Haemophilus influenzae type b (Hib) é uma importante bactéria Gram-negativa causadora de pneumonia, meningite e septicemia em crianças abaixo dos 5 anos de idade. A prevenção contra a doença pode ser alcançada pela imunização da população com vacina conjugada polissacarídeo-proteína, uma vez que a vacina de polissacarídeo não é eficiente. As vacinas conjugadas disponíveis comercialmente custam para o governo brasileiro cerca de US2,7 a dose, sendo necessárias no mínimo 3 doses para imunização completa. O presente estudo desenvolveu um novo método de conjugação de polissacarídeo capsular de Hib (PRP) com toxóide tetânico (TT). O método hidrazona baseia-se em 3 etapas simples: oxidação e derivatização de PRP com espaçador molecular e conjugação com TT na presença de uma carbodiimida e de um éster amino reativo. Após um estudo detalhado de cada etapa do método hidrazona, o novo processo mostrou excelentes resultados de rendimento mesmo após escalonamento. A imunogenicidade e o índice de avidez do conjugado hidrazona foram avaliados e os resultados encontrados foram comparáveis a vacina comercial Hiberix®. A técnica de HPSEC mostrou-se eficaz e o perfil cromatográfico do conjugado hidrazona foi muito similar ao da vacina Hiberix. Finalmente, o novo processo de conjugação de vacina permitiu o desenvolvimento de uma poderosa tecnologia constituindo uma excelente opção para o governo brasileiro. / Haemophilus influenzae type b (Hib) is an important encapsulated bacteria, which causes pneumonia, meningitis and septicaemia in infants. Prevention against infection is achieved by the currently available polysaccharide protein conjugate vaccine. However, due to its high production costs (around U$ 2,7 per dose) this formulation cannot be used in mass immunization programs in Brazil. In the present study, we developed a new method for the conjugation of Hib polysaccharide (PRP) and tetanus toxoid (TT). The hydrazone method is based on 3 singles steps: PRP oxidation, PRP derivatization with linker spacer and conjugation with TT in the presence of carbodiimide and an amino reactive ester. After detailed study of each step of method, the new process showed very good yield of conjugation even when it was scaled-up. The immunogenicity and the avidity index of hydrazone conjugate were evaluated and the results were comparable with those obtained with the commercial vaccine Hiberix®. The HPLC hydrazone profile was very similar to HPLC Hiberix profile. Finally, the new conjugation process allows the development of a powerful vaccine technology, constituting an excellent choice for the brazilian government.

OBJECTIVE: The principal objective of this study was to measure various aspects of immunity in children with invasive infections due to Haemophilus influenzae type b and Staphylococcus aureus. These serious infections are a significant cause of childhood morbidity and mortality in all populations and affect healthy as well as compromised children. Evidence suggests that imbalances or deficiencies in certain aspects of immunity such as IgG subclasses, the capacity to make specific subclass antibodies, antibody affinities, complement isotypes, immunoglobulin allotypes or mannose binding protein may place certain children at risk for developing invasive disease. Investigation of these factors in a group of children with infection necessitated that normal ranges be established for children of comparable ages from the same population. A secondary objective of this study has therefore been to establish normal percentiles for the IgG subclasses in age, race and sex matched healthy controls. METHODS: Patients admitted to the Red Cross War Memorial Children's Hospital with septic meningitis due to Haemophilus influenzae type b and osteomyelitis/septic arthritis due to Haemophilus influenzae type b or Staphylococcus aureus formed the study population. Section A of this thesis describes the methods for establishing, validating and standardizing ELISAs for measuring the IgG subclasses (lgGl, IgG2, IgG3 and IgG4) and subclass antibodies specific to Haemophilus influenzae polyribosylribitol phosphate, Staphylococcus aureus teichoic acid and tetanus toxoid. The relative affinity of antibodies in these ELISAs was determined by the incorporation of diethylamine (DEA). In order to determine the immunoglobulin allotypes ELISAs were developed to measure the G1m(f), G2m(n) and Km(3) allotypes. The frequency of these allotypic markers in the different ethnic groups was established. The relationship between immunoglobulin allotypes and IgG subclass values were investigated in both patient and control groups. RESULTS: ELISA assays to measure IgG subclasses; IgG, IgG 1 and IgG4 tetanus toxoid antibodies; IgG, IgG 1 and IgG2 H. influenzae type b polyribosylribitol phosphate capsular polysaccharide antibodies; IgG, IgG1 and IgG2 S. aureus teichoic acid antibodies and G1m(f), G2m(n) and Km(3) allotypes were successfully established. Where possible the assays were standardized with reference sera and specimens were exchanged with international laboratories. Age, race and sex related percentile charts and tables of normal ranges for IgG and IgG subclasses of Black and Coloured children were established. The IgG and IgG 1 values were higher than those previously reported for children in developed countries. Black children with H. influenzae meningitis had significantly lower IgG 1, IgG2 and IgG3 levels compared to the controls and although similar trends were seen for IgG and IgG4 levels they were not statistically significant. Coloured children with H. influenzae meningitis and Coloured and Black children with H. influenzae osteomyelitis/septic arthritis also showed a similar tendency of lower IgG and IgG subclass levels than the controls but these trends were also not significantly different. All patients responded to tetanus toxoid antigen suggesting normal immunocompetence to protein antigens. H. influenzae type b capsular polysaccharide antibodies were low in children with H. influenzae type b meningitis and osteomyelitis/septic arthritis and did not increase during the illness. IgG and IgG 1 teichoic acid antibodies were raised in patients with S. aureus osteomyelitis/septic arthritis although no further rise in these antibodies was seen when measured several weeks after the illness. The antibody affinity ELISAs showed that IgG 1 tetanus toxoid antibody had a greater affinity than IgG4 tetanus toxoid antibody, the IgG 1 and IgG2 H. influenzae capsular polysaccharide antibodies were of similar affinity and the IgG 1 teichoic acid antibody was of higher affinity than the IgG2 antibody. The G1m(f) and G2m(n) positive allotypes were uncommon in Black but common in the Coloured populations whereas Km(3) was common in both groups. There was a significantly decreased frequency of the G2m(n) positive allotype in Coloured patients with H. influenzae type b meningitis and H. influenzae type b osteomyelitis/septic arthritis which was not found in patients with S. aureus osteomyelitis/septic arthritis. In both Coloured and Black children with H. influenzae meningitis there was a significantly decreased frequency of the Km(3) allotype. No differences in C4 isotypes and mannose binding protein levels were evident in the patient and control groups. CONCLUSION: This study has developed simple, specific and reproducible ELISAs to measure IgG subclasses and subclass antibodies specific to tetanus toxoid, H. influenzae polyribosylribitol phosphate and S. aureus teichoic acid. Age, sex and race related normal ranges for IgG subclasses in the local Black and Coloured populations have been established. Black children with H. influenzae type b meningitis had significantly lower IgG 1, IgG2 and IgG3 levels compared to the controls. There was a clear association between a decrease of the G2m(n) allotype and the Km(3) allotype and susceptibility to invasive infections caused by H. influenzae.

Submitted by Priscila Nascimento (pnascimento@icict.fiocruz.br) on 2012-11-23T15:50:27Z No. of bitstreams: 1 elo-de-oliveira-rodrigues.pdf: 697936 bytes, checksum: fb3899c2c6d659498d8e8ab0a1a0b33a (MD5) / Made available in DSpace on 2012-11-23T15:50:27Z (GMT). No. of bitstreams: 1 elo-de-oliveira-rodrigues.pdf: 697936 bytes, checksum: fb3899c2c6d659498d8e8ab0a1a0b33a (MD5) Previous issue date: 2009 / Fundação Oswaldo Cruz. Instituto de Tecnologia em Imunobiológicos. Rio de Janeiro, RJ, Brasil / Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Rio de Janeiro, RJ, Brasil. / O Instituto de Tecnologia em Imunobiológicos, Biomanguinhos, é uma unidade da FIOCRUZ produtora de vacinas, biofármacos e reativos. O Departamento de Controle de Qualidade, pertencente a unidade de Biomanguinhos, é responsável pelos diversos ensaios físico-químicos, microbiológicos e biológicos para liberação dos produtos finais, produtos intermediários e matérias-primas. Devido à necessidade de normalizar seus ensaios referentes a produtos finais e intermediários, várias medidas têm sido tomadas como: calibração e qualificação de equipamentos, validação de métodos analíticos, aquisição de padrões, entre outras atividades de melhoria. Uma das dificuldades encontradas pelos laboratórios de controle de qualidade é a aquisição de padrões que tenham características semelhantes aos produtos produzidos em Biomanguinhos. A disponibilidade de materiais de referência/padrões que atendam às necessidades do laboratório e a dificuldade em obtê-los, além dos custos elevados, os tornam impeditivos para uso nos ensaios rotineiros. Esta dissertação tem como objetivo estabelecer a prática da produção de material de referência in housepara os métodos que são utilizados para o controle de qualidade de produtos obtidos em Biomanguinhos. O planejamento eelaboração do material de referência de trabalho serão realizados considerando-se todas as condições necessárias para que a substância candidata atenda às normas vigentes relacionadas à normalização de métodos de controle de qualidade. A implantação da metodologiae dos requisitos necessários para obtenção do material serão descritos neste trabalho. O material “candidato” a material de referência é opolirribosil-ribitol fosfato, o PRRP, que após conjugação com a proteína monomérica tetânica, torna-se o princípio ativo da vacina contra Haemophilus influenzae, a vacina Hib. A avaliação do material de referência candidato é baseada nos estudos de caracterização, homogeneidade e estabilidade, utilizando-se ferramentas estatísticas adequadas, visando à atribuição do seu valor com uma incerteza de medição associada, atendendo aos propósitos desejados e agregando maior confiabilidade aos produtos analisados pelo laboratório. Além do uso interno, há a intenção de produzir este material de referência emitindo certificado de acordo com as normas vigentes, e assim fornecê-lo também para o INCQS, órgão da FIOCRUZ responsável pelo controle de qualidade nacional de vacinas e medicamentos. / The Institute of Technology in Immunobiologicals, Biomanguinhos, is a vaccine, biopharmaceuticals, and diagnostic kits production unit that belongs to Fiocruz. The Quality Control Department is responsible for the many physical-chemical, microbiological, and biological assays performed to release the final and intermediate products and the raw materials. Due to the need of standardization of the assays, some measures have been being taken, such as equipments’ calibration and qualification, validation of analytical methods, and standards purchase. One of the challenges faced by the quality control laboratories is the acquisition of standards that have the same characteristics as the Biomanguinhos products. The low availability of standards and reference materials that attend the laboratories’ needs and the difficulties in obtaining these products, besides the high costs, make their use in the laboratories routine almost impossible. This thesis intends to establish the production practice for the in-house reference materials used in Biomanguinhos’ quality control assays. The planning and elaboration of the reference materials will be made according to the current legislation that concerns the standardization of quality control methods. The deployment of the methodology and of the requirements for the material obtainment will be discussed in this work. The ‘candidate’ to be a reference material is the polyrribosil ribitol phosphate (PRRP) that, after conjugation with the tetanical monomeric protein, becomes the active substance of the Haemophilus influenzaevaccine (Hib). The evaluation of the candidate material is based in characterization, homogeneity and stability studies, using suitable statistical tools, in order to assign its value with an associated measurement uncertainty. It aggregates reliability to the products analyzed in the laboratories. Besides the internal use, the purpose of this work is to certify the reference material in accordance with the current regulations, so that itcan be more trustable and therefore be used by INCQS, Fiocruz unit responsible for the nationalquality control of vaccines and other pharmaceutical products.

Indiana University-Purdue University Indianapolis (IUPUI) / Haemophilus ducreyi causes chancroid, a sexually transmitted genital ulcerative disease that facilitates the transmission of HIV-1. H. ducreyi also causes non-sexually transmitted cutaneous ulcers in children in tropical regions. During human infection, H. ducreyi is subject to a variety of stresses. The stringent response is a bacterial stress response system induced by nutrient limiting conditions and mediated by guanosine tetra- and pentaphosphate [(p)ppGpp] and the transcriptional regulator DksA. (p)ppGpp and DksA jointly interact with RNA polymerase to regulate genes critical for bacterial survival. We hypothesized that the stringent response is required for H. ducreyi virulence in humans. A ΔrelAΔspoT mutant, which is unable to synthesize (p)ppGpp, was partially attenuated for abscess formation in human volunteers. Loss of (p)ppGpp increased bacterial resistance to phagocytosis and stationary phase survival; however, the mutant was more sensitive to oxidative stress. A ΔdksA mutant was also partially attenuated in humans. The ΔdksA mutant behaved like the (p)ppGpp mutant in stationary phase survival and sensitivity to oxidative stress, but exhibited decreased resistance to phagocytosis. Both mutants had decreased adherence to fibroblasts, but the mechanisms underlying the adherence defect were distinct. To better understand the roles of (p)ppGpp and DksA in regulating gene expression, we performed transcriptome analysis of the parent and mutant strains. (p)ppGpp and DksA deficiency resulted in dysregulation of multiple genes including several known virulence determinants. At stationary phase, (p)ppGpp and DksA targets were not identical but significantly overlapped; as the mutants were phenotypically distinct, this finding underscores both the unique and joint roles DksA and (p)ppGpp play in regulation of H. ducreyi virulence. We conclude that (p)ppGpp and DksA play significant roles in H. ducreyi pathogenesis. This is the first study to show that the stringent response has a direct role in the ability of a bacterial pathogen to cause disease in humans.

Sialic acid (SA) is a known major virulence factor of Haemophilus influenza (Hi). This study aims to analyse the functions of some candidate sialometabolism genes, and to further our current understanding on the Hi sialometabolism gene regulation. Two candidate sialometabolism genes (HI0227 and HI0148) and their adjacent ORFs (HI0228, HI0148.1 and HI0149) were studied. HI0148.1 and HI0149 are transcribed as a single gene in screened NTHi strains, and we refer to the combined ORF as NTHI0236 (the designation in strain 86-028NP). Across Hi strains screened, the sequences of HI0227, HI0148 and NTHI0236 are conserved. However, the sequence of HI0228 is heterogeneous. Mutants that lack the functions of HI0227, HI0228, HI0148 and NTHI0236 were compared to their respective wild type parent strains for ability to grow on SA (in aerobic and microaerophilic conditions), their ability to sialylate LPS and their ability to resist complement mediated killing. The mutants did not exhibit major differences in the tested aspects of sialometabolism compared to their respective wild type strains. Changes observed in some of the mutants in serum bactericidal assays and LPS profiles were due to the effect of phase variable genes. The sialometabolism functions of HI0227, HI0148, and NTHI0236 remain obscure, and we postulate that HI0228 is a pseudogene. Investigation of Hi sialometabolism gene regulation was conducted using mutants that lack different steps of the Neu5Ac catabolism pathway and the Neu5Ac activation pathway. The expression of nanE and siaP, respectively representing the Neu5Ac catabolism and transport operons, were assessed using RT-PCR and qPCR. We investigated a temporal/concentration effect of Neu5Ac on the expression of sialometabolism operons, which highlights the importance of studying the Hi sialometabolism gene regulation as a dynamic process. We further demonstrated that GlcN-6P, a Neu5Ac intermediate from the catabolism pathway, is likely the SIS sugar that interacts with SiaR, the repressor protein of the Hi sialometabolism operons. We postulate that upon binding of GlcN-6P to SiaR, the SiaR-mediated repression on the Hi sialometabolism operons is relieved, resulting in the induction of the expression of Neu5Ac catabolism and transport genes.

Réalisé au sein du LISBP (Laboratoire d’Ingénierie des Systèmes Biologiques et des Procédés, INSA Toulouse), ce travail porte sur l’étude du germe pathogène Haemophilus influenzae de type b (Hib). L’objectif du travail est l’amélioration de la compréhension du métabolisme cellulaire et de la synthèse du polyoside capsulaire chez Hib, utilisé pour la fabrication du vaccin. Ainsi, une étude bibliographique associée à une analyse in silico et à une démarche expérimentale rationnelle a permis de développer un milieu chimiquement défini répondant aux critères de robustesse du procédé et de qualité du produit. Par ce travail, il a pu être défini les facteurs nutritionnels et environnementaux influents sur la production de biomasse et de PRP. Plus généralement, ce travail a permis une meilleure compréhension de la physiologie cellulaire. Une étude Bioinformatique et Transcriptomique a permis l’établissement de cartes métaboliques spécifiques de la souche Hib de notre étude, et de mieux comprendre l’influence du milieu de culture sur la production de polyoside capsulaire. En complément, une étude Fluxomique a permis de développer les connaissances sur le métabolisme général de la souche et plus particulièrement sur la voie de synthèse du polyoside.Sur un plan plus appliqué, une corrélation directe entre la consommation de glucose et la production de polyoside a pu être établie, et ce, dans un environnement maîtrisé de culture en fermenteur. La combinaison du travail développé tant sur le procédé que sur le milieu de culture chimiquement défini ou la sélection de souche, a permis, à l’échelle laboratoire, une multiplication par 6,2 de la production de polyoside capsulaire (7,8 en spécifique) par rapport à la condition initiale de l’étude en milieu complexe. Ce résultat a alors pu être validé chez Sanofi-pasteur à l’échelle pré-industrielle (1000L), tout en conservant une qualité du produit conforme aux critères de production pharmaceutique / This work, undertaken at LISBP (Laboratoire d’Ingénierie des Systèmes Biologiques et des Procédés, INSA Toulouse), concerns the study of the pathogen Haemophilus influenzae type b (Hib). The objective was to improve the understanding of cellular metabolism and the synthesis of the capsular polysaccharide in Hib, used for vaccine production. A literature review coupled with a rational experimental approach has enabled a chemically defined medium that meets the criteria of process robustness and product quality to be developed. This work has defined the key nutritional and environmental factors affecting biomass and PRP production. Bioinformatics and Transcriptomic studies have allowed the specific metabolic characteristics of the Hib strain to be mapped and enabled a better understanding of the influence of culture medium on capsular polysaccharide production to be obtained. A Fluxomics study points to specific organization of the central pathways and more specifically the interaction between the pentose phosphate pathway and the pathway of polysaccharide biosynthesis. On a more applied aspect, direct correlation between glucose consumption and production of polysaccharide was established in a batch culture. The combined knowledge obtained in this study enabled a 6.2-fold increase in production of capsular polysaccharide (7.8 in specific production) to be obtained in laboratory scale installations as compared to the initial fermentation process using complex media. This result was then validated at Sanofi-pasteur at the pilot-plant level (1000L), and shown to maintain product quality as defined by pharmaceutical production criteria

Antibiotic resistance in bacteria is a growing threat to global health. Many of the genes responsible for resistance are carried on mobile genetic elements which can be transferred laterally between strains and species. The most important of these are conjugative and mobilisable elements including plasmids and integrating and conjugating elements, ICEs. Haemophi/us influenzae is an important human pathogen, which was first identified as carrying antibiotic resistance genes in the 1970s. Much of this resistance is encoded by ICEHin1056, which is present in H. influenzae strains worldwide. The aims of this study were to describe features of the biology of ICEHin1056, with particular reference to the genetic site and control mechanisms responsible for instigating conjugative transfer. The origin of transfer has been localised to a sequence on ICEHin1056 and an environmental stressor initiating conjugative transfer, oxidative stress, has been identified. In addition, detailed phylogenetic analysis has demonstrated ICEHin1056 to be part of a much larger family of mobile genetic elements, widely distributed in proteobacteria and carrying accessory genes responsible for survival in adverse environments, virulence and antibiotic resistance. The ICEs in the family have conserved homology of gene content and synteny of gene arrangement over deep evolutionary time, challenging the accepted paradigm of modular mosaicism of mobile genetic elements. A key event in increasing dissemination of the ICE, acquisition of a phage type integrase gene has also been identified. The findings presented provide significant insight into the behaviour of ICEs and may in future allow predictions about the spread of virulence factors and antibiotic resistance genes, with important implications for human and animal health.

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.