High-yield expression of a viral peptide animal vaccine in transgenic tobacco chloroplasts

The 2L21 peptide, which confers protection to dogs against challenge with virulent canine parvovirus (CPV), was expressed in tobacco chloroplasts as a C-terminal translational fusion with the cholera toxin B subunit (CTB) or the green fluorescent protein (GFP). Expression of recombinant proteins was dependent on plant age. A very high-yield production was achieved in mature plants at the time of full flowering (310 mg CTB-2L21 protein per plant). Both young and senescent plants accumulated lower amounts of recombinant proteins than mature plants. This shows the importance of the time of harvest when scaling up the process. The maximum level of CTB-2L21 was 7.49 mg/g fresh weight (equivalent to 31.1% of total soluble protein, TSP) and that of GFP-2L21 was 5.96 mg/g fresh weight (equivalent to 22.6% of TSP). The 2L21 inserted epitope could be detected with a CPV-neutralizing monoclonal antibody, indicating that the epitope is correctly presented at the C-terminus of the fusion proteins. The resulting chimera CTB-2L21 protein retained pentamerization and G(M1)-ganglioside binding characteristics of the native CTB and induced antibodies able to recognize VP2 protein from CPV. To our knowledge, this is the first report of an animal vaccine epitope expression in transgenic chloroplasts. The high expression of antigens in chloroplasts would reduce the amount of plant material required for vaccination (approximately 100 mg for a dose of 500 microg antigen) and would permit encapsulation of freeze-dried material or pill formation.     

Use of the viral 2A peptide for bicistronic expression in transgenic mice

Background  

Transgenic animals are widely used in biomedical research and biotechnology. Multicistronic constructs, in which several proteins are encoded by a single messenger RNA, are commonly used in genetically engineered animals. This is currently done by using an internal ribosomal entry site to separate the different coding regions. 2A peptides result in the co-translational 'cleavage' of proteins and are an attractive alternative to the internal ribosomal entry site. They are more reliable than the internal ribosomal entry site and lead to expression of multiple cistrons at equimolar levels. They work in a wide variety of eukaryotic cells, but to date have not been demonstrated to function in transgenic mice in an inheritable manner.     

Use of viral vectors for vaccine production in plants

The small size of plant viral genomes, the ease with which they can be manipulated, and the simplicity of the infection process is making the viral vectors an attractive alternative to the transgenic systems for the expression of foreign proteins in plants. One use of these virus expression systems is for vaccine production. There are two basic types of viral system that have been developed for the production of immunogenic peptides and proteins in plants: epitope presentation and polypeptide expression systems. In this review, we discuss advances made in this field.     

Current trends in edible vaccine development using transgenic plants

Immunology textbooks currently report orally administered antigens as inducing immune tolerance rather than immune stimulation. Nevertheless, current plant-based edible vaccine technology, if sufficiently developed, may offer several advantages. For example, it is easy to apply, store, and transport. It could also induce both mucosal and systemic immune responses, which cannot be achieved using an injection vaccine. Plant-based vaccines are also anticipated to prove quite useful in the animal industry, since the cost of injection is a significant burden in the animal industry. Although no commercial plant-based edible vaccines are currently available, several candidate vaccines are undergoing clinical trials. Consequently, many scientists are anticipating that a commercial plant-based edible vaccine will be available in the near future.     

Cytosolic localization in transgenic plants of therolC peptide fromAgrobacterium rhizogenes

TherolC gene ofAgrobacterium rhizogenes codes for a peptide with an apparent molecular weight of approximately 20 kDa. Immunolocalization of therolC peptide, in leaves of transgenic plants which are genetic mosaics for the expression of therolC gene, is restricted to the phenotypically altered sectors. Subcellular fractionation of homogenates from 35S-rolC transgenic leaves shows the cytosolic localization of therolC peptide.     

Production of complex viral glycoproteins in plants as vaccine immunogens

Plant molecular farming offers a cost‐effective and scalable approach to the expression of recombinant proteins which has been proposed as an alternative to conventional production platforms for developing countries. In recent years, numerous proofs of concept have established that plants can produce biologically active recombinant proteins and immunologically relevant vaccine antigens that are comparable to those made in conventional expression systems. Driving many of these advances is the remarkable plasticity of the plant proteome which enables extensive engineering of the host cell, as well as the development of improved expression vectors facilitating higher levels of protein production. To date, the only plant‐derived viral glycoprotein to be tested in humans is the influenza haemagglutinin which expresses at ~50 mg/kg. However, many other viral glycoproteins that have potential as vaccine immunogens only accumulate at low levels in planta. A critical consideration for the production of many of these proteins in heterologous expression systems is the complexity of post‐translational modifications, such as control of folding, glycosylation and disulphide bridging, which is required to reproduce the native glycoprotein structure. In this review, we will address potential shortcomings of plant expression systems and discuss strategies to optimally exploit the technology for the production of immunologically relevant and structurally authentic glycoproteins for use as vaccine immunogens.     

The expression of a heat-inducible chimeric gene in transgenic tobacco plants

Summary A chimeric gene under the control of the hsp70 promoter of Drosophila is heat regulated in roots, stems and leaves, but not in pollen of transgenic tobacco plants. For these and other parameters, it behaves similarly to plant heat-shock genes.     

Benefits and risks of antibody and vaccine production in transgenic plants

Phytopharming, the production of protein biologicals in recombinant plant systems, has shown great promise in studies performed over the past 13 years. A secretory antibody purified from transgenic tobacco was tested successfully in humans, and prevented bacterial re-colonization after topical application in the mouth. Rapid production of patient-tailored anti-lymphoma antibodies in recombinant Tobamovirus-infected tobacco may provide effective cancer therapy. Many different candidate vaccines from bacterial and viral sources have been expressed in transgenic plants, and three human clinical trials with oral delivery of transgenic plant tissues have shown exciting results. The use of crop plants with agricultural practice could allow cheap production of valuable proteins, while providing enhanced safety by avoidance of animal viruses or other contaminants. However development of this technology must carefully consider the means to ensure the separation of food and medicinal products when crop plants are used for phytopharming.     

Photoregulated expression of a pea rbcS gene in leaves of transgenic plants

A 2.4-kb pea genomic fragment, containing a member (rbcS-E9) of the multigene family encoding the small subunit (rbcS) of ribulose-1,5-bisphosphate carboxylase, was inserted into a non-oncogenic, Ti-plasmid vector and introduced into the genomes of Petunia hybrida (Mitchell) and Nicotiana tabacum (SR1) plants by in vitro transformation. Petunia and tobacco plants containing the introduced pea rbcS-E9 gene were regenerated from protoplasts. In these transgenic plants the rbcS-E9 gene is transcribed accurately using its own promoter and its expression is light-induced and organ-specific. A deletion mutant with 352 bp of 5'-upstream sequence still retains photoinducibility and leaf-specific expression. Clonal analysis of independent transgenic petunia plants revealed that chromosomal positions in the recipient plant genome affect the quantitative but not qualitative aspects of rbcS-E9 expression.     

Arabidopsis thaliana small subunit leader and transit peptide enhance the expression ofBacillus thuringiensis proteins in transgenic plants

The expression of the modified gene for a truncated form of thecryIA(c) gene, encoding the insecticidal portion of the lepidopteran-active CryIA(c) protein fromBacillus thuringiensis var.kurstaki (B.t.k.) HD73, under control of theArabidopsis thaliana ribulose-1,5-bisphosphate carboxylase (Rubisco) small subunitats1A promoter with and without its associated transit peptide was analyzed in transgenic tobacco plants. Examination of leaf tissue revealed that theats1A promoter with its transit peptide sequence fused to the truncated CryIA(c) protein provided a 10-fold to 20-fold increase incryIA(c) mRNA and protein levels compared to gene constructs in which the cauliflower mosaic virus 35S promoter with a duplication of the enhancer region (CaMV-En35S) was used to express the samecryIA(c) gene. Transient expression assays in tobacco protoplasts and the whole plant results support the conclusion that the transit peptide plus untranslated sequences upstream of that region are both required for the increase in expression of the CryIA(c) protein. Furthermore, the CaMV-En35S promoter can be used with theArabidopsis ats1A untranslated leader and transit peptide to increase expression of this protein. While subcellular fractionation revealed that the truncated CryIA(c) protein fused to theats1A transit peptide is located in the chloroplast, the increase in gene expression is independent of targeting of the CryIA(c) protein to the chloroplast. The results reported here provide new insight into the role of 5 untranslated leader sequences and translational fusions to increase heterologous gene expression, and they demonstrate the utility of this approach in the development of insect-resistant crops.     

转基因植物表达药用蛋白的研究进展

基因工程技术的进步使得转基因植物广泛应用于工业、农业各个领域,尤其在医药制造领域。研究成果表明,转基因植物作为生物反应器在制备药用蛋白,如重组疫苗、重组动物抗体、细胞因子等方面较其他表达系统,如微生物及动物表达系统具有成本低、应用安全等优势,但在工业化技术方面仍存在障碍。     

Patterns of green fluorescent protein expression in transgenic plants

Modified forms of genes encoding green fluorescent protein (GFP) can be macroscopically detected when expressed in whole plants. This technology has opened up new uses for GFP such as monitoring transgene presence and expression in the environment once it is linked or fused to a gene of interest. When whole-plant or whole-organ GFP visualization is required, GFP should be predictably expressed and reliably fluorescent. In this study the whole plant expression and fluorescence patterns of a mGFP5er gene driven by the cauliflower mosaic virus 35S promoter was studied in intact GFP-expressing transgenic tobacco (Nicotiana tabacum cv. Xanthi). It was shown that GFP synthesis levels in single plant organs were similar to GUS activity levels from published data when driven by the same promoter. Under the control of the 35S promoter, high expression of GFP can be used to visualize stems, young leaves, flowers, and organs where the 35S promoter is most active. Modified forms of GFP could replace GUS as the visual marker gene of choice.     

Variability of organ-specific gene expression in transgenic tobacco plants

Variability of expression of introduced marker genes was analysed in a large number of tobacco regenerants from anAgrobacterium-mediated transformation. In spite of standardization of sampling, considerable variation of GUS and NPTII expression was observed between individual transformants at different times of analysis and in different parts of the same plant. Organ-specificity of root versus leaf expression conferred by the par promoter from the haemoglobin gene ofParasponia andersonii in front of thegus gene showed a continuous spectrum. GUS expression in roots was found in 128 out of 140 plants; expression in leaves was found in 46 plants, and was always lower than in the corresponding roots. NPTII expression regulated by the nos promoter also showed a continuous spectrum. Expression levels were generally higher in roots than in leaves. Plants with high GUS expression in leaves showed high NPTII activity as well. A positive correlation between the level of NPTII expression and the numbers of integrated gene copies was noted. Chromosomal position effects and physiological determination are suggested as triggers for the variations. The transformed regenerated tobacco plants were largely comparable to clonal variants.     

Expression of protective antigen in transgenic plants: a step towards edible vaccine against anthrax

Protective antigen (PA) is the most potent molecule for vaccination against anthrax. In the present study, we have successfully integrated protective antigen gene in nuclear genome of tobacco plants by Agrobacterium mediated leaf-disc transformation method. Expression of protective antigen gene was detected by immunoblot analysis using antisera raised against purified PA. A distinct band of approximately 83kDa lighted up in the protein extracted from transformed plants while there was no such band in untransformed plants. The plant expressed PA showed biological activity just like native PA, which was demonstrated by cytolytic assay on macrophage like cell lines with lethal factor. This study establishes for the first time expression of PA gene in a plant system and thus marks the first milestone towards developing edible vaccine against anthrax.     

Good manufacturing practices production of a purification-free oral cholera vaccine expressed in transgenic rice plants

Key message

The first Good Manufacturing Practices production of a purification-free rice-based oral cholera vaccine (MucoRice-CTB) from transgenic plants in a closed cultivation system yielded a product meeting regulatory requirements.

Abstract

Despite our knowledge of their advantages, plant-based vaccines remain unavailable for human use in both developing and industrialized countries. A leading, practical obstacle to their widespread use is producing plant-based vaccines that meet governmental regulatory requirements. Here, we report the first production according to current Good Manufacturing Practices of a rice-based vaccine, the cholera vaccine MucoRice-CTB, at an academic institution. To this end, we established specifications and methods for the master seed bank (MSB) of MucoRice-CTB, which was previously generated as a selection-marker-free line, evaluated its propagation, and given that the stored seeds must be renewed periodically. The production of MucoRice-CTB incorporated a closed hydroponic system for cultivating the transgenic plants, to minimize variations in expression and quality during vaccine manufacture. This type of molecular farming factory can be operated year-round, generating three harvests annually, and is cost- and production-effective. Rice was polished to a ratio of 95 % and then powdered to produce the MucoRice-CTB drug substance, and the identity, potency, and safety of the MucoRice-CTB product met pre-established release requirements. The formulation of MucoRice-CTB made by fine-powdering of drug substance and packaged in an aluminum pouch is being evaluated in a physician-initiated phase I study.

     

Development of a new tobamovirus-based viral vector for protein expression in plants

Plants are becoming an interesting alternative system for the heterologous production of pharmaceutical proteins, providing a more scalable, cost-effective, and biologically safer option than the current expression systems. The development of plant virus expression vectors has allowed rapid and high-level transient expression of recombinant genes, and, in turn, provided an attractive plant-based production platform. Here we report the development of vectors based on the tobamovirus Pepper mild mottle virus (PMMoV) to be used in transient expression of foreign genes. In this PMMoV vector, a middle part of the viral coat protein gene was replaced by the green fluorescent protein (GFP) gene, and this recombinant genome was assembled in a binary vector suitable for plant agroinoculation. The accumulation of GFP was evaluated by observation of green fluorescent signals under UV light and by western blotting. Furthermore, by using this vector, the multiepitope gene for chikungunya virus was successfully expressed and confirmed by western blotting. This PMMoV-based vector represents an alternative system for a high-level production of heterologous protein in plants.

     

Isolation of a polyubiquitin promoter and its expression in transgenic potato plants.

A polyubiquitin clone (ubi7) was isolated from a potato (Solanum tuberosum) genomic library using a copy-specific probe from a stress-induced ubiquitin cDNA. The genomic clone contained a 569-bp intron immediately 5' to the initiation codon for the first ubiquitin-coding unit. Two chimeric beta-glucuronidase (GUS) fusion transgenes were introduced into potato. The first contained GUS fused to a 1156-bp promoter fragment containing only 5' flanking and 5' untranslated sequences from ubi7. The second transgene contained GUS translationally fused to the carboxy terminus of the first ubiquitin-coding unit and thus included the intron present in the 5' untranslated region of the polyubiquitin gene. Both ubi7-GUS transgenes were activated by wounding in tuber tissue and in leaves by application of exogenous methyl jasmonate. They were also expressed constitutively in the potato tuber peel (outer 1-2 mm). Both transgenes were actively expressed in mature leaves. Exceptionally high levels of expression were observed in senescent leaves. Transgenic clones containing the ubi7 intron and the first ubiquitin-coding unit showed GUS expression levels at least 10 times higher than clones containing GUS fused to the intronless promoter.