Bordetella pertussis e B. parapertussis são agentes etiológicos da coqueluche. A IL-12 liga a imunidade inata e adaptativa. Investigamos alguns mecanismos que controlam a síntese de IL-12 em macrófagos medulares murinos (MfDM) ativados in vitro com estas duas espécies de bactérias. Demonstramos que IL-12p40 e TNF-a foram produzidos pelos MfDM ativados com qualquer uma das bactérias. A síntese de IL-12p40 foi dependente de TNF-a, MyD88 e NFkB e independente de MAPK p38 e ERK 1/2. Durante a estimulação com B. pertussis a produção de IL-12p40 foi dependente de TLR-4, mas com B. parapertussis envolveu outras vias independentes de MyD88 e TLR-4. Estas bactérias não induziram a síntese de IL-12p70, necessitando de sinais moleculares adicionais de IFN-g, que aumentou a síntese desta citocina. A produção de IL-12 p70 aumentou após o bloqueio das vias PI3K, MAPK p38 e ERK1/2 assim como após a adição exógena de PT sobre MfDM ativados com B. parapertussis. Portanto, diversas vias de sinais dependentes e independentes de TLR-4 controlam a produção de IL-12 neste modelo. / Bordetella pertussis and B. parapertussis are etiological agents of whooping cough. IL-12 links the innate and adaptive immunity. We investigated the ability of both bacteria to modulate IL-12 by in vitro activation of bone marrow derived macrophages (MfDM). We demonstrated that IL-12p40 and TNF-a were produced after stimulation of cells with either bacterium. IL-12p40 production was dependent on TNF-a, MyD88 and NFkB but independent of MAPK p38 and ERK 1/2. During B. pertussis activation the production of IL-12p40 was dependent on TLR-4, while B. parapertussis activation was MyD88 and TLR-4 independent. However, the bacteria alone did not induce IL-12p70 synthesis, requiring IFN-g as an additional signal. Evidences indicated MAPK p38, ERK1/2 and PI3K during B. pertussis and B. parapertussis activation, as well as the exogenous addition of PT to B. parapertussis activated MfDM, was critical for the up regulation of IL-12p70. This finding indicates that different TLR-4 dependent and independent signaling pathways may control the production of IL-12 in this model.

As vacinas pertussis celulares apresentam certa reatogenicidade e as acelulares, menos reatogênicas, têm seu uso restrito, devido a seu alto custo. O Instituto Butantan desenvolveu uma vacina pertussis celular (Plow), com baixo teor de lipopolissacarídeo e outra acelular (Pa), por metodologia simples e econômica. Essas preparações, combinadas aos toxóides diftérico e tetânico (DTPlow e DTPa, respectivamente), foram comparadas à DTP tradicional, com diferentes adjuvantes: vitamina A, surfactante pulmonar, BCG, monofosforil lipídeo A (MPL) e Al(OH)3. A resposta humoral em camundongos foi semelhante para as diferentes formulações e independente do adjuvante utilizado. As vacinas induziram níveis equilibrados de IgG1/IgG2a anti-pertussis e a DTPlow mostrou-se menos reatogênica, induzindo níveis significativamente menores de IL-6 sérica. A adição de MPL sugeriu tendência de proteção contra a colonização nasotraqueal no grupo imunizado com DTPa e levou à indução de IFN-g nos grupos imunizados com DTP e DTPa, sugerindo possível atividade imunomodulatória para Th1. / The whole cell pertussis vaccines present some reactogenicity and the acellular, less reactogenic, have prohibitive use due to its high cost. Instituto Butantan developed a less reactogenic whole cell pertussis vaccine (Plow), with low lipopolysaccharide content and an acellular vaccine (Pa), by simple and economic methodology. These preparations, combined to diphtheria and tetanus toxoids (DTPlow and DTPa, respectively), were compared with the traditional DTP, with different adjuvants: vitamin A, pulmonary surfactant, BCG, monophosphoryl lipid A (MPL) and Al(OH)3. The humoral immune response induced in mice by the different vaccine formulations, was similar and independent of the adjuvant used. The vaccines induced balanced levels of IgG1/IgG2a anti-pertussis and DTPlow showed to be less rectogenic, inducing lower levels of serum IL-6. The use of MPL suggested to induce higher protection against nasotracheal colonization in DTPa group and induced IFN-g in the DTP and DTPa groups, suggesting a possible immunemodulatory activity for Th1.

La coqueluche est une pathologie due à la bactérie Bordetella pertussis qui touche les voies respiratoires des patients infectés causant toux, leucocytose, fièvre, et dont les symptômes peuvent aller jusqu’au décès chez les individus les plus à risque (nouveau-nés et enfants immunodéprimés en particulier). Ciblée par différents programmes vaccinaux depuis de nombreuses années, cette pathologie sévit à nouveau dans de nombreux pays développés où le nombre de cas augmente fortement depuis la fin des années 2000. Cette résurgence montre la nécessité de développer de nouvelles stratégies afin de comprendre les mécanismes de l’infection par B. pertussis. Dans ce contexte, la recherche préclinique apparaît comme essentielle pour comprendre la physiopathologie de la coqueluche. De nombreux modèles animaux ont été décrits pour l’étude de la coqueluche mais aucun de ces modèles ne permet de reproduire l’ensemble du spectre des symptômes cliniques de la pathologie, notamment la toux. Cependant, au cours des dernières années un modèle d’infection par Bordetella pertussis chez le jeune babouin a été développé aux Etats-Unis et permet de reproduire la pathologie observée chez l’homme, notamment concernant la toux et la transmission. Ce modèle semble ainsi très prometteur pour l’étude de la physiopathologie de la coqueluche.Cependant, de nombreuses inconnues subsistent dans ce modèle, notamment concernant la colonisation bactérienne et les interactions entre la bactérie et l’hôte. Nous avons ainsi cherché dans cette étude à évaluer d’une part l’impact de différents facteurs comme l’âge des animaux, la dose d’infection ainsi que la voie d’exposition sur la pathologie déclarée par les babouins suite à l’infection par la souche B1917 de B. pertussis afin de pouvoir proposer un parallèle avec les données cliniques disponibles. Nous avons également développé l’utilisation de techniques d’imagerie in vivo comme l’endomicroscopie confocale couplée à la bronchoscopie afin d’étudier la localisation et la cinétique de colonisation et certaines interactions du pathogène dans le tractus respiratoire inférieur au cours de la pathologie. Cette étude nous a ainsi permis d’approfondir les connaissances de physiopathologie de la coqueluche dans ce modèle babouin et consolidera cet outil précieux pour l’évaluation des futures stratégies de prévention contre cette pathologie. / Whooping cough, or pertussis, is a respiratory disease caused by Bordetella pertussis bacterial colonization of human airways. Main symptoms are cough, leukocytosis, fever and may even be lethal for some patients (e.g. newborn infants and immuno-deficient patients). Despite a good vaccination coverage worldwide against pertussis, whooping cough cases have been re-increasing in several developed countries in the past twenty years. This resurgence points out the crucial need to develop new control strategies and to better understand pertussis pathophysiology, notably using appropriate animal models. Numerous preclinical models including mice, rats, rabbits and swine have been described for B. pertussis infection studies. However, none of these models reproduce the full spectrum of clinical pertussis symptoms, especially cough. The recent baboon model of whooping cough described in the last few years in the US appears to be a very relevant model for pertussis pathophysiology studies as these animals reproduced all clinical symptoms as observed in humans including cough.However, many aspects of bacterial colonization and interactions with the host have yet to be described in this model.We have then evaluated diverse parameters such as animal age, the inoculum dose and the exposition route on the pathology symptoms and immune responses developed by baboons following B. pertussis B1917 strain inoculation in order to draw a parallel with human clinical data. We also developed in this model in vivo imaging techniques like confocal endomicroscopy coupled with bronchoscopy in order to evaluate bacterial colonization kinetics, localization and some interactions in the lower respiratory tract of infected baboons. Then, this study brought additional data on whooping cough physiopathology in this baboon model, which will be crucial for evaluating future prevention strategies against pertussis disease

<p>Uprkos rezultatima postignutih imunizacijom, veliki ka&scaron;alj je i danas vodeći uzrok smrti među zaraznim bolestima protiv kojih se sprovodi imunizacija. Postojanje različitih vrsta nadzora, prisutnih nedostataka laboratorijske dijagnostike oboljenja, slabosti u dostupnim definicijama slučaja velikog ka&scaron;lja i neprepoznavanje oboljenja, otežavaju realno sagledavanje opterećenja velikim ka&scaron;ljem i poređenje postignutih rezultata u različitim zemljama. Postojeće definicije slučaja velikog ka&scaron;lja nisu prihvatljive u svim uzrastima. Cilj istraživanja je bio da se na reprezentativnom uzorku Grada Novog Sada utvrdi: stopa incidencije velikog ka&scaron;lja tokom jedne godine savremenog nadzora; senzitivnost i specifičnost definicija slučaja velikog ka&scaron;lja, datih od strane Globalne pertusis inicijative za tri uzrasne grupe. U istraživanje je uključeno 213 ispitanika pacijenata iz sentinelnog nadzora nad velikim ka&scaron;ljem Doma zdravlja Novi Sad i 107 ispitanika pacijenata iz hospitalnog nadzora sa iste teritorije koji su ispunjavali predložene definicije slučaja za tri uzrasne grupe (0-3 meseca; 4 meseca-9 godina života; 10 godina i stariji). Podaci za istraživanje dobijeni su popunjavanjem anketnog upitnika. Laboratorijsko testiranje sumnji na veliki ka&scaron;alj sprovedeno je u Centru za mikrobiologiju Instituta za javno zdravlje Vojvodine. Kod pacijenata sa ka&scaron;ljem u trajanju do 21 dan testiranje je vr&scaron;eno upotrebom PCR metoda, a kod pacijenata sa ka&scaron;ljem u trajanju vi&scaron;e od 21 dan, dokazivanje oboljenja vr&scaron;eno je upotrebom ELISA IgA i IgG serolo&scaron;kih testova. Potvrđenim slučajem velikog ka&scaron;lja smatran je pacijent sa ispunjenom definicijom slučaja oboljenja i laboratorijskom potvrdom oboljenja, PCR ili ELISA testom. Procenjena stopa incidencije velikog ka&scaron;lja u Novom Sadu je 237,7/100.000, a stopa incidencije hospitalizovanih u Novom Sadu je 16,4/100.000. Najvi&scaron;e stope incidencije velikog ka&scaron;lja u sentinelnom nadzoru registruju se u uzrastu 10-14, a u hospitalnom u uzrastu od 7 do 9 godina. Senzitivnost, specifičnost i stepen verovatnoće pozitivnog rezultata testiranog simptoma/znaka iz predloženih definicija slučaja se razlikuju po uzrastima i po pojedinim simptomima/znacima. Predložene definicije slučaja u uzrastima od 4 meseca do 9 godina i u uzrastu od 10 godina i starijih imaju veću verovatnoću otkrivanja obolelih u hospitalnom u odnosu na sentinelni nadzor za pojedine simptome/znakove. Budući da je tokom istraživanja u sentinelnom nadzoru oboljenje potvrđeno kod svakog petog, a u hospitalnom kod svakog drugog testiranog pacijenta, predložene definicije slučaja se mogu koristiti u nadzoru nad velikim ka&scaron;ljem.</p> / <p>Despite all results achieved by immunization, pertussis is still the leading cause of death among vaccine preventable diseases. Different types of surveillance and laboratory confirmation of pertussis, weakness of existing case definitions for pertussis and broad spectrum clinical manifestation of disease, complicate overview of disease and result comparison of surveillance in different countries. The current pertussis case definition is not acceptable for all age groups of patients. The aim of this research was to determine: the pertussis incidence rate in population of Novi Sad during one year of modern surveillance; the sensitivity and specificity of clinical case definition for pertussis, given by the Global Pertussis Initiative for three age groups, with a representative sample of population in the City of Novi Sad. 213 patients from sentinel surveillance of pertussis Novi Sad Health Centre and 107 hospitalized patients from Novi Sad, who fulfilled criteria of case definition proposed for the three age groups (0-3 months, 4 months-9 years; 10 years of age and older), were included in the research. Research data obtained from a questionnaire. Laboratory testing of suspected cases were conducted at the Centre for Microbiology, Institute of Public Health of Vojvodina. For patients with coughing less than 21 days PCR method was used, and for patients with cough lasting more than 21 days, laboratory confirmation of disease was performed using ELISA IgA and IgG serological tests. Confirmed case of pertussis consider to be a patient with symptoms/signs according to proposed case definition and with laboratory confirmation of the pertussis, PCR or ELISA. Estimated incidence rate of pertussis for population in the city of Novi Sad was 237.7/100,000, and the pertussis incidence rate in hospitalized patient in Novi Sad was 16.4/100,000. The highest incidence rate of pertussis in the sentinel surveillance was registered in the age group 10-14 and in the hospital surveillance in the group 7 to 9 years of age. The values of sensitivity, specificity and positive likelihood ratio of symptoms/signs from the proposed case definition were calculated and they different by age and by certain symptoms/signs from proposed case definition. Certain symptoms/signs of the proposed case definition have a higher probability of detection among patients aged 4 months to nine, and at the age of ten year and older, in the hospital versus those in sentinel surveillance for pertussis. During research from the patients who were tested in sentinel surveillance every fifth was laboratory confirmed case and in the hospital surveillance every second patient was confirmed case, so the proposed case definitions can be used in the surveillance of pertussis.</p>

Bordetella pertussis is a strictly human pathogen colonizing the upper respiratory tract, causing a respiratory disease known as whooping cough or pertussis. The introduction of whole-cell vaccines and acellular vaccines, resulted in a significant reduction in the incidence of disease and reduce the fatalities associated with infection. However, epidemiological data show a significant increase in the incidence of the disease in recent decades. The increasing incidence is mainly attributed to the transition from the whole- cell vaccine to an acellular vaccine. Based on research from recent years has shown that acellular vaccines have many drawbacks, and it is therefore necessary to change the vaccination strategy. One possible solution to the situation is the development of a new generation of whole-cell vaccines with reduced reactogenicity. The new whole-cell vaccine was prepared by a genetically modified B. pertussis strain. B. pertussis was modified using allelic exchange to develop strain encoding enzymatically inactive pertussis toxin, modified lipid A and lacking dermonecrotic toxin. This combination of genetic modifications in mice led to a decrease in reactogenicity test vaccine in vivo. In case of intranasal infection whole-cell vaccine containing genetically modified strain is providing...

Whooping cough (pertussis) is a highly contagious acute respiratory disease caused by the strict human pathogen Bordetella pertussis, a gram-negative coccobacillus. The worldwide mass-vaccination was started in 1940s and to date, a number of whole-cell (Pw) and acellular pertussis vaccine (Pa) formulations were developed. Yet the current vaccines are incapable of providing sustained, lifelong immunity and eliminating subclinical infections, which pose a threat especially for unimmunized infants as well as adolescents and adults. Thus, finding new protein candidates with high immune protective capacities is necessary to enhance the clinical efficacy of current acellular pertussis (Pa) vaccines. In this study, iron-superoxide dismutase (FeSOD) protein was investigated for its capacity of conferring protectivity as well as stimulating humoral and cellular responses against B. pertussis infection in a mouse model. For this purpose, sodB gene, which encodes iron-superoxide dismutase FeSOD protein, was amplified from the genomic DNA of the universal B. pertussis strain &lsquo / Tohama I&rsquo / and sequentially cloned to pGEM&reg / -T subcloning and pET-28a(+) expression vectors. Afterwards sodb/pET28a(+) construct was introduced to E. coli BL21(DE3) cells and the gene was overexpressed therein via IPTG induction. The expressed FeSOD protein was then purified by affinity chromatography and its previously reported immunogenicity was confirmed by Western blot. After filter-sterilization, the protein was adsorbed to alum [Al(OH)3] adjuvant and introduced to BALB/c twice at three weeks intervals intraperitoneally at a concentration of 20 &mu / g purified FeSOD protein/mouse. Another group of mice were immunized in tandem with heat-inactivated whole-cell suspension of B. pertussis. Ten days after the second immunization, mice were intranasally challenged with the local &lsquo / Saadet&rsquo / strain of B. pertussis. Next the lungs of groups of mice were excised, homogenized and plated as serial dilutions on days 5, 8 and 14 post-challenge, and viable lung CFU counts were carried out. Whole cell immunization conferred complete bacterial clearance following B. pertussis intranasal infection while FeSOD immunization failed to attain such protection. In addition to the protectivity assay, ELISA was performed to assess the humoral (i.e. IgG) immune response triggered upon FeSOD- and whole-cell immunizations and a statistically significant increase in anti-FeSOD IgG production was observed in FeSOD-immunized group. Finally, cellular immune response was tested via cytokine (IFN-&gamma / ) assay, in which spleens of mice were excised, splenocytes were cultured and the level of IFN-&gamma / production upon FeSOD addition to the cultures was measured via ELISA. This test showed that whole-cell immunization triggered IFN-&gamma / production at significant levels while FeSOD-immunization did not / indicating the failure of alum-adsorbed FeSOD immunization in inducing cell-mediated immune response.

Bordetella pertussis est l’agent responsable de la coqueluche. Pour coloniser le tractus respiratoire humain, cette bactérie à Gram négatif, aérobie stricte, produit de nombreux facteurs de virulence dont l’expression est sous la dépendance du système à deux composants BvgAS. BvgS est un senseur-kinase dimérique. Chaque monomère est constitué de trois domaines putatifs de perception - deux domaines Venus flytrap périplasmiques et un domaine PAS cytoplasmique -, suivis du domaine enzymatique et deux autres domaines, de phospho-transfert et receveur, impliqués dans la cascade de phosphorylation. L’expression du régulon de virulence est activée suite à la phosphorylation par BvgS du régulateur de réponse BvgA. BvgS est en mode kinase à l’état basal, et la perception de basses températures ou de signaux chimiques comme les ions sulfate ou nicotinate cause son passage en mode phosphatase. L’étude présentée dans ce manuscrit vise à caractériser le senseur-kinase BvgS en analysant les domaines putatifs de perception ainsi que la transduction de signal qui s’effectue au sein de la molécule. L’étude de la portion périplasmique a permis de mettre en évidence, à l’état basal, un gradient de dynamique décroissant. En se fixant au domaine VFT2 proximal à la membrane, le nicotinate induirait une diminution de la dynamique du second lobe du VFT1, causée par la formation d’un bloc compact entre le domaine VFT2 et le deuxième lobe du domaine VFT1. Cette rigidification exercerait une tension sur les hélices α qui précèdent les segments transmembranaires, provoquant une transition de la portion cytoplasmique vers l’état phosphatase. La perception de modulateurs par le domaine VFT2 - ou possiblement la fixation d’un ligand dans la cavité du VFT1- modifierait cette dynamique et causerait le changement d’activité de BvgS. Ainsi, nous proposons un modèle dans lequel le VFT1 est considéré comme le moteur du système, lui impulsant une dynamique qui serait relayée ou atténuée par le domaine VFT2. Une recherche de ligands antagonistes pour le domaine VFT1 a été entreprise, selon l’idée que la fixation d’un ligand réduirait la dynamique de ce dernier. Au sein du dimère, des connecteurs prédits pour former des enroulements d’hélices α (‘coiled coil’) relient entre eux les domaines VFT et PAS, et les domaines PAS et kinase de BvgS. La transduction d’information entre les domaines périplasmiques et le site enzymatique de BvgS a été analysée par mutagénèse dirigée et ‘cysteine scanning’. Des contacts proches sont observés entre les hélices constituant le segment transmembranaire, qui ne semblent pas être modifiés après perception de modulateur. Nous suggérons donc un modèle de piston symétrique pour la transmission d’information au travers de la membrane. Le coiled coil putatif précédant le domaine PAS présente une certaine dynamique rotationnelle à l’état basal. La perception de modulateurs semble induire l’écartement de ces hélices, ce qui pourrait permettre un changement de l’interface des domaines PAS. L’étude de la topologie du domaine PAS confirme une modification de cette interface entre les modes kinase et phosphatase de BvgS. Enfin, le coiled coil reliant les domaines PAS et kinase est sujet à une forte dynamique rotationnelle à l’état basal, en accord avec un modèle de régulation de l’activité kinase proposé dans d’autres systèmes. Suite à la perception de modulateur, une rigidification marquée de ce coiled coil est observée, permettant le passage en mode phosphatase. L’existence de deux états dynamiques différents de ce coiled coil a également été mise en évidence en absence du domaine PAS.Ces études ont permis d’avancer dans la compréhension de BvgS et de proposer un modèle de la signalisation au sein de ce senseur-kinase, qui pourrait s’appliquer aux autres membres de la famille de BvgS. / Bordetella pertussis is the agent of an acute and highly contagious respiratory disease, whooping cough. In order to colonize the human respiratory tract, this strictly aerobic Gram negative bacterium produces many virulence factors, the expression of which is regulated by the BvgAS two-component system. BvgS is a sensor-kinase composed of three putative domains of perception –two periplasmic Venus flytrap domains and a cytoplasmic PAS domain -, followed by the enzymatic domain and two other domains called phosphotransfert and receiver involved in the phophorelay. The expression of the virulence regulon is activated after the phosphorylation by BvgS of the response regulator BvgA. BvgS is in a kinase mode at the basal state, and the perception of low temperatures or chemical signals like sulfate ions or nicotinate causes a shift to the phosphatase state. The study presented in this manuscript has focused on the characterization of the BvgS sensor-kinase. We have analyzed its putative domains of perception and the mechanisms of signal transduction.Investigations into the dynamics of the periplasmic moiety has provided evidence for a decreasing gradient of dynamics from N to C-terminus at the basal state. Nicotinate binding to the membrane-proximal VFT2 domains decreases the dynamics of the second lobe of VFT1. Tighter interactions between the latter and the VFT2 domain cause a tension on the α helices that precede the transmembrane segments, triggering the transition to the phosphatase state of the enzymatic portion. Perception of modulator by the VFT2 domains –or possibly binding of a ligand in the VFT1 cavity- thus appears to modify periplasmic dynamics, which shifts BvgS activity. We propose that the VFT1 domains are the motor for BvgS activity, and their dynamics are relayed or attenuated by the VFT2 domains. A search for antagonistic VFT1 ligands has been undertaken, along the idea that ligand binding may reduce their dynamics.The VFT and PAS domains, and the PAS and kinase domains are joined to each other by long α helices predicted to form coiled coils. We performed directed mutagenesis and cysteine scanning analyses to decipher signal transduction between the periplasmic domains and the enzymatic moiety of BvgS. The close contacts between the helices of the transmembrane segment are not modified after perception of the modulator, suggesting that signal transduction across the membrane is mediated by symmetrical piston motions. The putative coiled coil before the PAS domain shows rotational dynamics at the basal state. Modulator perception causes the helices to splay, and this motion may modify the PAS domains interface. Our topology analyses of the PAS domain confirm that changes occur at this interface between the kinase and phosphatase states of BvgS. Finally, the coiled coil between the PAS and kinase domains presents a strong rotational dynamics at the basal state, which is consistent with the model of regulation of kinase activity proposed for other sensor-kinases. After perception of a modulator, this coiled coil becomes more rigid, allowing the shift to the phosphatase state. The occurrence of two states of dynamics for this coiled coil has also been demonstrated in the absence of the PAS domain.These studies have advanced our understanding of BvgS and allow us to propose a model of signaling by this sensor-kinase, which may apply more broadly to other family members.

The thesis deals with the problem of the occurrence of whooping cough in the South Bohemian Region in the period of 2003-2012. Despite the high level of vaccination the incidence of this illness after a significant fall in the 1980s has been growing constantly since 1993. This trend evidently also applies in other developed countries with a high rate of vaccination. The main aim is to draw up an overview of the occurrence of whooping cough in the South Bohemian Region in the monitored period of the last 10 years, compare this occurrence with other regions in the Czech Republic and map out the changes which have occurred in whooping cough vaccination. Quantitative and qualitative research was chosen to process the empirical part. Data for quantitative research was acquired from the database of infectious diseases (EPIDAT), and consisted of reported cases of whooping cough. Another essential group of data were demographic figures about the number and age structure of the population. The second qualitative part was drawn up on the basis of a literary search. I acquired additional information by communicating with individual territorial workplaces of the Regional Hygiene Station of the South Bohemian Region based in České Budějovice. Using the processed data I discovered that the incidence of whooping cough in the South Bohemian Region does not have an ascending trend in 2003-2012 unlike the trend in the whole of the Czech Republic. The highest occurrence of whooping cough in the Czech Republic in the monitored period was reported in the 10-14 age group. The research also showed that in the period of 2003-2012 individual regions differed greatly in the occurrence and can be divided into two groups ? regions with the highest reached incidence below 10 cases/100,000 people and regions with incidence with the highest reached incidence above 10 cases/100,000 people which includes the South Bohemian Region. There have been many changes in the vaccination of whooping cough and the most important was the transition from whole cell vaccine to acellular in 2007 and adding a 6th booster dose between the 10th and 11th year in 2009. The greatest attention should be paid above all to increase information or test the knowledge of the lay public, especially women preparing for pregnancy and people taking care of newborn babies and infants about the occurrence, potential risks and possibilities of vaccination against this infection even at an adult age. In my opinion a further necessity is the consolidation of diagnostics, definition of a case and procedures of surveillance throughout the Czech Republic, education of paediatricians and general practitioners about the illness and topicality of the problem, an appeal to report the occurrence of this infection to epidemiologists, observing the isolation of the patient, monitoring possible epidemiological connections between cases and making use of the offer of the NRL (National Reference Laboratory) services against whooping cough and diphtheria. This thesis can be used in practice as a summary of information material for the lay and professional public about the occurrence of whooping cough in the South Bohemian Region and throughout the Czech Republic and overview of vaccination against whooping cough. It can be an incentive for further anti-epidemic measures and the need for further research into this problem.

A coqueluche é uma doença contagiosa, causada por Bordetella pertussis e B. parapertussis e as vacinas de células inteiras (WCPs) contra pertussis, embora eficazes, foram associadas a efeitos indesejáveis. Já as vacinas pertussis acelulares são menos reatogênicas, mas caras, o que as torna inviáveis para países em desenvolvimento. Nesse estudo, avaliamos a resposta imune induzida por uma vacina pertussis celular com menor teor de LOS (Plow), desenvolvida pelo Instituto Butantan. Para isso, camundongos C57BL-6 foram imunizados com WCP tradicional ou Plow, formulada com MPL-A de B. pertussis ou com Hidróxido de Alumínio e vacinas tríplice bacterianas com componente pertussis acelular, Pertacel ou Adacel. Após o esquema de imunização, foi avaliada a resposta imune humoral e celular contra a B. pertussis, e B. parapertussis, além de resposta inata a um antígeno não relacionado, BCG. Ensaio de transmissão também foi realizado, após desafio com B. pertussis ou B. parapertussis. Nossos resultados consolidam a avaliação da resposta imune humoral e celular induzida pela Plow, além de apresentar resultados bastante interessantes relativos à atividade da Plow na diminuição da transmissão bacteriana, tanto de pertussis quanto de parapertussis. / Bordetella pertussis and B. parapertussis are the causative agents of whooping cough. Whole cell pertussis (wPs) vaccines, although effective, were associated with undesirable effects. On the other hand, aP vaccines are less reactogenic, but expensive, which made their use unable for developing countries. In this study, we assessed the immunological response, induced by a whole cell pertussis vaccine with low LOS content (Plow), developed by Instituto Butantan. To this, C57BL-6 mice were immunized with traditional whole cell pertussis vaccine or Plow, administrated with Monophosporil lipid A from B.pertussis or Aluminum hydroxide, and diphtheria-tetanus-acellular pertussis vaccines, Pertacel or Adacel. After the immunization scheme, were evaluated humoral and cellular immune responses against B. pertussis and B. parapertussis, in addition to innate response to an unrelated antigen, BCG. Transmission assay was also performed, after B. pertussis or B. parapertussis challenge. Our results consolidated the immune humoral and cellular responses induced by Plow, besides interesting results with regards the efficacy of the vaccine in decreasing the transmission of B. pertussis and B. parapertussis in mice.

Introdução: A vacina tríplice acelular de adultos (dTpa) foi introduzida no Programa Nacional de Imunizações (PNI) em novembro de 2014, sendo recomendada para gestantes e profissionais de saúde (PS) que têm contato com gestantes e recém-nascidos. De abril a dezembro de 2015, foram implementadas várias estratégias para aumentar a cobertura vacinal entre os profissionais do Instituto Central do Hospital das Clínicas da FMUSP. Objetivos: Avaliar a cobertura vacinal entre os PS após implementação de cada estratégia e ao término de um ano; avaliar as variáveis associadas à vacinação; e avaliar os principais motivos de não vacinação entre os PS com indicação para tal. Métodos: Estratégias implementadas: divulgação, no boletim do hospital, de texto relembrando da necessidade de vacinação de coqueluche; reforço da necessidade da vacinação, via correio eletrônico, para as chefias de enfermagem das Divisões de Clínica Obstétrica, Neonatologia e Anestesia; aulas sobre a vacina dTpa nas reuniões científicas das Divisões de Clínica Obstétrica e Neonatologia; e vacinação ativa dos profissionais na Divisão de Clínica Obstétrica, Neonatologia e Anestesia. A cobertura vacinal foi avaliada ao fim de cada mês até abril de 2016, por meio do sistema informatizado de vacinação usado no CRIE-HC. Foi usado o modelo de regressão de Poisson com variância robusta para avaliação das variáveis associadas com a vacinação com dTpa. As razões de prevalência foram calculadas e seus intervalos de confiança de 95% estimados. Para avaliar os motivos de não vacinação, foram realizadas ligações telefônicas para os profissionais que não receberam a vacina e aplicado questionário padronizado. Resultados: Entre os 515 PS elegíveis para vacinação, 59 não possuíam registro no sistema informatizado de vacinação e foram excluídos. Assim, este estudo incluiu 456 PS. Após as intervenções, a cobertura vacinal com dTpa aumentou de 2,9% para 41,2%. As coberturas vacinais após a implementação de cada estratégia foram: 3,7% após publicação no Boletim do hospital; 10,5% após mensagem de correio eletrônico para as chefias de enfermagem; 16,2% após aula sobre a vacina em reuniões científicas das Divisões de Clínica Obstétrica e Neonatologia; 27,9% após vacinação ativa na Divisão de Clínica Obstétrica; 40,6% após vacinação ativa na Divisão de Neonatologia e 41,2% após vacinação ativa na Divisão de Anestesia. Na análise multivariada, ser médico (a), trabalhar nas Divisões de Clínica Obstétrica ou Anestesia e ter recebido a vacina de influenza de 2015 foram associados à vacinação com dTpa. Foi feito contato telefônico com 39 profissionais que não receberam a vacina em nosso serviço; apenas 9 (23%) referiram ter recebido a vacina em outros serviços; e dos 30 não vacinados, 27 (90%) alegaram desconhecimento da recomendação. Conclusões: Conhecimento sobre a doença e a recomendação de vacinação são importantes para aumentar a cobertura vacinal entre PS. Porém, mesmo sabendo do efeito cumulativo na cobertura vacinal a cada estratégia realizada, a vacinação ativa dos PS em seus locais de trabalho parece ter sido a estratégia que mais contribuiu para o aumento da cobertura. A cobertura vacinal final de dTpa permanece baixa e maiores esforços são necessários para aumentá-la / Introduction: The acellular pertussis vaccine for adults (Tdap) was introduced in the Brazilian National Immunization Program (PNI) in November 2014, being recommended for pregnant women and healthcare workers (HCWs) who have contact with pregnant women and newborns. From April to December 2015, interventions to raise Tdap coverage among HCWs of the Instituto Central do Hospital das Clínicas were implemented. Objective: To evaluate the cumulative vaccine coverage after each intervention; identify factors associated to Tdap vaccination among HCWs; and evaluate the main reasons for HCWs not receiving Tdap. Methods: Interventions implemented: a note on the hospital\'s internal newsletter, reminding HCWs of the importance of pertussis vaccination; email to the nurse´s teams leaders strengthening vaccine recommendations; lectures on pertussis and Tdap for physicians at the clinical meetings of the Obstetrics and Neonatology Clinics; on-site vaccination by mobile teams at the Obstetrics, Neonatology, and Anesthesiology Clinics. The vaccine coverage was evaluated at the end of each month until April-2016. A multivariate Poisson regression model with robust error variance was used to evaluate variables associated with Tdap vaccination. Prevalence ratios (PR) and their 95%CI were estimated. To evaluate the reasons for HCWs not to be vaccinated, those who have not received Tdap were called by phone and a standard questionnaire was applied. Results: Among 515 HCWs eligible for immunization, 59 professionals were not registered in the vaccination data system and were excluded because information about Tdap vaccine could not be achieved. The study included 456 HCWs. After the interventions, Tdap coverage raised from 2.9% to 41.2%. The vaccine coverage after each intervention was: 3.7% after a note on the hospital\'s internal newsletter; 10.5% after email to the nurse´s teams leaders strengthening vaccine recommendations; 16.2% after lectures on pertussis and Tdap for physicians at the clinical meetings of the Obstetrics and Neonatology Clinics; 27.9% after on-site vaccination by mobile teams at the Obstetrics Clinic; 40.6% after on-site vaccination at the Neonatology Clinic and 41.2% after on-site vaccination at the Anesthesiology Clinic. In the multiple analysis, occupation, working place and having received influenza vaccination in 2015 were independently associated to Tdap vaccination. Thirty-nine HCWs that have not received Tdap were contacted by phone: 90% of them claimed they did not know the vaccine recommendation. Conclusions: Knowledge about pertussis and the recommendation of vaccination are important to raise vaccine coverage between HCWs. Even knowing the cumulative effect of each strategy on vaccine coverage, HCWs vaccination in their workplaces seems to be the most effective strategy in raising coverage. The final Tdap coverage remains low and greater efforts are needed to increase it