RESUMEN
Using culture filtrate Ag-specific mAbs generated from mycobacteria-infected H-2b haplotype mice, we have previously identified three genes in the Mycobacterium tuberculosis genome, encoding proteins homologous to the periplasmic ATP-binding cassette phosphate-binding receptor PstS of the phosphate-specific transport system of E. coli. To define the potential vaccinal properties of these phosphate-binding proteins, female C57BL/6 mice were injected i.m. with plasmid DNA encoding PstS-1, PstS-2, or PstS-3 proteins from M. tuberculosis and immunogenicity and protective efficacy against i.v. challenge with M. tuberculosis H37Rv was analyzed. Significant levels of highly Ag-specific Abs and Th1-type cytokines IL-2 and IFN-gamma could be detected following vaccination with each of the three genes. However, only mice vaccinated with PstS-3 DNA demonstrated significant and sustained reduction in bacterial CFU numbers in spleen and lungs for 3 mo after M. tuberculosis challenge, as compared with CFU counts in mice vaccinated with control DNA. Vaccination with PstS-2 DNA induced a modest reduction in CFU counts in spleen only, whereas vaccination with PstS-1 DNA was completely ineffective in reducing bacterial multiplication. In conclusion, our results indicate that DNA vaccination is a powerful and easy method for comparative screening of potentially protective Ags from M. tuberculosis and that the PstS-3 protein is a promising new subunit vaccine candidate.
Asunto(s)
Transportadoras de Casetes de Unión a ATP/inmunología , Antígenos Bacterianos/inmunología , Proteínas Bacterianas , Vacunas Bacterianas/inmunología , Proteínas de Escherichia coli , Mycobacterium tuberculosis/inmunología , Proteínas de Unión Periplasmáticas , Fosfatos/metabolismo , Tuberculosis/inmunología , Vacunas de ADN/inmunología , Transportadoras de Casetes de Unión a ATP/genética , Animales , Anticuerpos Antibacterianos/biosíntesis , Antígenos Bacterianos/genética , Vacunas Bacterianas/genética , Reacciones Cruzadas , Femenino , Interleucina-2/biosíntesis , Pulmón/inmunología , Pulmón/microbiología , Ratones , Ratones Endogámicos C57BL , Mycobacterium/inmunología , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/crecimiento & desarrollo , Proteínas de Unión a Fosfato , Plásmidos/inmunología , Bazo/inmunología , Bazo/metabolismo , Bazo/microbiología , Tuberculosis/microbiología , Tuberculosis/prevención & control , Vacunas de ADN/genéticaRESUMEN
Two bacillus Calmette-Guérin (BCG)-susceptible mouse strains, BALB/c and C57BL/6, were infected intravenously with Mycobacterium intracellulare, M. avium or M. scrofulaceum and monitored during 3 months for mycobacterial replication and antibody and Th1-type cytokine production in response to cytoplasmic and secreted antigens from M. bovis BCG. Whereas initial colony-forming unit (CFU) counts of M. intracellulare and M. avium were higher in lungs than in spleen, the opposite was observed for M. scrofulaceum. Mycobacterium intracellulare was the most virulent species and its replication could not be controlled in either mouse strain. It also induced the strongest antibody response. Mycobacterium avium was eliminated in both mouse strains and M. scrofulaceum finally was eliminated in C57BL/6 but multiplied in spleen from BALB/c mice. Significant sustained interleukin-2 and interferon-gamma production towards BCG antigens was only found in M. scrofulaceum infection. As in BCG-vaccination, M. scrofulaceum-infected C57BL/6 mice demonstrated a higher response towards whole BCG culture filtrate, BCG extract and purified antigen 85 complex (Ag85) from BCG than did BALB/c mice. The data suggest that the presence of M. scrofulaceum in the environment may possibly interfere in genetically predisposed subjects with BCG vaccine and its protective efficacy against M. tuberculosis.
Asunto(s)
Anticuerpos Antibacterianos/biosíntesis , Antígenos Bacterianos/inmunología , Vacuna BCG/inmunología , Complejo Mycobacterium avium/inmunología , Mycobacterium avium/inmunología , Mycobacterium scrofulaceum/inmunología , Mycobacterium/patogenicidad , Células TH1/inmunología , Animales , Reacciones Cruzadas , Interferón gamma/biosíntesis , Interleucina-2/biosíntesis , Pulmón/microbiología , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Mycobacterium/crecimiento & desarrollo , Mycobacterium/inmunología , Bazo/inmunologíaRESUMEN
BALB/c and C57BL/6 mice were injected intramuscularly with plasmid DNA encoding the three components of the immunodominant 30-32 kDa antigen 85 complex (Ag85A, Ag85B, and Ag85C) from Mycobacterium tuberculosis culture filtrate, in order to investigate the utility of nucleic acid vaccination for induction of immune responses against mycobacterial antigens. Ag85A and Ag85B encoding plasmids induced a robust Th1-like response towards native Ag85, characterized by elevated levels of interleukin (IL)-2, interferon-gamma, and TNF-alpha. Levels of IL-4, IL-6, and IL-10 were low or undetectable. Plasmid encoding Ag85C was not effective. Cytotoxic T cell activity was also generated in in vitro restimulated splenocyte cultures from Ag85A and Ag85B DNA vaccinated mice. Finally, Ag85A and Ag85B DNA vaccination conferred significant protection against mycobacterial replication in lungs from B6 mice, subsequently challenged. Therefore, this technique may be useful for the definition of protective antigens of M. tuberculosis and the development of a more effective tuberculosis vaccine.
Asunto(s)
Antígenos Bacterianos/inmunología , Vacuna BCG/inmunología , Mycobacterium bovis/inmunología , Vacunas de ADN/inmunología , Animales , Anticuerpos Antibacterianos/biosíntesis , Antígenos Bacterianos/genética , Vacuna BCG/administración & dosificación , Vacuna BCG/genética , División Celular/efectos de los fármacos , Citocinas/biosíntesis , ADN Bacteriano/inmunología , Inyecciones Intramusculares , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Mycobacterium bovis/citología , Linfocitos T Citotóxicos/inmunología , Células TH1/inmunología , Tuberculosis/inmunología , Tuberculosis/prevención & control , Vacunas de ADN/administración & dosificación , Vacunas de ADN/genéticaRESUMEN
CD8+ T cells are essential for protection against mycobacteria, as is clearly demonstrated by the fatal outcome of experimental infection of beta-2 microglobulin knockout mice. However, the mechanisms and antigens (Ags) leading to CD8+ T-cell activation and regulation have been poorly characterized. Here we show that, upon immunization of major histocompatibility complex (MHC)-congenic mice with Mycobacterium bovis bacillus Calmette-Guérin (BCG), a cytotoxic response against BCG culture filtrate (CF) Ags (CFAgs) is induced in H-2b and H-2bxd haplotypes but not in H-2d haplotype. This response is mediated by CD8+ T cells and absolutely requires the activation of CD4+ T cells and their secretion of interleukin 2. The lack of cytotoxic response in H-2d mice cannot be explained by impaired cytokine production or by a defect in Ag presentation by H-2d macrophages. Using the MHC class I mutant B6.C-H-2bm13 mouse strain, we demonstrate that cytotoxic T lymphocytes (CTLs) recognize CFAgs exclusively in association with D(b) molecules. These Ags are cross-reactive in mycobacteria, since BCG-induced CTLs also recognize macrophages pulsed with CF from Mycobacterium tuberculosis H37Rv and H37Ra and from two virulent strains of M. bovis. Moreover, immunization with Mycobacterium kansasii induces CTLs able to lyse macrophages pulsed with BCG CF. Finally, we have found that these Ags can be characterized as hydrophilic proteins, since they do not bind to phenyl-Sepharose CL-4B. Our results indicate that MHC-linked genes exert a profound influence on the generation of CD8+ CTLs following BCG vaccination.
Asunto(s)
Antígenos Bacterianos/inmunología , Citotoxicidad Inmunológica , Activación de Linfocitos , Mycobacterium bovis/crecimiento & desarrollo , Mycobacterium bovis/inmunología , Linfocitos T Citotóxicos/inmunología , Linfocitos T Citotóxicos/microbiología , Animales , Presentación de Antígeno/genética , Antígenos Bacterianos/aislamiento & purificación , Vacuna BCG/inmunología , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD8-positivos , Sistema Libre de Células/inmunología , Reacciones Cruzadas , Medios de Cultivo , Epítopos/inmunología , Femenino , Antígenos H-2/genética , Antígenos H-2/inmunología , Interleucina-2/metabolismo , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C3H , Ratones Endogámicos C57BL , Ratones Endogámicos DBA , Mycobacterium bovis/químicaRESUMEN
Tuberculosis is the most widespread and lethal infectious disease affecting humans. Immunization of mice with plasmid DNA constructs encoding one of the secreted components of Mycobacterium tuberculosis, antigen 85 (Ag85), induced substantial humoral and cell-mediated immune responses and conferred significant protection against challenge with live M. tuberculosis and M. bovis bacille Calmette-Guérin (BCG). These results indicate that immunization with DNA encoding a mycobacterial antigen provides an efficient and simple method for generating protective immunity and that this technique may be useful for defining the protective antigens of M. tuberculosis, leading to the development of a more effective vaccine.
Asunto(s)
Antígenos Bacterianos/genética , Vacuna BCG/inmunología , ADN Bacteriano/inmunología , Mycobacterium tuberculosis/inmunología , Tuberculosis/prevención & control , Animales , Anticuerpos Antibacterianos/sangre , Formación de Anticuerpos , Antígenos Bacterianos/inmunología , Vacuna BCG/administración & dosificación , Citocinas/inmunología , ADN Bacteriano/administración & dosificación , Modelos Animales de Enfermedad , Inmunidad Celular , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Mycobacterium tuberculosis/genética , Linfocitos T/inmunología , Células Tumorales CultivadasRESUMEN
TH1 cytokine secretion was examined in response to synthetic peptides of the 85A component of the major secreted, fibronectin-binding antigen 85 complex from Mycobacterium tuberculosis in seven different mouse strains infected with live M. bovis BCG. Twenty-eight overlapping 20-mer peptides covering the complete mature 295-amino-acid (AA) protein were synthesized. Significant interleukin-2 (IL-2) and gamma interferon (IFN-gamma) secretion could be measured following in vitro stimulation of spleen cells with these peptides. H-2d haplotype mice reacted preferentially against the amino-terminal half of the protein, i.e., against peptide 5 (AA 41 to 60) and especially against peptide 11 (AA 101 to 120), which contained an I-Ed binding motif. H-2b haplotype mice, on the other hand, reacted against peptides from both amino- and carboxy-terminal halves of the protein, peptide 25 (AA 241 to 260) being the most potent stimulator of IL-2 and IFN-gamma production. (BALB/c x C57BL/6)F1 animals with the H-2d/b haplotype weakly recognized peptides specific for both parental lines. Finally, CBA/J (H-2k) and major histocompatibility complex class II mutant B6.C.bm12 mice, carrying a mutant I-A beta bm12 allele on an H-2b background, reacted only very weakly to the 85A peptides. Reactive T cells isolated from lungs of BCG-infected H-2b haplotype mice recognized the same epitopes as spleen cells, especially peptide 25. These data confirm previous findings regarding the powerful IL-2 and IFN-gamma-inducing properties of antigen 85 during infection with live M. bovis BCG.
Asunto(s)
Antígenos Bacterianos/genética , Mycobacterium bovis/inmunología , Linfocitos T Colaboradores-Inductores/inmunología , Secuencia de Aminoácidos , Animales , Citocinas/biosíntesis , Citocinas/metabolismo , Epítopos/genética , Femenino , Pulmón/inmunología , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Endogámicos CBA , Ratones Endogámicos DBA , Datos de Secuencia Molecular , Mycobacterium bovis/genética , Mapeo Peptídico , Péptidos/genética , Péptidos/inmunología , Especificidad de la Especie , Bazo/inmunología , Tuberculosis/inmunología , Tuberculosis/prevención & controlRESUMEN
We have collected sera from 4053 patients of different parts of Belgium. Sera were randomly selected whatever the kind of pathology. Anti-Helicobacter (Campylobacter) pylori IgG were determined with an ELISA technique using whole formalized bacteria. The results suggest that the mean antibody titres differ between various areas, with an overall higher prevalence in the north-western part of the country.