Expression of the native cholera toxin B subunit gene and assembly as functional oligomers in transgenic tobacco chloroplasts.
(1/22)
The B subunits of enterotoxigenic Escherichia coli (LTB) and cholera toxin of Vibrio cholerae (CTB) are candidate vaccine antigens. Integration of an unmodified CTB-coding sequence into chloroplast genomes (up to 10,000 copies per cell), resulted in the accumulation of up to 4.1 % of total soluble tobacco leaf protein as functional oligomers (410-fold higher expression levels than that of the unmodified LTB gene expressed via the nuclear genome). However, expression levels reported are an underestimation of actual accumulation of CTB in transgenic chloroplasts, due to aggregation of the oligomeric forms in unboiled samples similar to the aggregation observed for purified bacterial antigen. PCR and Southern blot analyses confirmed stable integration of the CTB gene into the chloroplast genome. Western blot analysis showed that the chloroplast- synthesized CTB assembled into oligomers and were antigenically identical with purified native CTB. Also, binding assays confirmed that chloroplast-synthesized CTB binds to the intestinal membrane GM1-ganglioside receptor, indicating correct folding and disulfide bond formation of CTB pentamers within transgenic chloroplasts. In contrast to stunted nuclear transgenic plants, chloroplast transgenic plants were morphologically indistinguishable from untransformed plants, when CTB was constitutively expressed in chloroplasts. Introduced genes were inherited stably in subsequent generations, as confirmed by PCR and Southern blot analyses. Increased production of an efficient transmucosal carrier molecule and delivery system, like CTB, in transgenic chloroplasts makes plant-based oral vaccines and fusion proteins with CTB needing oral administration commercially feasible. Successful expression of foreign genes in transgenic chromoplasts and availability of marker-free chloroplast transformation techniques augurs well for development of vaccines in edible parts of transgenic plants. Furthermore, since the quaternary structure of many proteins is essential for their function, this investigation demonstrates the potential for other foreign multimeric proteins to be properly expressed and assembled in transgenic chloroplasts. (+info)
Milestones in chloroplast genetic engineering: an environmentally friendly era in biotechnology.
(2/22)
Chloroplast genomes defied the laws of Mendelian inheritance at the dawn of plant genetics, and continue to defy the mainstream approach to biotechnology, leading the field in an environmentally friendly direction. Recent success in engineering the chloroplast genome for resistance to herbicides, insects, disease and drought, and for production of biopharmaceuticals, has opened the door to a new era in biotechnology. The successful engineering of tomato chromoplasts for high-level transgene expression in fruits, coupled to hyper-expression of vaccine antigens, and the use of plant-derived antibiotic-free selectable markers, augur well for oral delivery of edible vaccines and biopharmaceuticals that are currently beyond the reach of those who need them most. (+info)
Production of human papillomavirus type 16 virus-like particles in transgenic plants.
(3/22)
Cervical cancer is linked to infection with human papillomaviruses (HPV) and is the third most common cancer among women worldwide. There is a strong demand for the development of an HPV preventive vaccine. Transgenic plants expressing the HPV major capsid protein L1 could be a system to produce virus-like particles for prophylactic vaccination or could even be used as edible vaccines to induce an L1-specific prophylactic immune response. Here, we describe the generation of transgenic tobacco and potato plants carrying the HPV type 16 major structural gene L1 under the control of the cauliflower mosaic virus 35S promoter. All attempts to express either the original, unmodified L1 gene or an L1 gene with a codon usage optimized for expression in plants failed. Surprisingly, small amounts of the protein were detected using an L1 gene optimized for expression in human cells. However, Northern blot analysis revealed that most of the L1 transcripts were degraded. Introduction of the translational enhancer Omega derived from the tobacco mosaic virus strongly increased transcript stability and resulted in accumulation of L1 protein to approximately 0.5 to 0.2% of total soluble protein in transgenic tobacco and potato plants, respectively. The plant-derived L1 protein displayed conformation-specific epitopes and assembled into virus-like particles. Furthermore, we did not find any indications of protein modification of the L1 protein produced in plants. Plant-derived L1 was as immunogenic as L1 expressed in baculovirus-infected insect cells. Feeding of tubers from transgenic potatoes to mice induced an anti-L1 antibody response in 3 out of 24 mice, although this response was only transient in two of the mice. Our data, however, indicate that an anti-L1 response was primed in about half of the 24 animals. (+info)
Oral immunization with rotavirus VP7 expressed in transgenic potatoes induced high titers of mucosal neutralizing IgA.
(4/22)
Rotaviruses (RV) are a common cause of severe diarrhea in young children, resulting in nearly one million deaths worldwide annually. Rotavirus VP7 was the rotavirus neutralizing protein. Previous study reported that VP7 DNA vaccine can induce high levels of IgG in mice but cannot protect mice against challenge (Choi, A.H., Basu, M., Rae, M.N., McNeal, M.M., Ward, R.L., 1998. Virology 250, 230-240). We found that rotavirus VP7 could maintain its neutralizing immunity when it was transformed into the potato genome. Mice immunized with the transformed tubers successfully elicited serum IgG and mucosal IgA specific for VP7. The mucosal IgA titer was as high as 1000, while serum IgG titer was only 600. Neutralizing assays indicated that IgA could neutralize rotavirus. These results indicate the potential usefulness of plants for production and delivery of edible rotavirus vaccines. (+info)
Transformation of an edible crop with the pagA gene of Bacillus anthracis.
(5/22)
Vaccination against anthrax is the most important strategy to combat the disease. This study describes a generation of edible transgenic crop expressing, functional protective antigen (PA). In vitro studies showed that the plant-expressed antigen is qualitatively similar to recombinant PA. Immunization studies in mouse animal models indicated the generation of PA-specific neutralizing antibodies and stressed the need for improving expression levels to generate higher antibody titers. Genetic engineering of a plant organelle offers immense scope for increasing levels of antigen expression. An AT-rich PA gene (pagA) coding for the 83-kDa PA molecule was thus cloned and expressed in tobacco chloroplasts. Biolistics was used for the transformation of a chloroplast genome under a set of optimized conditions. The expression of the pagA gene with 69% AT content was highly favored by an AT-rich chloroplast genome. A multifold expression level of functional PA was obtained as compared with the nuclear transgenic tobacco plants. This report describes for the first time a comprehensive study on generating transgenic plants expressing PA, which may serve as a source of an edible vaccine against anthrax. Two important achievements of expressing PA in an edible crop and use of chloroplast technology to enhance the expression levels are discussed here. (+info)
Immunogenicity of a plant-derived edible rotavirus subunit vaccine transformed over fifty generations.
(6/22)
Major efforts have been put forth for the development of effective rotavirus vaccines including transgenic plant vaccines. Previous studies have reported that rotavirus VP7 maintains its neutralizing immunity when it is transformed into the potato genome. The present study was aimed at investigating the hereditary stability of VP7-transformed potatoes over fifty generations. The VP7 gene was stably transcribed and expressed in potato cells as detected by RT-PCR and Western blotting. Humeral and mucosal responses were successfully induced in BALB/c mice fed with the fiftieth generation transformed potato tubers. There were no significant differences in serum IgG and fecal IgA between the mice fed with the first and fiftieth generation potatoes (P>0.05). Profiles of cytokines such as IFN-gamma, IL-2, IL-4, IL-5 and TGF-beta in immunized mice showed a naive T-cells bias to Th1 and Th3 polarization. Moreover, specific CTL responses were also detected in C57BL/6 mice fed with transformed potatoes. This research represents a significant step towards the development of rotavirus vaccines derived from a transgenic plant that can be obtained by long-term and large-scale vegetative reproduction. To our knowledge, this is the first finding regarding vaccines derived from plants that can be propagated for many generations. (+info)
Edible vaccines: current status and future.
(7/22)
Edible vaccines hold great promise as a cost-effective, easy-to-administer, easy-to-store, fail-safe and socioculturally readily acceptable vaccine delivery system, especially for the poor developing countries. It involves introduction of selected desired genes into plants and then inducing these altered plants to manufacture the encoded proteins. Introduced as a concept about a decade ago, it has become a reality today. A variety of delivery systems have been developed. Initially thought to be useful only for preventing infectious diseases, it has also found application in prevention of autoimmune diseases, birth control, cancer therapy, etc. Edible vaccines are currently being developed for a number of human and animal diseases. There is growing acceptance of transgenic crops in both industrial and developing countries. Resistance to genetically modified foods may affect the future of edible vaccines. They have passed the major hurdles in the path of an emerging vaccine technology. Various technical obstacles, regulatory and non-scientific challenges, though all seem surmountable, need to be overcome. This review attempts to discuss the current status and future of this new preventive modality. (+info)
An efficient plant viral expression system generating orally immunogenic Norwalk virus-like particles.
(8/22)