RESUMO
Pertussis resurgence had been attributed to waning vaccine immunity and Bordetella pertussis adaptation to escape vaccine-induced immunity. Circulating bacteria differ genotypically from strains used in production of pertussis vaccine. Pertactin-deficient strains are highly prevalent in countries that use acellular vaccine (aP), suggesting strong aP-imposed selection of circulating bacteria. To corroborate this hypothesis, systematic studies on pertactin prevalence of infection in countries using whole-cell vaccine are needed. We provide pertussis epidemiologic data and molecular characterization of B. pertussis isolates from Buenos Aires, Argentina, during 2000-2017. This area used primary vaccination with whole-cell vaccine. Since 2002, pertussis case incidences increased at regular 4-year outbreaks; most cases were in infants <1 year of age. Of the B. pertussis isolates analyzed, 90.6% (317/350) contained the ptxP3-ptxA1-prn2-fim3-2 allelic profile. Immunoblotting and sequencing techniques detected only the 2 pertactin-deficient isolates. The low prevalence of pertactin-deficient strains in Argentina suggests that loss of pertactin gene expression might be driven by aP vaccine.
Assuntos
Proteínas da Membrana Bacteriana Externa/genética , Bordetella pertussis/classificação , Bordetella pertussis/genética , Deleção de Genes , Fatores de Virulência de Bordetella/genética , Coqueluche/epidemiologia , Coqueluche/microbiologia , Argentina/epidemiologia , Proteínas da Membrana Bacteriana Externa/imunologia , Bordetella pertussis/imunologia , Criança , Pré-Escolar , Genótipo , Humanos , Lactente , Vacina contra Coqueluche/administração & dosagem , Vacina contra Coqueluche/imunologia , Vigilância em Saúde Pública , Sorogrupo , Fatores de Virulência de Bordetella/imunologia , Coqueluche/diagnóstico , Coqueluche/prevenção & controleRESUMO
Pertussis is a severe respiratory disease caused by infection with the bacterial pathogen Bordetella pertussis The disease affects individuals of all ages but is particularly severe and sometimes fatal in unvaccinated young infants. Other Bordetella species cause diseases in humans, animals, and birds. Scientific, clinical, public health, vaccine company, and regulatory agency experts on these pathogens and diseases gathered in Buenos Aires, Argentina from 5 to 8 April 2016 for the 11th International Bordetella Symposium to discuss recent advances in our understanding of the biology of these organisms, the diseases they cause, and the development of new vaccines and other strategies to prevent these diseases. Highlights of the meeting included pertussis epidemiology in developing nations, genomic analysis of Bordetella biology and evolution, regulation of virulence factor expression, new model systems to study Bordetella biology and disease, effects of different vaccines on immune responses, maternal immunization as a strategy to prevent newborn disease, and novel vaccine development for pertussis. In addition, the group approved the formation of an International Bordetella Society to promote research and information exchange on bordetellae and to organize future meetings. A new Bordetella.org website will also be developed to facilitate these goals.
Assuntos
Bordetella pertussis/imunologia , Bordetella pertussis/fisiologia , Vacina contra Coqueluche/imunologia , Coqueluche/imunologia , Animais , Argentina/epidemiologia , Proteínas da Membrana Bacteriana Externa/imunologia , Humanos , Lactente , Vacinação , Fatores de Virulência de Bordetella/imunologia , Coqueluche/epidemiologia , Coqueluche/microbiologiaRESUMO
Pertussis has resurged during the last two decades in different countries. In particular in the 2010-2013 period large outbreaks were detected in US, Australia, UK and The Netherlands with significant mortality in infants. The epidemiological situation of pertussis points out the need to develop new vaccines and in this regard we previously developed a new vaccine based on outer membrane vesicles (OMVs) which have been shown to be safe and to induce protection in mice. Here we have further investigated the properties of OMVs vaccines; in particular we studied the contribution of pertussis toxin (PTx) and pertactin (Prn) in OMVs-mediated protection against pertussis. PTx-deficient OMVs and Prn-deficient OMVs were obtained from defective Bordetella pertussis mutants. The absence of PTx or Prn did compromise the protective capacity of the OMVs formulated as Tdap vaccine. Whereas the protective efficacy of the PTx-deficient OMVs in mice was comparable to Prn-deficient OMVs, the protective capacity of both of them was significantly impaired when it was compared with the wild type OMVs. Interestingly, using OMVs obtained from a B. pertussis strain which does not express any of the virulence factors but expresses the avirulent phenotype; we observed that the protective ability of such OMVs was lower than that of OMVs obtained from virulent B. pertussis phase. However, it was surprising that although the protective capacity of avirulent OMVs was lower, they were still protective in the used mice model. These results allow us to hypothesize that OMVs from avirulent phase shares protective components with all OMVs assayed. Using an immune proteomic strategy we identified some common components that could play an important role in protection against pertussis.
Assuntos
Proteínas da Membrana Bacteriana Externa/imunologia , Toxina Pertussis/imunologia , Vacina contra Coqueluche/imunologia , Fatores de Virulência de Bordetella/imunologia , Coqueluche/prevenção & controle , Animais , Antígenos de Bactérias/imunologia , Feminino , Camundongos Endogâmicos BALB CRESUMO
Pertussis is a serious infectious disease of the respiratory tract caused by the gram-negative bacteria Bordetella pertussis. There has been a reemergence of this disease within the population of several countries that have well established vaccination programs. Analyzes of clinical isolates suggest an antigenic divergence between the vaccine-based strains to the circulating strains. Although antibodies against P.69 are involved in the observed protective immunity, the sequences recognized as antigenic determinants in P.133, the precursor for P.69, P.3.4 and P.30, have not be determined. Here, the precise mapping of linear B-cell epitopes within the predicted P.133 pertactin sequences was accomplished using the SPOT-synthesis of peptide arrays onto cellulose membranes and screening with murine sera generated by vaccination with either the Pertussis cellular (miPc) or Pertussis acellular (miPa) vaccine. A total of 23 major epitopes were identified by sera from miPc vaccinated mice, while thirteen were identified by sera from miPa vaccinated mice. Of these epitopes, 12 epitopes were specifically identified by antibodies produced in response to the miPc vaccine and two were specific to the miPa vaccine. These epitopes were distributed throughout the pertactin sequence but a significant number were concentrated to the P.30 Prn segment. An analysis of the epitope correlation homologies indicated that the variations from the observed mutations in pertactin would not constitute a problem using these vaccines. In addition, the mapping of epitopes demonstrated a higher number of linear B-cell epitopes immunized with the Pc vaccine than the Pa vaccine.
Assuntos
Proteínas da Membrana Bacteriana Externa/imunologia , Mapeamento de Epitopos , Epitopos de Linfócito B/imunologia , Vacina contra Coqueluche/imunologia , Fatores de Virulência de Bordetella/imunologia , Sequência de Aminoácidos , Animais , Anticorpos Antibacterianos/sangue , Bordetella pertussis/imunologia , Reações Cruzadas , Camundongos , Dados de Sequência Molecular , Estrutura Terciária de Proteína , Vacinas Acelulares/imunologiaRESUMO
This study examined the immunogenic properties of the fusion protein fimbria 2 of Bordetella pertussis (Fim2)-cholera toxin B subunit (CTB) in the intranasal murine model of infection. To this end B. pertussis Fim2 coding sequence was cloned downstream of the cholera toxin B subunit coding sequence. The expression and assembly of the fusion protein into pentameric structures (CTB-Fim2) were evaluated by SDS-PAGE and monosialotetrahexosylgaglioside (GM1-ganglioside) enzyme-linked immunosorbent assay (ELISA). To evaluate the protective capacity of CTB-Fim2, an intraperitoneal or intranasal mouse immunization schedule was performed with 50 µg of CTB-Fim2. Recombinant (rFim2) or purified (BpFim2) Fim2, CTB, and phosphate-buffered saline (PBS) were used as controls. The results showed that mice immunized with BpFim2 or CTB-Fim2 intraperitoneally or intranasally presented a significant reduction in bacterial lung counts compared to control groups (P < 0.01 or P < 0.001 , resp.). Moreover, intranasal immunization with CTB-Fim2 induced significant levels of Fim2-specific IgG in serum and bronchoalveolar lavage (BAL) and Fim2-specific IgA in BAL. Analysis of IgG isotypes and cytokines mRNA levels showed that CTB-Fim2 results in a mixed Th1/Th2 (T-helper) response. The data presented here provide support for CTB-Fim2 as a promising recombinant antigen against Bordetella pertussis infection.
Assuntos
Antígenos de Bactérias/imunologia , Bordetella pertussis/imunologia , Toxina da Cólera/imunologia , Proteínas de Fímbrias/imunologia , Imunização , Proteínas Recombinantes de Fusão/imunologia , Fatores de Virulência de Bordetella/imunologia , Coqueluche/microbiologia , Coqueluche/prevenção & controle , Animais , Líquido da Lavagem Broncoalveolar , Citocinas/genética , Citocinas/metabolismo , Feminino , Imunidade Humoral , Imunoglobulina A/sangue , Imunoglobulina G/sangue , Pulmão/metabolismo , Pulmão/microbiologia , Pulmão/patologia , Camundongos Endogâmicos BALB C , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas Recombinantes de Fusão/isolamento & purificação , Coqueluche/sangue , Coqueluche/imunologiaRESUMO
Whooping cough remains a health problem despite high vaccination coverage. It has been recommended that development of new strategies provide long-lasting immunity. The aim of this work was to evaluate the potential of proteoliposomes (PL) extracted from Bordetella pertussis as a vaccine candidate against whooping cough. The size of the B. pertussis PL was estimated to be 96.7 ± 50.9 nm by Scanning Correlation Spectroscopy and the polydispersity index was 0.268. Western blots using monoclonal antibodies revealed the presence of pertussis toxin, pertactin, and fimbriae 3. The Limulus Amebocyte Lisate (LAL) assay showed endotoxin levels lower than those reported for whole cell pertussis licensed vaccines, while the Pyrogen Test indicated 75 ng/mL/Kg. The PL showed high protection capacity in mouse challenge models. There was 89.7% survival in the intracerebral challenge and total reduction of the number of CFU in the intranasal challenge. No significant differences (p > 0.05) were observed between mice immunized with B. pertussis PL and the Cuban DTwP vaccine, whichever challenge model used. These results encouraged us to continue the development of the B. pertussis PL as a component of a new combined vaccine formulated with tetanus and diphtheria toxoids or as a booster dose for adolescents and adults.
Assuntos
Vacinas Bacterianas/imunologia , Bordetella pertussis/imunologia , Proteolipídeos/imunologia , Coqueluche/prevenção & controle , Animais , Proteínas da Membrana Bacteriana Externa/imunologia , Vacinas Bacterianas/administração & dosagem , Feminino , Proteínas de Fímbrias/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Toxina Pertussis/imunologia , Fatores de Virulência de Bordetella/imunologia , Coqueluche/imunologiaRESUMO
Filamentous haemagglutinin adhesin (FHA) is an important virulence factor from Bordetella pertussis related to the adhesion and spread of the bacteria through the respiratory tract. Three distinct domains have been characterized in mature FHA, and among them, the FHA(442-863) fragment was suggested to be responsible for the heparin-binding activity. In this study, we cloned the gene encoding the HEP fragment (FHA(430-873)) in a Lactobacillus casei-inducible expression vector based on the lactose operon. The recombinant bacteria, transformed with the resulting construct (L. casei-HEP), were able to express the heterologous protein depending on the sugar added to the culture. Subcutaneous inoculation of L. casei-HEP in Balb/C mice, using the cholera toxin B subunit as adjuvant, induced systemic anti-HEP antibodies that were able to inhibit in vitro erythrocyte haemagglutination induced by FHA. This is the first example of a B. pertussis antigen produced in lactic acid bacteria and opens new perspectives for alternative vaccine strategies against whooping cough.
Assuntos
Adesinas Bacterianas/imunologia , Anticorpos Antibacterianos/imunologia , Bordetella pertussis/imunologia , Hemaglutinação/imunologia , Lacticaseibacillus casei/genética , Fatores de Virulência de Bordetella/imunologia , Coqueluche/prevenção & controle , Adesinas Bacterianas/genética , Adjuvantes Imunológicos , Animais , Anticorpos Antibacterianos/genética , Feminino , Imunidade nas Mucosas , Óperon Lac , Camundongos , Camundongos Endogâmicos BALB C , Vacina contra Coqueluche/imunologia , Estrutura Terciária de Proteína , Proteínas Recombinantes/imunologia , Transformação Bacteriana , Vacinas Sintéticas/imunologia , Fatores de Virulência de Bordetella/genéticaRESUMO
Filamentous hemagglutinin adhesin (FHA) is important for the adherence of Bordetella pertussis to the host ciliary epithelial cells of the respiratory tract. Several binding domains have been characterized in the FHA molecule. For example, an putative heparin-binding domain of FHA was previously located in the FHA(442-863) region. In this work, the HEP fragment, corresponding to FHA(430-873) was amplified by PCR and subcloned in an Escherichia coli expression plasmid. Purified recombinant HEP was used to produce polyclonal antibodies in mice that were able to recognize HEP and FHA in ELISA and in Western-blot assays. Although recombinant HEP displayed low ability to bind heparin and no hemagglutination activity, the anti-HEP antibodies were able to inhibit FHA mediated hemagglutination activity in goose erythrocytes. These results indicate that other amino acid residues that are not present in the FHA(430-873) fragment may be necessary for heparin binding. Further studies to address the immunogenic response against HEP are also required.
Assuntos
Adesinas Bacterianas/genética , Adesinas Bacterianas/imunologia , Anticorpos Antibacterianos/imunologia , Bordetella pertussis/imunologia , Hemaglutinação/imunologia , Hemaglutininas/genética , Hemaglutininas/imunologia , Fatores de Virulência de Bordetella/genética , Fatores de Virulência de Bordetella/imunologia , Adesinas Bacterianas/química , Animais , Especificidade de Anticorpos , Clonagem Molecular , Eritrócitos/imunologia , Eritrócitos/microbiologia , Feminino , Hemaglutininas/química , Heparina/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Estrutura Terciária de Proteína , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Fatores de Virulência de Bordetella/química , Coqueluche/prevenção & controleRESUMO
BACKGROUND: New vaccination strategies are needed to control the increasing problem of pertussis in teenagers and adults. AIM: To determine the immunogenicity and reactogenicity of a diphtheria-tetanus-acellular pertussis (dTpa) vaccine with reduced antigen content. MATERIAL AND METHODS: A single dose of the dTpa vaccine was administered to 60 children 10 to 11 years old and 60 healthy adults. At the moment of vaccination and one month later, antibody levels were measured against 3 B pertussis antigens: anti-pertussis toxin (PT), anti-pertactin (PRN) and anti-filamentous hemagglutinin (FHA), as well as anti-tetanus and anti-diphtheria antibodies. Local and general symptoms were registered during 14 days following vaccine administration. RESULTS: Antibody response for PT, FHA and PRN was 98.3%, 100% and 100% in adults and 98.2%, 100% and 98.2% in children. Seropositivity for all pertussis antigens was 100% in adults and in children one month after vaccination. Geometric mean titers (GMT) significantly increased in adults and children. The seroprotection level achieved for tetanus and diphtheria antibodies one month after vaccination was 96.7% for adults and 100% for children, respectively. No serious adverse events were reported during the study. Among local symptoms pain was the most frequent (88-90%), but it was mostly mild or moderate. Solicited general symptoms observed for children and adults, respectively, included headache (37% and 53%), fatigue (18% and 35%) gastrointestinal symptoms (18% and 25%) and fever (8% and 3%). Only one vaccinee had fever above 39 degrees C. CONCLUSIONS: The dTpa vaccine showed an adequate safety profile and induced an intense immunological response to all antigens in adults and children aged 10-11.
Assuntos
Anticorpos Antibacterianos/sangue , Antígenos de Bactérias/imunologia , Vacinas contra Difteria, Tétano e Coqueluche Acelular/imunologia , Adulto , Antígenos de Bactérias/análise , Criança , Vacinas contra Difteria, Tétano e Coqueluche Acelular/efeitos adversos , Feminino , Seguimentos , Hemaglutininas , Humanos , Masculino , Pessoa de Meia-Idade , Fatores de Virulência de Bordetella/imunologiaRESUMO
The recent development of acellular pertussis vaccines has been a significant improvement in the conventional whole-cell diphtheria-pertussis-tetanus toxoid vaccines, but high production costs will limit its widespread use in developing countries. Since Mycobacterium bovis BCG vaccination against tuberculosis is used in most developing countries, a recombinant BCG-pertussis vaccine could be a more viable alternative. We have constructed recombinant BCG (rBCG) strains expressing the genetically detoxified S1 subunit of pertussis toxin 9K/129G (S1PT) in fusion with either the beta-lactamase signal sequence or the whole beta-lactamase protein, under control of the upregulated M. fortuitum beta-lactamase promoter, pBlaF*. Expression levels were higher in the fusion with the whole beta-lactamase protein, and both were localized to the mycobacterial cell wall. The expression vectors were relatively stable in vivo, since at two months 85% of the BCG recovered from the spleens of vaccinated mice maintained kanamycin resistance. Spleen cells from rBCG-S1PT-vaccinated mice showed elevated gamma interferon (IFN-gamma) and low interleukin-4 (IL-4) production, as well as increased proliferation, upon pertussis toxin (PT) stimulation, characterizing a strong antigen-specific Th1-dominant cellular response. The rBCG-S1PT strains induced a low humoral response against PT after 2 months. Mice immunized with rBCG-S1PT strains displayed high-level protection against an intracerebral challenge with live Bordetella pertussis, which correlated with the induction of a PT-specific cellular immune response, reinforcing the importance of cell-mediated immunity in the protection against B. pertussis infection. Our results suggest that rBCG-expressing pertussis antigens could constitute an effective, low-cost combined vaccine against tuberculosis and pertussis.