfield et al., 2001; Lamphear et al., 2002). An additional advantage of oral vaccines for livestock animal applications is that the carcass is not damaged by injections. Inexpensive vaccination programmes using oral plant vaccines for domestic animals could also combat human disease agents such as enterohaemorrhagic E. coli which infect cattle and can spread to humans through poorly

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processed meat. Oral plant vaccines may also allow the practical and inexpensive immunisation of wild animal populations, which may act as reservoirs for disease, as with rabies. Most likely, the first application of oral plant vaccines will be in the area of animal health, since, the trials to demonstrate safety and efficacy are much shorter than for human vaccines. They may also demonstrate their value in combination with established components, just as the B subunit of cholera toxin produced in recombinant bacteria has been tested in combination with chemically killed strains (Qadri et al., 2000). Alternatively, plant vaccines may first be used as a single oral component in an immunisation course also involving injections, for example, as a hepatitis B booster vaccine, where it may serve to widen compliance or as a measles vaccine to protect infants prior to the time at which it is efficacious to administer the established live attenuated vaccine. Ultimately, oral plant combination vaccines should be developed that can protect against multiple pathogens. Furthermore, their inexpensive costs of production and administration advantages make oral vaccines excellent candidates for situations where many doses are needed for protection, as may be the case with HIV.

5. Unintended consequences While there are many potential advantages for plant made vaccines, there also exist the potential for unintended consequences. The two most frequently cited are the potential to induce tolerance and the potential for the vaccines to inadvertently enter the food chain. Tolerance to specific antigens can be induced through repeated exposures regardless of how the antigen is administered (Challacombe and Tomasi, 1980; Tomasi, 1980). Oral tolerance mechanisms have been studied as they relate to food allergens and autoimmune diseases (Weiner, 1997; Hachimura, 2000; Stanley, 2002; Strobel, 2002). There is no reason to believe that plant-based vaccines will be any different in respect to inducing tolerance compared to vaccines produced in other systems. However, all vaccines undergo extensive testing, with oversight from regulatory agencies, to define the correct dosage and the appropriate schedule of boosting. Therefore, tolerance from prescribed doses is highly unlikely. Plant-based vaccines do have the potential to induce tolerance if the vaccine inadvertently enters the food chain and repeated exposures are experienced without our knowledge. Currently, many vaccines are made in animals, yeast and eggs. These production systems are also used to produce food. The reason we do not see vaccines currently in our food supply is that in generating vaccines, a completely different production system is followed. There exists an array of procedures to ensure that the vaccines are contained throughout the entire production phase with

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regulatory oversight. These procedures ensure the safety of commodity food, and at the same time, the quality of vaccines. This situation is not unlike that proposed for the use of plant-based vaccines. If the plants were grown as commodity crops, then there would be potential for exposure leading to tolerance. However, plant-based vaccines are grown under contained conditions with regulatory oversight, similar to vaccines produced in other production systems. Plant-based vaccines represent only a very small percentage of any total food crop (, 0.1%) and would be produced in a closed loop system, thereby, keeping them separate from the food chain. While it is unlikely that there would be any vaccine in a food crop, we cannot set a zero tolerance standard, which is both theoretically and practically impossible. We can, however, set a limit for exposure in food crops based on safety models. Risk models can be used to show that the amount of vaccine that could potentially end up in a food crop would be orders of magnitude lower than that shown to be needed to induce oral tolerance, if, in some rare case, an individual is exposed.

Acknowledgements We thank Barry Lamphear for critical comments on the manuscript.

References Arakawa, T., Chong, D.K.X., Merritt, J.L., Langridge, W.H.R., 1997. Expression of cholera toxin B subunit oligomers in transgenic potato plants. Transgenic Res. 6, 403– 413. Arakawa, T., Chong, D.K.X., Langridge, W.H.R., 1998. Efficacy of a food plant-based oral cholera toxin B subunit vaccine. Nat. Biotechnol. 16, 292– 297. Archer, D.B., 1993. Enzyme production by recombinant Aspergillus. In: Murooka, Y., Imanaka, T. (Eds.), Recombinant Microbes for Industrial and Agricultural Applications, Marcel Dekker, New York, NY, pp. 373 –393. Aziz, M.A., Singh, S., Kumar, P.A., Bhatnagar, R., 2002. Expression of protective antigen in transgenic plants: a step towards edible vaccine against anthrax. Biochem. Biophys. Res. Commun. 299, 345–351. Bailey, M.R., 2000. A model system for edible vaccination using recombinant avidin produced in corn seed. Master of Science thesis, Texas A&M University. Belanger, H., Fleysh, N., Cox, S., Bartman, G., Deka, D., Trudel, M., Koprowski, H., Yusibov, V., 2000. Human respiratory syncytial virus vaccine antigen produced in plants. FASEB J. 14, 2323–2328. Brennan, F.R., Jones, T.D., Longstaff, M., Chapman, S., Bellaby, T., Smith, H., Xu, F., Hamilton, W.D.O., Flock, J.-I., 1999a. Immunogenicity of peptides derived from a fibronectin-binding protein of S. aureus expressed on two different plant viruses. Vaccine 17, 1846–1857. Brennan, F.R., Jones, T.D., Gilleland, L.B., Bellaby, T., Xu, F., North, P.C., Thompson, A., Staczek, J., Lin, T., Johnson, J.E., Hamilton, W.D.O., Gilleland, H.E. Jr, 1999b. Pseudomonas aeruginosa outer-membrane protein F epitopes are highly immunogenic in mice when expressed on a plant virus. Microbiology 145, 211–220. Brennan, F.R., Bellaby, T., Helliwell, S.M., Jones, T.D., Kamstrup, S.,

Dalsgaard, K., Flock, J.-I., Hamilton, W.D.O., 1999c. Chimeric plant virus particles administered nasally or orally induce systemic and mucosal immune responses in mice. J. Virol. 73, 930 –938. Brennan, F.R., Gilleland, L.B., Staczek, J., Bendig, M.M., Hamilton, W.D.O., Gilleland, H.E. Jr, 1999d. A chimeric plant virus vaccine protects mice against a bacterial infection. Microbiology 145, 2061–2067. Carrillo, C., Wigdorovitz, A., Oliveros, J.C., Zamorano, P.I., Sadir, A.M., Gomez, N., Salinas, J., Escribano, J.M., Borca, M.V., 1998. Protective immune response to foot-and-mouth disease virus with VP1 expressed in transgenic plants. J. Virol. 72, 1688–1690. Carrillo, C., Wigdorovitz, A., Trono, K., Dus Santos, M.J., Castanon, S., Sadir, A.M., Ordas, R., Escribano, J.M., Borca, M.V., 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. Carter, J.E. III, Langridge, W.H.R., 2002. Plant-based vaccines for protection against infectious and autoimmune diseases. Crit. Rev. Plant Sci. 21, 93–109. Castanon, S., Marin, M.S., Martin-Alonso, J.M., Boga, R., Casais, R., Humara, J.M., Ordas, R.J., Parra, F., 1999. Immunization with potato plants expressing VP60 protein protects against rabbit hemorrhagic disease virus. J. Virol. 73, 4452–4455. Challacombe, S.J., Tomasi, T.B. Jr, 1980. Systemic tolerance and secretory immunity after oral immunization. J. Exp. Med. 152, 1459–1472. Chikwamba, R., McMurray, J., Shou, H., Frame, B., Pegg, S.-E., Scott, P., Mason, H., Wang, K., 2002a. Expression of a synthetic E. coli heatlabile enterotoxin B sub-unit (LT-B) in maize. Mol. Breed. 10, 253 –265. Chikwamba, R., Cunnick, J., Hathaway, D., McMurray, J., Mason, H., Wang, K., 2002b. A functional antigen in a practical crop: LT-B producing maize protects mice against Escherichia coli heat labile enterotoxin (LT) and cholera toxin (CT). Transgenic Res. 11, 479 –493. Dalsgaard, K., Uttenthal, A., Jones, T.D., Xu, F., Merryweather, A., Hamilton, W.D., Langeveld, J.P., Boshuizen, R.S., Kamstrup, S., Lomonossoff, G.P., Porta, C., Vela, C., Casal, J.I., Meloen, R.H., Rodgers, P.B., 1997. Plant-derived vaccine protects target animals against a viral disease. Nat. Biotechnol. 15, 248 –252. Daniell, H., Streatfield, S.J., Wycoff, K., 2001a. Medical molecular farming: production of antibodies, biopharmaceuticals and edible vaccines in plants. Trends Plant Sci. 6, 219–226. Daniell, H., Lee, S.-B., Panchai, T., Wiebe, P.O., 2001b. Expression of the native cholera toxin B subunit gene and assembly of functional oligomers in transgenic tobacco chloroplasts. J. Mol. Biol. 311, 1001–1009. da Silva, J.V., Garcia, A.B., Flores, V.M.Q., de Macedo, Z.S., MedinaAcosta, E., 2002. Phytosecretion of enteropathogenic Escherichia coli pilin subunit A in transgenic tobacco and its suitability for early life vaccinology. Vaccine 20, 2091–2101. Durrani, Z., McInerney, T.L., McLain, L., Jones, T., Bellaby, T., Brennan, F.R., Dimmock, N.J., 1998. Intranasal immunization with a plant virus expressing a peptide from HIV-1 gp41 stimulates better mucosal and systemic HIV-1-specific IgA and IgG than oral immunization. J. Immunol. Methods 220, 93–103. Dus Santos, M.J., Wigdorovitz, A., Trono, K., Rios, R.D., Franzone, P.M., Gil, F., Moreno, J., Carrillo, C., Escribano, J.M., Borca, M.V., 2002. A novel methodology to develop a foot and mouth disease virus (FMDV) peptide-based vaccine in transgenic plants. Vaccine 20, 1141–1147. Ehsani, P., Khabiri, A., Domansky, N.N., 1997. Polypeptides of hepatitis B surface antigen produced in transgenic potato. Gene 190, 107–111. Eldridge, J.H., Staas, J.K., Meulbroek, J.A., Tice, T.R., Gilley, R.M., 1991. Biodegradable and biocompatible poly(DL -lactide-co-glycolide) microspheres as an adjuvant for staphylococcal enterotoxin B toxoid which enhance the level of toxin-neutralizing antibodies. Infect. Immun. 59, 2978–2985. Fernandez-Fernandez, M.R., Martinez-Torrecuadrada, J.L., Casal, J.I.,

S.J. Streatfield, J.A. Howard / International Journal for Parasitology 33 (2003) 479–493 Garcia, J.A., 1998. Development of an antigen presentation system based on the plum pox potyvirus. FEBS Lett. 427, 229–235. Fernandez-Fernandez, M.R., Mourino, M., Rivera, J., Rodriguez, R., PlanaDuran, J., Garcia, J.A., 2001. Protection of rabbits against rabbit hemorrhagic disease virus by immunization with the VP60 protein expressed in plants with a potyvirus-based vector. Virology 280, 283–291. Franconi, R., Di Bonito, P., Dibello, F., Accardi, L., Muller, A., Cirilli, A., Simeone, P., Dona, M.G., Venuti, A., Giorgi, C., 2002. Plant-derived human papillomavirus 16 E7 oncoprotein induces immune response and specific tumor protection. Cancer Res. 62, 3654–3658. Gil, F., Brun, A., Wigdorovitz, A., Catala, R., Martinez-Torrecuadrada, J.L., Casal, I., Salinas, J., Borca, M.V., Escribano, J.M., 2001. Highyield expression of a viral peptide vaccine in transgenic plants. FEBS Lett. 488, 13 –17. Gilleland, H.E. Jr, Gilleland, L.B., Staczek, J., Harty, R.N., Garcia-Sastre, A., Palese, P., Brennan, F.R., Hamilton, W.D.O., Bendahmane, M., Beachy, R.N., 2000. Chimeric animal and plant viruses expressing epitopes of outer membrane protein F as a combined vaccine against Pseudomonas aeruginosa lung infection. FEMS Immunol. Med. Microbiol. 27, 291–297. Gomez, N., Carrillo, C., Salinas, J., Parra, F., Borca, M.V., Escribano, J.M., 1998. Expression of immunogenic glycoprotein S polypeptides from transmissible gastroenteritis coronavirus in transgenic plants. Virology 249, 352 –358. Gomez, N., Wigdorovitz, A., Castanon, S., Gil, F., Ordas, R., Borca, M.V., Escribano, J.M., 2000. Oral immunogenicity of the plant derived spike protein from swine-transmissible gastroenteritis coronavirus. Arch. Virol. 145, 1725– 1732. Hachimura, S., 2000. Oral tolerance and its mechanisms: from the viewpoint of antigen structures. Clin. Gastroenterol. 15, 2-1-b. Haq, T.A., Mason, H.S., Clements, J.D., Arntzen, C.J., 1995. Oral immunization with a recombinant bacterial antigen produced in transgenic plants. Science 268, 714– 716. Harashima, S., 1994. Heterologous protein production by yeast host– vector systems. Bioprocess Technol. 19, 137–158. Hood, E.E., Witcher, D.R., Maddock, S., Meyer, T., Baszczynski, C., Bailey, M., Flynn, P., Register, J., Marshall, L., Bond, D., Kulisek, E., Kusnadi, A., Evangelista, R., Nikolov, Z., Wooge, C., Mehigh, R.J., Hernan, R., Kappel, W.K., Ritland, D., Li, C.-P., Howard, J.A., 1997. Commercial production of avidin from transgenic maize: characterization of transformant, production, processing, extraction and purification. Mol. Breed. 3, 291–306. Hood, E.E., Howard, J.A. (Eds.), 2002. Plants as Factories for Protein Production, Kluwer Academic Publishers, The Netherlands. Huang, Z., Dry, I., Webster, D., Strugnell, R., Wesselingh, S., 2001. Plantderived measles virus hemagglutinin protein induces neutralizing antibodies in mice. Vaccine 19, 2163–2171. Jackson, S., Mestecky, J., Childers, N.K., Michalek, S.M., 1990. Liposomes containing anti-idiotypic antibodies: an oral vaccine to induce protective secretory immune responses specific for pathogens of mucosal surfaces. Infect. Immun. 58, 1932–1936. Jani, D., Singh Meena, L., Mohammad Rizwan-ul-Haq, Q., Singh, Y., Sharma, A.K., Tyagi, A.K., 2002. Expression of cholera toxin B subunit in transgenic tomato plants. Transgenic Res. 11, 447–454. Jilka, J.M., Hood, E.E., Dose, R., Howard, J.A., 1999. The benefits of proteins produced in transgenic plants. AgBiotechNet 1, 1–4. Joelson, T., Akerblom, L., Oxelfelt, P., Strandberg, B., Tomenius, K., Morris, T.J., 1997. Presentation of a foreign peptide on the surface of tomato bushy stunt virus. J. Gen. Virol. 78, 1213–1217. Kapusta, J., Modelska, A., Figlerowicz, M., Pniewski, T., Letellier, M., Lisowa, O., Yubisov, V., Koprowski, H., Plucienniczak, A., Legocki, A.B., 1999. A plant-derived edible vaccine against hepatitis B virus. FASEB J. 13, 1796–1799. Kong, Q., Richter, L., Yang, Y.F., Arntzen, C.J., Mason, H.S., Thanavala, Y., 2001. Oral immunization with hepatitis B surface antigen expressed in transgenic plants. Proc. Natl Acad. Sci. USA 98, 11539–11544.

491

Koo, M., Bendahmane, M., Lettieri, G.A., Paoletti, A.D., Lane, T.E., Fitchen, J.H., Buchmeier, M.J., Beachy, R.N., 1999. Protective immunity against murine hepatitis virus (MHV) induced by intranasal or subcutaneous administration of hybrids of tobacco mosaic virus that carries an MHV epitope. Proc. Natl Acad. Sci. USA 96, 7774–7779. Korban, S.S., 2002. Targeting and expression of antigenic proteins in transgenic plants for production of edible oral vaccines. In Vitro Cell. Dev. Biol. Plant 38, 231 –236. Kusnadi, A.R., Nikolov, Z.L., Howard, J.A., 1997. Production of recombinant proteins in transgenic plants: practical considerations. Biotechnol. Bioeng. 56, 473–484. Kusnadi, A.R., Hood, E.E., Witcher, D.R., Howard, J.A., Nikolov, Z.L., 1998. Production and purification of two recombinant proteins from transgenic corn. Biotechnol. Prog. 14, 149 –155. Lamphear, B.J., Streatfield, S.J., Jilka, J.M., Brooks, C.A., Barker, D.K., Turner, D.D., Delaney, D.E., Garcia, M., Wiggins, W., Woodard, S.L., Hood, E.E., Tizard, I.R., Lawhorn, B., Howard, J.A., 2002. Delivery of subunit vaccines in maize seed. J. Controlled Release 85, 169 –180. Langeveld, J.P.M., Brennan, F.R., Martinez-Torrecuadrada, J.L., Jones, T.D., Boshuizen, R.S., Vela, C., Casal, J.I., Kamstrup, S., Dalsgaard, K., Meloen, R.H., Bendig, M.M., Hamilton, W.D.O., 2001. Inactivated recombinant plant virus protects dogs from a lethal challenge with canine parvovirus. Vaccine 19, 3661–3670. Lauterslager, T.G.M., Florack, D.E.A., van der Wal, T.J., Molthoff, J.W., Langeveld, J.P.M., Bosch, D., Boersma, W.J.A., Hilgers, L.A.Th., 2001. Oral immunization of naive and primed animals with transgenic potato tubers expressing LT-B. Vaccine 19, 2749–2755. Lee, R.W.H., Strommer, J., Hodgins, D., Shewen, P.E., Niu, Y., Lo, R.Y.C., 2001. Towards development of an edible vaccine against bovine pneumonic pasteurellosis using transgenic white clover expressing a Mannheimia haemolytica A1 leukotoxin 50 fusion protein. Infect. Immune. 69, 5786–5793. Mallett, C.P., Hale, T.L., Kaminski, R.W., Larsen, T., Orr, N., Cohen, D., Lowell, G.H., 1995. Intranasal or intragastric immunization with proteosome-Shigella lipopolysaccharide vaccines protects against lethal pneumonia in a murine model of Shigella infection. Infect. Immun. 63, 2382–2386. Marino, M.M., 1991. In: Prokop, A., Bajpai, R.K., Ho, C.S. (Eds.), Recombinant DNA Technology and Applications, McGraw Hill, New York, NY, pp. 29– 65. Marusic, C., Rizza, P., Lattanzi, L., Mancini, C., Spada, M., Belardelli, F., Benvenuto, E., Capone, I., 2001. Chimeric plant virus particles as immunogens for inducing murine and human responses against human immunodeficiency virus type 1. J. Virol. 75, 8434– 8439. Mason, H.S., Lam, D.M., Arntzen, C.J., 1992. Expression of hepatitis B surface antigen in transgenic plants. Proc. Natl Acad. Sci. USA 89, 11745–11749. Mason, H.S., Ball, J.M., Shi, J.J., Jiang, X., Estes, M.K., Arntzen, C.J., 1996. Expression of Norwalk virus capsid protein in transgenic tobacco and potato and its oral immunogenicity in mice. Proc. Natl Acad. Sci. USA 93, 5335–5340. Mason, H.S., Haq, T.A., Clements, J.D., Arntzen, C.J., 1998. Edible vaccine protects mice against Escherichia coli heat-labile enterotoxin (LT): potatoes expressing a synthetic LT-B gene. Vaccine 16, 1336–1343. Matsumura, T., Itchoda, N., Tsunemitsu, H., 2002. Production of immunogenic VP6 protein of bovine group A rotavirus in transgenic potato plants. Arch. Virol. 147, 1263– 1270. McCormick, A.A., Kumagai, M.H., Hanley, K., Turpen, T.H., Hakim, I., Grill, L.K., Tuse, D., Levy, S., Levy, R., 1999. Rapid production of specific vaccines for lymphoma by expression of tumor-derived singlechain Fv epitopes in tobacco plants. Proc. Natl. Acad. Sci. USA 96, 703– 708. McGarvey, P.B., Hammond, J., Dienelt, M.M., Hooper, D.C., Fu, Z.F., Dietzschold, B., Koprowski, H., Michaels, F.H., 1995. Expression of the rabies virus glycoprotein in transgenic tomatoes. Biotechnology 13, 1484–1487.

492

S.J. Streatfield, J.A. Howard / International Journal for Parasitology 33 (2003) 479–493

McInerney, T.L., Brennan, F.R., Jones, T.D., Dimmock, N.J., 1999. Analysis of the ability of five adjuvants to enhance immune responses to a chimeric plant virus displaying an HIV-1 peptide. Vaccine 17, 1359–1368. McLain, L., Durrani, Z., Wisniewski, L.A., Porta, C., Lomonossoff, G.P., Dimmock, N.J., 1996. Stimulation of neutralizing antibodies to human immunodeficiency virus type 1 in three strains of mice immunized with a 22 amino acid peptide of gp41 expressed on the surface of a plant virus. Vaccine 14, 799–810. Modelska, A., Dietzschold, B., Sleysh, N., Fu, Z.F., Steplewski, K., Hooper, D.C., Koprowski, H., Yusibov, V., 1998. Immunization against rabies with plant-derived antigen. Proc. Natl Acad. Sci. USA 95, 2481–2485. Nemchinov, L.G., Liang, T.J., Rifaat, M.M., Mazyad, H.M., Hadidi, A., Keith, J.M., 2000. Development of a plant-derived subunit vaccine candidate against hepatitis C virus. Arch. Virol. 145, 2557– 2573. Nicholas, B.L., Brennan, F.R., Martinez-Torrecuadrada, J.L., Casal, J.I., Hamilton, W.D., Wakelin, D., 2002. Characterization of the immune response to canine parvovirus induced by vaccination with chimaeric plant viruses. Vaccine 20, 2727–2734. O’Brien, G.J., Bryant, C.J., Voogd, C., Greenberg, H.B., Gardner, R.C., Bellamy, A.R., 2000. Rotavirus VP6 expressed by PVX vectors in Nicotiana benthamiana coats PVX rods and also assembles into viruslike particles. Virology 270, 444 –453. Porta, C., Spall, V.E., Loveland, J., Johnson, J.E., Barker, P.J., Lomonossoff, G.P., 1994. Development of cowpea mosaic virus as a high-yielding system for the presentation of foreign peptides. Virology 202, 949–955. Qadri, F., Wenneras, C., Ahmed, F., Asaduzzaman, M., Saha, D., Albert, M.J., Sack, R.B., Svennerholm, A.-M., 2000. Safety and immunogenicity of an oral, inactivated enterotoxigenic Escherichia coli plus cholera toxin B subunit vaccine in Bangladeshi adults and children. Vaccine 18, 2704–2712. Richter, L.J., Thanavala, Y., Arntzen, C.J., Mason, H.S., 2000. Production of hepatitis B surface antigen in transgenic plants for oral immunization. Nat. Biotechnol. 18, 1167–1171. Ryan, E.T., Butterton, J.R., Smith, R.N., Carroll, P.A., Crean, T.I., Calderwood, S.B., 1997. Protective immunity against Clostridium difficile toxin A induced by oral immunization with a live, attenuated Vibrio cholerae vector strain. Infect. Immun. 65, 2941–2949. Sandhu, J.S., Krasnyanski, S.F., Domier, L.L., Korban, S.S., Osadjan, M.D., Buetow, D.E., 2000. Oral immunization of mice with transgenic tomato fruit expressing respiratory syncytial virus-F protein induces a systemic immune response. Transgenic Res. 9, 127–135. Smith, M.L., Keegan, M.E., Mason, H.S., Shuler, M.L., 2002a. Factors important in the extraction, stability and in vitro assembly of the hepatitis B surface antigen derived from recombinant plant systems. Biotechnol. Prog. 18, 538–550. Smith, M.L., Mason, H.S., Shuler, M.L., 2002b. Hepatitis B surface antigen (HbsAg) expression in plant cell culture: kinetics of antigen accumulation in batch culture and its intracellular form. Biotechnol. Bioeng. 80, 812–822. Staczek, J., Bendahmane, M., Gilleland, L.B., Beachy, R.N., Gilleland, H.E. Jr., 2000. Immunization with a chimeric tobacco mosaic virus containing an epitope of outer membrane protein F of Pseudomonas aeruginosa provides protection against challenge with P. aeruginosa. Vaccine 18, 2266–2274. Stanley, S., 2002. Oral tolerance of food. Curr. Allergy Asthma Rep. 2, 73–77. Streatfield, S.J., Jilka, J.M., Hood, E.E., Turner, D.D., Bailey, M.R., Mayor, J.M., Woodard, S.L., Beifuss, K., Horn, M.E., Delaney, D.E., Tizard, I.R., Howard, J.A., 2001. Plant-based vaccines: unique advantages. Vaccine 19, 2742–2748. Streatfield, S.J., Mayor, J.M., Barker, D.K., Brooks, C., Lamphear, B.J., Woodard, S.L., Beifuss, K.K., Vicuna, D.V., Massey, L.A., Horn, M.E., Delaney, D.D., Nikolov, Z.L., Hood, E.E., Jilka, J.M., Howard, J.A., 2002. Development of an edible subunit vaccine in corn against

enterotoxigenic strains of Escherichia coli. In Vitro Cell. Dev. Biol. Plant 38, 11–17. Streatfield, S.J., Lane, J.R., Brooks, C.A., Barker, D.K., Poage, M.L., Mayor, J.M., Lamphear, B.J., Drees, C.F., Jilka, J.M., Hood, E.E., Howard, J.A., 2003. Corn as a production system for human and animal vaccines. Vaccine 21, 812 –815. Strobel, S., 2002. Oral tolerance, systemic immunoregulation and autoimmunity. Ann. N.Y. Acad. Sci. 958, 47 –58. Tackaberry, E.S., Dudani, A.K., Prior, F., Tocchi, M., Sardana, R., Altosaar, I., Ganz, P.R., 1999. Development of biopharmaceuticals in plant expression systems: cloning, expression and immunological reactivity of human cytomegalovirus glycoprotein B (UL55) in seeds of transgenic tobacco. Vaccine 17, 3020– 3029. Tacket, C.O., Mason, H.S., Losonsky, G., Clements, J.D., Levine, M.M., Arntzen, C.J., 1998. Immunogenicity in humans of a recombinant bacterial antigen delivered in a transgenic potato. Nat. Med. 4, 607 –609. Tacket, C.O., Mason, H.S., Losonsky, G., Estes, M.K., Levine, M.M., Arntzen, C.J., 2000. Human immune responses to a novel Norwalk virus vaccine delivered in transgenic potatoes. J. Infect. Dis. 182, 302 –305. Thanavala, Y., Yang, Y.-F., Lyons, P., Mason, H.S., Arntzen, C., 1995. Immunogenicity of transgenic plant-derived hepatitis B surface antigen. Proc. Natl Acad. Sci. USA 92, 3358–3361. Tomasi, T.B. Jr, 1980. Oral tolerance. Transplantation 29, 353– 356. Tuboly, T., Yu, W., Bailey, A., Degrandis, S., Du, S., Erickson, L., Nagy, E., 2000. Immunogenicity of porcine transmissible gastroenteritis virus spike protein expressed in plants. Vaccine 18, 2023–2028. Turpen, T.H., Reinl, S.J., Charoenvit, Y., Hoffman, S.L., Fallarme, V., Grill, L.K., 1995. Malarial epitopes expressed on the surface of recombinant tobacco mosaic virus. Biotechnology (N Y) 13, 53–57. Usha, R., Rohll, J.B., Spall, V.E., Shanks, M., Maule, A.J., Johnson, J.E., Lomonossoff, G.P., 1993. Expression of an animal virus antigenic site on the surface of a plant virus particle. Virology 197, 366–374. Van De Verg, L., Herrington, D.A., Murphy, J.R., Wasserman, S.S., Formal, S.B., Levine, M.M., 1990. Specific immunoglobulin Asecreting cells in peripheral blood of humans following oral immunization with a bivalent Salmonella typhi – Shigella sonnei vaccine or infection by pathogenic S. sonnei. Infect. Immun. 58, 2002–2004. Webster, D.E., Cooney, M.L., Huang, Z., Drew, D.R., Ramshaw, I.A., Dry, I.B., Strugnell, R.A., Martin, J.L., Wesselingh, S.L., 2002. Successful boosting of a DNA measles immunization with an oral plant-derived measles virus vaccine. J.Virol. 76, 7910–7912. Weiner, H.L., 1997. Oral tolerance: immune mechanisms and treatment of autoimmune diseases. Immunol. Today 18, 335–343. Wigdorovitz, A., Perez Filgueira, D.M., Robertson, N., Carrillo, C., Sadir, A.M., Morris, T.J., Borca, M.V., 1999a. Protection of mice against challenge with foot and mouth disease virus (FMDV) by immunization with foliar extracts from plants infected with recombinant tobacco mosaic virus expressing the FMDV structural protein VP1. Virology 264, 85–91. Wigdorovitz, A., Carrillo, C., Dus Santos, M.J., Trono, K., Peralta, A., Gomez, M.C., Rios, R.D., Franzone, P.M., Sadir, A.M., Escribano, J.M., Borca, M.V., 1999b. Induction of a protective antibody response to foot and mouth disease in mice following oral or parenteral immunization with alfalfa transgenic plants expressing the viral structural protein VP1. Virology 255, 347–353. Witcher, D.R., Hood, E.E., Peterson, D., Bailey, M., Bond, D., Kusnadi, A., Evangelista, R., Nikolov, Z., Wooge, C., Mehigh, R., Kappel, W., Register, J., Howard, J.A., 1998. Commercial production of b-glucuronidase (GUS): a model system for the production of proteins in plants. Mol. Breed. 4, 301 –312. Yu, J., Langridge, W.H.R., 2001. A plant-based multicomponent vaccine protects mice from enteric diseases. Nat. Biotechnol. 19, 548–552. Yusibov, V., Modelska, A., Steplewski, K., Agadjanyan, M., Weiner, D., Hooper, D.C., Koprowski, H., 1997. Antigens produced in plants by

S.J. Streatfield, J.A. Howard / International Journal for Parasitology 33 (2003) 479–493 infection with chimeric plant viruses immunize against rabies virus and HIV-1. Proc. Natl Acad. Sci. USA 94, 5784–5788. Yusibov, V., Hooper, D.C., Spitsin, S.V., Fleysh, N., Kean, R.B., Mikheeva, T., Deka, D., Karasev, A., Cox, S., Randall, J., Koprowski, H., 2002. Expression in plants and immunogenicity of plant virus-based experimental rabies vaccine. Vaccine 20, 3155– 3164. Zhang, G., Leung, C., Murdin, L., Rovinski, B., White, K.A., 2000. In planta expression of HIV-1 p24 protein using an RNA plant virusbased expression vector. Mol. Biotechnol. 14, 99 –107.

493

Zhang, G.G., Rodrigues, L., Rovinski, B., White, K.A., 2002. Production of HIV-1 p24 protein in transgenic tobacco plants. Mol. Biotechnol. 20, 131– 136. Zhong, G.-Y., Peterson, D., Delaney, D.E., Bailey, M., Witcher, D.R., Register, J.C. III, Bond, D., Li, C.-P., Marshall, L., Kulisek, E., Ritland, D., Meyer, T., Hood, E.E., Howard, J.A., 1999. Commercial production of aprotinin in transgenic maize seeds. Mol. Breed. 5, 345 –356.