Activation of cytotoxic T cells in human requires specific binding of antigenic peptides to human leukocyte antigen (HLA) molecules. HLA is the most polymorphic protein in the human body, currently 1814 different alleles collected in the HLA sequence database at the European Bioinformatics Institute. Most of the HLA molecules recognise different peptides. Also, some peptides can be recognised by several of HLA molecules. In the present project, all available class I HLA alleles are classified into supertypes. Super - binding motifs for peptides binding to some supertypes are defined where binding data are available. A variety of chemometric techniques are used in the project, including 2D and 3D QSAR techniques and different variable selection methods like SIMCA, GOLPE and genetic algorithm. Principal component analysis combined with molecular interaction fields calculation by the program GRID is used in the class I HLA classification. This thesis defines an HLA-A3 supermotif using two QSAR methods: the 3D-QSAR method CoMSIA, and a recently developed 2D-QSAR method, which is named the additive method. Four alleles with high phenotype frequency were included in the study: HLA-A*0301, HLA-A*1101, HLA-A*3101 and HLA- A*6801. An A*020T binding motif is also defined using amino acid descriptors and variable selection methods. Novel peptides have been designed according to the motifs and the binding affinity is tested experimentally. The results of the additive method are used in the online server, MHCPred, to predict binding affinity of unknown peptides. In HLA classification, the HLA-A, B and C molecules are classified into supertypes separately. A total of eight supertypes are observed for class I HLA, including A2, A3, A24, B7, B27, B44, CI and C4 supertype. Using the HLA classification, any newly discovered class I HLA molecule can be grouped into a supertype easily, thus simplifying the experimental function characterisation process.

Although much has been unravelled with regards to the mechanisms of proteolysis of exogenously derived antigen for presentation via histocompatibility class-II (MHC-II), key questions remain unresolved. The exact role of each proteolytic enzyme in this process is not understood. The aspartic proteinase cathepsin E is hypothesised to play an important role. The aim of this study is to examine this by the use of novel aspartic proteinase inhibitors based upon the aspartic proteinase inhibitor pepstatin A. The first two inhibitors designed and synthesised included a mannose sugar molecule to improve solubility, a linker section and a single pepstatin molecule. The mannose derivative 3'-succinimidoxycarbonyl- propyl 2,3,4,6-tetra-O-acetyl-a-D-mannopyranoside was synthesised and linked via an amide bond to either N--butyloxycarbonyl) propane-1,3-diamine or 7V-(/-butyloxycarbonyl) cystamine, the latter of these containing a cellularly cleavable disulphide bond. After deprotection, the amines of these two molecules were further reacted with pepstatin succinimide to form the precursors to the inhibitors Af-(3-(a-D-mannopyranosyloxy) propylcarbonyl),7V' (pepstat-inyl) propane- 1,3-diamine (Mannose Pepstatin Conjugate 1, MPC1) and Ar-(3-(a-D-mannopyranosyloxy) propylcarbonyl),W-(pepstatinyl) cyst-amine (MPC2), the final products being formed by selective removal of the mannopyranosyl acetyl protecting groups. The purified inhibitors were tested in a well- characterised in vitro model of antigen processing, in which ovalbumin is processed and presented by a B-cell line A20 to ovalbumin-specific T-cells, DO11-10. Further studies aimed to address the role of cathepsin E within dendritic cells (DCs), one of the key cells involved in initiating and propagating immune response to antigen. Further inhibitors were designed with the aim of improving solubility and cellular targeting. These inhibitors contained multiple mannose sugars attached either to a protein backbone bovine serum albumin (MPC5 and 6) or a poly-lysine backbone (MPC3 and 4). As for MPC2, a cleavable linker was included in the design of MPC4 and MPC6, to facilitate intracellular release of pepstatin from the carrier. The full synthesis of MPC5 and MPC6 was achieved by the coupling of either AiodoacetylXTV'-Cpepstatinyl) propane-1,3-diamine (MPC5) or N- (iodoacetylW-Cpepstatinyl) cystamine (MPC6) to the single free sulfhydryl of mannosylated bovine serum albumin (BSA) protein. The mannosylated BSA protein was achieved by the thiourea bond formation between the multiple lysine residues contained within BSA and the amine-reactive mannose derivative 4-isothiocyanatophenyl ot-D-mannopyranoside. Biochemical tests showed that between 22 and 24 mannose units were coupled to each BSA protein. The inhibitors MPC5 and MPC6 were tested for their ability to block processing and presentation of ovalbumin by mouse bone-marrow derived DCs. Since pepstatin A inhibits not only cathepsin E but also the lysosomal proteinase cathepsin D, the inhibitors were further tested on DCs prepared from mice deficient in cathepsin D. The results suggest that cathepsin E has an important and non-redundant role in antigen processing of ovalbumin by dendritic cells. This work was supported by funds supplied by the Department of Immunology and Molecular Pathology, UCL, and the Department of Chemistry, UCL. In addition, an Interdisciplinary Research Scholarship was provided by the UCL Graduate School, and a MONBUSHO Fellowship was provided by the Japanese Science & Education Ministry / British Council.

Invading organisms are detected by the innate immune system, through the recognition of conserved microbial structures. Innate responses are known to influence the development of adaptive immune responses, which are crucial for preventing infection and eliminating pathogens. Characterising the signals that initiate the induction of efficient cellular and humoral adaptive immune responses is particularly relevant for the rational design of new vaccines. The aims of this study were first to assess the ability of a broad range of conserved microbial stimuli to induce CD8+ T cells responses by cross-priming and enhance antibody responses against exogenous soluble protein antigens, and secondly to investigate the mechanisms by which microbial stimuli induced cross-priming. Stimulation of Toll like receptors (TLR) is believed to play a major role in the activation of innate and subsequent adaptive responses. All TLR agonists tested enhanced antigen-specific antibody responses, and in particular zymosan (TLR2/6), poly(I:C) (TLR3), LPS from E. coli (TLR4) and CpG DNA (TLR9) promoted IgG2a responses, which are thought to contribute effectively to protective mechanisms against pathogens in mice. However, only poly(I:C), LPS from E. coli and CpG DNA were able to stimulate the induction of cross-priming, whereas zymosan, peptidoglycan (TLR2/) and R-848 (TLR7) were ineffective. It is known that LPS from different bacteria species can elicit different immune responses. LPSs from Klebsiella pneumoniae and from Neisseria meningitidis, but not the unconventional LPS from Porphyromonas gingivalis, were able to induce cross-priming. Microbial mannose structures, as present in yeast mannan, Influenza hemagglutinin and polymannose LPS, were demonstrated for the first time to be able to induce functional cross-priming. In addition, mannan and polymannose LPS were found to enhance antigen-specific antibody responses and promote IgG2a responses. IFN-a/p and signalling through costimulatory molecules play a central role in the licensing of cross-priming. Experiments using knock-out mice showed that in all cases licensing of cross- priming was dependent on IFN-a/pR signalling, albeit to a varying degree. In contrast, signalling through CD40 was not required for induction of cross-priming by mannan and polymannose LPS. Induction of cross-priming by LPS from E. coli and by mannan was TLR4-dependent, whereas induction by polymannose LPS was TLR4-independent. This study thus identifies LPS from some bacteria species as cross-priming-inducing stimuli. It also confirms that activation of TLR can initiate induction of cross- priming, while indicating the existence of TLR-independent pathways. In addition, this work illustrates the importance of IFN-oc/pR signalling as a cross-priming licensing stimulus.

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