Práce poskytuje základní právní a ekonomické informace o sponzorování a jeho postavení v komunikačním mixu firem. Kromě toho objasňuje důvody, proč je sponzorování v současné době pro společnosti natolik atraktivní.

<p>As an opportunistic Gram-negative pathogen, Pseudomonas aeruginosa must be able to adapt to changes and survive stressors in its environment during the course of infection. To aid survival in the hostile host environment, P. aeruginosa has evolved a myriad of virulence factors including the production of an exopolysaccharide capsule, as well as secretion of degradative proteases and lipases that also function as defense mechanisms. Outer membrane vesicles (OMVs) acts as a secretion system to disseminate virulence factors and function as a general bacterial stress response to remove accumulated periplasmic waste. Despite the growing insights of the field into the potential functions of OMVs, the mechanism for formation remains to be fully elucidated. The three proposed mechanisms for OMV formation in P. aeruginosa are mediated by the Pseudomonas quinolone signal PQS, the AlgU envelope stress response pathway, and the periplasmic chaperone MucD. This report investigates how P. aeruginosa responds to sublethal physiological stressors with regards to OMV production levels and whether the proposed mechanisms for OMV formation are required for stress-induced OMV formation. We concluded that exposure to cell wall directed stressors increased OMV production and activity of the sigma factor that controls MucD expression, AlgU. AlgU was shown to be sufficient to induced OMV production upon overexpression; however, stress-induced OMV production was not dependent on activation of AlgU as vesiculation could be induced in strains lacking AlgU. Furthermore, MucD levels were not inversely proportional to OMV production under acute stress, and the ability to produce PQS was not required for OMV production. Finally, an investigation of the response of P. aeruginosa to oxidative stress revealed that hydrogen peroxide-induced OMV production requires the presence of B-band but not A-band lipopolysaccharide. We also demonstrated that the ability for P. aeruginosa to sense oxidative stress via OxyR, was important for hydrogen peroxide-induced OMV production, by a yet to be determined method. Together these results demonstrate that current proposed mechanisms for OMV formation do not universally apply under all stress conditions, and that additional mechanisms for OMV formation are still to be identified and fully elucidated during acute stress in P. aeruginosa.</p> / Dissertation

Zugl.: Wien, Fachhochsch., Diplomarbeit, 2009

Vesículas de membrana externa, OMV, são formadas a partir de evaginações da membrana externa de bactérias Gram-negativas e têm ganhado interesse em suas funções biológicas por serem uma alternativa ao desenvolvimento de novas estratégias e combinações vacinais. OMV da bactéria comensal Neisseria lactamica induzem anticorpos que possuem reatividade cruzada com N. meningitidis e podem ser, além de antígeno para a doença meningocócica, um potencial adjuvante de mucosa. O objetivo deste trabalho é estabelecer condições de cultivo de N. lactamica para a obtenção de OMV e avaliar a função adjuvante destas OMV em combinação com o antígeno de superfície, PspA5, de Streptococcus pneumoniae. Foram realizados cultivos descontínuos em biorreatores de 5L por 10-15h. O meio de Catlin, MC, teve a concentração do substrato fonte de carbono (lactato) e aminoácidos modificada, além do acréscimo de extrato de levedura. Foram monitorados temperatura, pH, agitação e oxigênio dissolvido. Amostras foram coletadas a cada hora para análise de biomassa, consumo de nutrientes, produção de ácidos e concentração das OMV. Foram determinados os parâmetros cinéticos de produtividade máxima de células (ProdXmáx) e de produto (ProdPmáx), fatores de conversão (Yx/s, Yp/s e Yp/x) e velocidade de crescimento (Xmáx). O meio MC3LA2AA2YE (12h), com 18,0 g/L de lactato e o dobro da concentração original dos aminoácidos do MC foi o melhor, quando comparado aos demais para a obtenção de OMV. N. lactamica, cultivada nesta condição, apresentou produtividade máxima de OMV de 30,66 mg OMV/L.h, e concentração de OMV de 340,43 mg/L, na 11&#170; hora de cultivo. Os ensaios imunológicos foram realizados com a formulação de OMV puras ou OMV tratadas com detergente, para a retirada de parte do lipooligossacarídeo (LOS), em combinação com a proteína PspA5 de S. pneumoniae. O esquema de imunização foi de duas doses via intranasal, em modelo murino. Foram avaliados a indução de anticorpos IgG anti-PspA5 e o potencial protetor das formulações. Os grupos vacinais com adjuvante apresentaram indução de anticorpos IgG anti-PspA5 de aproximadamente 105ng/mL e sobrevivência de 100&#37;, 75&#37; e 66,7&#37;, respectivamente, para PspA5-OMVp, PspA5-OMVt0,5&#37;, e PspA5-OMVt0,3&#37; Os resultados evidenciam atividade adjuvante determinante das OMV em combinação com a proteína heteróloga PspA5 e proteção contra o desafio de Streptococcus pneumoniae em modelo murino. / Outer membrane vesicles, OMV, are formed and released from all Gram-negative bacteria´s outer membrane. Those vesicles have gained interest from scientific community due to their biological functions, as they can be a potential alternative to the development of new vaccine strategies and formulations. OMV from the commensal bacteria Neisseria lactamica induce antibodies that present cross reactivity with N. meningitidis and may be a potential mucosal adjuvant in addition to antigen for a meningococcal disease. The objective of this work is to define culture conditions of N. lactamica in order to obtain OMV, and evaluate the adjuvant function of their OMV in combination to the surface antigen, PspA5, from Streptococcus pneumoniae. Discontinuous batches cultures were carried out in 5L bioreactors for 10-15h. Catlin medium, MC, had its carbon substrate concentration (lactate) and its amino acids concentration modified according to each experiment, plus the addition of yeast extracts. Temperature, pH, agitation and dissolved oxygen were monitored. Samples were collected hourly for analysis of biomass, nutrient consumption, acid production and OMV yield. Maximal cell production products (ProdXmáx) and product (ProdPmáx), conversion factors (Yx/s, Yp/s e Yp/x) and growth rate (Xmáx) were obtained. The MC3LA2AA2YE medium (12h), with 18.0 g / L lactate and double the original MC amino acid concentrations, was the best formulation to obtain OMV. N. lactamica, cultivated in this condition, presented maximum OMV productivity of 30.66 mg OMV/L.h and OMV concentration of 340.43 mg/L, at the 11th hour of cultivation. Immunological assays were performed with formulations with native OMV or OMV treated with detergent to remove part of the lipooligosaccharide (LOS), in combination with S. pneumoniae PspA5 protein. Two-dose of intranasal immunization were administered in mice. An induction of anti-PspA5 IgG antibodies and the protective potential of the formulations were evaluated. Adjuvanted vaccine groups showed induction of anti-PspA5 IgG antibodies of approximately 105 ng/mL and 100&#37;, 75&#37; and 66.7&#37; survival, respectively for PspA5-OMVp, PspA5-OMVt0,5&#37;, e PspA5-OMVt0,3&#37;. The results obtained in this project show a significant adjuvant activity of Neisseria lactamica OMV in combination with heterologous protein PspA5 and protection against Streptococcus pneumoniae challenge in mice model.

No description available.

Human Papilloma Viruses (HPVs) are a large family of viruses with a capsid constituted by the L1 and L2 proteins, which bind to receptors of the basal epithelial cells, thus promoting virus entry. The majority of sexually active people become exposed to HPV, which is the most common cause of cervical cancer affecting more than 600.000 women every year. Moreover, every year more than 13.000 new cases of HPV-related cancers, including anal, penile and head and neck cancers, are diagnosed in men. Three vaccines are available based on the L1 capsid protein, which self-assembles and forms virus-like particles (VLPs) when expressed in yeast and insect cells. Although very effective, these vaccines are HPV type-restricted, and their costs limit broad vaccination campaigns, especially in low- and middle- income countries. Recently, vaccine candidates based on the conserved L2 epitope from serotypes 16, 18, 31, 33, 35, 6, 51 and 59 were shown to elicit broadly neutralizing anti-HPV antibodies, reaching a protection around 90% against all the HPV serotypes. During my research activity, we have tested whether E. coli Outer Membrane Vesicles (OMVs) could be successfully decorated with L2 polytopes and whether the engineered OMVs could induce neutralizing antibodies. OMVs represent an attractive vaccine platform for their intrinsic adjuvanticity and their low production costs. We show that strings of L2 epitopes could be efficiently expressed on the surface of the OMVs and a polypeptide constituted by the L2 epitopes from serotypes 18, 33, 35 and 59 provided broad cross-protective activity against a large panel of HPV serotypes as judged by the in vitro pseudovirus neutralization assay. In order to better characterize the vesicle and in perspective of future clinical studies of our HPV candidate vaccine, we also worked on the setting-up of a simple and reproducible production process at laboratory scale ready to be transferred at industrial level.Moreover, we focused our attention on the strategy used for the engineering of the OMVs with the L2 epitopes and in particular on the carrier used for the delivery of the fusion construct in the surface of the vesicle. More in detail, since part of the carrier is a human cancer epitope, we tested whether a similar scaffold, with less homologies to the human gene could maintain the same properties in terms of: i) expression level of the fused epitopes in the OMVs, ii) localization on the surface of the vesicle and iii) 9 immunogenicity and efficiency to stimulate the immune system in order to produce anti L2 antibodies. Considering all the results described in this work combined with the points of strength of the OMV-based vaccine platform, as the simplicity of the production process, the yields of vaccine doses and the low cost/dose, our data provide a very promising prototype of universal anti-HPV vaccine.

Brucella abortus is a zoonotic agent that primarily infects cattle and causes brucellosis. B. abortus strain RB51 is a live, attenuated vaccine licensed for cattle. However, there is no available vaccine to prevent human brucellosis. Outer membrane vesicles have been tested as potential vaccines to prevent diseases caused by bacterial species. OMV are constantly released from Gram-negative bacteria. They are comprised principally of the outer membrane components and periplasmic proteins from the bacterial cell envelope. The research in this thesis examined the adjuvant property of non-replicative, metabolically active irradiated strain RB51 and the protective ability of OMV derived from strain RB51. Irradiated B. abortus strain RB51 was assessed for its ability to act as an adjuvant to induce protection against malaria. It was found that irradiated B. abortus strain RB51 administered along with fasciclin related adhesive protein (FRAP) to mice induced a protective immune response and a significant decrease in parasitemia after challenge with Plasmodium berghei. Strain RB51 and strain RB51 over-producing Cu/Zn superoxide dismutase (Cu/Zn SOD) were used to produce OMV. Western blotting and SDS-PAGE gel staining confirmed the presence of OMV and the over-production of Cu/Zn SOD. OMV were delivered to mice using an intraperitoneal route and, in some cases, with aluminum hydroxide adjuvant. The immune response was assessed by antibody isotyping with respect to OMV and measuring splenic clearance (i.e. protection) from a B. abortus strain 2308 challenge. The results demonstrate that OMV from B. abortus strain RB51 or strain RB51 over producing Cu/Zn SOD produced a Th1 polarized immune response as measured by specific OMV antibodies and cytokines but no statistically significant protection was observed. / Master of Science

Ce travail de thèse comporte deux volets ayant pour trait commun l’analyse a posteriori d’une épidémie d’infection invasive à méningocoque (IIM) survenue en Normandie entre 2003 et 2012 et liée à l’expansion d’un clone hypervirulent particulier (B:14:P1.7,16/ST-32 ). Le premier travail (publié dans Virulence : Sevestre et al 2018 ;9 :923-929) s’est focalisé sur les déterminants de la virulence de « B14 » en comparant 6 isolats identifiés les uns d’IIM, les autres de portage pharyngé sain (ces derniers exprimant ou non la capsule). Apparemment identiques selon le typage classique (immunotypage et génotypage par MLST), ces 3 groupes bactériens se sont révélés distincts après analyse génomique et comparaison gène par gène (plus de 600 gènes au profil génétique variable entre les groupes) conduisant à identifier le rôle majeur de l’acquisition du fer dans la virulence et en particulier celui du système HmbR, un récepteur de l’hémoglobine. Dans un modèle murin (souris transgéniques rendues sensibles à l’infection humaine) les 3 groupes de souches sont aussi apparus distincts, avec une hiérarchie des marqueurs d’infectiosité (titres bactériens, taux de cytokines). La restauration du système HmbR (souches de portage capsulées « Off » dérivées en « On ») a restauré le pouvoir invasif in vitro et chez l’animal. Si le fer était déjà connu comme facteur de virulence pour différentes espèces bactériennes, l’originalité ici est d’avoir identifié le rôle de la variation de phase du gène hmbR au sein d’un même clone épidémique, permettant l’adaptation au portage, condition sine qua non de la transmission d’individu en individu. Le second travail (publié dans Vaccine : Sevestre et al 2017 ;35 :4029-4033) s’est intéressé à la durabilité et à l’ampleur de la protection vaccinale du MenBvac®, vaccin à base de vésicules de membranes externes (Outer Membrane Vesicles, OMV) utilisé jadis pour contrôler l’épidémie. Ceci a pu être réalisé grâce à deux cohortes d’enfants vaccinés par un schéma à 4 doses et prélevés pour les uns 1 an après la dernière dose et pour les autres 4 an après. L’immunogénicité (étude de l’activité bactéricide du sérum vis-à-vis du clone ciblé) s’est avérée de durabilité moyenne avec 48% des enfants protégés à 1 an et 31% à 4 ans, un résultat en phase avec les données de la littérature sur les vaccins OMV. Un effet bactéricide fut observé très au-delà de « B14 », du fait d’une immunité croisée aux souches avec une homologie au moins partielle de la porine PorA (principal déterminant antigénique des vaccins OMV) soit pour le MenBvac® 15% des clones virulents B actuellement circulants en France, un résultat davantage original car ayant jusqu’alors que peu investigué. / This thesis work includes two studies which both contribute to analyze a posteriori an outbreak of invasive meningococcal diseases (IMD) that had occurred in Normandy from 2003 to 2012 due to the expansion of a single hypervirulent clone (B:14:P1.7,16/ST-32 ). The first work (published in Virulence: Sevestre et al 2018 ;9 :923-929) focused on the virulence determinants of “B14” by comparing 6 isolates, either from IMD or from asymptomatic carriage (these latter expressing or not the capsule). Apparently identical on the basis of classical typing methods (immunotyping and MLST genotyping), these 3 groups of isolates were markedly different by whole genome analysis and on gene by gene comparison (more than 600 genes presenting a variable genetic profile). This analysis leaded to identify the crucial implication of iron acquisition in virulence and in particular the place of the HmbR system, an hemoglobin receptor. In a murine model (transgenic mice made susceptible to infection), these 3 groups also appeared separated, with a distinct infectivity hierarchy (bacterial counts, levels of cytokines). The restoration of the HmbR system in the capsulated carriage isolates (switch from Off phase to On phase) also restored their invasiveness in vitro and in vivo. Even if iron is already known to be a determining factor in the virulence of many bacterial species, our results clearly indicate the importance of the hmbR phase variation among clonal epidemic isolates, allowing adaptation to carriage, sine qua non condition for people to people transmission. The second work (published in Vaccine: Sevestre et al 2017 ;35 :4029-4033) concerned the durability and the cross-protection of the MenBvac®, an OMV vaccine (Outer Membrane Vesicles), used in the past to control the outbreak. This work has been done thanks to 2 cohorts of children vaccinated with 4 doses and sampled either 1 year or 4 years after the last dose. The efficacy (serum bactericidal activity against the epidemic strain) was short lasting, with 48% of children protected after 1 year and 31% after 4 years, a result in accordance with OMV literature. A bactericidal effect was observed far beyond “B14”, by cross-immunity with strains harboring homologies, even partials of the porin PorA (main antigenic determinant in OMV vaccine), indicating a coverage for 15% of virulent isolates B circulating in France, an original result as until then not so far investigated.

La vaccination est une des meilleures procédures de prévention coût-efficaces. Des couvertures vaccinales non adéquates présentent des problèmes de Santé publique considérables. Réduire ou éliminer les occasions manquées de vaccination (OMV) dans les régions les plus vulnérables permettrait d’y augmenter la couverture vaccinale. L’Inde a un des plus grands programmes de vaccination dans le monde, cependant il y existe d’importants gradients de couvertures vaccinales d’une région à l’autre. Objectifs : Cette étude visait à estimer la prévalence des OMV chez des jeunes enfants en zone rurale de Hardoi en Inde et identifier les potentiels déterminants et facteurs prédictifs des OMV. Méthodes : Les données secondaires d’une étude pré-post ont été utilisées pour mener une étude transversale. Les OMV ont été définies selon la définition de l’Organisation mondiale de la santé. Deux sources d’information sur le statut vaccinal ont été considérées : mémoire des mère ou carnet de vaccination (M/C) pour les analyses principales; et carnet de vaccination seulement (CS) en analyse de sensibilité. La prévalence des OMV dans la première année de vie (OMV-1AV) chez les enfants de 12 à moins de 24 mois et celle des OMV pendant la période optimale de vaccination (OMV-PO) chez les 0 à moins de 24 mois ont été calculées par sexe et bloc administratif. Les potentiels déterminants des OMV ont été identifiés à l’aide de modèles hiérarchiques. Des modèles prédictifs ont été construits pour identifier les facteurs qui permettraient de mieux cibler les enfants plus à risque d’OMV: leur pouvoir prédictif a été évalué avec la statistique c. Résultats : La prévalence des OMV-1AV selon la source M/C est de 19,3% ; celle selon CS est de 76,0%. La prévalence des OMV-PO selon M/C est de 14,6% alors qu’elle est de 65,7% selon CS. Pour les OMV-1AV et les OMV-PO, la prévalence variait d’un vaccin à l’autre mais seulement selon CS. Les déterminants des OMV varient selon la source d’information sur le statut vaccinal. Les principaux potentiels déterminants selon M/C sont: problèmes logistiques (OMV-1AV Rapport de cotes (RC) = 3,38; OMV-PO RC = 2,59); malaise ressenti chez l’enfant (OMV-1AV RC = 0,37; OMV-PO RC = 0,52); refus des vaccinateurs de vacciner sans avoir le carnet de vaccination (OMV-1AV RC = 5,66; OMV-1AV RC = 5,23); effets secondaires (OMV RC = 8,24; OMV-PO RC = 5,62); et le fait qu’un membre de la famille s’oppose à la vaccination de l’enfant; (OMV-1AV RC = 4,03; OMV-PO RC = 4,61). Des modèles prédictifs efficaces ont été construits et présentaient des statistiques c variant entre 0,72 et 0,79. Certains facteurs permettaient d’améliorer le pouvoir prédictif des modèles sans être nécessairement des potentiels déterminant des OMV tel que le temps de déplacement à pied entre le ménage et le centre de vaccination. Retombées : Les résultats suggèrent que la situation des OMV est complexe que ce soit du point de vue de la source d’information sur le statut vaccinal, de l’identification de leurs potentiels déterminants ou sur la capacité à cibler les individus les plus à risque. Les divergences au niveau des estimations de la prévalence selon la source d’information soulignent l’importance d’assurer un meilleur contrôle de la validité des sources d’information afin de maximiser l’exactitude des informations fournies. / Introduction: Missed opportunities for vaccination (MOV) affect vaccination coverages and contribute to create considerable vaccination gradient between and within regions. In India, despite major vaccination accomplishments, important vaccination gradients persist. MOV have been reported but the situation is not well known in many parts of the country. Aim: Quantify MOV in children in rural Hardoi district and identify potential determinants and predictive factors. Methods: We defined MOV using the definition of the World Health Organization. Our outcomes were missed opportunities for vaccination in first year of life (MOV-FYL) and missed opportunities for on-time vaccination (MO-OTV). We used a cross-sectional design. Vaccination status was verified according to two sources: by mothers’ recalls OR children vaccination card for the main analysis; and by vaccination card only for sensitivity analysis. We calculated the prevalence of both outcomes in a clustered population of 0 to under 24month-old children recruited in a census-like manner from rural area in Hardoi, India. We used multilevel binary logistic regression to identify potential determinants of MOV and multivariable logistic regression to built prediction models. Results: The prevalence was 19.30% and 14.39% for MOV-FYL and MO-OTV respectively. There were little variations across child sex and vaccines. However, among vaccination cardholders, the prevalence was 75.99% and 65.73% for MOV-FYL and MO-OTV respectively and varied across vaccines. Marked potential determinants using the main source of information about vaccination status were: logistics problems (MOV-FYL Odds Ratio (OR) = 3.38; MO-OTV OR = 2.59); child feeling unwell (MOV-FYL OR = 0.37; MO-OTV OR = 0.52); the refusal of health provider to vaccinate without the vaccination card (MOV-FYL OR = 5.66; MO-OTV OR = 5.23); side effects (MOV-FYL OR = 8.24; MO-OTV OR = 5.62); and family member not allowing vaccination (MOV-FYL OR = 4.03; MO-OTV OR = 4.61). Predictive models for MOV-FYL and MO-OTV yielded c statistics around 0.72 and 0.79 respectively and had the best sensitivity/specificity balance when used in a population with 15%-20% probability of MOV. Conclusion: Our study revealed that quantifying the prevalence of MOV is rather complexed. The source of information about vaccination status is key to obtain the best estimates, hence the knowledge on the reliability of the information from the card or obtained from recalls is a must. Many potential modifiable determinants should be explored and there is potential for predictability: interventions should be developed to reduce risks of MOV in targeted individuals, increase vaccination coverage and reduce vaccination gradients.