Evaluation of hairy roots from Solanaceous species for efficient production of a functional subunit vaccine

Vaccination is pivotal for the reduction of morbidity and mortality caused by infectious diseases. Many diseases remain prevalent however, due to limitations of current vaccine production systems, which have necessitated ongoing evaluation of novel or improved vaccine production systems. Desired qualities of new vaccine production systems include: cost-effective high vaccine yield, increased safety and improved delivery methods, improved storage and transport; and improved immunogenicity and efficacy of the vaccines. The use of plants and plant tissues as bio-reactor platforms for the production of vaccines are an alternative system developed with the aim of achieving many of the above stated desired characteristics. Their advantages include: pre-existing transgenic crop infrastructure which can be adapted to allow for cost-effective large scale production of recombinant proteins; production of protein structures with appropriate perhaps complex folding and posttranslational modifications; efficient storage and transport at ambient temperatures; decreased risk from contamination with animal/microbial biohazardous material; the potential for oral administration when using non-toxic plant species. Unfortunately, plant-made pharmaceutical production systems also have limitations. The chief limitation associated with plant-made pharmaceuticals is the generally low net-yield of final products from plants; legislation and community concerns that cause ever increasing restrictions; and regulations on production methods pertaining to the environmental contamination with transgenes and/or potential health risks of consumption of transgenic material. The research presented in this thesis examines several approaches to ameliorate certain restrictions of current plant-made pharmaceutical systems, to potentially overcome current limitations of plant-made vaccine production technologies.Specifically, a selection of plant species; Nicotiana tabacum (a model plant species used widey for biotechnological studies), Solanum lycopersicum (a globally grown edible plant species) and Petunia parodii (a potentially non-toxic plant species not commonly present in the human diet) were used. Each species was evaluated for their potential to seed hairy root cultures by infection with Agrobacterium rhizogenes and resultant hairy roots were then assessed for the ability to produce a model vaccine antigen -the B-subunit of the heat-labile toxin (LTB) from Enterotoxigenic Escherichia coli. The initial phase of the studies presented in this thesis, demonstrated that hairy root cultures of each test species had the ability to produce correctly folded, structurally functional LTB protein. In general however, the petunia cultures showed the most promise by producing the highest levels of LTB per unit biomass and exhibiting the least amount of antigen-mediated growth inhibition. Furthermore, the study demonstrated that hairy roots of each test species exhibited the ability to continuously secrete LTB antigen into the culture medium which could potentially be utilised for increasing product yields. In addition, hairy roots of N. tabacum were able to be regenerated into whole plants (by stimulation with plant hormones) whose seeds were able to be stored and used to re-establish LTB producing hairy root cultures from subsequent generations, which is potentially advantageous for efficient storage and transport of the vaccine. Subsequent phases of the study involved the characterisation of the putatively wound/jasmonate inducible (cost-effective inducible methods) quinolinate phosophoribosyl transferase 2 (NtQPT2) promoter sequence (derived from N. tabacum), for its ability to drive LTB production in the different previously characterised test species, in comparison to the commonly used constitutive CaMV35s promoter from the cauliflower mosaic virus. In hairy cultures of all investigated species, basal NtQPT2 driven LTB production was observed to be significantly (p≤0.05) less than those produced by CaMV35s driven expression, and the possibility of this being advantageous is discussed. Induction of NtQPT2 mediated LTB production by methyl jasmonate, was not shown in any of the root cultures tested with potential reasons for this also being discussed.However, regenerated LTB producing tobacco plants (under the regulation of NtQPT2 promoter) after wounding treatment elicited a 500% increase in LTB production over LTBproducing plants regulated by the CaMV35s promoter, indicating that this promoter could have applications for future plant-made vaccine production systems. In the final part to this thesis, a collaborative study was performed, where P. parodii hairy roots producing LTB were utilised for comparison with other plant-made vaccine production systems (transgenic fruit, transiently infected leaf material and cell cultures), and assessed for their ability to elicit immune responses in animal oral immunisation trials. The transgenic petunia hairy root material was fed to mice (model monogastric system) and sheep (model ruminant system) and in both cases was able to elicit a detectable mucosal and systemic immune response. The combined observations of the studies presented in this thesis, could potentially prove useful for increasing efficiency and efficacy of current plant-made vaccine production systems -involving hairy root cultures (in particular those of Petunia parodii), not only as a system for vaccine expression but also as a potential oral delivery platform. The studies also highlight the potential uses for the NtQPT2 promoter for producing toxigenic products which can be costeffectively induced in roots of plants to achieve greater product yields after wounding