A LEA protein for improving abiotic stress tolerance and vaccine production in transgenic plants

The use of transgenic plants to produce novel products has great biotechnological potential as relatively inexpensive inputs (e.g. light, water, and nutrients) are required in return for potentially valuable outputs (e.g. bioactive metabolites, diagnostic proteins and vaccines). Extensive research is ongoing in this area internationally with the aim of producing plant-made vaccines (PMV) of importance for both animals and humans. Avian influenza (AI) infection is endemic among birds, and although relatively uncommon, infection may result in humans due to close contact with infected animals. Development of PMV against AI in birds would provide economic protection for the international poultry industry and decrease the AI reserviour in the bird population thereby reducing the chance of human infection directly or indirectly from this source. The current study involved two distinct experimental investigative approaches with the common aim of improving efficacy of a PMV against Al virus. In part 1, immunogenicity of the HA antigen produced by stably transformed Nicotiana plants was investigated. The haemagglutinin (HA) glycoprotein of AI virus is an important target immunogen, and several reports show animals that received purified doses of transiently transformed Nicotiana benthamiana-derived influenza vaccines were protected against subsequent viral challenge. Commercial tobacco (N. tabacum) has many advantages as a plant expression host, including well established genetic engineering protocols; capacity to produce large biomass under cultivated conditions; and being a non-food crop, there is minimal likelihood of contaminating the food chain with transgene. However, synthesis of pharmaceutical proteins in traditional tobacco cultivars may have potential negative connotations associated with the presence of nicotine-alkaloids. The presence of these alkaloids in a PMV would produce additional and possibly cost-prohibitive regulatory hurdles, particularly if extensive purification of proteins is not feasible on a commercial scale. A major focus of part 1 of the current project therefore was to investigate the production of HA protein in stably transformed Nicotiana plants possessing minimum alkaloid content. Genetic constructs incorporating two separate promoters and also plant-optimised and native HA coding regions were investigated. Levels of HA in transgenic plants of low alkaloid tobacco (LAFC53), resulting from constructs containing the strong chimeric promoter ΔMAS, were found to have significantly higher (p< 0.05) HA accumulation compared to those obtained using the CsVMV promoter. The use of a plant-optimised HA coding sequence did not result in significantly higher levels of HA expression compared to the native viral HA sequence however. In further experiments, sexual hybridisation of selected HA containing N. tabacum transgenics with a A622-RNAi N. glauca transgenic produced previously in this laboratory resulted in self-sterile hybrids that were completely devoid of alkaloids. Soluble leaf extract from these hybrids proved to be equally immunogenic in mice trials, as purified, or alkaloid-containing HA extracts containing equivalent amount of HA. In part 2 of this project, the possibility of utilising stress-activated genes from desiccation tolerant plants to enhance capacity for PMV production was examined. Plant material producing PMV may require downstream processing such as freeze-drying to concentrate and prolong the shelf-life of the expressed pharmaceutical protein. This contributes to the cost of production, is time consuming and may lead to a reduction in final protein yield due to the instability of proteins during freeze-drying and/or storage under ambient conditions. Recent studies reveal that late embryogenesis abundant (LEA) proteins can act as molecular chaperones and can protect surrounding proteins from aggregation and hence may stabilise the protein structure during freezing and dehydration via freeze-drying. In part 2 of this project, constructs containing a LEA gene (SDG2i) from the resurrection grass Sporobolus stapfianus were co-infiltration with HA antigen constructs into N. benthamiana to assess capacity to reduce loss of HA antigens during downstream processes such as freeze-drying and oven-drying. Results obtained were inconclusive however and further experiments are required to determine the potential for LEA proteins to protect a PMV during post-harvest processing. In vitro results did show that the SDG2i protein protects the activity of lactate dehydrogenase from negative effects of heat and cold treatment however. Constitutive expression of the SDG2i in Arabidopsis also enhanced the tolerance of transgenic plants during salt and sugar stresses, possibly via modulation of the abscisic acid hormone response. This study laid the foundation by establishing optimal genetic conditions for HA expression and antigenicity in Nicotiana species and characterising the possible protection afforded by the presence of the end product of the SDG2i gene. Future experiments should be aimed at answering whether the SDG2i gene can improve stress tolerance of transgenic plants producing a PMV.