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.

Haemophilus parasuis es un miembro de la familia Pasteurellaceae y un importante patógeno del tracto respiratorio porcino, causante de la enfermedad de Glässer. En la actualidad, esta enfermedad es una de las que tiene mayor incidencia en el sector porcino con una elevada repercusión económica. Por este motivo, es necesario estudiar la biología molecular de este patógeno para poder desarrollar vacunas más eficientes que las existentes hasta el presente. Para abordar dicho estudio es imprescindible disponer de algún proceso de transferencia genética eficaz en H. parasuis. No obstante, dado el desconocimiento que se tenía de dicho patógeno, no se conocía ningún mecanismo natural o de laboratorio que permitiera introducir DNA en H. parasuis. Por ello, el primer objetivo del presente trabajo ha sido desarrollar un método de transferencia genética en H. parasuis que permita estudiar posteriormente los genes implicados en su virulencia y en la colonización del hospedador.En la presente tesis se ha demostrado por vez primera que H. parasuis es capaz de transformar de forma natural si el DNA exógeno contiene la secuencia ACCGAACTC, la cual es muy similar al motivo ACCGCACTT descrito en H. influenzae como Uptake Signal Sequence. Además, se ha optimizado este proceso, determinando las concentraciones más adecuadas de células bacterianas, AMPc y DNA transformante, así como el tiempo de incubación. Un segundo objetivo de este trabajo ha sido averiguar el papel de la timidilato sintasa, enzima clave en la síntesis de desoxitimidina monofosfato (dTMP), en la virulencia de H. parasuis. Para ello, se ha construido un mutante knockout en el gen thyA de H. parasuis y se ha determinado la capacidad de colonización y la inmunogeniciad de la cepa salvaje y de dicho mutante thyA en un modelo animal de cobayas. Los resultados han mostrado que la cepa defectiva en la enzima timidilato sintasa tienen una menor capacidad de colonización, a pesar de que es capaz de inducir una respuesta inmune en el animal. Por ello, el mutante thyA de H. parasuis puede ser un buen candidato para el desarrollo de futuras vacunas.Finalmente, en este trabajo se ha estudiado la importancia de la proteína Fur en la virulencia de H. parasuis. Dicha proteína es un regulador pleiotrópico que, entre otras funciones, controla los mecanismos implicados en la captación de hierro. Para abordar esta parte del trabajo se propuso la construcción de un mutante de H. parasuis defectivo en la proteína Fur. Los resultados obtenidos han mostrado claramente que dicha proteína es esencial para la viabilidad de esta bacteria. Así, se ha determinado el entorno genético del gen fur y se ha confirmado la no viabilidad de los mutantes fur mediante la obtención de cepas merodiploides. / Haemophilus parasuis, is a member of the family Pasteurellaceae and an important respiratory-tract pathogen of swine, which is the etiological agent of Glässer's disease. At present, the H. parasuis infections produce significant mortality and morbidity in pig farms, giving rise to important economic losses in this industry. For this reason, it is necessary to study the molecular biology of this pathogen to develop vaccines more efficient. Because no genetic manipulation system is available for H. parasuis so far, the first objective of this work was to develop a method of gene transfer to study the genes involved in virulence of this organism. In this work has been demonstrated for the first time that H. parasuis is capable of transforming exogenous DNA which contains ACCGAACTC sequence. This sequence is very similar to the motive ACCGCACTT described in H. influenzae as Uptake Signal Sequence. In addition, this process has been optimized, determining the most appropriate concentrations of bacterial cells, cAMP and transforming DNA, as well as the incubation time.A second objective of this study was to determine the role of thymidylate synthase enzyme in the virulence of H. parasuis. This enzyme is essential for dTMP synthesis and, consequently, for DNA replication. To do so, a H. parasuis thyA mutant was constructed in order to analyze its colonization characteristics and its capacity to generate serum bactericidal activity in infected guinea pigs. The data showed that colonization by the H. parasuis thyA mutant was much less than that of the wild-type strain. Nevertheless, the mutant generated a strong immunogenic response similar to the wild-type strain. Therefore, the H. parasuis thyA mutant can be a good candidate to develop future vaccines. Finally, this work has studied the importance of the Fur protein in the virulence of H. parasuis. This protein is a global regulator which, among other duties, controls the mechanisms involved in iron uptake. To perform this part of the study was proposed the construction of a H. parasuis fur mutant. The results have clearly shown that this protein is essential for the viability of this bacterium. Thus, it has been determined the genetic surrounding of fur gene and has confirmed the non-viability of fur mutants by obtaining H. parasuis fur-defective merodiploid strains.

Diss. (sammanfattning) Uppsala : Sveriges lantbruksuniv. / Härtill 4 uppsatser.

Submitted by Hiolanda Rêgo (hiolandarego@gmail.com) on 2016-07-29T17:20:06Z No. of bitstreams: 1 Tese_Med_Josilene Borges Torres Lima.pdf: 812403 bytes, checksum: f3a47d8a2a2a13648f9abf0ee0d4b9aa (MD5) / Approved for entry into archive by Delba Rosa (delba@ufba.br) on 2016-08-22T12:47:57Z (GMT) No. of bitstreams: 1 Tese_Med_Josilene Borges Torres Lima.pdf: 812403 bytes, checksum: f3a47d8a2a2a13648f9abf0ee0d4b9aa (MD5) / Made available in DSpace on 2016-08-22T12:47:57Z (GMT). No. of bitstreams: 1 Tese_Med_Josilene Borges Torres Lima.pdf: 812403 bytes, checksum: f3a47d8a2a2a13648f9abf0ee0d4b9aa (MD5) / A introdução de vacinas conjugadas contra o Haemophilus influenzae sorotipo “b” (Hib) foi um avanço de grande impacto na Saúde Pública, praticamente eliminando a meningite por Hib nos países onde foi implementada. O presente trabalho descreveu a meningite por H. influenzae em Salvador, Bahia, estudando o impacto da vacina, e caracterizou os isolados do sorotipo não “b”, identificados durante vigilância ativa populacional. A população estudada foi composta de pacientes identificados por cultura positiva para H. influenzae, e os dados clínicos obtidos através de entrevista e revisão de prontuário. O sorotipo dos isolados foi determinado utilizando antisoros, e reação em cadeia pela polimerase (PCR). A tipagem molecular foi realizada pelas técnicas de eletroforese em campo pulsátil e multilocus sequence typing; e para identificação de mecanismo de virulência, foi utilizada PCR e sequenciamento da região de deleção IS1016-bexA. Para cálculos de incidência foram utilizados números de casos da região Metropolitana de Salvador, e a população do censo do ano de 2000. Após 5 anos de introdução da vacina anti-Hib, a incidência da meningite pelo H. influenzae teve uma redução de 97,6% na população total, e de 97,5% em menores de cinco anos. Em 11 anos de vigilância, foram identificados 40 casos de meningite por isolados do sorotipo não b, sendo 80% após o uso da vacina. Na caracterização molecular, os isolados do sorotipo “a” foram agrupados em dois clones, sendo que um deles apresentava o fator de virulência, a deleção parcial IS1016-bexA, o qual foi associado com elevada mortalidade (OR 10,8 e IC 0,7-345). Estudos de epidemiologia molecular são importantes para estimar a eficácia da vacina, bem como monitorar a emergência de isolados de outros sorotipos para avaliar a necessidade de intervenção na composição da vacina.

Thèse numérisée par la Direction des bibliothèques de l'Université de Montréal.

() is a commensal organism of the gastrointestinal tract. It rarely causes hepatobiliary infections; however, in the presence of underlying inflammation, immunosuppression, or malignancy, it can cause hepatobiliary infection via an ascending route. Herein, we report a case of pyogenic liver abscess secondary to associated with cholangiocarcinoma, which was treated with ceftriaxone and metronidazole.

Ältere Menschen sind gegenüber invasiven Infektionen und Sepsis besonders vulnerabel mit ungünstiger Prognose. Staphylococcus aureus und Haemophilus influenzae können beide invasive Infektionen verursachen. Oft geht eine asymptomatische Besiedelung einer Infektion voraus und ist ein Risikofaktor für eine invasive Infektion. Daher wurde eine bizentrische Querschnittstudie in den Regionen Aachen und Würzburg durchgeführt, um die Prävalenz von H. influenzae, S. aureus und MRSA (Methicillin resistenter S. aureus) bei asymptomatischen Senioren zu bestimmen, wie auch Risikofaktoren für eine Besiedelung. Von Oktober 2012 bis Mai 2013 wurden 677 Erwachsenen im Alter von 65 Jahren oder älter eingeschlossen, die zu Hause oder in Seniorenheimen lebten. Die Prävalenz von H. influenzae bei älteren Menschen war mit einer Trägerrate von nur 1,9% ([95% CI: 1,0 - 3,3%]; 13/677) sehr niedrig. Trägerisolate waren überwiegend nicht typisierbare H. influenzae, zeigten eine hohe clonale Diversität und waren alle Ampicillin-sensibel. Die Prävalenz von S. aureus war mit 28,5% ([95% CI: 25,1 - 32,1%]; 193/677) hoch, wie für die deutsche Allgemeinbevölkerung bekannt, während MRSA bei weniger als 1% der Teilnehmer gefunden wurde (0,7% [95% CI: 0,2 - 1,7%]; 5/677). Die Prävalenz von H. influenzae, S. aureus und MRSA unterschied sich nicht signifikant zwischen selbständig zu Hause lebenden Senioren und Pflegeheimbewohnern. Ältere, selbständig lebende Menschen mit höherem Bildungsniveau hatten signifikant höhere Kolonisierungsraten mit S. aureus (adjusted OR: 1,905 [95% CI: 1,248 - 2,908]; p = 0,003). Bei Pflegeheimbewohnern war eine Kolonisierung signifikant mit Verheiratet sein assoziiert (adjusted OR: 3,367 [95% CI: 1,502 - 7,546]; p = 0,003). Diese Ergebnisse unterstreichen die Bedeutung von sozio-demographischen Faktoren für eine Kolonisierung mit S. aureus und schließen eine Lücke bei epidemiologischen Daten zu H. influenzae. / Elderly people are especially vulnerable to invasive infections and sepsis with often poor outcome. Staphyloccus aureus and Haemophilus influenzae both can cause invasive infections. Asymptomatic colonization often precedes infection and poses a risk for invasive infection. Therefore, a bi-centric cross-sectional carrier study was conducted in the regions of Aachen and Wuerzburg, Germany, to determine the prevalence of H. influenzae, S. aureus and MRSA (methicillin resistant S. aureus) in asymptomatic elderly people and to identify risk factors for colonization. From October 2012 to May 2013 677 adults aged 65 years and older were included, living at home or in nursing homes. In contrast to children and younger adults the prevalence of H. influenzae was very low among elderly people with a carriage rate of only 1.9% ([95% CI: 1.0 - 3.3%]; 13/677). Carrier isolates were predominantly non typeable H. influenzae, showed a high clonal diversity and were all susceptible to ampicillin. The prevalence of S. aureus was expectedly high as known for the German general population (28.5% [95% CI: 25.1 - 32.1%]; 193/677), while MRSA was found in less than 1% of the individuals (0.7% [95% CI: 0.2 - 1.7%]; 5/677). The prevalence of H. influenzae, S. aureus und MRSA did not differ significantly between community dwellers and nursing home residents. Elderly community-dwellers with higher education level had significantly higher colonization rates with S. aureus (adjusted OR: 1.905 [95% CI: 1.248 - 2.908]; p = 0.003). Among nursing home residents, colonization was significantly associated with being married (adjusted OR: 3.367 [1.502 - 7.546]; p = 0.003). These results underline the importance of socio-demographic factors for colonization with S. aureus and close a gap in epidemiological data on H. influenzae.

Bacterial transformation is defined as a mechanism of genetic exchange whereby a population of bacteria can obtain genetic informa-tion as a result of cellular uptake and integration of extracellular deoxyribonucleic acid released from other bacteria by natural or in-duced lysis. In order for a transformable strain of bacteria to take up DNA and to undergo transformation, the cells must be in a physiolog-ical state called competence. A cell is referred to as competent if it has the ability to bind extracellular DNA irreversibly and subsequently to integrate and express the DNA.

No description available.