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Plants are remarkably plastic with respect to the type of foreign proteins they are able to express: diverse proteins of viral, bacterial, fungal, invertebrate and vertebrate origin have been successfully expressed in plants [1]. Transgenic plants have some characteristic features that make them particularly well suited for cost-effective bioproduction of proteins for pharmaceutical uses. These include: (a) low production costs, (b) reduced time to market, (c) unlimited supply, (d) eukaryotic protein processing and (e) safety. Cost advantages are based not only on the low cost of biomass production, but also costs associated with research and development, scale-up, reduced requirements for quality assurance as plants are not hosts to major mammalian pathogens and storage and delivery is also simple. Plant-based strategies also have advantages in the pace at which feasibility testing can be done at R & D successes can be scaled up and brought to market. Many of the therapeutic proteins require posttranslational processing for bioactivity. Though certain modification steps are lacking or differ in plants e. g. glycan composition, there is remarkable conservation of these protein processing steps between plants and animals such that the majority of the human proteins that have been produced in plants show significant structural, biochemical and functional equivalency to proteins from humans or animal cell cultures.
Plants as antigen delivery systems
In the context of using plants as antigen production and delivery systems, there are two main options for engineering a plant. The first is to integrate the DNA encoding the gene of interest into the nuclear genome to generate stably transformed transgenic plants expressing the antigen constitutively or in specific tissues. The second is to integrate the genetic material encoding immunologically active protein or peptide into the genome of a plant virus used to infect plants.
When virus-based production systems are used for antigen production, the chimeric virus particles or free foreign proteins must usually be purified from infected leaf tissues, which can be costly and complicated process. However, production of antigenic proteins in edible tissues of transgenic plants presents the very attractive possibility that these tissues can be consumed directly, providing an "edible oral vaccine" which may obviate the need to purify the vaccine protein.
The mucosal immune system
The success of an edible vaccine requires induction of mucosal immune system (MIS). The MIS is the first line of defense of the surfaces where most human and animal pathogens initiate infection, that is, the mucosal surfaces found lining the digestive tract, respiratory tract and urino-reproductive tract. It is obvious that injected vaccines do not generate satisfactory MIS and that in order to elicit sufficient mucosal immunity, the vaccine should be delivered directly to the mucosal surface [2]. The common mucosal immune system (CMIS) can be divided into two functionally distinct compartments, namely, namely inductive sites and effector sites. This network is highly integrated and finely regulated, and the outcome of mucosal tissue encounter with foreign antigens and pathogens can range from mucosal and serum antibodies, T-cell CMI, and cytotoxic lymphocyte (CTL) responses, on the one hand, to systemic anergy, a response commonly termed mucosal tolerance, on the other. This physiological division is of paramount importance in the design of vaccines effective in the induction of protective immunity within the mucosal immune system and, in particular, its humoral branch. In the mucosal system, major effector molecule, secretory IgA (sIgA), is by far the most abundant immunoglobulin class in the body and are resistant to intestinal proteases. An oral vaccine must survive the proteases and low pH of the stomach to reach the effector sites in the intestines. Nevertheless, the default response of the intestinal immune system to food antigens is tolerance [3]. There is sufficient evidence to show that all ingested antigens are seen as food. Oral antigens that bind to epithelial cells (such as E. coli fimbriae or bacterial toxins from V. cholerae and enterotoxigenic E. coli) or that replicate (such as bacterial pathogens) is immunogenic rather than tolerogenic. Tolerance is dose-, schedule-, and antigen-specific [4]. The challenge of edible vaccine approach lies in the fact that it stimulates an immunogenic response to the vaccine protein and maintains the tolerogenic response to the food vehicle.
Plant systems for recombinant vaccine production
The choice of which plant species to use for production and delivery of an oral vaccine can readily be tailored to the subject to be vaccinated. Few important food plants for which transformations have been reported are: alfalfa, banana, maize, potato, soybean, tomato, and wheat. Plant model systems such as; tobacco, potato, tomato and maize have been employed for recombinant protein expression. Each of these species is readily transformed, and can be propagated and regenerated in tissue culture by established protocols. Transgenic tobacco engineered to express a recombinant vaccine protein is not ideal material for oral delivery due to high levels of alkaloids and nicotine. The main disadvantage with potato tubers is that they are not high in protein content and thus level of recombinant protein must be higher on a percentage total protein basis than tissues containing more protein, and the same is true for tomatoes. But the advantage with tomatoes is that humans can consume them raw.
Level of transgene expression
Economical use of plants as a production system requires maximum transgene expression and recombinant protein accumulation. Augmenting protein stability, mRNA stability and/ translatability, and enhancing promoter strength can increase yields of recombinant proteins. A strategy based on tissue specific expression would allow the accumulation of recombinant protein only in tissue to be fed as a vaccine. Even the use of "plant optimized" synthetic genes has been shown to enhance the accumulation of several recombinant proteins dramatically [5].
Posttranslation modifications
An important issue in the expression of vaccine antigens is glycosylation as carbohydrate groups contribute to immunogenicity. Plants also glycosylate proteins but the rules for N- and O-linked glycosylation in plants are different. The capability of plants to glycosylate heterologous proteins will need to be determined for each antigen.
Transgenic edible plant vaccines against bacterial pathogens
The surface protein antigen A (spaA) from Streptococcus mutans produced in transgenic tobacco was the first description of an antigen being produced in plants, which appeared in a European patent [6]. The concept of edible vaccine was first proposed by Arntzen and Mason in 1992. The study by Haq et al. [7] provided the first "proof of concept" demonstrating that bacterial antigen, binding subunit of Escherichia coli heat-labile enterotoxin (LT-B) expressed in transgenic tobacco and potato is immunologically active and produces both serum and mucosal antibodies against LT-B protein. Oral feeding of mice with transgenic potatoes expressing LT-B resulted in oral immune responses and mice fed with transgenic tubers (three weekly doses of 20-50mg LT-B) were partially protected against challenge with holotoxin LT [8]. More recently human volunteers, ingested 50-100g raw tubers per dose at 0, 7 and 21 days [9], developed serum and mucosal immune responses.
Transgenic edible plant vaccines against viral pathogens
Hepatitis B
Hepatitis B surface antigen (HbsAg) expressed in transgenic tobacco [10] and immunogenicity of the HBsAg purified from transgenic tobacco leaves was evaluated in mice [11]. The results clearly demonstrated that B- and T-cell epitopes of HBsAg are preserved. Oral immunization of mice with HBsAg expressed in transgenic potato plants was analyzed [12]. Mice fed with transgenic potato expressed HBsAg produced HBsAg-specifc serum antibodies that exceeded the protective level and on parenteral boosting, generated a strong long-lasting secondary immune response.
Norwalk virus
Transgenic tobacco and potato plants were generated expressing Norwalk virus capsid protein (NVCP) [13]. Expression of NVCP in tobacco leaves resulted in production of virus like particles. Plant-derived antigen delivered orally to mice stimalted the production of humoral and mucosal antibody responses. The study was further extended to analyze oral immunogenicity of NVCP, assembled as virus like particles, in humans [14]. Twenty volunteers received 2-3 doses each consisting of 150g of raw, peeled, diced potato that contained 215-751mg of NVCP. 95% (19/20) volunteers developed increased numbers of specific IgA antibody- secreting cells, 20% (4/20) developed specific serum IgG and 30% (6/20) developed specific stool IgA. To sum up, 19 of 20 volunteers developed some kind of immune response.
Respiratory syncytial virus (RSV)
Transgenic tomato plants expressing RSV fusion (F) protein were developed [15]. Two different constructs were used; F gene placed either under the control of constitutively expressed CaMV 35S promoter (pJSS3) or the fruit specific E8 promoter (pJSS4). However, the average levels of RSV-F antigen in fruits of transgenic plants transformed with constructs driven by E8 and CaMV 35S promoter were similar, 12.68±2.55 and 9.01±1.87mg/g fruit fresh weight, respectively. Oral immunization of mice with ripe transgenic tomato fruits led to the induction of both serum and mucosal RSV-F specific antibodies. Increase in serum antibody titre was observed upon exposure of mice to inactivated RSV antigen.
Measles virus
Measles virus hemagglutinin protein was expressed in transgenic tobacco [16]. Three different expression constructs were tested; pBinH (H alone), pBinH/KDEL (addition of a C-terminal endoplasmic reticulum-retention signal sequence SEKDEL) and pBinSP/H/KDEL (further addition of an authentic N-terminal plant signal peptide). Plants transformed with pBinH/KDEL expressed highest levels of recombinant H protein. Intraperitoneal immunization of mice with transgenic plant-derived H protein produced serum anti-H antibodies that neutralized measles virus in vitro. Further, gavaging with transgenic tobacco leaf extract also developed neutralizing serum anti-H antibodies.
Rabies virus
Rabies virus glycoprotein (G-protein) was expressed in transgenic potatoes [17]. The G-protein expressed in tomato appeared as two distinct bands with apparent molecular mass of 60 and 62 kDa as compared to the expected 66kDa. The differences in sizes may be due to altered glycosylation and/or specific enzymatic cleavage of sugar or amino acid residues. Results from immunoprecipitation and Western blots demonstrate that important immunological epitopes are presented in the plant produced G-protein. The expression level was estimated to be between 1-10ng/mg total soluble protein or 0.001%.
Rotavirus
Rotavirus NSP4 enterotoxin epitope protein was fused with cholera toxin B subunit and transgenic potato plants were generated [18]. The plant expressed CTB-NSP4 oligomerized as demonstrated by immunoblot analysis and retained enterocyte receptor GM1 ganglioside binding affinity. The expression was in the range of 0.01-0.1% of total soluble tuber protein.
Foot and mouth disease virus (FMDV)
The first report showing protection against an animal viral disease by immunization with an antigen expressed in a transgenic plant was by Carrillo et al. [19]. The structural protein VP1 of foot-and-mouth disease virus was expressed in Arabidopsis thaliana. Mice immunized with leaf extract elicited specific antibody response and all the immunized mice were protected against challenge with virulent FMDV. Further, VP1 expressed in transgenic alfalfa, induced protective immune response to FMDV in mice upon oral or parenteral immunization [20]. In order to increase the level of expression of the transgene, the same group developed transgenic potato plants containing VP1 gene under the control of either single (pRok2) or double (pRok3) copy of CaMV 35S promoter [21]. They failed to detect any significant differences in immune responses obtained following immunization with pRok2 or pRok3. Recently, transgenic alfalfa plants expressing the immunogenic peptide VP135-160 fused to bGUS protein were generated [22]. The FMDV epitope expressed in plants is highly immunogenic in mice as revealed by a strong anti-FMDV response, and complete protection against experimental challenge with the virulent virus.
Transmissible gastroenteritis coronavirus (TGEV)
Arabidopsis plants were genetically transformed to express immunogenic glycoprotein S polypeptide from TGEV [23]. Mice immunized with leaf extract from transgenic plants developed specific-antibodies to TGEV, immunoprecipitated the virus-induced proteins, and neutralized the virus infectivity. Oral immunogenicity of the spike protein expressed in potato was examined [24]. Potato plants were transformed with N-terminal of spike protein containing major antigenic sites. Intraperitoneal immunization of mice with transgenic plant extract induced serum IgG specific for TGEV. Further, mice fed with potato tubers expressing the recombinant protein developed specific immune response. Independent of the immunization route used, no neutralizing antibodies were detected. The differences in neutralization activity between sera from mice immunized with Arabidopsis-derived of potato-derived antigen could be influenced by the posttranslational processing of the glycoprotein depending on the plant used, or due to plant proteins that, accompanying the recombinant protein. Tuboly et al. [25] reported the immunogenicity of porcine transmissible gastroenteritis virus spike protein expressed in tobacco plants. Pigs immunized with transgenic plant extract developed TGEV-specific immune response as demonstrated by virus neutralization.
Conclusions
The expression of antigens in vegetables and fruits has opened up a new avenue for the development of oral vaccines. The potentially low cost of production and scale-up to agricultural levels that plant promise, should provide a source for antibodies, vaccines and therapeutic molecules for the population of the whole world.
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