Particle-mediated DNA delivery (PMDD) is a technique that uses compressed helium gas to accelerate microscopic gold beads coated with DNA into the skin. As a vaccination tool, it provides an attractive approach for generating antigen specific immunotherapy. As the number of applications for this technology in pre-clinical disease models grows, the need for in vitro surrogate models, which in part, can replace in vivo testing, is becoming increasingly important. Additionally, before it can be utilised successfully in man, a number of critical parameters which contribute to overall efficacy in vivo need to be more fully evaluated. To these ends, an in vitro model which featured PMDD as the means of plasmid delivery was developed. The relationship between the efficiency of PMDD to stimulate antigen specific CD8+ T cell responses in vitro and in vivo under conditions of single and dual plasmid immunisation was explored. Important aspects of the technology such as the association between antigen dose and resulting response were investigated. It was found that the in vitro model correlated well with the early stage primary response in vivo but this correlation deteriorated post boost. In addition, the relative contribution of antigenic competition and co-operation in shaping the resulting response was assessed after co-immunising with two independent antigens. Under the experimental conditions investigated, the dominant type of interaction was found to be competitive. Results suggested that competition occurred at the level of the T cell. Competition was most pronounced when both antigens were delivered in a linked manner and when both antigens were presented on the same APC. These results highlight the requirement for optimising key technical parameters in PMDD. Additionally, it demonstrates that interclonal T cell competition is a functionally relevant phenomenon and that it can occur when standard DNA vaccination strategies are employed.

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Interferon alpha 2b (IFN-a2b) is a soluble cytokine and a member of the type I interferon (IFN) family of which there are fourteen members. IFN-a2b is used in the treatment of hairy cell leukaemia and both hepatitis B and C. IFN-a2b actions are mediated through specific receptor binding which initiates a series of signalling cascades leading to the transcription of genes carrying the promoters containing the interferon stimulated response element (ISRE). Due to these actions the IFNs as a whole are an ideal family of proteins to be investigated further with the possibility of the production of life saving drug products. Cobra Biomanufacturing have a keen interest in IFN-a2b especially due to its beneficial effects. This project is therefore an investigation of the IFN-a2b molecule from expression to final purified product. The refolding of the IFN-a2b was the main concern of this project due to it being the `bottleneck' for many protein based processes. The investigation of natively folded IFN-a2b led to the conclusion that IFN-a2b is a stable protein which remains folded under reducing conditions. The addition of guanidine-HCI or urea to the native protein reveals differences in the success of unfolding. These differences were also witnessed during the solubilisation of the IFNa2b inclusion bodies. The refolding is only successful when the inclusion bodies have been solubilised in guanidine-HCI. Due to the differing refolding results gained, which depended on the denaturant used to solubilise the inclusion bodies, the solubilised inclusion bodies themselves were investigated. This revealed partial structure present in the urea solubilised inclusion bodies; however this was confirmed as being not native. The implications of this for biomanufacturing are that if the starting conditions are not correct even as early as the solubilisation of the inclusion bodies then the following process will result in incorrectly folded product.

Previous work has shown that repetitive intranasal (i.n.) administration of the myelin basic protein peptide MBP Acl-9[4Y] induces tolerance in the TCR transgenic Tg4 EAE model. Chronic antigen stimulation of C04+ T cells was found to induce an anergic IL-l0-secreting regulatory phenotype in Th-I cells, protecting animals from EAE. Alternative routes for peptide administration were explored in the Tg4 model, to increase our understanding of the factors influencing peptide immunotherapy. Epicutaneously (e. c.) administered MBP Acl- 9[4Y] was slowly trafficked to the lymph nodes and spleen where C04+ T cells were activated. However, repetitive e.c. MBP Acl-9[4Y] administration did not reliably induce characteristics of C04+ T cell tolerance, attributed to the difficulty of controlling the e.c. administered MBP Acl-9 [4 Y] dose. Use of the subcutaneous (s.c.) route circumvented this problem, and titrated MBP Acl-9[4Y] doses were administered s.c. to Tg4 mice. Tolerance induced was proportional to the s.c. MBP Acl-9[4Y] dose administered, with higher doses better inducing an anergic, suppressive C04+ T cell phenotype, upregulation of IL-l0 secretion and protection from EAE. However, further increasing the s.c. peptide dose induced severe adverse effects in Tg4 (and Tg4 Rag-l -!-) mice, concomitant with high systemic inflammatory cytokine levels. Novel application of the technique of dose escalation to self-peptide immunotherapy allowed delivery of high peptide doses s.c. to Tg4 Rag-l +/+ and -/- mice without adverse effects. Escalating to higher s.c. peptide doses better induced tolerance, providing long-term protection of Tg4 Rag-l -/- mice from the spontaneous development of EAE. This demonstrates that low peptide doses administered s.c. during the escalation stage of treatment modulated the response of a monoclonal C04+ T cell population to subsequent high s.c. peptide doses. Collectively, these results show that route of antigen administration contributes to the outcome of peptide immunotherapy, which is also closely related to the peptide dose administered. Furthermore, we propose that dose escalation is essential for the safe and effective translation of peptide immunotherapy of auto immune disease into the clinic.

Tumour escapes from immunosurvillence though tumour-mediated suppression. Tumour disrupts dendritic cells (DC) and macrophage functions and interferes with developing anti-tumour microenvironment. Indeed, DC dysfunction in cancer is a limiting factor in cancer immunotherapy. The hypothesis that alternation of DC MAPK pathway is a strategy by which tumours evade immunosurvillence was tested. Our results revealed that, tumours exploit DC through manipulation of ERK signalling pathway which resulting in inhibition of IL-12 production. In addition, tumours suppress the fibroblasts induced IL-23 production by DC. Through Interfering with IL- 12 and IL-23 production, tumours down regulate immediate inflammatory response responsible for defending against the newly developed malignancy. Similarly, our results on monocyte/ macrophage lineage showed that tumour alternate their function away from pro-inflammatory activity and closer to the regulatory profile.

Exosomes are small membrane-bound vesicles of endosomal origin secreted by many cell types including tumour cells. Their potential use in cancer immunotherapy has been suggested by the discovery that tumour-derived exosomes can be a source of tumour associated antigens (T AA) and protect against tumour challenge in mice. The initial excitement has been tempered more recently however, by reports showing that exosomes can have inhibitory effects on T cells and natural killer (NK) cells. Heat-shock proteins are endogenous danger signals that activate the immune system; their expression is increased by heat-shock stress or v-irradiation. In this thesis, heat-shock stress and v-irradiation are investigated as mechanisms for increasing the immunogenicity of human and murine melanoma-derived exosomes. Exosomes derived from untreated or irradiated human melanoma cells did not activate DC, although heat-shock exosomes induced a small increase in expression of MHC-II, CD40 and inflammatory cytokines. Although exosome- induced maturation was far from optimal compared to LPS, exosomes did not inhibit the LPS-induced maturation of DC and could therefore be used to load DC with TAA for immunotherapy. The use of exosomes for priming a naive T cell response was investigated in a human in vitro system. Control, irradiated and heat-shock exosomes induced similar levels of cytotoxicity against melanoma cell lines suggesting that they were equally effective in generating an immune response. Murine melanoma-derived exosomes were investigated as anti-cancer vaccines in vivo in a mouse model. Neither control nor stressed exosomes, +/- DC were able to protect mice against subsequent tumour challenge. However, Treg depletion in vivo mediated rejection of tumour challenge; vaccination with exosomes inhibited this immune response, although heat-shock exosomes appeared least inhibitory, suggesting they may be more immunogenic than control or irradiated exosomes. Finally, the immunogenicity of exosomes might be increased by upregulating expression of TAA or immune adjuvants. NKG7 is a tetraspanin that is similar to CD63, a protein found enriched in exosomes. EGFP was successfully targeted into exosomes by linking it to the protein NKG7. EGFP-expressing exosomes induced strong maturation of DC, indicating increased immunogenicity. Therefore, the immunogenicity of exosomes can be enhanced by using a targeting mechanism to increase their expression of immune adjuvants.

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