Constructing and transient expression of a gene cassette containing edible vaccine elements and shigellosis, anthrax and cholera recombinant antigens in tomato.

Shigella dysenteriae causing shigellosis is one of the diseases that threaten the health of human society in the developing countries. In Shigella, IpaD gene is one of the key pathogenic genes causing strong mucosal immune system reactions. Anthrax disease is caused by Bacillus anthracis. PA protective antigen is one of the subunits in anthrax toxin complex responsible for the transfer of other subunits into the cytosol of host cells. The 20 kDa subunit of PA (PA20) has the property of immunogenicity. CTxB or B subunit of Vibrio cholerae toxin (CT) is a non-toxic protein and has the function to transfer toxic subunit into cytosol of the host cells by binding to GM1 receptor. The aim of this study was to fuse PA20, ipaD and CTxB and transform tomato plants by this cassette in order to produce an oral vaccine against shigellosis, anthrax and cholera. CTxB was used for these two antigens as an immune adjuvant. IpaD and PA20 genes were cloned in pBI121 containing the CTxB gene and Extensin signal peptide. In order to evaluate the transient expression of Shigellosis, Anthrax and Cholera antigens, agro-infiltrated tomato tissues were inoculated with Agrobacterium tumefaciens containing the gene cassette. Cloning was confirmed by PCR, enzymatic digestion and sequencing techniques. Expression of the antigens was examined by SDS-PAGE, dot blot and ELISA. Maturate green fruits demonstrated the highest expression of the recombinant proteins. The first phase of this study was carried out for cloning and expressing of CtxB, ipaD and PA20 antigens in tomato. In the next phase, we aim to analyze the immunogenicity of this vaccine candidate in laboratory animals.

A CGMMV genome-replicon vector with partial sequences of coat protein gene efficiently expresses GFP in Nicotiana benthamiana.

A highly infectious clone of Cucumber green mottle mosaic virus (CGMMV), a cucurbit-infecting tobamovirus was utilized for designing of gene expression vectors. Two versions of vector were examined for their efficacy in expressing the green fluorescent protein (GFP) in Nicotiana benthamiana. When the GFP gene was inserted at the stop codon of coat protein (CP) gene of the CGMMV genome without any read-through codon, systemic expression of GFP, as well as virion formation and systemic symptoms expression were obtained in N. benthamiana. The qRT-PCR analysis showed 23 fold increase of GFP over actin at 10days post inoculation (dpi), which increased to 45 fold at 14dpi and thereafter the GFP expression was significantly declined. Further, we show that when the most of the CP sequence is deleted retaining only the first 105 nucleotides, the shortened vector containing GFP in frame of original CP open reading frame (ORF) resulted in 234 fold increase of GFP expression over actin at 5dpi in N. benthamiana without the formation of virions and disease symptoms. Our study demonstrated that a simple manipulation of CP gene in the CGMMV genome while preserving the translational frame of CP resulted in developing a virus-free, rapid and efficient foreign protein expression system in the plant. The CGMMV based vectors developed in this study may be potentially useful for the production of edible vaccines in cucurbits.

[Plants as an alternative source of therapeutic proteins]. / Rosliny jako alternatywne zródlo bialek terapeutycznych.

In recent years, there has been an increased interest of researchers in developing efficient plant heterologous expression systems of proteins for a wide range of applications. It represents an alternative to the traditional strategy utilizing bacterial, yeast, insect or mammalian cells. New techniques of identification and characterization and effective methods of plant genetic transformation allow the range of recombinant protein products to be expanded. Great expectations are associated with the use of plants as bioreactors for the production of specific proteins of therapeutic interest. This strategy offers a number of advantages, the most important being: the possibility of a significant reduction in production costs, the safety of the products obtained and full eukaryotic post-translational modifications of proteins. A group of proteins of special interest is pharmaceuticals, and a number of successful experiments have confirmed the possibility of obtaining heterogeneous proteins with therapeutic potential: monoclonal antibodies, vaccine antigens, and a variety of cytokines. This work is focused on selected recombinant proteins belonging to those groups expression of which was achieved in plant cells. These proteins may be used in the future for therapy or prevention of viral, bacterial or cancer diseases.

Feasibility of Pisum sativum as an expression system for pharmaceuticals.

Based on its high protein content and excellent storage capacity, pea (Pisum sativum), as well as other plants, is considered to be a suitable production platform for protein-based pharmaceuticals. Its capacity to produce high proportions of active recombinant proteins (up to 2% total soluble protein corresponding to approximately 8 mg/g fresh weight) has been proven using pea-derived strong seed-specific promoters. The active antigens produced were also stable for more than 4 years. Pea can be used as a feed additive, up to a proportion of 30% to total feed, despite the presence of lectins. Thus, a low dosage of recombinant pea-based pharmaceuticals is non-hazardous. In addition, it is independent of N-fertilisation, has excellent biosafety characteristics and is accessible to gene transfer. Growth systems with a capacity for high yield are available for the greenhouse (5 t/ha) and, to a limited extent, also in the field (2.3 t/ha). The practicable establishment of pea seed banks allows a continuous production process. Although the use of a pea system is limited by complex transformation procedures, these advantages render pea a promising plant for the production of pharmaceuticals.

Expression of a cholera toxin B subunit-neutralizing epitope of the porcine epidemic diarrhea virus fusion gene in transgenic lettuce (Lactuca sativa L.).

Transgenic plants have been used as a safe and economic expression system for the production of edible vaccines. A synthetic cholera toxin B subunit gene (CTB) was fused with a synthetic neutralizing epitope gene of the porcine epidemic diarrhea virus (sCTB-sCOE), and the sCTB-sCOE fusion gene was introduced into a plant expression vector under the control of the ubiquitin promoter. This plant expression vector was transformed into lettuce (Lactuca sativa L.) using the Agrobacterium-mediated transformation method. Stable integration and transcriptional expression of the sCTB-sCOE fusion gene was confirmed using genomic DNA PCR analysis and northern blot analysis, respectively. The results of western blot analysis with anti-cholera toxin and anti-COE antibody showed the synthesis and assembly of CTB-COE fusion protein into oligomeric structures with pentameric sizing. The biological activity of CTB-COE fusion protein to its receptor, G(M1)-ganglioside, in transgenic plants was confirmed via G(M1)-ELISA with anti-cholera toxin and anti-COE antibody. Based on G(M1)-ELISA, the expression level of CTB-COE fusion proteins reached 0.0065% of the total soluble protein in transgenic lettuce leaf tissues. Transgenic lettuce successfully expressing CTB-COE fusion protein will be tested to induce efficient immune responses against porcine epidemic diarrhea virus infection by administration with raw material.

Oral immunogenicity of tomato-derived sDPT polypeptide containing Corynebacterium diphtheriae, Bordetella pertussis and Clostridium tetani exotoxin epitopes.

DPT vaccine, designed to immunize against diphtheria, pertussis, and tetanus, has been shown to be effective in humans. Nevertheless, dissatisfaction with the whole-cell preparations is due to the reactogenicity, which has to lead to the development of new safer formulations. Previously, we described the expression in tomato of a plant-optimized synthetic gene encoding the recombinant polypeptide sDPT, containing mainly immunoprotective epitopes of the diphtheria, pertussis and tetanus exotoxins and two adjuvants. In this study, we examined whether the ingestion of tomato-derived sDPT protein induces specific antibodies in mice after three weekly doses scheme. A positive group immunized with DPT toxoids was included. Specific antibody levels were assessed in serum, gut and lung. Sera tested for IgG antibody response to pertussis, tetanus and diphtheria toxin showed responses to the foreign antigens; interestingly, the response to diphtheria epitope was similar to those observed in the positive group. We found higher IgG1 than IgG2a responses in serum. A modest IgG response was observed in the tracheopulmonary fluid. High response of IgA against tetanus toxin was evident in gut, which was statistically comparable to that obtained in the positive group. The levels of response in these groups were higher than those in mice that received wild-type tomato. These findings support the concept of using transgenic tomatoes expressing sDPT polypeptide as model for edible vaccine against diphtheria, pertussis, and tetanus.

The mucosal immune response to plant-derived vaccines.

Transgenic plants present enormous potential as one of the most cost-effective and safe systems for large-scale production of proteins for industrial, pharmaceutical, veterinary and agricultural uses. Heat-stable plant-derived vaccines that are administered orally could in effect enhance vaccine coverage in children and infants, particularly in developing countries. Here we discuss the current status of plant-derived vaccines and their potential to champion the battle against infectious diseases in the least developed countries.

Plant-made vaccines for humans and animals.

The concept of using plants to produce high-value pharmaceuticals such as vaccines is 20 years old this year and is only now on the brink of realisation as an established technology. The original reliance on transgenic plants has largely given way to transient expression; proofs of concept for human and animal vaccines and of efficacy for animal vaccines have been established; several plant-produced vaccines have been through Phase I clinical trials in humans and more are scheduled; regulatory requirements are more clear than ever, and more facilities exist for manufacture of clinic-grade materials. The original concept of cheap edible vaccines has given way to a realisation that formulated products are required, which may well be injectable. The technology has proven its worth as a means of cheap, easily scalable production of materials: it now needs to find its niche in competition with established technologies. The realised achievements in the field as well as promising new developments will be reviewed, such as rapid-response vaccines for emerging viruses with pandemic potential and bioterror agents.

Transgenic lettuce producing a candidate protein for vaccine against edema disease.

Pig edema disease is a bacterial disease caused by Shiga toxin 2e-producing Escherichia coli belonging mainly to serotypes O138, O139, and O141. The B subunit of Shiga toxin 2e (Stx2eB) is a candidate protein for use in a vaccine against edema disease. We produced this protein in transgenic lettuce (Lactuca sativa), an edible plant that can be cultivated in a factory setting. In a transient expression system, we found that NtADH 5'-untranslated region (5'-UTR) functions as a translational enhancer in lettuce cells, and that Stx2eB accumulates most efficiently in the endoplasmic reticulum (ER) of lettuce cells. Stx2eB was produced in stable transgenic lettuce plants expressing a modified Stx2eB gene fused with the NtADH 5'-UTR and sequence encoding ER localization signals.

Generation and immunogenicity of Japanese encephalitis virus envelope protein expressed in transgenic rice.

Transgenic plants have become attractive as bioreactors to produce heterologous proteins that can be developed as edible vaccines. In the present study, transgenic rice expressing the envelope protein (E) of Japanese encephalitis virus (JEV), under the control of a dual cauliflower mosaic virus (CaMV 35S) promoter, was generated by Agrobacterium-mediated transformation. Southern blot, Northern blot, Western blot and ELISA analyses confirmed that the E gene was integrated into transgenic rice and was expressed in the leaves at levels of 1.1-1.9 microg/mg of total soluble protein. After intraperitoneal immunization of mice with crude protein extracts from transgenic rice plants, JEV-specific neutralizing antibody could be detected. Moreover, E-specific mucosal immune responses could be detected in mice after oral immunization with protein extracts from transgenic rice plants. These results show the potential of using a transgenic rice-based expression system as an alternative bioreactor for JEV subunit vaccine.

Bioproduction of therapeutic proteins in the 21st century and the role of plants and plant cells as production platforms.

In the last decade, the technique to genetically modify crop plants has gained more and more interest in terms of bioproduction of heterologous proteins. Plants have been discovered as a possible source for large amounts of cost effective recombinant protein. Main application fields are therapeutics for use in animal and human health, diagnostics, and technical enzymes. This review is focused on the recent progress in this field of molecular farming. After a comparison with hitherto established protein production systems, the advantages of plants as an alternative production system are discussed. An overview about the different host plants and possible expression strategies is given and the progress in commercialization of the techniques is highlighted. Finally, the role of plant cell cultures for the production of recombinant proteins is discussed.

Research advances on transgenic plant vaccines.

In recent years, with the development of genetics molecular biology and plant biotechnology, the vaccination (e.g. genetic engineering subunit vaccine, living vector vaccine, nucleic acid vaccine) programs are taking on a prosperous evolvement. In particular, the technology of the use of transgenic plants to produce human or animal therapeutic vaccines receives increasing attention. Expressing vaccine candidates in vegetables and fruits open up a new avenue for producing oral/edible vaccines. Transgenic plant vaccine disquisitions exhibit a tempting latent exploiting foreground. There are a lot of advantages for transgenic plant vaccines, such as low cost, easiness of storage, and convenient immune-inoculation. Some productions converged in edible tissues, so they can be consumed directly without isolation and purification. Up to now, many transgenic plant vaccine productions have been investigated and developed. In this review, recent advances on plant-derived recombinant protein expression systems, infectious targets, and delivery systems are presented. Some issues of high concern such as biosafety and public health are also discussed. Special attention is given to the prospects and limitations on transgenic plant vaccines.

Risk analysis for plant-made vaccines.

The production of vaccines in transgenic plants was first proposed in 1990 however no product has yet reached commercialization. There are several risks during the production and delivery stages of this technology, with potential impact on the environment and on human health. Risks to the environment include gene transfer and exposure to antigens or selectable marker proteins. Risks to human health include oral tolerance, allergenicity, inconsistent dosage, worker exposure and unintended exposure to antigens or selectable marker proteins in the food chain. These risks are controllable through appropriate regulatory measures at all stages of production and distribution of a potential plant-made vaccine. Successful use of this technology is highly dependant on stewardship and active risk management by the developers of this technology, and through quality standards for production, which will be set by regulatory agencies. Regulatory agencies can also negatively affect the future viability of this technology by requiring that all risks must be controlled, or by applying conventional regulations which are overly cumbersome for a plant production and oral delivery system. The value of new or replacement vaccines produced in plant cells and delivered orally must be considered alongside the probability and severity of potential risks in their production and use, and the cost of not deploying this technology--the risk of continuing with the status quo alternative.

Transgenic plants for the production of veterinary vaccines.

The expression of antigens in transgenic plants has been increasingly used in the development of experimental vaccines, particularly oriented to the development of edible vaccines. Hence, this technology becomes highly suitable to express immunogenic proteins from pathogens. Foot and mouth disease virus, bovine rotavirus and bovine viral diarrhoea virus are considered to be the most important causative agents of economic loss of cattle production in Argentina, and they are thus optimal candidates for alternative means of immunization. Here, we present a review of our results corresponding to the expression of immunogenic proteins from these three viruses in alfalfa transgenic plants, and we discuss the possibility of using them for the development of plant-based vaccines.

Is there a role for plant-made vaccines in the prevention of HIV/AIDS?

Although educational programs have had some impact, immunization against HIV will be necessary to control the AIDS pandemic. To be effective, vaccination will need to be accessible and affordable, directed against multiple antigens, and delivered in multiple doses. Plant-based vaccines that are heat-stable and easy to produce and administer are suited to this type of strategy. Pilot studies by a number of groups have demonstrated that plant viral expression systems can produce HIV antigens in quantities that are appropriate for use in vaccines. In addition, these plant-made HIV antigens have been shown to be immunogenic. However, given the need for potent cross-clade humoral and T-cell immunity for protection against HIV, and the uncertainty surrounding the efficacy of protein subunit vaccines, it is most likely that plant-made HIV vaccines will find their niche as booster immunizations in prime-boost vaccination schedules.

The next 15 years: taking plant-made vaccines beyond proof of concept.

Significant potential advantages are associated with the production of vaccines in transgenic plants; however, no commercial product has emerged. An analysis of the strengths, weaknesses, opportunities and threats for plant-made vaccine technology is provided. The use of this technology for human vaccines will require significant investment and developmental efforts that cannot be supported entirely by the academic sector and is not currently supported financially by industry. A focus on downstream aspects to define potential products, conduct of additional basic clinical testing, and the incorporation of multidisciplinary strategic planning would accelerate the potential for commercialization in this field. Estimates of production cost per dose and volume of production are highly variable for a model vaccine produced in transgenic tomato, and can be influenced by the optimization of many factors. Commercialization of plant-made vaccine technology is likely to be led by the agricultural biotechnology sector rather than the pharmaceutical sector due to the disruptive nature of the technology and the complex intellectual property landscape. The next major milestones will be conduct of a phase II human clinical trial and demonstration of protection in humans. The achievement of these milestones would be accelerated by further basic investigation into mucosal immunity, the codevelopment of oral adjuvants, and the integration of quality control standards and good manufacturing practices for the production of preclinical and clinical batch materials.

Oral hepatitis B vaccine candidates produced and delivered in plant material.

Hepatitis B is a major global health problem; approximately two billion people are infected with the virus worldwide, despite the fact that safe and efficacious vaccines have been developed and used for nearly 20 years. Prohibitive costs for vaccine purchase and administration restrict uptake in many developing nations. Agencies such as the Global Alliance for Vaccination and Immunization are helping to make current vaccines more available, but reduced costs would greatly aid this effort. Oral delivery is an option to reduce the expense of administering hepatitis B vaccines. It may also improve compliance, and orally delivered vaccines may be more efficacious among poor responders to current vaccines. However, to induce protective efficacy, oral administration may require encapsulation of antigen and delivery of large doses. Plant-based expression systems offer an oral delivery alternative with low production costs, and they also encapsulate the antigen. Some plant-based systems also stabilize antigen and therefore reduce storage and distribution costs. The hepatitis B major surface antigen has been expressed in several plant systems. A variety of regulatory sequences and subcellular targets have been used to achieve expression suitable for early stage clinical trials. However, further increase in expression will be necessary for practical and efficacious products. Appropriate processing can yield palatable products with uniform antigen concentration. The antigen expressed in plant systems shows extensive disulphide cross-linking and oligomerization and forms virus-like particles. Oral delivery of the antigen in plant material can induce a serum antibody response, prime the immune system for a subsequent injection of antigen and give a boosted response to a prior injection. Small scale clinical trials in which the antigen has been delivered orally in edible plant material indicate safety and immunogenicity.