Pseudomonas aeruginosa (P. aeruginosa) is a frequently isolated pathogen that causes septicaemia and chronic respiratory infection. It exhibits a higher mortality rate than other gram-negative bacteria and the need for effective immunotherapy is emphasised by the frequency of antibiotic resistance associated with this organism. Mucosal immunisation with a whole killed cell P. aeruginosa vaccine has previously demonstrated a significant immune response in both rodent studies and human trials. This study is a continuation of that research, with the major goal being the identification of a purified protein antigen that could form the basis of a mucosal vaccine against P. aeruginosa. Specifically, the aims of this study were the development of purification protocols for the isolation of previously untested protein antigens, assessment of the efficacy of these antigens to enhance bacterial clearance in an animal model of acute respiratory infection, determination of the immune parameters that are associated with the resolution of P. aeruginosa respiratory infection and finally, cloning of an identified antigen which demonstrated vaccine efficacy. Protocols were established to isolate proteins for use as antigens in immune response studies. The proteins purified in this study were Pa 13, Azurin, acyl carrier protein (ACP), Amidase, Aminopeptidase, KatA and Pa70. These proteins were used to immunise rats by intestinal intra-Peyer's patch (IPP) inoculation and intratracheal (IT) boost. The immunisation protocol employed was designed to target mucosal antigen-specific immune responses where the route of immunisation, Peyer's patch (PP) intestinal inoculation, is akin to the oral delivery of antigens to the gut-associated lymphoid tissue (96). Investigations of a previously uncharacterised antigen, Pa60, later identified this protein as the P. aeruginosa catalase, KatA. This study demonstrated enhanced bacterial clearance of both homologous and heterologous challenge following immunisation with KatA. The level of clearance demonstrated by KatA was promising when compared to that of killed whole cell immunisation. KatA was cloned and studies with the recombinant protein showed enhanced bacterial clearance commensurate with that of the native protein. Immunisations with other proteins identified four additional antigens which enhanced bacterial clearance; Pa13, Pa40, Pa45 and Pa70. Amino acid sequence analysis indicated that Pa13 may be a novel protein, whereas Pa40 was determined to be amidase and Pa45, aminopeptidase. Pa70 was not successfully sequenced. These proteins were effective in significantly enhancing bacterial clearance of homologous P. aeruginosa challenge. For KatA, Pa13 and Pa70, clearance was associated with a marked phagocytic cell recruitment. In contrast, amidase and aminopeptidase demonstrated clearance with a minimal cellular response. Proteins; azurin and ACP were non-protective, failing to clear a live P aeruginosa challenge. Analysis of the antigen-specific responses of these nonprotective proteins and comparison with those antigens which enhanced bacterial clearance were used to determine factors that may contribute to the resolution of an acute pulmonary infection. The study has demonstrated that mucosal immunisation using purified protein antigens can enhance the clearance of pulmonary infection with P. aeruginosa. It has also contributed to the understanding of immune responses to newfound antigens of P. aeruginosa and identified antigen-specific responses which confirm their potential as vaccine candidates.

This research contributes to understandings of the occurrence of and associations between skeletal evidence of vitamin D deficiency and chronic respiratory infections across the life course based on human skeletal material from the Roman period sites of Isola Sacra in Italy (1st - 3rd centuries AD) and Ancaster in the United Kingdom (3rd - 4th centuries AD). Modern clinical data demonstrate a positive association between these two conditions that affects the ways in which they are experienced today, and may extend into the past. Macroscopic, radiographic, and histological evidence for skeletal manifestations of vitamin D deficiency and chronic respiratory infections were considered in the context of archaeological and historical evidence available for the Roman period in order to elucidate patterns in disease occurrence that reflect the unique local biologies of these two assemblages. Differing prevalence values for active and healed lesions caused by both conditions, as well as variation in age at death distributions and the relationship of lesions associated with vitamin D deficiency and chronic respiratory infections with one another and with age at death, provide information on the experience of both conditions and the potential interactions between them. Skeletal lesions caused by both conditions are present in individuals throughout the life course at Ancaster and Isola Sacra, with particular implications for disease experiences during infancy, adolescence, and pregnancy in the Roman period. These results point to a picture of morbidity and mortality at Ancaster that involves longer term survival of and more efficient immune responses to chronic disease processes, with higher levels of skeletal lesions indicating the presence of more "survivors" at this site. The combination of lower frequencies of skeletal lesions and higher mortality at Isola Sacra, on the other hand, suggests that fewer individuals may have survived to the point where they were able to mount a skeletal response to disease. / Dissertation / Doctor of Philosophy (PhD)