Optimising delivery of oral plant-made vaccines

Compared to prokaryotic or other eukaryotic expression systems, plants provide many benefits for the production of recombinant proteins. Extensive research has dramatically increased the yield and quality of proteins expressed in planta, yet far less is known about the complex physical and immunological characteristics associated with using plants as both expression and oral delivery vehicles for antigenic proteins. The research presented in this thesis investigates the oral delivery and immunological presentation of antigens in the gut. The type of plant tissue (leaf, hairy roots, and fruit) was shown to alter the yield of the heat- labile enterotoxin B-subunit (LTB) immunogen from the mucosal pathogen enterotoxigenic Escherichia coli, and the site of antigen release and the resulting mucosal immune response in mice (Chapter 2). As different expression system and host species have broadly different characteristics, a simpler experimental system was used where the LTB protein was engineered to accumulate in different subcellular locations within the same species and tissue (Chapter 3). Correctly folded LTB was localised to the apoplast, endoplasmic reticulum, non- lytic vacuoles, and protein bodies when expressed in the leaves of Nicotiana benthamiana. This study suggests the site of subcellular targeting not only influences the yield and quality of protein accumulation, but also the release characteristics during simulated digestion in vitro and the resulting mucosal immunogenicity when delivered orally to mice. In addition to the physical characteristics of antigen release during digestion, many immunostimulatory agents are endogenous to plants. The concentration of the known adjuvant of Solanum lycopersicum, α-tomatine was characterised in fruit expressing Norwalk virus capsid protein virus-like particles (Chapter 5). When freeze-dried transgenic fruit at two ripening stages with different concentrations of endogenous α-tomatine was fed to mice, no variation in the seroconversion or magnitude of humoral or mucosal response was observed. Nor was there any variation in response when fruit was formulated with escalating doses of purified α-tomatine. These data suggest the concentration of α-tomatine alone does not potentiate the mucosal immunogenicity to orally-delivered VLPs. The primary goal of this research has been to characterise and optimise the delivery and immunogenicity of antigens within plant cells. These experiments highlight the importance of considering the immunological context when using plant cells to deliver vaccine antigens. While the host factors investigated in this thesis are amenable to rational design, they are still only a small part of the highly complex plant-based production and delivery system which requires improved characterisation before progressing beyond proof-of-concept clinical trials.