Дисертації з теми "Streptococcus pneumoniae – immunologie"

Les acides gras à chaîne courte (AGCC) sont des métabolites produits principalement par le microbiote intestinal. Ils jouent un rôle important dans la régulation des réponses immunitaires et inflammatoires. L'acétate, le principal AGCC, est décrit pour disséminer dans l’organisme et réguler la fonction d’organes distaux tels que les poumons. Des travaux récents indiquent une fonction dans le contrôle des agents pathogènes, notamment d’origine bactérienne. Notre groupe a précédemment démontré que l'acétate augmente l’élimination de Streptococcus pneumoniae dans le cadre d'une infection secondaire post-virale. Cette protection est médiée par les macrophages alvéolaires, la première ligne de défense pulmonaire. Ainsi, notre objectif était d'évaluer l'effet de l'acétate sur l’activité bactéricide des macrophages alvéolaires et d’identifier les mécanismes impliqués dans cette réponse. Nous montrons ici que la supplémentation en acétate dans l'eau de boisson module la sécrétion de protéines de défense par les cellules pulmonaires murines et conduit à une réduction de la charge de S. pneumoniae. Nous montrons par analyse transcriptomique (RNAseq) que l’acétate induit une signature spécifique de défense de l’hôte au sein des macrophages alvéolaires conditionnés en présence de S. pneumoniae. Cet effet s’accompagne par l’augmentation de l’activité bactéricide des macrophages mediée pour l'oxyde nitrique (NO). L’augmentation de NO induit par acétate dépendait de l'augmentation des niveaux d'IL-1β. De manière surprenante, la production d'IL-1β déclenchée par l'acétate est indépendante de son récepteur de surface (Free-Fatty Acid Receptor 2) et des enzymes responsables de son métabolisme (Acetyl-CoA Synthetases 1/2). En contrepartie, l'acétate a modulé le profil glycolytique des macrophages induisant l’activation de HIF-1α, qui aboutit à la transcription de l’IL-1β. De plus, l'augmentation de la sécrétion de l’IL-1β déclenchée par l'acétate reposait sur l'activation de l'inflammasome NLRP3. En conclusion, nous avons identifié un nouveau mécanisme conduisant à l’élimination des bactéries par les macrophages alvéolaires traité avec l’acétate. L'acétate augmente la production et la sécrétion d'IL-1β selon un mécanisme dépendant de l'axe glycolyse/HIF-1α et de NLRP3, respectivement. Par conséquent, des niveaux plus élevés d'IL-1β conduit à une augmentation de la production de NO et une meilleure activité bactéricide des macrophages
Short chain fatty acids (SCFAs) are metabolites produced mainly by the gut microbiota with a known role in immune regulation. Acetate, the major SCFA, is described to disseminate to distal organs such as the lungs. Moreover, the literature supports that acetate modulates inflammation and improves bacterial clearance. Our group has previously demonstrated that acetate improves Streptococcus pneumoniae clearance in the context of a secondary post-viral infection. This protection is mediated by alveolar macrophages, the first line of pulmonary immune defense. Thus, our aim was to evaluate the effect of acetate on the killing ability of alveolar macrophages and to delineate the mechanisms involved in this response. Here we show that acetate supplementation in drinking water modulated the secretion of host defense proteins by murine pulmonary cells and led to reduced S. pneumoniae loads in the lungs. To understand the mechanisms of bacterial clearance, alveolar macrophages were used. Transcriptomic analysis (RNAseq) revealed that acetate induced a specific signature of host defense in S. pneumoniae conditioned macrophages. This associates with the improved killing ability of acetate treated macrophages mediated by nitric oxide (NO) production. Increased NO concentration triggered by acetate was dependent on augmentation of IL-1β levels. Surprisingly, IL-1β production led by acetate was neither dependent on its cell surface receptor (Free-Fatty Acid Receptor 2), nor on the enzymes responsible for its metabolism (Acetyl-CoA Synthetase 1 and 2). Alternatively, acetate enhanced the glycolytic profile of macrophages resulting in greater HIF-1α activity which culminated in higher transcription of IL-1β. Moreover, the increased secretion of IL-1β triggered by acetate relied on NLRP3 inflammasome activation. In conclusion, we unravel a new mechanism of bacterial killing by acetate-activated macrophages. We show that acetate increased IL-1β production and secretion in a mechanism dependent on the axis glycolysis/HIF-1α and NLRP3, respectively. Consequently, higher levels of IL-1β resulted in augmented NO production and improved killing ability of alveolar macrophages

Streptococcus pneumoniae est le pathogène bactérien le plus important des voies respiratoires (pneumonie, bronchite et otite moyenne) chez les adultes et les enfants. Cette bactérie est responsable d’une morbidité et une mortalité importantes. Bien que la pénicilline présente une activité contre de nombreux isolats de S. pneumoniae, la résistance à cet antibiotique est aujourd'hui, fréquemment rencontrés à la fois à l'hôpital et dans la communauté. La résistance à la pénicilline chez Streptococcus pneumoniae est causée par un bloc de gènes codant pour des versions altérées de protéines liant la pénicilline (PLP). Néanmoins, S. pneumoniae a également développé des mécanismes de résistance à la pénicilline indépendants des PLPs altérées. L'objectif principal de cette thèse était d’utiliser des approches génomiques pour comprendre le génotype et le phénotype de résistance aux ß-lactamines chez S. pneumoniae. Le travail présenté dans cette thèse a indiqué que des mutations dans les PLPs ne sont pas suffisantes pour obtenir une résistance de haut niveau à la pénicilline et au céfotaxime. Cette étude a indiqué également que la sélection de la résistance à la pénicilline chez S. pneumoniae peut impliquer l’acquisition de mutations conférant une tolérance à l’accumulation d’oxydants causée par les antibiotiques. Cette tolérance peut se traduire par une augmentation de la survie qui permet possiblement la sélection des déterminants majeurs de résistance tels que des mutations dans les PLPs. Dans le cas des souches cliniques résistantes à la pénicilline, nous présentons également un nouveau rôle pour une alpha-amylase cytoplasmique conférant une résistance modérée à la pénicilline en présence d'altération des PLPs. Par ailleurs, nos travaux sur la résistance au céfotaxime chez S. pneumoniae a permis la découverte de nouveaux gènes impliquées dans la résistance au céfotaxime, y compris les gènes spr1333, spr0981, spr1704 et spr1098 qui codent respectivement pour un peptidoglycan GlcNAc déacetylase, une glycosyltransférase, un transporteur ABC et une sortase. Nos travaux génomiques ont permis de découvrir de nouveaux gènes de résistance aux β-lactamines chez S. pneumoniae.
Streptococcus pneumoniae is the most important bacterial pathogen of the respiratory tract (pneumonitis, bronchitis and otitis media) in adults and children resulting in significant morbidity and mortality. Although penicillin shows activity against many isolates of S. pneumoniae, resistance to this antibiotic is now frequently encountered, both at the hospital and in the community. Penicillin resistance in Streptococcus pneumoniae is mediated by a mosaic of genes encoding altered penicillin-binding proteins (PBPs). Nonetheless, S. pneumoniae has also developed non-PBP mechanisms implicated in penicillin resistance. The principal objective of this thesis was to use global sequencing approaches to understand ß-lactam resistance genotype and phenotype in S. pneumoniae. The work presented in this thesis indicated that mutations in PBPs are not sufficient to achieve high level resistance to penicillin and cefotaxime. This study also indicates that the selection of resistance to penicillin in S. pneumoniae involves the acquisition of mutations conferring tolerance to the antibiotic-induced accumulation of oxidants. This tolerance can translate into an increased survival that putatively enables the selection of major resistance determinants such as mutations in PBPs. In the case of clinical isolates, we also report a new role for a cytoplasmic alpha amylase in conferring moderate resistance to penicillin in the presence of altered PBPs. Furthermore, our works on cefotaxime resistance has allowed the discovery of novel cefotaxime resistance genes in S. pneumoniae including spr1333, spr0981, spr1704 and spr1098 coding respectively for a peptidoglycan GlcNAc deacetylase, a glycosyltransferase, an ABC transporter, and a sortase were implicated in resistance to cefotaxime. Our genomic approaches were useful to discover novel β-lactam resistance genes in S. pneumoniae.

Les immunoglobulines jouent un rôle clé dans l'interaction hôte-pathogène. Les immunoglobulines A (IgA) sous forme d'IgA sécrétoires sont en particulier impliquées dans la défense des épithéliums pulmonaire et digestif. Le FcaRI (ou CD89) est un récepteur spécifique liant la fraction Fc des IgA qui est responsable de l'activité biologique de ces immunoglobulines. Une fois lié à son ligand, le FcaRI transmet un signal intracellulaire via l'adaptateur Fcy qui est porteur d'un motif ITAM (Immunoreceptor tyrosine-based activation motif) sur sa portion intra cellulaire. L'adaptateur Fcy est également capable de transduire un signal inhibiteur dénommé ITAM inhibiteur (ITAMi). Nous montrons ici que le Streptococcus pneumoniae est capable de lier directement (sans opsonine) le FcaRI recombinant, à la fois en solution et in vitro, et que cette interaction est inhibée par l'adjonction de FcaRI soluble recombinant. Nous avons également montré que l'adjonction de Streptococcus pneumoniae sur des macrophages transgéniques pour le FcaRI induit une activation cellulaire et une production de cytokines pro inflammatoires. Dans une seconde partie nous avons démontré que, dans un modèle murin de pneumopathie à Streptococcus pneumoniae, la présence de FcaRI constitue un avantage de survie. Cette dernière est associée à une diminution des lésions histologiques pulmonaires et une moindre invasion bactérienne pulmonaire. Au final nous concluons que la famille des Fc récepteurs est capable de lier des pathogènes de manière indépendante des opsonines. Le FcaRI pourrait ainsi avoir une fonction supplémentaire en agissant comme un senseur antibactérien
An innate immune response is essential for survival of the host upon infection. Opsonin-independent bacteria recognition for tell activation is an important mechanism for bacteria clearance. Here, we show that the IgA receptor FcaRI binds Streptococcus pneumoniae directly, independently of IgA and mediates immunoreceptor tyrosine-based activation motif (ITAMa) signaling. These interactions increased bacteria phagocytosis and cytokine release and induced reactive oxygen species production by bone marrow-derived macrophages from FcaRI-transgenic mice. In these mice, using two differen models of sepsis, the presence of FcaRI was associated with host protection as evidenced by enhanced local bacterial containment, decreased tissue damage and increased survival. This work reveals the significant involvement of the ITAM-bearing FcaRI in innate immunity

L'utilisation de lignées de souris dérivées d’individus sauvages nous a permis d’identifier une nouvelle sous-population de lymphocytes B, nommée "Bw", qui se distingue par de nombreux critères des sous-populations de cellules B déjà connues chez la souris. Alors que la présence des cellules B-1a CD5pos est quasiment restreinte aux lignées de souris appartenant à la sous-espèce Mus musculus domesticus, les lymphocytes Bw sont conservés à travers l’évolution du genre Mus. Les souris de laboratoire ont hérité la population B-1a CD5pos de la sous-espèce M. M. Domesticus. Les lymphocytes Bw péritonéaux présentent le phénotype caractéristique suivant : CD19posCD5negMac-1posB220highIgMhighIgDhighCD43negCD9neg. Les cellules Bw sont retrouvées, à des fréquences variables dans la rate, les ganglions, la cavité péritonéale ainsi que les PBL. Le répertoire anticorps de ces lymphocytes est enrichi en spécificités autoréactives contre des antigènes tels que l’ADN, la tubuline ou l’actine, ainsi qu’envers des globules rouges traités par de la broméline. En réponse au LPS et au CpG, ligands des TLR 4 et 9 respectivement, les cellules Bw produisent plus d’anticorps anti-PC que les cellules B-2. Il en est de même suite à une immunisation par la bactérie S. Pneumoniae. Les précurseurs issus de la moelle osseuse sont capables, au même titre que les précurseurs foetaux, de se différencier en cellules Bw, contrairement aux précurseurs des cellules B-1 qui sont enrichis dans le foie foetal. Ce travail révèle la présence d’une nouvelle population de cellules B Mac-1pos, les lymphocytes Bw, conservée à travers l’évolution du genre Mus et impliquée dans des réponses immunitaires innées.

The pathogen Streptococcus pneumoniae encounters different levels of oxygen during the infection cycle including colonisation, pneumonia, bateraemia and meningitis. These different anatomical niches require high levels of genome changes to sense and respond to those external environmental stimuli. The bacterial gene expression is known to be affected by oxygen, and it must react properly for survival and for developing invasive pneumococcal desiseases (IPDs). Microarray techniques have allowed scanning the whole pneumococcal genome during growth in different tensions of oxygen mimicking in vivo conditions. It was found that oxygenated growth conditions have significantly elevated several key virulence genes. This was further confirmed with qRT-PCR for a selection of genes implicated in pathogenicity. Moreover, post-transcriptional stages have been also investigated such as protein production, biofilm formation, biological activities and adherence assays for several virulence factors performed under the effect of presence or absence of oxygen. The data illustrate that 420 out of 2,236 genes (17 % of the entire TIGR4 genome) were differentially expressed in the presence of oxygen compared to its absence. 262 genes (11 %) were over-expressed when pneumococci were grown in oxygenated conditions relative to transcriptional profile in anaerobic growth conditions, indicating the magnitude of roles played by oxygen on pneumococcal gene expression. Anaerobic growth of TIGR4 showed down-regulation of 158 genes (7 %). Oxygen modulates induction of ply, pspC and other seven genes involved in pili structuring subunits (rlrA, rrgA, rrgB and rrgC) and assembling enzymes (srtB, srtC and srtD). This may suggest that the pneumococcal population grown under atmospheric environment is equipped with greater capability to progress IPDs compared to anaerobically grown bacteria. In addition to this, pneumococcal adhesion in vitro for TIGR4 grown in oxygenated or anaerobic growth conditions revealed a significant increase in those grown in oxygenated growth conditions, indicating that oxygen may play a key role in bacterial-host attachment. Interestingly, ablation of pspC has resulted in similar adhesion percentages of TIGR4 grown under both conditions, oxygenated and anaerobic. Furthermore, several genes involved in metabolism were up-regulated in oxygenated environment, particularly transporters, which are considered highly important for a bacterium that lacks an electron transport chain, catalase and tricarboxylic acid. Additionally, the results showed phenotypic characterisation and changes in cells morphology from pneumococcal growth curves for several strainswith different genome backgrounds grown under different levels of oxygen concentrations. Further investigation of the pathogen biology revealed differences in pneumolysin production and activity. These findings highlight that virulence genes expression is induced once the micro-organism is exposed to oxygenated environment, and data analysis has demonstrated potential links between pneumococcal metabolism and their ability to cause diseases.

Streptococcus pneumoniae is a commensal human pathogen and the causative agent of invasive pneumococcal disease. Carriage of the pneumococcus in the nasopharynx of humans is thought to be mediated by biofilm formation. Isogenic populations of S. pneumoniae grown under biofilm conditions frequently give rise to morphological colony variants, including small colony variant (SCV) phenotypes. This work employs phenotypic characterisation and whole genome sequencing coupled with ultra-pure liquid chromatography mass spectrophotometry (UPLC/MSE) of biofilm-derived S. pneumoniae serotype 22F pneumococcal colony morphology variants to investigate the diversification during biofilm formation. Phenotypic profiling revealed that SCVs exhibit reduced growth rates, reduced capsule expression, altered metabolic profiles and increased biofilm formation compared to the parent strain. Whole genome sequencing of 12 SCVs from independent biofilm experiments revealed that all SCVs studied had mutations within the DNA-directed RNA polymerase delta subunit (RpoE). Mutations included four large-scale deletions ranging from 51-264 basepairs (bp), one insertion resulting in a coding frameshift and seven nonsense single nucleotide substitutions that result in a truncated gene product. UPLC/MSE of the SCVs revealed up-regulation of a common sub-set of stress-inducible proteins which are part of an interaction network consisting of the 60 kDa chaperonin, chaperone protein DnaK, cell division protein FtsZ and manganese superoxide dismutase. This work links mutations in the rpoE gene to SCV formation and enhanced biofilm development in S. pneumoniae, with important implications for colonisation, carriage and persistence of the organism. Furthermore, consistent mutation within the pneumococcal rpoE gene presents an unprecedented level of parallel evolution in pneumococcal biofilm development. This work has given insight into the genetic diversity which may arise during pneumococcal colonisation which in turn may help inform future drug and vaccine design.

A murine model of acquired immunodeficiency syndrome (AIDS) was developed by infecting C57BL/6 mice with murine leukemia retrovirus LP-BM5. Murine AIDS shares many features with human AIDS. Murine and human AIDS cause impairment of cell-mediated and humoral immune responses, and increase risk of opportunistic infection such as Streptococcus pneumoniae. Cofactors such as ethanol may determine the severity of the retrovirus infection and the rate of progression to AIDS. Changes in nutritional status due to retrovirus infection or ethanol consumption, can play an important role in immunomodulation in the animal. Immunomodulation observed in animals with chronic ethanol ingestion is associated with age of the animal, the nutritional composition of the diet, and the amount of ethanol consumed. Young mice are more sensitive to the immunomodulating effects of ethanol and diet than mature mice. The percentage of B cells in mature mice was significantly increased with consumption of nutritionally superoptimal diet containing ethanol while ethanol ingestion with a nutritionally inadequate diet severely decreased the percentage of B cells when compared to control or pair-feeding. Cytokine secretion, and natural killer cell and phagocytic activities were modulated by ethanol as well as by the nutritional quality of the diet. Both retrovirus infection and ethanol consumption affected survival rate after Streptococcus pneumoniae infection in mice. Chronic ethanol consumption, but not retrovirus infection resulted in significant reduction in serum level of anti pneumococcal polysaccharide antibody which in combination with complement system make up an important part of host defense against S. pneumoniae. However, retrovirus infection significantly reduced resistance to S. pneumoniae. Retrovirus infected mice fed a diet containing a high concentration of ethanol for short term exhibited a greater resistance to S. pneumoniae infection than mice fed diets with low concentration or no ethanol. S. pneumoniae antigen immunization improved survival of the mice infected with S. pneumoniae. In conclusion, ethanol and nutritional adequacy of diet induced immunomodulation of the host. Ethanol consumption during retroviral infection may accelerate the progression of murine AIDS through changes in the lymphoid cells and resistance to S. pneumoniae infection.

The human bacterial pathogen, Streptococcus pneumoniae (the pneumococcus), has been shown to modulate different parts of the innate immune response of its host, however its ability to modulate the adaptive immune response remains largely uninvestigated. Furthermore, the importance of the adaptive arm of the immune system in responding to Streptococcus pneumoniae has only recently begun to be elucidated. I therefore investigated a potentially novel pneumococcal immunomodulatory mechanism involving the effect of the pneumococcal toxin, pneumolysin, upon the cells at the heart of the adaptive immune response; the CD4 T cell. I generated purified pneumolysin and a purified pneumolysin mutant called F433 to allow me to examine this potential effect. I found that pneumolysin inhibits in vitro antigen specific murine CD4 T cell proliferation and cytokine production and that this effect is not observed with the F433 mutant pneumolysin. Furthermore, I demonstrated that pneumolysin accomplished this inhibitory activity by inducing apoptosis of activated CD4 T cells and suggest that lipid rafts may be involved in this process since we also demonstrated that pneumolysin preferentially binds to lipid rafts. Finally I demonstrated that pneumolysin is able to inhibit the in vivo accumulation of T cells and also inhibits in vivo antibody production. I propose that the immunomodulatory mechanism I have described may play an important role during pneumococcal infection and that this warrants further investigation. I propose that detailed in vivo studies are required to demonstrate that this mechanism functions during infection and to elucidate the effect this has upon the course of infection.

Streptococcus pneumoniae (S. pneumoniae) is a Gram-positive, encapsulated bacterium capable of causing significant morbidity and mortality throughout the world. A hallmark of S. pneumoniae infection is infiltration of neutrophils (PMNs) that assist in controlling the spread infection but may also contribute to pathology. Paradoxically, studies have shown that limiting PMN infiltration into the lumen of the lung during infection actually betters clinical outcome in experimental S. pneumoniae infection. The final step in PMN luminal trafficking is a Hepoxilin A3 (HXA3)-dependent migration across the pulmonary epithelium. HXA3 is a PMN chemoattractant that forms gradients along the polarized epithelial face, drawing PMNs from the basolateral to the apical surface during proinflammatory responses. HXA3 requires assistance of an integral- membrane protein transporter to escape the cell and form the gradient. The pulmonary HXA3 transporter is currently unidentified. In this work, we identify the pulmonary HXA3 transporter as the ATP-Binding Cassette Transporter (ABC transporter) Multi-drug Resistance Associated Protein 2 (ABCC2, MRP2). We demonstrate that MRP1 and MRP2 are divergent ABC- transporters that control transepithelial PMN migration through efflux of a distinct anti-inflammatory substance and the pro-inflammatory HXA3 in the context of Streptococcus pneumoniae infection. Enrichment of MRP2 on the plasma membrane requires detection of the bacterial virulence factors pneumolysin (PLY) and hydrogen peroxide. PLY and hydrogen peroxide not only coordinate MRP2 apical membrane enrichment but also influence HXA3-dependent PMN transepithelial migration. They influence migration through stimulation of epithelial intracellular calcium increases that are crucial for HXA3 production as well as MRP2 translocation to the plasma membrane. PLY and hydrogen peroxide are not sufficient in their signaling alone, however, and require at least one additional bacterial signal to induce HXA3/MRP2 proinflammatory activities.

Given that Streptococcus pneumoniae can cause life-threatening pulmonary and systemic infection, an apparent paradox is that the bacterium resides, usually harmlessly, in the nasopharynx of many people. Humoral immunity is thought to be the primary defense against serious pneumococcal infection, and we hypothesized that nasopharyngeal colonization of mice results in the generation of an antibody response that provides long-term protection against lung infection. We found that survival of of C57L/6 mice after intranasal inoculation with wild-type serotype 4 strain TIGR4 pneumococci required B cells but not T cells, suggesting that nasopharyngeal colonization elicited a protective humoral immune response. In fact, intranasal inoculation resulted in detectable pneumococcal-specific antibody responses, and protected mice against a subsequent high-dose S. pneumoniae pulmonary challenge. B cells were required for this response, and transfer of immune sera from i.n. colonized mice, or monoclonal antibodies against phosphorylcholine, a common surface antigen of S. pneumoniae, was sufficient to confer protection. IgA, which is thought to participate in mucosal immunity, contributed to but was not absolutely required for protection from pulmonary challenge. Protection induced by i.n. colonization lasted at least ten weeks. Although it was partially dependent on T cells, depletion of CD4+ T cells at the time of challenge did not alter protection, suggesting that T cells did not provide essential help in activation of conventional memory cells. Peritoneal B1b cells and radiation-resistant, long-lived antibody secreting cells have previously been shown to secrete anti-pneumococcal antibodies and mediate protection against systemic infection following immunization with killed bacteria or capsular polysaccharide [1, 2]. We found that peritoneal cells were not sufficient for colonization-induced protection, but sub-lethally irradiated mice largely survived pulmonary challenge. Thus, our results are consistent with the hypothesis that nasopharyngeal colonization, a common occurrence in humans, is capable of eliciting extended protection against invasive pneumococcal disease by generating long-lived antibody-secreting cells.

C-reactive protein (CRP) binds to Streptococcus pneumoniae through the phosphocholine groups present in the cell wall and subsequently activates the complement system to kill the pathogen. To escape the attack of complement, pneumococci recruit a complement inhibitory protein, factor H, on their surface. It has been shown that CRP protects mice against pneumococcal infection only when injected within 2 hours after administering pneumococci. We hypothesized that CRP is not protective when injected at later times because, by then, factor H is recruited by pneumococci. In the current study, we evaluated the protective effects of an engineered CRP molecule (E-CRP) which does not bind to factor H in fluid phase but binds to factor H-coated pneumococci. We found that E-CRP, unlike native CRP, protected mice regardless of the timing of administering E-CRP by drastically reducing bacteremia and increasing survival of mice. Next, we established another murine model of pneumococcal infection using the antibiotic clarithromycin. We found that the combination of E-CRP and clarithromycin was more protective against infection when compared to the protective effects of either E-CRP alone or clarithromycin alone. These findings suggest that the structure of native CRP has to be altered to display its full anti-pneumococcal activity and that CRP and antibiotic act synergistically to protect against pneumococcal infection by decreasing bacteremia. These data also have implications for infections with other bacterial species that use factor H to evade the attack of complement. Additionally, the administration of E-CRP may be therapeutically beneficial to treat infections with antibiotic-resistant bacteria.

Introdução: A vacina pneumocócica é recomendada para crianças com IRC, pelo maior risco de adquirir a forma invasiva da doença. O Streptococcus pneumoniae é o principal agente etiológico causador de pneumonia e otite média aguda, e a segunda causa de meningite na criança. A vacina conjugada 7 - valente tem se mostrado imunogênica e com efeito duradouro em crianças normais, porém em pacientes com IRC, devido às alterações imunológicas associadas à uremia, pode resultar uma resposta sub - ótima e de vida curta. Objetivo: Determinar as concentrações de anticorpos da classe IgG contra os sorotipos 4,6B, 9V, 14, 18C, 19F e 23 F do Streptococcus pneumoniae, antes e depois da administração da vacina antipneumocócica conjugada 7 - valente, em crianças portadoras de insuficiência renal crônica em tratamento conservador e diálise, e comparar a resposta entre eles. Métodos: Selecionamos 48 crianças, com idade de 1 ano a 9 anos, portadoras de IRC, com depuração de creatinina menor ou igual a 70 ml/min/1,73m 2 , calculada pela fórmula de Schwartz, divididos em: grupo 1 (G1, tratamento conservador), e grupo 2 (G2,tratamento dialítico). Os pacientes receberam duas doses da vacina conjugada 7 - valente, com intervalo de 60 dias. A sorologia foi colhida antes da primeira dose, e 30 a 60 dias após o reforço. Os títulos de anticorpos para os sorotipos presentes na vacina (4, 6B, 9V, 14, 18C, 19F, 23F) foram determinados pelo método imunoenzimático (ELISA). Resultados: A análise da concentração pré - vacinal de IgG mostrou maior percentual dos pacientes do grupo 1 e 2 com IgG <0,6 para os sorotipos 4, 9 e 18, e maior percentual com IgG maior ou igual a 1,3 ug/ml para os sorotipos 14 e 19. Para avaliar a concentração de IgG pós - vacinal foi utilizado o cálculo de freqüências com intervalo de confiança 95%(IC95), para os 7 sorotipos nos 2 grupos, utilizando-se 3 critérios : Critério A: IgG pós vacinal maior ou igual a 1,3 ug/ml, por este critério verificou-se freqüência de resposta do G1 de 0,650 (IC95: 0,407-0,864) a 1,0 (IC95: 0,663 - 1,0) e do G2 de 0,777 (IC95: 0,399 -0,971) a 1,0 (IC95: 0,663 - 1,0), critério B: ou seja, delta (pré/pós) maior ou igual a 4X, freqüência de resposta G1 de 0,458 (IC95: 0,255 - 0,671) a 0,708 (IC95%: 0,488 - 0,873) e do G2 de 0,458 (IC95%: 0,255 - 0,671) a 0,708 (IC95%: 0,488 - 0,973), e critério C:isto é, delta (pré/pós) maior ou igual a 4X e IgG maior ou igual a 1,3 ug/ml, freqüência de resposta do G1 de 0,416 (IC95%: 0,221 - 0,633) a 0,625 (IC95%: 0,406 - 0,811) e do G2 de 0,416 (IC95%: 0,221 - 0,633) a 0,666 (IC95%: 0,447 - 0,843). G1 e G2 mostraram comportamento semelhante na análise das freqüências de resposta, segundo os 3 critérios. Conclusão: Este estudo é pioneiro na avaliação da vacina antipneumocócica conjugada em crianças com IRC, e demonstrou uma boa resposta para cada critério analisado separadamente, com comportamento semelhante nos 2 grupos. A dificuldade inerente à definição de critérios de soroconversão, demonstra a necessidade de estudos multicêntricos com acompanhamento clínico e laboratorial em longo prazo para avaliar a sustentabilidade da soroconversão e a imunogenicidade da vacina.
Introduction: The pneumococcal vaccine is recommended for children with CRF due to their increased risk in acquiring the invasive form of the disease. Streptococcus pneumoniae is the main etiological agent that causes pneumonia and acute middle otitis and the second cause of meningitis in children. The 7-valent conjugated pneumococcal vaccine has been shown to be immunogenic and to have a lasting effect in normal children; however, in children with CRF, the vaccine can produce a suboptimal and short-lasting response due to the immunological alterations associated to uremia. Objective: to determine pneumococcal IgG antibodies to serotypes 4, 6B, 9V, 14, 18C, 19F e 23 F before and after 7 - valent conjugated pneumococcal vaccine, in children with chronic renal failure in conservative treatment and dialysis. Methods: 48 children with CRF, aged 1 to 9 years, with a creatinine clearance of menor ou igual a 70 ml/min/1.73 calculated by the Schwartz formula, were selected for the present study and divided in two groups: Group 1: conservative treatment and Group 2: dialytic treatment. The patients received two doses of the 7- valent conjugated vaccine, with a 60-day interval between them. Serological samples were collected before the first dose and 30 to 60 days after the second one. Antibody titers for the serotypes present in the vaccine (4, 6B, 9V, 14, 18C, 19F, 23F) were determined by the immunoenzymatic method (ELISA). Results: The analysis of the pre-vaccinal IgG concentration showed a higher percentage of patients from Groups 1 and 2 with IgG < 0.6 for the serotypes 4, 9 and 18, and a higher percentage with pre-vaccinal IgG maior ou igual a 1.3 ug/ml for the serotypes 14 and 19. To assess the post-vaccinal IgG concentration, the calculation of frequencies with 95% confidence interval (95CI) was employed for the 7 serotypes in both groups, using three criteria: Criterion A: post-vaccinal IgG maior ou igual a 1.3 ug/ml, this criterion verified the frequency of response from G1 0,650 (IC95: 0,407-0,864) to 1,0 (IC95: 0,663 - 1,0) and G2 0,777 (IC95: 0,399 - 0,971) to 1,0 (IC95: 0,663 - 1,0), criterion B: i.e., delta (pre/post) maior ou igual a 4X, frequency of response from G1 0,458 (95CI: 0.255 ? 0.671) to 0.708 (95CI: 0.488 - 0.873) and G2 0,458 (IC95: 0,255 - 0,671) to 0,708 (IC95: 0,488 - 0,873); criterion C: i.e., delta (pre/post) ?4X and IgG maior ou igual a 1.3 ug/ml, frequency of response from G1 0.416 (95CI: 0.221 - 0.633) to 0,625(95CI: 0,406 - 0,811) and G2 0,416 (9CI: 0,221 - 0,633) to 0.666 (95CI: 0.447 - 0.843). Groups 1 and 2 showed similar behavior at the analysis of response frequencies according to the three criteria. Conclusion: this is the first study to assess the conjugated pneumococcal vaccine in children with CRF, which showed good response, with a similar behavior in both groups. The inherent difficulty in defining criteria for seroconversion demonstrates the need for multicentric studies with long-term clinical and laboratory follow up, in order to assess the seroconversion duration and the vaccine immunogenicity.

The anti-pneumococcal function of native C-reactive protein (CRP) involves its binding to phosphocholine molecules present on Streptococcus pneumoniae and subsequent activation of the complement system. However, when pneumococci recruit complement inhibitory protein factor H on their surface, they escape complement attack. Non-native forms of CRP have been shown to bind immobilized factor H. Accordingly, we hypothesized that modified CRP would bind to factor H on pneumococci, masking its complement inhibitory activity, allowing native CRP to exert its anti-pneumococcal function. As reported previously, native CRP protected mice from lethal pneumococcal infection when injected 30 minutes before infection but not when injected 24 hours after infection. However, a combination of native and mutant CRP was found to protect mice even when administered 24 hours after infection. Therefore, it is concluded that while native CRP is protective only against early-stage infection, a combination of native and mutant CRP offers protection against late-stage infection.

[Truncated abstract] Pneumococcal exposure is high and life-long in developing countries including Papua New Guinea (PNG), with children under 2 years of age being at most risk for early upper respiratory tract pneumococcal carriage and infection. Deaths from pneumococcal diseases such as pneumonia and meningitis are common and likely the result of an absence of vaccination programmes. The need for effective and affordable pneumococcal vaccines has led to the testing of protein antigens including pneumolysin (Ply) and pneumococcal surface protein A (PspA) as novel vaccine antigens. Little is known on the immune responses to these proteins in humans, particularly in high-risk populations where such vaccines will be of most benefit. In this study, we examined the roles of naturally acquired antibody and cellular immune responses in mothers and newborns to Ply and PspA family 1 (PspA1) and family 2 (PspA2) in protection against or risk for early carriage in a high-risk PNG population. Antibodies to Ply, PspA1 and PspA2 were measured in plasmas of 241 mothers and 115 newborns (cords) from PNG, and 50 Australian mothers using an enzyme-linked immunosorbent assay (ELISA). Pernasal swabs were collected from PNG mothers at the time of delivery, one month post-partum, and weekly within the first month of life from their newborns to determine pneumococcal carriage. Cellular immune responses to Ply, PspA1 and PspA2, the TLR2/TLR4 ligands, LTA and LPS and to PHA were measured in cord blood mononuclear cells (CBMC) of 84 PNG versus 33 Australian newborns. Innate and T-cell cytokine responses in the PNG newborns were then analysed to determine their effect on infant pneumococcal carriage. ... No protective effect against infant pneumococcal carriage was observed with maternal and cord IgG levels for all antigens. Maternal carriage at time of delivery increased the risk for infant pneumococcal carriage in the first month of life (HR: 1.93, 95% CI 1.36 – 2.73, p = 0.001) with 70% of infants being colonised. Papua New Guinean newborns produced higher innate IL-10 and IFN-¿ (p = 0.003) and TNF-a (p < 0.001) to Ply compared to Australian newborns with no significant differences observed for IL-6 or IL-12. IFN-¿ responses to LPS and LTA (p = 0.005 and p < 0.001) were higher in PNG than Australian newborns, while IL-6, IL-10 (p < 0.001) and TNF-a (p = 0.002) to LPS with LTA-induced IL-6 and IL-10 (p < 0.001) were higher in Australian newborns. T-cell IL-5, IL-10, IL-13, IFN-¿, IL-6 and TNF-a response levels to PspA and PHA stimulation were significantly high in PNG newborns. No differences were observed for cytokine responses to Ply and PspA between PNG infant pneumococci carriers and non-carriers. Papua New Guinean infants are colonised by pneumococci very early in life and this may be influenced by high maternal carriage rates. PspA- and Ply-IgG levels are high in PNG mothers and undergo cross placental transfer but do not appear to be protective against early pneumococcal carriage. In PNG newborns, PspA elicits T-cell responses, while Ply drives more innate cellular responses, neither were demonstrated to have a protective effect against early carriage though further work is required to better define these and their relation to immune development in early childhood.