Unsolved problems in plastid transformation.

Plants have been proved as a novel production platform for a wide range of biologically important compounds such as enzymes, therapeutic proteins, antibiotics, and proteins with immunological properties. In this context, plastid genetic engineering can be potentially used to produce recombinant proteins. However, several challenges still remain to be overcome if the full potential of plastid transformation technology is to be realized. They include the development of plastid transformation systems for species other than tobacco, the expression of transgenes in non-green plastids, the increase of protein accumulation and the appearance of pleiotropic effects. In this paper, we discuss the novel tools recently developed to overcome some limitations of chloroplast transformation.

Production of foreign proteins using plastid transformation.

In the past decades, the progress made in plant biotechnology has made possible the use of plants as a novel production platform for a wide range of molecules. In this context, the transformation of the plastid genome has given a huge boost to prove that plants are a promising system to produce recombinant proteins. In this review, we provide a background on plastid genetics and on the principles of this technology in higher plants. Further, we discuss the most recent biotechnological applications of plastid transformation for the production of enzymes, therapeutic proteins, antibiotics, and proteins with immunological properties. We also discuss the potential of plastid biotechnology and the novel tools developed to overcome some limitations of chloroplast transformation.

Recombinant plant-expressed tumour-associated MUC1 peptide is immunogenic and capable of breaking tolerance in MUC1.Tg mice.

The human epithelial mucin MUC1 is a heavily glycosylated transmembrane protein that is overexpressed and aberrantly glycosylated on over 90% of human breast cancers. The altered glycosylation of MUC1 reveals an immunodominant peptide along its tandem repeat (TR) that has been used as a target for tumour immunotherapy. In this study, we used the MUC1 TR peptide as a test antigen to determine whether a plant-expressed human tumour-associated antigen can be successfully expressed in a plant system and whether it will be able to break self-antigen tolerance in a MUC1-tolerant mouse model. We report the expression of MUC1 TR peptide fused to the mucosal-targeting Escherichia coli enterotoxin B subunit (LTB-MUC1) in a plant host. Utilizing a rapid viral replicon transient expression system, we obtained high yields of LTB-MUC1. Importantly, the LTB-MUC1 fusion protein displayed post-translational modifications that affected its antigenicity. Glycan analysis revealed that LTB-MUC1 was glycosylated and a MUC1-specific monoclonal antibody detected only the glycosylated forms. A thorough saccharide analysis revealed that the glycans are tri-arabinans linked to hydroxyprolines within the MUC1 tandem repeat sequence. We immunized MUC1-tolerant mice (MUC1.Tg) with transiently expressed LTB-MUC1, and observed production of anti-MUC1 serum antibodies, indicating breach of tolerance. The results indicate that a plant-derived human tumour-associated antigen is equivalent to the human antigen in the context of immune recognition.

Transgenic chloroplasts are efficient sites for high-yield production of the vaccinia virus envelope protein A27L in plant cellsdagger.

Orthopoxviruses (OPVs) have recently received increasing attention because of their potential use in bioterrorism and the occurrence of zoonotic OPV outbreaks, highlighting the need for the development of safe and cost-effective vaccines against smallpox and related viruses. In this respect, the production of subunit protein-based vaccines in transgenic plants is an attractive approach. For this purpose, the A27L immunogenic protein of vaccinia virus was expressed in tobacco using stable transformation of the nuclear or plastid genome. The vaccinia virus protein was expressed in the stroma of transplastomic plants in soluble form and accumulated to about 18% of total soluble protein (equivalent to approximately 1.7 mg/g fresh weight). This level of A27L accumulation was 500-fold higher than that in nuclear transformed plants, and did not decline during leaf development. Transplastomic plants showed a partial reduction in growth and were chlorotic, but reached maturity and set fertile seeds. Analysis by immunofluorescence microscopy indicated altered chlorophyll distribution. Chloroplast-synthesized A27L formed oligomers, suggesting correct folding and quaternary structure, and was recognized by serum from a patient recently infected by a zoonotic OPV. Taken together, these results demonstrate that chloroplasts are an attractive production vehicle for the expression of OPV subunit vaccines.

Plants as biofactories for the production of subunit vaccines against bio-security-related bacteria and viruses.

The development of new generation vaccines is an imperative tool to counteract accidental or intended release of bio-threat agents, such as Bacillus anthracis, Yersinia pestis and variola virus, and to control natural outbreaks. In the past few years, numerous data accumulated on the immunogenicity and safety of plant-made vaccines against bio-security-related organisms. In addition, expression levels achieved for these antigenic proteins are practical for the production of sufficient material for large-scale vaccination programs. These data demonstrated that the plant-based approach is feasible for manufacturing recombinant vaccines against bio-terror agents that could be mass-produced at reasonable cost.

Plant-made subunit vaccine against pneumonic and bubonic plague is orally immunogenic in mice.

Yersinia pestis, the causative agent of plague, is an extremely virulent bacterium but there are no approved vaccines for protection against it. Our goal was to produce a vaccine that would address: ease of delivery, mucosal efficacy, safety, rapid scalability, and cost. We developed a novel production and delivery system for a plague vaccine of a Y. pestis F1-V antigen fusion protein expressed in tomato. Immunogenicity of the F1-V transgenic tomatoes was confirmed in mice that were primed subcutaneously with bacterially-produced F1-V and boosted orally with transgenic tomato fruit. Expression of the plague antigens in fruit allowed producing an oral vaccine candidate without protein purification and with minimal processing technology.

Oral immunogenicity of a plant-made, subunit, tuberculosis vaccine.

Transgenic plants are a novel way to produce and deliver oral vaccines. Arabidopsis thaliana material shown previously to express the tuberculosis (TB) antigen ESAT-6 fused to the B subunit of Escherichia coli heat-labile enterotoxin (LTB) was fed to mice and the resulting immune response investigated. The plant-made LTB-ESAT-6 fusion protein induced antigen-specific responses from CD4+ cells and increased IFN-gamma production, indicating a Th1 response. In addition, a Th2 response was induced in the Peyer's patch. This is the first report of an orally delivered, subunit, tuberculosis vaccine priming an antigen-specific, Th1 response.

Expression systems and developments in plant-made vaccines.

Delivery of vaccines to mucosal surfaces can elicit humoral and cell-mediated responses of the mucosal and systemic immune systems, evoke less pain and discomfort than parenteral delivery, and eliminate needle-associated risks. Transgenic plants are an ideal means by which to produce oral vaccines, as the rigid walls of the plant cell protect antigenic proteins from the acidic environment of the stomach, enabling intact antigen to reach the gut associated lymphoid tissue. In the past few years, new techniques (such as chloroplast transformation and food processing) have improved antigen concentration in transgenic plants. In addition, adjuvants and targeting proteins have increased the immunogenicity of mucosally administered plant-made vaccines. These studies have moved plant-made vaccines closer to the development phase.

Targeting of plant-derived vaccine antigens to immunoresponsive mucosal sites.

Most pathogenic microorganisms enter their host via the mucosal surfaces lining the digestive, respiratory and urino-reproductive tracts of the body. The most efficient means of protecting these surfaces is through mucosal immunization. Transgenic plants are safe and inexpensive vehicles to produce and mucosally deliver protective antigens. However, the application of this technology is limited by the poor response of the immune system to non-particulate, subunit vaccines. Co-delivery of therapeutic proteins with targeting proteins, such as the B subunit of the Escherichia coli heat labile enterotoxin (LTB), could increase the effectiveness of such antigens.