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Heterogeneity in immunodominant outer membrane proteins has been proposed as a significant factor in the failure of an NTHi infection to induce immune protection against subsequent infections. This study has examined the vaccine potential of three outer membrane proteins in an attempt to identify conserved regions that could be targeted by an immune response after vaccination. The three proteins investigated were: TbpB, P5 and P48 (HI0164). The optimal route of immunisation in clearing a bolus inoculum of NTHi to the lung in the rat has been shown to be a combination of gut sensitisation with a respiratory boost and this regime was used in the present study. A panel of NTHi isolates was assessed to determine the frequency with which strains were able to bind transferrin and thus be targeted by a TbpBspecific immune response. A high proportion of strains was able to bind transferrin with similar frequencies in isolates associated with infection and those from normal throat swabs. A protocol was developed to purify nonlipidated recombinant TbpB from NTHi using a glutathione-Stransferase (GST)-rTbpB fusion protein and Glutathione-Sepharose affinity chromatography. Mucosal-directed immunisation with rTbpB significantly enhanced clearance of an NTHi challenge to the lung, however, whilst rTbpB-specific antibodies were cross-reactive on Western immunoblots, the cross-reactivity was variable in both transferrin binding inhibition assays and bactericidal activity. This suggested that the rTbpB-specific humoral response would be variable in the recognition of heterologous NTHi isolates. The secondary structure of P5 has been controversial with several reports suggesting that P5 was a fimbrin protein composed of coiled coils. In this present study the interstrain variation in P5 amongst isolates from diverse anatomical sites, as well as computer prediction methods and spectrophotometric analysis, generated a model of P5 based on the homologous E. coli protein, OmpA. This model suggested a B-barrel conformation with no evidence of coiled coils. Synthetic peptides corresponding to conserved regions of P5 that were thought to be surface exposed, as well as a region (H3) with some homology to a protective epitope in the P. aeruginosa protein, OprF, were then combined with a "promiscuous" T cell epitope from the measles virus F protein (MVF) and used for immunisation studies. Whilst variable protection was seen with the peptides, the MVF/H3 peptide was the most efficacious of the antigens assessed in this study in enhancing clearance of NTHi. This occurred in the absence of detectable peptide- or PS-specific antibody leading to the suggestion that cell mediated responses may have played an important role in enhancing clearance in this model. The highly conserved nature of the region in P5 represented by the H3 peptide suggests that further study should be focused on this peptide as a potential NTHi vaccine candidate. The last antigen, P48, is homologous to a A. pleuropneumoniae antigen, AopA, which has been proposed to have potential as a vaccine component against pleuropneumonia in pigs. Sequence analysis of the gene encoding P48 from several isolates showed that this protein was well conserved. Recombinant P48 was purified from a GST-rP48 fusion protein and used for immunisation, which also conferred significant protection. However, immunisation with rP48 was not as efficacious as immunisation with the MVF/H3 peptide. Whilst immunisation with rP48 induced high antibody titres, no bactericidal activity could be detected indicating that bactericidal antibody had not contributed to the observed clearance. In addition, the rP48- specific serum IgG was predominantly of the IgG2a isotype suggesting that Thl cell mediated responses had been induced by immunisation with rP48.

One of the major challenges of vaccine development is the conservation of immunogenicity and protective efficacy through the stages of design, production, formulation and delivery. The critical issue is that how and in what form an antigen is taken up by antigen presenting cells for proteolytic processing and presentation to the immune system bound to MHC can have dramatic effects on the activation of Th cells to drive clonal responses and induction of immunological memory. Nontypeable Haemophilus influenzae (NTHi) is a pathogenic commensal of the human respiratory tract that causes diseases with enormous socioeoconomic burdens. There is no licensed vaccine, although the potential for vaccination with outer membrane components to reduce the incidence of disease caused by NTHi has recently been demonstrated in clinical trials. The issue of immunomodulation was explored in this thesis in the context of the further evaluation of a leading NTHi vaccine candidate, the outer membrane protein OMP26. The efficacy of recombinant OMP26 (rOMP26) against NTHi challenge has been previously demonstrated in mice, rats and chinchillas. In rats, efficacy was shown to be restricted to the precursor form (containing the signal peptide) and not the mature form of rOMP26. The immunodulatory effects of changes to the rOMP26 structure were further investigated in this thesis. A range of structural variants of rOMP26 were constructed in view of reducing extraneous plasmid-derived sequence from the antigen and to introduce a unique cysteine residue as a potential conjugate site for multivalent vaccine development (Chapter 2). It was demonstrated that minor structural changes to rOMP26 such as the addition, deletion, modification or relative positioning of a single amino acid or bulky group, designed to increase the efficiency of production or introduce (cysteine) conjugation sites, altered the expression of the protein in E. coli and the immunogenicity in Balb/C mice. Furthermore, in contradiction to the published report (El-Adhami et al. 1999) and a new study in rats (Chapter 3), there was no positive effect of the signal peptide in mice, with precursor and mature forms of rOMP26 equally immunogenic (Chapter 2). Following confirmation of the need to retain the signal peptide for the immunogenicity of rOMP26 in rats, a precursor form (rOMP26VTAL) in which the conserved n-region of the signal peptide was deleted, and shown to reduce the efficiency of the cleavage of the signal peptide by signal peptidase during protein overexpression in E. coli (Chapter 3). Not only did this deletion result in an increase the yield and stability of the purified precursor protein, but rOMP26VTAL was highly immunogenic and enhanced the clearance of NTHi from the lungs of challenged rats. The potential for signal peptides to be exploited as an immune-enhancing moiety in a proteinaceous vaccine is discussed. Following the development of rOMP26VTAL as a production optimised variant of rOMP26, the next step was to test the feasibility of rOMP26VTAL as a component of a multivalent vaccine (Chapter 4). Two chimeras were constructed with LB1(f)2,1,3, a trivalent synthetic B-cell epitope from the extracellular loop 3 region of the P5 fimbrin protein of NTHi, positioned at the N- or C-terminus of rOMP26VTAL. The solubility of rOMP26VTAL was affected by the fusion, with both chimera constructs expressed only in the insoluble fraction, thus requiring a denaturing protocol for purification. Although rLB1(f)2,1,3-OMP26VTAL was expressed and purified as a more stable protein and in greater yield than rOMP26VTAL-LB1(f)2,1,3, the relative positioning of the fusion was important and rOMP26VTAL-LB1(f)2,1,3 was significantly more immunogenic in rats than rLB1(f)2,1,3-OMP26VTAL. In addition, rOMP26VTALLB1( f)2,1,3, but not rLB1(f)2,1,3-OMP26VTAL induced a significant degree of bacterial clearance following pulmonary challenge with NTHi, in levels comparable to the highly efficacious rOMP26VTAL construct. In the third part of the thesis, bacterial ghosts were evaluated as a novel mucosal delivery technology for rOMP26VTAL and rOMP26VTAL-LB1(f)2,1,3, (Chapter 5). To mimic the natural presentation of OMP26 and P5 fimbrin antigens on the cell surface of NTHi, an OmpA� sandwich fusion surface display system was developed for the outer membrane expression of the OMP26 constructs in E. coli ghosts. Following gut immunisation, but not intranasal immunisation even when co-administered with the cholera toxin�derived adjuvant CTA1-DD, bacterial ghosts were successful at presenting OMP26VTAL and rOMP26VTAL-LB1(f)2,1,3 to the immune system for the induction of enhanced clearance of NTHi in the rat pulmonary challenge model. Although this study was the first to demonstrate enhanced bacterial clearance induced by heterologous antigens expressed in the outer membrane of bacterial ghosts, future studies with ghosts will require optimisation of the expression levels of the OmpA� fusion proteins possibly to avoid cross-reactive responses related to high doses of ghosts in the inoculum. This thesis presents data that both supports the further evaluation of rOMP26 constructs for clinical trials, and has demonstrated the significant effects of structural changes, method of production and delivery system can have on the immunogenicity of a candidate vaccine. Such knowledge will contribute to and provide some new approaches for enhancing the efficiency of vaccine development against a range of diseases including those caused by NTHi. Major Outcomes: 1. Demonstration that the immunogenicity of rOMP26 antigen constructs is affected by structural modifications and their positioning within the construct, and by the delivery system. 2. Development of rOMP26VTAL, an rOMP26 construct with the KNIAK sequence deletion of the signal peptide n-region. This protein retains the immunogenicity and protective efficacy of rOMP26, but is produced with reduced cleavage of the signal peptide, resulting in higher yields and a stable protein. Lacks extraneous plasmidderived multiple cloning site sequence, and is produced in high yield as a stable protein. 3. Construction of a NTHi rOMP26VTAL-LB1(f)2,1,3 chimera antigen that induced enhanced clearance of NTHi in an acute pulmonary challenge model in rats. 4. Development of an OmpA� surface display system for the expression of rOMP26 antigen constructs in the outer membrane of E. coli/bacterial ghosts 5. Bacterial ghosts were successful as delivery vehicles for rOMP26 candidate vaccine constructs when delivered in the gut.

Pulmonary infection caused by nontypeable Haemophilus influenzae (NTHi) is a significant cause of morbidity and mortality in both industrialised and developing countries. Previous work from this group resulted in the development of a respiratory model in rodents which has precipitated studies into the pathogenesis of infection by NTHi and investigation of the humoral and cellular mechanisms by which the bacteria are cleared from the lung. Comparison of mucosally immunised with non-immunised animals has demonstrated that not only are bacteria cleared more rapidly from the lungs, but there is a more rapid response and resolution of inflammatory factors in the mucosally immunised animals following challenge with NTHi. This inflammatory response is partially regulated by the ability of the mucosally immunised animals to rapidly produce, then control the production of tumour necrosis factor (TNF)-a. The TNF-a is produced by both macrophages and type I pneumocytes in the alveoli and also by the endothelial cells lining the blood vessels in the lungs. Immunocytochemical studies have identified cellular subsets accumulating in the lung at various time points following infection. Marked differences in cellular infiltration into the lung tissue were noted between immunised and non-immunised animals after challenge with NTHi. Immunised animals demonstrated an early influx of macrophages, CD8+ T cells and Y8+ T cells, followed by enhanced expression of the MHC-II marker, cellular infiltration by polymorphonuclear leukocytes and finally an increased number of both B cells and CD4+ T cells. In contrast, non-immunised animals did not demonstrate any proliferation nor extravasation of lymphocytes or increased expression of MHC-II before total bacterial clearance had occurred. Polymorphonuclear leukocyte infiltration occurred in the non-immunised animals, however at a later time than that seen in immunised animals. Challenging rodents to establish persistent infection highlighted the inappropriately aggressive white blood cell response to an initial challenge when bacteria may be masked by other substances, followed by the inability to amplify the polymorphonuclear leukocyte response on repeated challenge with NTHi. This hyporesponsiveness in the macrophage population, shown by lack of detectable TNF-a production, concomitant with low numbers of NTHi resulted in a continuously high number of macrophages in the alveoli and the possibility of increased damage to the lung tissue. The requirement for cell surface TNF-a and CD8+ T cells to enhance the clearance of NTHi from the lungs further strengthens previous in vitro and in vivo findings of the possible significance of cellular invasion as a mechanism of pathogenicity for NTHi. This thesis has contributed to the understanding of both the immune response to and the pathogenicity mechanisms of pulmonary infection with NTHi. Kinetic studies identifying cellular responses and cytokine levels have emphasised the ability of mucosal immunisation to increase the rate of immune response and resolution of inflammation to NTHi infection in the lung. Observations demonstrating a requirement for macrophages and CD8+ T cells in mechanisms associated with enhancing NTHi clearance from the lung will lead to further investigations.

Nontypeable Haemophilus influenzae (NTHi), Streptococcus pneumoniae and Moraxella catarrhalis are common bacterial agents of otitis media which is a major cause of morbidity in young children. Mucosal immune responses are an integral part of the immune defense against middle ear infection and it is known that certain populations, including Australian Aboriginal children, are highly susceptible to disease. The current study focussed on the development of the mucosal immunity to the three bacterial pathogens in Aboriginal and non-Aboriginal children from birth to two years of age, living in the Kalgoorlie-Boulder region of Western Australia. Salivary and breast milk IgA levels were measured by the enzyme Linked immunosorbent assay. The measured IgA levels, combined with socio-economic, demographic and bacteriological data were analyzed statistically to determine the influential factors on the mucosal IgA response in these children over time. This study found that each antigen-specific IgA examined followed a distinct ontogeny pattern and IgA responses differed significantly according to age, indigenous status and feeding type. Indoors smoke exposure, maternal smoking, and sibling day care attendance had some impact on salivary IgA levels in the children. However, household crowding and the presence of older siblings had the most significant impact on salivary IgA levels for children of different age groups. These two factors were correlated to increased nasophayrngeal colonization by H. influenzae, S. pneumoniae and M. catarrhalis and colonization status was also found to influence salivary IgA levels in the children. No correlation between maternal breast milk IgA levels and child salivary IgA levels was observed. The results suggest that the degree of exposure to environmental factors rather than immunological deficit is responsible for the observed differences in salivary IgA responses between Aboriginal and non-Aboriginal children and modifying these factors could lead to a reduction in the burden of otitis media experienced by the children. Further studies correlating specific salivary IgA levels to diseases such as otitis media will reveal the role of specific salivary IgA responses in the prevention of infection by respiratory pathogens.

Nontypeable Haemophilus influenzas (NTHi) is recognised as a significant human pathogen causing mild to severe respiratory tract infections. At present, no vaccine is available for prevention of infection caused by this pathogen. Several outer membrane proteins (OMPs) of NTHi and its lipooligosaccharide have been investigated as possible vaccine antigens against NTHi infections. Previous investigations in our laboratory have shown that OMP26 from an NTHi 289 strain was able to significantly enhance pulmonary clearance of NTHi in a rat model in which animals were immunised via intestinal Peyer's patches and then boosted intratracheally (Kyd and Cripps, 1998; El- Adhami et al., 1999). In recent studies, the OMP26, when used as a parenteral immunogen, was also highly effective at inducing immune responses that led to significantly enhanced clearance of the chinchilla nasopharynx (Kyd et al., 2003). These studies indicate significant potential of the OMP26 as a candidate vaccine antigen and warrant further investigations for development of a vaccine against NTHi. This thesis focussed on the immunological and structural characterisation of the NTHi vaccine candidate, OMP26. Peptides of OMP26 were used as tools to localise the immunologically important regions of the OMP26. Two different E. coli expression systems, the GST gene fusion and the 6xHis tagged systems, were employed to construct the OMP26 peptides. It was found in this study that, despite efforts to optimise the system, the GST-fusion protein system failed to produce consistent results for the purification and storage of the OMP26 peptides. In contrast, the 6xHis tagged system exhibited more reliable outcomes in the production of the recombinant OMP26 peptides and the stability of the stored purified peptides. As such, the purified OMP26 peptides from the 6xHis tagged system were chosen to map major regions of immunological significance for the OMP26 protein. The regions of the OMP26 which are involved in the induction of the acquired immune responses have been identified in the present study. Based on the antigen specific lymphocyte proliferation assay, the dominant T cell epitopes for OMP26 were located between amino acid residues 95 and 197 (T3+T4 region). These identified T cell epitopes exhibited the capability of efficient T cell activation, suggesting that the epitopes within the T3+T4 region potentially had the highest affinity for binding to the MHC molecules than did any other OMP26 region. Using two different assay systems, ELISA and BIA, the predominant B cell epitopes of OMP26 were located between amino acid residues 45 and 145 (T2+T3 region). This region was also found to be immunodominant across all animal species tested, and with all immunisation regimens used. Flow cytometry analysis also revealed that these particular epitopes were expressed on the surface of NTHi cells. By integration of the data obtained from these current experimental studies and the computational analysis of the OMP26 sequence, two hypothetical models of the OMP26 were also proposed in this study. The significant outcomes obtained in this thesis provide a better understanding of the specificity of the host immune responses to the OMP26 protein These findings provide great benefit not only for the development of a future NTHi vaccine but for the development of the peptide-based immunodiagnostic reagents as well. These diagnostic reagents will be valuable, in particular, for the evaluation of efficacy of an NTHi vaccine in humans that may include OMP26 or specific conformational structures. Future studies are still required to further define the minimum epitope length required for the B and T cell responses identified in this study. The significance of these responses in immune protection against NTHi infection also requires further investigations. Human immune responses also need to be determined, but this can only be achieved following clinical trial studies.

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