RESUMO
Factor H binding protein (fHbp) is a lipoprotein of Neisseria meningitidis important for the survival of the bacterium in human blood and a component of two recently licensed vaccines against serogroup B meningococcus (MenB). Based on 866 different amino acid sequences this protein is divided into three variants or two families. Quantification of the protein is done by immunoassays such as ELISA or FACS that are susceptible to the sequence variation and expression level of the protein. Here, selected reaction monitoring mass spectrometry was used for the absolute quantification of fHbp in a large panel of strains representative of the population diversity of MenB. The analysis revealed that the level of fHbp expression can vary at least 15-fold and that variant 1 strains express significantly more protein than variant 2 or variant 3 strains. The susceptibility to complement-mediated killing correlated with the amount of protein expressed by the different meningococcal strains and this could be predicted from the nucleotide sequence of the promoter region. Finally, the absolute quantification allowed the calculation of the number of fHbp molecules per cell and to propose a mechanistic model of the engagement of C1q, the recognition component of the complement cascade.
Assuntos
Antígenos de Bactérias/metabolismo , Proteínas de Bactérias/metabolismo , Neisseria meningitidis Sorogrupo B/metabolismo , Sequência de Aminoácidos , Antígenos de Bactérias/genética , Antígenos de Bactérias/imunologia , Proteínas de Bactérias/genética , Proteínas de Bactérias/imunologia , Ensaio de Imunoadsorção Enzimática , Citometria de Fluxo , Variação Genética , Humanos , Espectrometria de Massas/métodos , Meningite Meningocócica/imunologia , Meningite Meningocócica/microbiologia , Vacinas Meningocócicas/imunologia , Neisseria meningitidis Sorogrupo B/classificação , Neisseria meningitidis Sorogrupo B/genética , Filogenia , Especificidade da EspécieRESUMO
The adhesion of Streptococcus pneumoniae is a key step during colonization of human respiratory tract mucosae. Here we demonstrate that pneumococcal type I pilus significantly increases the adhesiveness of poorly adhering highly capsulated strains in vitro. Interestingly, preincubation of bacteria with antibodies against the major pilus backbone subunit (RrgB) or the adhesin component (RrgA) impaired pneumococcal association to human epithelial cells. Screening for anti-RrgA monoclonal antibodies specifically affecting the adhesive capacity of S. pneumoniae led to the identification of the monoclonal 11B9/61 antibody, which greatly reduced pilus-dependent cell contact. Proteomic-based epitope mapping of 11B9/61 monoclonal antibody revealed a well-exposed epitope on the D2 domain of RrgA as the target of this functional antibody. The data presented here confirm the importance of pilus I for S. pneumoniae pathogenesis and the potential use of antipilus antibodies to prevent bacterial colonization.
Assuntos
Anticorpos Antibacterianos/imunologia , Anticorpos Monoclonais/imunologia , Aderência Bacteriana/efeitos dos fármacos , Células Epiteliais/microbiologia , Proteínas de Fímbrias/imunologia , Fímbrias Bacterianas/imunologia , Streptococcus pneumoniae/imunologia , Linhagem Celular , Mapeamento de Epitopos , Humanos , Fatores de Virulência/imunologiaRESUMO
AIM: This paper reports a case of all-on-four rehabilitation where bone density at implant sites was assessed both through preoperative computed tomographic (CT) scans and using a micromotor working as an intraoperative bone density measurement device. BACKGROUND: Implant-supported rehabilitation is a predictable treatment option for tooth replacement whose success depends on the clinician's experience, the implant characteristics and location and patient-related factors. Among the latter, bone density is a determinant for the achievement of primary implant stability and, eventually, for implant success. The ability to measure bone density at the placement site before implant insertion could be important in the clinical setting. CASE DESCRIPTION: A patient complaining of masticatory impairment was presented with a plan calling for extraction of all her compromised teeth, followed by implant rehabilitation. A week before surgery, she underwent CT examination, and the bone density on the CT scans was measured. When the implant osteotomies were created, the bone density was again measured with a micromotor endowed with an instantaneous torque-measuring system. The implant placement protocols were adapted for each implant, according to the intraoperative measurements, and the patient was rehabilitated following an all-on-four immediate loading protocol. CONCLUSION: The bone density device provided valuable information beyond that obtained from CT scans, allowing for site-specific, intraoperative assessment of bone density immediately before implant placement and an estimation of primary stability just after implant insertion. CLINICAL SIGNIFICANCE: Measuring jaw-bone density could help clinicians to select implant-placement protocols and loading strategies based on site-specific bone features.
Assuntos
Perda do Osso Alveolar/reabilitação , Densidade Óssea , Implantes Dentários , Tomografia Computadorizada por Raios X/instrumentação , Implantação Dentária Endóssea , Feminino , Humanos , Pessoa de Meia-Idade , Projetos PilotoRESUMO
Neisseria meningitidis is an obligate human commensal that commonly colonizes the oropharyngeal mucosa. Carriage is age dependent and very common in young adults. The relationships between carriage and invasive disease are not completely understood. In this work, we performed a longitudinal carrier study in adolescents and young adults (173 subjects). Overall, 32 subjects (18.5%) had results that were positive for meningococcal carriage in at least one visit (average monthly carriage rate, 12.1%). Only five subjects tested positive at all four visits. All meningococcal isolates were characterized by molecular and serological techniques. Multilocus sequence typing, PorA typing, and sequencing of the 4CMenB vaccine antigens were used to assess strain diversity. The majority of positive subjects were colonized by capsule null (34.4%) and capsular group B strains (28.1%), accounting for 23.5% and 29.4% of the total number of isolates, respectively. The fHbp and nhba genes were present in all isolates, while the nadA gene was present in 5% of the isolates. The genetic variability of the 4CMenB vaccine antigens in this collection was relatively high compared with that of other disease-causing strain panels. Indications about the persistence of the carriage state were limited to the time span of the study. All strains isolated from the same subject were identical or cumulated minor changes over time. The expression levels and antigenicities of the 4CMenB vaccine antigens in each strain were analyzed by the meningococcal antigen typing system (MATS), which revealed that expression can change over time in the same individual. Future analysis of antigen variability and expression in carrier strains after the introduction of the MenB vaccine will allow for a definition of its impact on nasopharyngeal/oropharyngeal carriage.
Assuntos
Técnicas de Tipagem Bacteriana , Portador Sadio/microbiologia , Infecções Meningocócicas/microbiologia , Tipagem Molecular , Neisseria meningitidis/classificação , Neisseria meningitidis/isolamento & purificação , Adolescente , Antígenos de Bactérias/análise , Portador Sadio/epidemiologia , DNA Bacteriano/genética , Feminino , Variação Genética , Genótipo , Humanos , Itália/epidemiologia , Estudos Longitudinais , Masculino , Infecções Meningocócicas/epidemiologia , Neisseria meningitidis/genética , Neisseria meningitidis/imunologia , Orofaringe/microbiologia , Sorotipagem , Adulto JovemRESUMO
RrgB is the major pilin which forms the pneumococcal pilus backbone. We report the high-resolution crystal structure of the full-length form of RrgB containing the IPQTG sorting motif. The RrgB fold is organized into four distinct domains, D1-D4, each of which is stabilized by an isopeptide bond. Crystal packing revealed a head-to-tail organization involving the interaction of the IPQTG motif into the D1 domain of two successive RrgB monomers. This fibrillar assembly, which fits into the electron microscopy density map of the native pilus, probably induces the formation of the D1 isopeptide bond as observed for the first time in the present study, since neither in published structures nor in soluble RrgB produced in Escherichia coli or in Streptococcus pneumoniae is the D1 bond present. Experiments performed in live bacteria confirmed that the intermolecular bond linking the RrgB subunits takes place between the IPQTG motif of one RrgB subunit and the Lys183 pilin motif residue of an adjacent RrgB subunit. In addition, we present data indicating that the D1 isopeptide bond is involved in RrgB stabilization. In conclusion, the crystal RrgB fibre is a compelling model for deciphering the molecular details required to generate the pneumococcal pilus.
Assuntos
Proteínas de Fímbrias/química , Proteínas de Fímbrias/metabolismo , Fímbrias Bacterianas/metabolismo , Multimerização Proteica , Streptococcus pneumoniae , Motivos de Aminoácidos/genética , Motivos de Aminoácidos/fisiologia , Cristalização , Cristalografia por Raios X , Proteínas de Fímbrias/genética , Fímbrias Bacterianas/química , Fímbrias Bacterianas/genética , Ligação de Hidrogênio , Fibras Minerais , Modelos Biológicos , Modelos Moleculares , Conformação Molecular , Mutagênese Sítio-Dirigida , Multimerização Proteica/genética , Streptococcus pneumoniae/genética , Streptococcus pneumoniae/metabolismoRESUMO
Streptococcus pneumoniae expresses on its surface adhesive pili, involved in bacterial attachment to epithelial cells and virulence. The pneumococcal pilus is composed of three proteins, RrgA, RrgB, and RrgC, each stabilized by intramolecular isopeptide bonds and covalently polymerized by means of intermolecular isopeptide bonds to form an extended fiber. RrgB is the pilus scaffold subunit and is protective in vivo in mouse models of sepsis and pneumonia, thus representing a potential vaccine candidate. The crystal structure of a major RrgB C-terminal portion featured an organization into three independently folded protein domains (D2-D4), whereas the N-terminal D1 domain (D1) remained unsolved. We have tested the four single recombinant RrgB domains in active and passive immunization studies and show that D1 is the most effective, providing a level of protection comparable with that of the full-length protein. To elucidate the structural features of D1, we solved the solution structure of the recombinant domain by NMR spectroscopy. The spectra analysis revealed that D1 has many flexible regions, does not contain any intramolecular isopeptide bond, and shares with the other domains an Ig-like fold. In addition, we demonstrated, by site-directed mutagenesis and complementation in S. pneumoniae, that the D1 domain contains the Lys residue (Lys-183) involved in the formation of the intermolecular isopeptide bonds and pilus polymerization. Finally, we present a model of the RrgB protein architecture along with the mapping of two surface-exposed linear epitopes recognized by protective antisera.
Assuntos
Proteínas de Fímbrias/química , Streptococcus pneumoniae/metabolismo , Animais , Proteínas de Bactérias/química , Adesão Celular , Modelos Animais de Doenças , Epitopos/química , Proteínas de Fímbrias/genética , Teste de Complementação Genética , Espectroscopia de Ressonância Magnética/métodos , Camundongos , Camundongos Endogâmicos BALB C , Mutagênese Sítio-Dirigida , Peptídeos/química , Conformação Proteica , Estrutura Terciária de Proteína , Sepse/metabolismoRESUMO
Streptococcus pneumoniae pilus 1 is present in 30 to 50% of invasive disease-causing strains and is composed of three subunits: the adhesin RrgA, the major backbone subunit RrgB, and the minor ancillary protein RrgC. RrgB exists in three distinct genetic variants and, when used to immunize mice, induces an immune response specific for each variant. To generate an antigen able to protect against the infection caused by all pilus-positive S. pneumoniae strains, we engineered a fusion protein containing the three RrgB variants (RrgB321). RrgB321 elicited antibodies against proteins from organisms in the three clades and protected mice against challenge with piliated pneumococcal strains. RrgB321 antisera mediated complement-dependent opsonophagocytosis of piliated strains at levels comparable to those achieved with the PCV7 glycoconjugate vaccine. These results suggest that a vaccine composed of RrgB321 has the potential to cover 30% or more of all pneumococcal strains and support the inclusion of this fusion protein in a multicomponent vaccine against S. pneumoniae.
Assuntos
Atividade Bactericida do Sangue , Proteínas de Fímbrias/imunologia , Fímbrias Bacterianas/imunologia , Proteínas Opsonizantes/sangue , Vacinas Pneumocócicas/imunologia , Proteínas Recombinantes de Fusão/imunologia , Streptococcus pneumoniae/imunologia , Animais , Anticorpos Antibacterianos/sangue , Proteínas do Sistema Complemento/imunologia , Feminino , Proteínas de Fímbrias/genética , Fímbrias Bacterianas/genética , Camundongos , Camundongos Endogâmicos BALB C , Fagocitose/imunologia , Infecções Pneumocócicas/imunologia , Infecções Pneumocócicas/prevenção & controle , Vacinas Pneumocócicas/administração & dosagem , Proteínas Recombinantes de Fusão/genética , Vacinas Sintéticas/administração & dosagem , Vacinas Sintéticas/imunologiaRESUMO
Non-typable Streptococcus pneumoniae (NTPn) strains are typically isolated from nasopharyngeal carriage or from conjunctivitis. Since the isolation of NTPn from invasive disease is rare, we characterized the genetic basis of the non-typability of two isolates obtained in Italy from two cases of bacteraemic pneumonia. MLST revealed that both NTPn belonged to ST191, which, according to the MLST database, is associated with serotype 7F. Sequencing of the capsular locus (cps) confirmed the presence of a 7F cps in both strains and revealed the existence of distinct single point mutations in the wchA gene (a glycosyltransferase), both leading to the translation of proteins truncated at the C terminus. To verify that these mutations were responsible for the non-typability of the isolates, a functional 7F WchA was overexpressed in both NTPn. The two NTPn along with their WchA-overexpressing derivatives were analysed by transmission electron microscopy and by high-resolution magic angle spinning NMR spectroscopy. Both NTPn were devoid of a polysaccharide capsule, and WchA overexpression was sufficient to restore the assembly of a serotype 7F capsule on the surface of the two NTPn. In conclusion, we identified two new naturally occurring point mutations that lead to non-typability in the pneumococcus, and demonstrated that WchA is essential for the biosynthesis of the serotype 7F capsule.
Assuntos
Proteínas de Bactérias/genética , Glicosiltransferases/genética , Infecções Pneumocócicas/microbiologia , Mutação Puntual , Streptococcus pneumoniae/enzimologia , Adulto , Sequência de Aminoácidos , Cápsulas Bacterianas/biossíntese , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Técnicas de Tipagem Bacteriana , Feminino , Glicosiltransferases/química , Glicosiltransferases/metabolismo , Humanos , Masculino , Dados de Sequência Molecular , Alinhamento de Sequência , Streptococcus pneumoniae/classificação , Streptococcus pneumoniae/genética , Streptococcus pneumoniae/isolamento & purificaçãoRESUMO
Thirty percent of Streptococcus pneumoniae isolates contain pilus islet 1, coding for a pilus composed of the backbone subunit RrgB and two ancillary proteins, RrgA and RrgC. RrgA is the major determinant of in vitro adhesion associated with pilus 1, is protective in vivo in mouse models, and exists in two variants (clades I and II). Mapping of the sequence variability onto the RrgA structure predicted from X-ray data showed that the diversity was restricted to the "head" of the protein, which contains the putative binding domains, whereas the elongated "stalk" was mostly conserved. To investigate whether this variability could influence the adhesive capacity of RrgA and to map the regions important for binding, two full-length protein variants and three recombinant RrgA portions were tested for adhesion to lung epithelial cells and to purified extracellular matrix (ECM) components. The two RrgA variants displayed similar binding abilities, whereas none of the recombinant fragments adhered at levels comparable to those of the full-length protein, suggesting that proper folding and structural arrangement are crucial to retain protein functionality. Furthermore, the two RrgA variants were shown to be cross-reactive in vitro and cross-protective in vivo in a murine model of passive immunization. Taken together, these data indicate that the region implicated in adhesion and the functional epitopes responsible for the protective ability of RrgA may be conserved and that the considerable level of variation found within the "head" domain of RrgA may have been generated by immunologic pressure without impairing the functional integrity of the pilus.
Assuntos
Adesinas Bacterianas/fisiologia , Fímbrias Bacterianas/fisiologia , Streptococcus pneumoniae/patogenicidade , Adesinas Bacterianas/genética , Sequência de Aminoácidos , Animais , Western Blotting , Proteção Cruzada/genética , Proteção Cruzada/fisiologia , Ensaio de Imunoadsorção Enzimática , Feminino , Fímbrias Bacterianas/genética , Citometria de Fluxo , Regulação Bacteriana da Expressão Gênica/genética , Regulação Bacteriana da Expressão Gênica/fisiologia , Imunização Passiva , Camundongos , Camundongos Endogâmicos BALB C , Infecções Pneumocócicas/microbiologia , Estrutura Terciária de Proteína/genética , Estrutura Terciária de Proteína/fisiologia , Proteínas Recombinantes/genética , Streptococcus pneumoniae/genética , Streptococcus pneumoniae/fisiologiaRESUMO
BACKGROUND: The multicomponent meningococcal serogroup B vaccine (4CMenB) is currently indicated for active immunization against invasive meningococcal disease caused by Neisseria meningitidis serogroup B (MenB). However, genes encoding the 4CMenB antigens are also variably present and expressed in strains belonging to other meningococcal serogroups. In this study, we evaluated the ability of antibodies raised by 4CMenB immunisation to induce complement-mediated bactericidal killing of non-MenB strains. METHODS: A total of 227 invasive non-MenB disease isolates were collected between 1 July 2007 and 30 June 2008 from England and Wales, France, and Germany; 41 isolates were collected during 2012 from Brazil. The isolates were subjected to genotypic analyses. A subset of 147 isolates (MenC, MenW and MenY) representative of the meningococcal genetic diversity of the total sample were tested in the human complement serum bactericidal antibody assay (hSBA) using sera from infants immunised with 4CMenB. RESULTS: Serogroup and clonal complex repertoires of non-MenB isolates were different for each country. For the European panel, MenC, MenW and MenY isolates belonged mainly to ST-11, ST-22 and ST-23 complexes, respectively. For the Brazilian panel, most MenC and MenW isolates belonged to the ST-103 and ST-11 complexes, respectively, and most MenY isolates were not assigned to clonal complexes. Of the 147 non-MenB isolates, 109 were killed in hSBA, resulting in an overall coverage of 74%. CONCLUSION: This is the first study in which 147 non-MenB serogroup isolates have been analysed in hSBA to evaluate the potential of a MenB vaccine to cover strains belonging to other serogroups. These data demonstrate that antibodies raised by 4CMenB are able to induce bactericidal killing of 109 non-MenB isolates, representative of non-MenB genetic and geographic diversity. These findings support previous evidence that 4CMenB immunisation can provide cross-protection against non-MenB strains in infants, which represents an added benefit of 4CMenB vaccination.
Assuntos
Infecções Meningocócicas , Vacinas Meningocócicas , Neisseria meningitidis Sorogrupo B , Antígenos de Bactérias/genética , Brasil , Inglaterra , França , Alemanha , Humanos , Lactente , Infecções Meningocócicas/prevenção & controle , Neisseria meningitidis Sorogrupo B/genética , Sorogrupo , Vacinação , País de GalesRESUMO
Streptococcus pneumoniae sortase A (SrtA) is a transpeptidase that is highly conserved among pneumococcal strains, whose involvement in adhesion/colonization has been reported. We found that intraperitoneal immunization with recombinant SrtA conferred to mice protection against S. pneumoniae intraperitoneal challenge and that the passive transfer of immune serum before intraperitoneal challenge was also protective. Moreover, by using the intranasal challenge model, we observed a significant reduction of bacteremia when mice were intraperitoneally immunized with SrtA, while a moderate decrease of lung infection was achieved by intranasal immunization, even though no influence on nasopharynx colonization was seen. Taken together, our results suggest that SrtA is a good candidate for inclusion in a multicomponent, protein-based, pneumococcal vaccine.
Assuntos
Aminoaciltransferases/imunologia , Proteínas de Bactérias/imunologia , Cisteína Endopeptidases/imunologia , Infecções Pneumocócicas/prevenção & controle , Vacinas Estreptocócicas/imunologia , Streptococcus pneumoniae/imunologia , Animais , Anticorpos Antibacterianos/uso terapêutico , Bacteriemia/prevenção & controle , Portador Sadio/prevenção & controle , Feminino , Imunização Passiva , Camundongos , Camundongos Endogâmicos BALB C , Nasofaringe/microbiologia , Pneumonia Pneumocócica/prevenção & controle , Vacinas de Subunidades Antigênicas/imunologia , Vacinas Sintéticas/imunologiaRESUMO
Analysis of publicly available genomes of Streptococcus pneumoniae has led to the identification of a new genomic element containing genes typical of gram-positive pilus islets (PIs). Here, we demonstrate that this genomic region, herein referred to as PI-2 (consisting of pitA, sipA, pitB, srtG1, and srtG2) codes for a second functional pilus in pneumococcus. Polymerization of the PI-2 pilus requires the backbone protein PitB as well as the sortase SrtG1 and the signal peptidase-like protein SipA. Presence of PI-2 correlates with the genotype as defined by multilocus sequence typing and clonal complex (CC). The PI-2-positive CCs are associated with serotypes 1, 2, 7F, 19A, and 19F, considered to be emerging serotypes in both industrialized and developing countries. Interestingly, strains belonging to CC271 (where sequence type 271 is the predicted founder of the CC) contain both PI-1 and PI-2, as revealed by genome analyses. In these strains both pili are surface exposed and independently assembled. Furthermore, in vitro experiments provide evidence that the pilus encoded by PI-2 of S. pneumoniae is involved in adherence. Thus, pneumococci encode at least two types of pili that play a role in the initial host cell contact to the respiratory tract and are potential antigens for inclusion in a new generation of pneumococcal vaccines.
Assuntos
Aderência Bacteriana , Fímbrias Bacterianas/fisiologia , Streptococcus pneumoniae/fisiologia , Linhagem Celular , Impressões Digitais de DNA , DNA Bacteriano/química , DNA Bacteriano/genética , Células Epiteliais/microbiologia , Fímbrias Bacterianas/genética , Fímbrias Bacterianas/ultraestrutura , Ordem dos Genes , Genes Bacterianos , Ilhas Genômicas , Genótipo , Humanos , Microscopia Eletrônica de Transmissão , Dados de Sequência Molecular , Infecções Pneumocócicas/microbiologia , Análise de Sequência de DNA , Sorotipagem , Streptococcus pneumoniae/classificação , Streptococcus pneumoniae/genética , Streptococcus pneumoniae/ultraestruturaRESUMO
Neisseria meningitidis is a common cause of bacterial meningitis in children and young adults worldwide. The 4CMenB vaccine (Bexsero), developed to combat meningococcal serogroup B (MenB) disease, contains subcapsular antigens that may induce immunity against strains of N. meningitidis, regardless of serogroup. Owing to differential levels of expression and peptide diversity in vaccine antigens across meningococcal strains, the meningococcal antigen typing system (MATS) was developed to estimate the potential MenB strain coverage of 4CMenB. Prior to introducing the 4CMenB vaccine into routine use, we sought to estimate the potential 4CMenB coverage against invasive MenB strains isolated in the Republic of Ireland (RoI) over four consecutive epidemiological years. MATS was applied to a panel of 105 invasive MenB strains isolated during July 2009 to June 2013. Sequence data characterizing the multilocus sequence typing (MLST) alleles and the major 4CMenB target peptides were extracted from isolate genome sequence data, hosted in the Bacterial Isolate Sequencing database (BIGSdb). MATS data indicated that 4CMenB may induce protective immunity against 69.5% (95% confidence interval [CI95%], 64.8% to 84.8%) of circulating MenB strains. Estimated coverage was highest against the most prevalent disease-causing lineage, cc41/44, where the most frequently observed sequence types, ST-154 and ST-41 (21% of isolates, collectively), were typically covered by three antigens. No significant temporal trends were observed. Overall, these data provide a baseline of strain coverage prior to the introduction of 4CMenB and indicate that a decrease in invasive meningococcal disease (IMD) is predicted following the introduction of 4CMenB into the routine infant immunization schedule in the RoI.IMPORTANCE The meningococcal antigen typing system (MATS) is an enzyme-linked immunosorbent assay (ELISA) that measures both the levels of expression and the immune reactivity of the three recombinant 4CMenB antigens. Together with PorA variable-region sequence data, this system provides an estimation of how susceptible MenB isolates are to killing by 4CMenB vaccine-induced antibodies. Assays based on subcapsular antigen phenotype analyses, such as MATS, are important in situations where conventional vaccine coverage estimations are not possible. Subcapsular antigens are typically highly diverse across strains, and vaccine coverage estimations would require unfeasibly large efficacy trials and screening of an exhaustive strain panel for antibody functional activity. Here, MATS was applied to all invasive meningococcal serogroup B (MenB) strains isolated over four consecutive epidemiological years (n = 105) and predicted reasonably high 4CMenB vaccine coverage in the Republic of Ireland.
Assuntos
Infecções Meningocócicas/prevenção & controle , Vacinas Meningocócicas/uso terapêutico , Cobertura Vacinal/estatística & dados numéricos , Vacinação/estatística & dados numéricos , Adolescente , Adulto , Distribuição por Idade , Idoso , Idoso de 80 Anos ou mais , Antígenos de Bactérias/genética , Criança , Pré-Escolar , Feminino , Humanos , Esquemas de Imunização , Lactente , Recém-Nascido , Irlanda/epidemiologia , Masculino , Infecções Meningocócicas/epidemiologia , Pessoa de Meia-Idade , Tipagem de Sequências Multilocus , Neisseria meningitidis Sorogrupo B/classificação , Adulto JovemRESUMO
OBJECTIVE: Although the incidence of meningococcal disease has been declining over the past decade in Portugal MenB meningococci is still an important cause of meningitis and sepsis. The aim of this study was to estimate the strain coverage of the 4CMenB vaccine in Portugal in order to support health policies for prevention and control of meningococcal disease. METHODS: Since 2002 the clinical and laboratory notification of meningococcal disease is mandatory in Portugal. National database includes since then all confirmed cases notified to the reference laboratory or to the Directorate of Health. Strains included in this study were all the invasive MenB isolated from the 1st July 2011 to the 30th June 2015, sent to the reference laboratory. To predict the vaccine strain coverage of the 4CMenB the expression and cross-reactivity of the surface antigens fHbp, NadA, NHBA were assessed by the Meningococcal Antigen Typing System (MATS) whereas PorA typing was performed by sequencing. The presence of at least one antigen with a Relative Potency (RP) greater than its MATS-positive bactericidal threshold RP value or the presence of PorA VR2 = 4 was considered to be predictive for a strain to be covered by the 4CMenB vaccine. RESULTS: The estimated 4CMenB strain coverage in Portugal was 67.9%. The percentage of strain coverage in each of the four epidemiological years ranged from 63.9% to 73.7%. Strains covered by one antigen represent 32.1% of the total of isolates, 29.2% of strains were covered by two antigens and 6.6% by three antigens. No strain had all the four antigens. Antigens that most contributed for coverage were NHBA and fHbp. Data from Portugal is in accordance with the MATS predicted strain coverage in five European countries (England and Wales, France, Germany, Italy and Norway) that pointed to 78% coverage for strains collected in the epidemiological year 2007-2008.
Assuntos
Infecções Meningocócicas/prevenção & controle , Vacinas Meningocócicas/imunologia , Adolescente , Adulto , Antígenos de Bactérias/metabolismo , Criança , Pré-Escolar , Feminino , Humanos , Imunogenicidade da Vacina , Lactente , Masculino , Infecções Meningocócicas/epidemiologia , Infecções Meningocócicas/imunologia , Pessoa de Meia-Idade , Modelos Biológicos , Portugal/epidemiologia , Adulto JovemRESUMO
BACKGROUND: The UK introduced 4CMenB-a multicomponent vaccine against serogroup B meningococcal disease-into the national infant immunisation programme in September, 2015. The Meningococcal Antigen Typing System (MATS) was used to estimate coverage by 4CMenB of invasive meningococcal group B isolates obtained during 2007-08 in England and Wales (MATS coverage). We aimed to repeat the MATS survey for invasive meningococcal group B isolates obtained during 2014-15, before 4CMenB introduction; compare strain coverage between 2007-08 and 2014-15; and investigate associations between MATS coverage, age, region, and disease outcomes. METHODS: Invasive serogroup B meningococcal isolates from cases in England, Wales, and Northern Ireland during 2014-15 were assayed using MATS and compared with 2007-08 data. MATS coverage was assessed by geographical region and age group. Clinical characteristics, risk factors, and outcomes were assessed according to MATS coverage for 2014-15 English cases. FINDINGS: In 2014-15, 165 of 251 (66%; 95% CI 52-80) meningococcal group B isolates were estimated by MATS to be covered by 4CMenB, compared with 391 of 535 (73%; 95% CI 57-87) in 2007-08. The proportion of MATS-positive isolates with one vaccine antigen increased from 23% (122 of 535) in 2007-08 to 31% (78 of 251) in 2014-15, whereas the proportion with more than one antigen fell from 50% (269 of 535) to 35% (87 of 251). This effect reflected changes in circulating strains, particularly ST-269 clonal complex strains. MATS coverage increased with age, varied by geographical region, and was associated with more severe disease. INTERPRETATION: In 2014-15, two-thirds of meningococcal group B isolates were predicted to be covered by 4CMenB. Temporal changes in MATS coverage underscore the need for continued monitoring of antigen expression and diversity, particularly in countries with 4CMenB programmes. FUNDING: Public Health England, GlaxoSmithKline.
Assuntos
Esquemas de Imunização , Infecções Meningocócicas/prevenção & controle , Vacinas Meningocócicas/administração & dosagem , Neisseria meningitidis Sorogrupo B/isolamento & purificação , Antígenos de Bactérias/sangue , Antígenos de Bactérias/imunologia , Pré-Escolar , Inglaterra , Humanos , Programas de Imunização , Lactente , Vacinas Meningocócicas/imunologia , Neisseria meningitidis Sorogrupo B/imunologia , Irlanda do Norte , País de GalesRESUMO
Neisseria meningitidis of serogroup B (MenB) is currently responsible for more than 70% of cases of invasive meningococcal disease (IMD) in Poland and Europe as a whole. The aim of this study was to estimate strain coverage of a multicomponent meningococcal serogroup B vaccine (4CMenB) in Poland; the meningococcal antigen typing system (MATS) was used to test a panel of 196 invasive MenB strains isolated in Poland in 2010 and 2011. The strains were also characterized by MLST and sequencing of porA, factor H-binding protein (fHbp), Neisserial heparin-binding antigen (nhba) and Neisserial adhesin A (nadA) genes. MATS and molecular data were analyzed independently and in combination. The MATS results predicted that 83.7% (95% CI: 78.6-91.0%) of isolates would be covered by the 4CMenB vaccine; 59.2% by one vaccine antigen, 19.9% by two and 4.6% by three antigens. Coverage by each antigen was as follows: fHbp 73.0% (95% CI: 68.9-77.5%), NHBA 28.6% (95% CI: 13.3-47.4%), NadA 1.0% (95% CI: 1.0-2.0%) and PorA 10.2%. Molecular analysis revealed that the most frequent clonal complexes (ccs) were cc32 (33.2%), cc18 (17.9%) and cc41/44 (15.8%) with estimated coverage of 98.5%, 88.6% and 93.5%, respectively. Consistent with findings for other European countries, our study predicts high coverage by the 4CMenB vaccine in Poland.
Assuntos
Vacinas Meningocócicas/imunologia , Neisseria meningitidis Sorogrupo B/classificação , Adesinas Bacterianas/genética , Adolescente , Adulto , Antígenos de Bactérias/genética , Antígenos de Bactérias/imunologia , Proteínas de Bactérias/genética , Técnicas de Tipagem Bacteriana , Criança , Pré-Escolar , Ensaio de Imunoadsorção Enzimática , Humanos , Lactente , Recém-Nascido , Infecções Meningocócicas/prevenção & controle , Tipagem de Sequências Multilocus , Polônia , Porinas/genética , Sorogrupo , Adulto JovemRESUMO
UNLABELLED: The pneumococcal Pilus-1 enhances attachment to epithelial cells in the respiratory tract and subsequent invasion. Pilus-1 expression is bi-stable and positively regulated by the RlrA transcriptional regulator. To delineate the role of pilus-1 in Experimental Otitis Media (EOM), we evaluated colonization and disease due to a Streptococcus pneumoniae (SP) wild type strain (Taiwan19F-14 wt) and its otherwise isogenic pilus-1 and pilus-2 deficient mutant (Taiwan19F-14 ΔPI-1/PI-2-) as well as potential for a chimeric protein (RrgB321) vaccine candidate for prevention of middle ear (ME) disease. METHODS: Chinchillas were challenged intranasally with either Taiwan19F-14 wt or Taiwan19F-14PI-1/PI-2 deficient mutant. ME status was assessed and direct cultures performed. New cohorts of animals were immunized with RrgB321 or alum. Intranasal challenge with Taiwan19F-14 wt [erythromycin susceptible E(S)] was performed. Subsequently, a second cohort of animals was immunized and challenged with either Taiwan19F-14 wt or a Pilus-1 over-expressing mutant [Taiwan19F-14+pMU1328_Pc-rlrA mutant; E resistant (R)] strain. Pilus-1 expression was analyzed in SP isolated from nasopharynx (NP) and ME fluids by flow cytometry. RESULTS: Culture positive EOM developed following challenge with either wild type SP (Taiwan19F-14) or its pilus-1 deficient mutant. Culture positive EOM developed following challenge with wild type in both RrgB321 immunized and control animals. Pilus-1 expression in ME fluids was significantly higher in controls compared to immunized chinchillas. In second cohort of immunized and control animals challenged with the over-expressing Pilus-1 mutant, delayed development of EOM in the immunized animals was observed. Pneumococci recovered from ME fluid of immunized animals were no longer E(R) signifying the loss of the pMU1328_Pc-rlrA plasmid. CONCLUSION: Pneumococcal pilus-1 was not essential for EOM. Regulation of Pilus-1 expression in ME fluids in the presence of anti RrgB321 antibody was essential for survival of S. pneumoniae. Pneumococci have evolved mechanisms of regulation of non-essential surface proteins to evade host defenses.
Assuntos
Proteínas de Bactérias/imunologia , Fímbrias Bacterianas/imunologia , Otite Média/imunologia , Otite Média/microbiologia , Vacinas Pneumocócicas/imunologia , Streptococcus pneumoniae/imunologia , Animais , Chinchila/imunologia , Chinchila/microbiologia , Contagem de Colônia Microbiana , Orelha Média/imunologia , Orelha Média/microbiologia , Imunização , Mutação/genética , Nasofaringe/imunologia , Nasofaringe/microbiologia , Otite Média/induzido quimicamente , Resultado do TratamentoRESUMO
BACKGROUND: Recent studies have identified Streptococcus pneumoniae serotype 11E and serovariant 11Av among isolates previously typed as 11A by classical serotyping methods. Serotype 11E and serovariant 11Av differ from serotype 11A by having totally or partially inactive wcjE, a gene in cps locus coding for an O-acetyl transferase. Serotype 11E is rare among carriage isolates but common among invasive isolates suggesting that it survives better during invasion. Aim of this work was to investigate the epidemiology of serotype 11A in a pneumococcal collection using a new serotyping approach based on High-Resolution Magic Angle Spinning Nuclear Magnetic Resonance (HR-MAS NMR) spectroscopy to distinguish serotypes 11A and 11E. METHODS: A collection of 48 (34 invasive and 14 carriage) S. pneumoniae isolates from Italy, previously identified as serotype 11A by the Quellung reaction, were investigated by wcjE sequencing, HR-MAS NMR spectroscopy and the reference flow cytometric serotyping assay (FCSA) based on monoclonal antibodies. RESULTS: HR-MAS NMR spectra from serotypes 11A and 11E showed different NMR peaks indicating that HR-MAS NMR could be used to distinguish these serotypes, although HR-MAS NMR could not distinguish serotype 11Av from serotype 11E unambiguously. Thirty-eight isolates were confirmed to be serotype 11A, 8 isolates with a mutated wcjE were serotype 11E, 1 isolate belonged to serovariant 11Av, and 1 isolate was a mixed population 11A/11Av. All 11E isolates were identified among invasive isolates. CONCLUSIONS: We proved that HR-MAS NMR can be of potential use for pneumococcal serotyping. The detection of serotype 11E among invasive isolates in our collection, supports previous epidemiological studies suggesting that mutations in wcjE can represent a mechanism promoting pneumococcal survival during invasion. The discovery of a spectrum of immunochemical diversity within established serotypes should stimulate efforts to develop new serotyping approaches.
Assuntos
Citometria de Fluxo/métodos , Espectroscopia de Ressonância Magnética/métodos , Sorogrupo , Sorotipagem/métodos , Streptococcus pneumoniae/classificação , Streptococcus pneumoniae/genética , Cápsulas Bacterianas/genética , Criança , Genótipo , HumanosRESUMO
Streptococcus pneumoniae is a major cause of morbidity and mortality worldwide. Pneumococcal strains are classified according to their capsular serotype and through a Multi-Locus Sequence Typing schema (MLST) based on the sequencing of seven housekeeping genes. However, strains with a defined allelic profile (Sequence Type, ST) can have different serotypes, suggesting that the micro-evolution of the MLST lineages leads to a considerable degree of phenotypic variability. To better investigate the genetic diversity within these lineages, we set-up and then validated an extended molecular typing schema (96-MLST) based on the sequencing of ninety-six genomic loci. 96-MLST loci were designed within core-genes in a collection of 39 complete genomes of S. pneumoniae. None of the capsular genes was included in the schema. When tested on a collection of 69 isolates, 96-MLST was able to partition strains with the same ST and diverse serotypes into groups that were homogenous for capsular serotype, improving our understanding of the evolution of epidemiologically relevant lineages. Phylogenetic sequence analysis showed that the capsular heterogeneity of three STs that were sampled more extensively could be traced back to a limited number of capsular switch events, indicating that changes of serotype occur occasionally during the short term expansion of clones. Moreover, a geographical structure of ST156 was identified, suggesting that the resolution guaranteed by this method is sufficient for phylogeographic studies. In conclusion, we showed that an extended typing schema was able to characterize the expansion of individual lineages in a complex species such as S. pneumoniae.
Assuntos
Cápsulas Bacterianas/genética , Tipagem de Sequências Multilocus , Sorotipagem , Streptococcus pneumoniae/classificação , Streptococcus pneumoniae/genética , Análise por Conglomerados , Loci Gênicos , Humanos , Filogenia , Streptococcus pneumoniae/isolamento & purificaçãoRESUMO
Streptococcus pneumoniae pili contribute to adherence and virulence. The regulation of pilus-1 expression is bistable, thus piliated strains contain a variable proportion of pilus-1-non-expressing bacteria. We investigated whether such proportion changes during colonization. Pilus-1-expressing bacteria were quantified in nasopharyngeal washes and pharyngeal tissues from mice that received intranasally bacterial populations with high (H), medium (M) or low (L) pilus-1 expression rates. In nasopharyngeal washes, at early colonization stages, pilus-1 expression rates decreased in H population, while increased in L and M; at later stages, expression rates decreased or remained low. Similar trends were observed in pharyngeal tissues, where, however, at late stages the expression rates were medium-high. In conclusion, pilus-1 is preferentially expressed at early colonization stages, consistently with its role in adhesion, while at later stages the expression is partially switched off. Pilus-1 expression rates observed in clinical isolates in vitro may not reflect the actual rates during colonization/infection.