Abstract
Allergic or atopic conditions can collectively affect more than 25% of the population, and current treatments are limited by their lack of disease modifying effects, which emphasises the immense benefits that could be offered by an effective therapy. α-Galactosylceramide (α-GalCer) is a molecule known to possess unique and potent immunostimulating properties that has demonstrated therapeutic efficacy as a vaccine against atopy in preclinical studies when administered parentally, however this route of delivery is not the most convenient for extensive clinical use. Oral administration of vaccines has been limited by the inherent instability of the active components under the conditions found in the gastrointestinal tract and insufficient induction of strong immune responses. Particulate formulations such as liposomes have been of interest in pharmaceutical research due to their capacity to improve both stability and immunogenicity of vaccine formulations. The aim of this thesis was to test the hypothesis that particulate formulations containing α-GalCer, which have been optimised for stability in the gastrointestinal conditions, could be used as a therapeutic oral delivery vehicle to stimulate regulatory immune responses and treat atopic conditions associated with hypersensitivity.
In order to improve the stability of liposomes in gastrointestinal conditions Chapter 2 investigated liposomes coated with silica nanoparticles, chitosan or polyethylene glycol. The in vitro evaluation of these formulations suggested that the electrostatically coated formulations may have greater antigen retention in simulated gastrointestinal fluids compared to the uncoated liposome formulation. Subsequent in vivo results in mouse models of atopy indicated that the formulations incorporating α-GalCer showed some potential to modulate antigen specific serum antibody levels and splenocyte proliferation and cytokine production. However, the responses generated were not of the desired regulatory type and could not be successfully reproduced. This indicated the significance of factors other than the stability of particles in inducing immune activation and the importance of addressing these other factors such as the ability to penetrate the mucus layer, uptake at the Peyer’s patches, and uptake by and activation of antigen presenting cells.
In Chapter 3, to address the potential shortcomings of the previous formulations, cationic liposomes, polymerised liposomes and poly-lactic-co-glycolic acid nanoparticles were optimised and evaluated as formulations intended to increased stability and antigen entrapment and to potentially initiate interactions with the mucus layer to order to increase cell uptake. All formulations exhibited satisfactory stability when evaluated in vitro in simulated gastrointestinal fluids and the cationic liposomes and poly-lactic-co-glycolic acid nanoparticles had improved antigen encapsulation. When tested in vivo in mouse models of atopy and airway hyper-responsiveness, the formulations appeared to induce changes in serum cytokines and in antigen-specific antibody levels, but these did not lead to changes in airway eosinophilia after challenge with the antigen. The impact of treatment with the α-GalCer formulations was variable, which reflects the difficulties associated with oral delivery of a protein vaccine and in stimulating a regulatory immune response. Even with these limitations, the cationic liposome formulation appeared to induce a systemic response seen through elevated serum IL-5 and TNF-α levels, as well as increases in serum antigen specific IgG1 and IgG2a levels, suggesting that immune stimulation is possible through the oral route using particulate formulations.
To further elucidate the process of immune stimulation by the cationic liposome formulation, experiments were conducted in Chapter 4 to investigate the fate of particles after oral administration and the subsequent stimulation of natural killer T cells. Using fluorescence microscopy, labelled cationic liposomes were observed within the subepithelial dome, just under the follicle-associated epithelium of the Peyer’s patch. It was difficult to quantify selective uptake of the liposomes into specific immune cells of the Peyer’s patch by fluorescence-activated cell sorting, but the CD11c+ cells represented a relatively large proportion of cells taking up formulation compared to the total cell proportion in the Peyer’s patch, suggesting that dendritic cells may be the prominent antigen presenting cells involved in uptake. In terms of immune stimulation, the α-GalCer loaded cationic liposomes stimulated both local and systemic immune responses, through increases in natural killer T cell populations and increased expression of IFN-γ and IL-4 in the Peyer’s patch, mesenteric lymph node and spleen. The liposome formulation also induced a greater degree of stimulation compared to non-liposomal α-GalCer, which emphasises the importance of the delivery vehicle in oral vaccine delivery.
This thesis evaluated the potential for inducing regulatory immune response through particulate delivery systems administered via the oral route. The results demonstrated that both local and systemic immune responses could be successfully induced by the adjuvant α-GalCer and that the particulate form can influence the degree of immune stimulation. The studies also emphasised the complexities of the gut associated lymphoid tissue and the uncertainties in the current understanding of how immune responses can be skewed or directed into a desired outcome. Despite the difficulty associated with oral vaccine delivery, the results from this thesis showed that there is potential for harnessing the unique properties of the gut associated lymphoid tissue and encourages further investigation to refine and optimise vaccine delivery through the oral route.