RESUMEN
Culture filtrate from Mycobacterium tuberculosis contains protective antigens of relevance for the generation of a new antituberculosis vaccine. We have identified two previously uncharacterized M. tuberculosis proteins (TB7.3 and TB10.4) from the highly active low-mass fraction of culture filtrate. The molecules were characterized, mapped in a two-dimensional electrophoresis reference map of short-term culture filtrate, and compared with another recently identified low-mass protein, CFP10 (F. X. Berthet, P. B. Rasmussen, I. Rosenkrands, P. Andersen, and B. Gicquel. Microbiology 144:3195-3203, 1998), and the well-described ESAT-6 antigen. Genetic analyses demonstrated that TB10.4 as well as CFP10 belongs to the ESAT-6 family of low-mass proteins, whereas TB7.3 is a low-molecular-mass protein outside this family. The proteins were expressed in Escherichia coli, and their immunogenicity was tested in cultures of peripheral blood mononuclear cells from human tuberculosis (TB) patients, Mycobacterium bovis BCG-vaccinated donors, and nonvaccinated donors. The two ESAT-6 family members, TB10.4 and CFP10, were very strongly recognized and induced gamma interferon release at the same level (CFP10) as or at an even higher level (TB10.4) than ESAT-6. The non-ESAT-6 family member, TB7.3, for comparison, was recognized at a much lower level. CFP10 was found to distinguish TB patients from BCG-vaccinated donors and is, together with ESAT-6, an interesting candidate for the diagnosis of TB. The striking immunodominance of antigens within the ESAT-6 family is discussed, and hypotheses are presented to explain this targeting of the immune response during TB infection.
Asunto(s)
Antígenos Bacterianos/química , Proteínas Bacterianas/inmunología , Mycobacterium tuberculosis/inmunología , Linfocitos T/inmunología , Secuencia de Aminoácidos , Animales , Antígenos Bacterianos/genética , Vacuna BCG/genética , Vacuna BCG/aislamiento & purificación , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Secuencia de Bases , Cartilla de ADN/genética , Humanos , Epítopos Inmunodominantes/química , Epítopos Inmunodominantes/genética , Técnicas In Vitro , Interferón gamma/biosíntesis , Activación de Linfocitos , Datos de Secuencia Molecular , Peso Molecular , Mycobacterium tuberculosis/genética , Conejos , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/inmunología , Homología de Secuencia de Aminoácido , Tuberculosis/inmunología , Tuberculosis/prevención & controlRESUMEN
The original Mycobacterium bovis Bacillus Calmette Guérin vaccine strain has developed into several different substrains which have been used for production of BCG vaccines throughout the world since 1921. Based on the latest genetic and antigenic knowledge, as well as the early literature reports on BCG vaccination, we are able to fit the different pieces of the BCG puzzle together and outline the origin of the different substrains of M. bovis BCG. The BCG vaccine substrains analysed demonstrate two distinct patterns, with an abrupt change consisting of a loss of several genes and altered biochemical characteristics in strains originating from Institut Pasteur after 1927. Further evidence from the literature is provided that a change occurred in virulence of the BCG parent strain at Institut Pasteur in the late 1920s. Based on this information a genealogical tree is proposed and discussed.
Asunto(s)
Vacuna BCG/historia , Mycobacterium bovis/inmunología , Tuberculosis/historia , Vacuna BCG/inmunología , Historia del Siglo XX , Humanos , Mycobacterium bovis/genéticaRESUMEN
Two antigens from Mycobacterium tuberculosis, ESAT-6 and MPT64, elicited delayed-type hypersensitivity (DTH) skin responses in outbred guinea pigs infected with M. tuberculosis by the aerosol and intravenous routes but not those sensitized with M. bovis BCG or M. avium. The DTH epitope of ESAT-6 was mapped to the C terminus. Nonresponders to the individual antigens were found, but all animals responded to a combination of ESAT-6 and MPT64 or their respective minimal target peptides. Correspondingly, these molecules could form the basis of a new skin test for tuberculosis.
Asunto(s)
Antígenos Bacterianos/inmunología , Hipersensibilidad Tardía/etiología , Mycobacterium tuberculosis/inmunología , Animales , Proteínas Bacterianas , Femenino , Cobayas , Pruebas CutáneasRESUMEN
Mycobacterium tuberculosis secretes several proteins into the extracellular environment, some of which are restricted to the M. tuberculosis complex. One of these antigens is MPT64. Recently, the authors showed that native as well as recombinant MPT64 is able to distinguish between an M. tuberculosis infection and a BCG Danish 1331 vaccination. Improved distinction between tuberculin purified protein derivative (PPD) sensitivity conferred by an M. tuberculosis infection and that induced by a BCG vaccination or infection with environmental mycobacteria would be useful in the control of tuberculosis. In this study, the authors report the mapping and characterization of a Dth-inducing epitope by the use of synthetic peptides in guinea-pigs vaccinated with BCG Danish 1331 or Tokyo. Studies with overlapping synthetic peptides have pinpointed the biological activity to a single Dth-inducing epitope at the carboxyterminal region of MPT64 consisting of 15 residues between amino acids Gly-173 and Ala-187, the core epitope (CE15). A fine mapping using truncated versions of CE15 indicates the epitope is restricted to 13 residues between amino acids Val-174 to Glu-186. However, the optimal Dth reactivity is obtained by CE15. Different modifications of CE15 revealed that a lysine tree construction improves the skin reactivity to a maximum level approaching that of the reactivity to tuberculin PPD.
Asunto(s)
Antígenos Bacterianos , Epítopos , Hipersensibilidad Tardía , Mycobacterium tuberculosis/inmunología , Secuencia de Aminoácidos , Animales , Antígenos Bacterianos/genética , Vacuna BCG/inmunología , Mapeo Epitopo , Epítopos/genética , Femenino , Cobayas , Indicadores y Reactivos , Datos de Secuencia Molecular , Mycobacterium tuberculosis/genética , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/inmunología , Pruebas Cutáneas/métodos , Especificidad de la Especie , Tuberculina/inmunología , Prueba de Tuberculina/métodos , Tuberculosis Pulmonar/diagnóstico , Tuberculosis Pulmonar/inmunologíaRESUMEN
The recall of long-lived immunity in a mouse model of tuberculosis (TB) is defined as an accelerated accumulation of reactive T cells in the target organs. We have recently identified Ag 85B and a 6-kilodalton early secretory antigenic target, designated ESAT-6, as key antigenic targets recognized by these cells. In the present study, preferential recognition of the ESAT-6 Ag during the recall of immunity was found to be shared by five of six genetically different strains of mice. Overlapping peptides spanning the sequence of ESAT-6 were used to map two T cell epitopes on this molecule. One epitope recognized in the context of H-2b,d was located in the N-terminal part of the molecule, whereas an epitope recognized in the context of H-2a,k covered amino acids 51 to 60. Shorter versions of the N-terminal epitope allowed the precise definition of a 13-amino acid core sequence recognized in the context of H-2b. The peptide covering the N-terminal epitope was immunogenic, and a T cell response with the same fine specificity as that induced during TB infection was generated by immunization with the peptide in IFA. In the C57BL/6j strain, this single epitope was recognized by an exceedingly high frequency of splenic T cells (approximately 1:1000), representing 25 to 35% of the total culture filtrate-reactive T cells recruited to the site of infection during the first phase of the recall response. These findings emphasize the relevance of this Ag in the immune response to TB and suggest that immunologic recognition in the first phase of infection is a highly restricted event dominated by a limited number of T cell clones.
Asunto(s)
Antígenos Bacterianos , Memoria Inmunológica , Mycobacterium tuberculosis/inmunología , Linfocitos T/inmunología , Secuencia de Aminoácidos , Animales , Antígenos Bacterianos/genética , Proteínas Bacterianas , Epítopos/genética , Femenino , Inmunización , Ratones , Ratones Endogámicos , Datos de Secuencia Molecular , Mycobacterium tuberculosis/genética , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/inmunología , Mapeo Peptídico , Proteínas Recombinantes/genética , Proteínas Recombinantes/inmunología , Especificidad de la Especie , Tuberculosis/inmunologíaRESUMEN
ESAT-6 is a secreted protein present in the short-term culture filtrate of Mycobacterium tuberculosis after growth on a synthetic Sauton medium. ESAT-6 has recently been demonstrated to induce strong T-cell responses in a mouse model of memory immunity after infection with M. tuberculosis. In Western blotting (immunoblotting), the monoclonal antibody HYB76-8, reacting with ESAT-6, gave a 6-kDa region was observed in filtrates from four of eight substrains of M. bovis BCG that produced high levels of MPB64, while no band occurred in the 6-kDa region with any of these BCG substrains. Southern blotting and PCR experiments with genomic mycobacterial DNA showed the presence of the esat-6 gene in reference strains and clinical isolates of M. tuberculosis as well as in virulent M. bovis. The esat-6 gene could not be demonstrated in any of the eight substrains of M. bovis BCG tested by these techniques. Two gene deletions that distinguish M. bovis BCG from virulent M. bovis have thus now been demonstrated. Deletion of mpb64 affects four of the eight substrains tested; deletion of esat-6 affects all of them. The reaction of HYB76-8 AT 26 kDa with four of the BCG substrains was demonstrated to result from cross-reactivity with MPB64. HYB76-8 was also shown to cross-react with the A, B, and C components of the antigen 85 complex and MPT51.
Asunto(s)
Antígenos Bacterianos/genética , Genes Bacterianos , Mycobacterium bovis/genética , Mycobacterium tuberculosis/genética , Secuencia de Aminoácidos , Anticuerpos Antibacterianos/inmunología , Anticuerpos Monoclonales/inmunología , Antígenos Bacterianos/inmunología , Proteínas Bacterianas , Secuencia de Bases , Western Blotting , Reacciones Cruzadas , Ensayo de Inmunoadsorción Enzimática , Humanos , Espectrometría de Masas , Datos de Secuencia Molecular , Peso Molecular , Mycobacterium bovis/patogenicidad , Mycobacterium tuberculosis/patogenicidad , Estándares de Referencia , Especificidad de la Especie , Tuberculosis/microbiología , VirulenciaRESUMEN
The gene encoding the immunogenic protein MPT64 found in culture filtrates of Mycobacterium tuberculosis H37Rv was expressed in Escherichia coli K-12 and purified as a recombinant protein. The purified recombinant MPT64 elicited delayed-type hypersensitivity (DTH) in outbred guinea pigs sensitized with Mycobacterium bovis BCG Tokyo. The skin reactions were comparable to those obtained with native MPT64. No skin reactions were observed when either recombinant MPT64 or native MPT64 was used in guinea pigs sensitized with M. bovis BCG Danish 1331. Amino- and carboxy-terminal deletion mutants of MPT64 were purified as fusion proteins for the mapping of DTH-inducing epitopes on recombinant MPT64 by use of the guinea pig skin test model. The part of the molecule responsible for the biological activity was located at the carboxy-terminal end. Further studies with overlapping synthetic peptides have pinpointed the biological activity at a single DTH-inducing epitope consisting of 15 residues between amino acids Gly-173 and Ala-187. Screening by PCR of 56 clinical isolates of M. tuberculosis from Danish and Tanzanian patients demonstrated the presence of mpt64 in all of the strains. These results point to MPT64 as a possible candidate for a skin test reagent specific for diagnosis of human tuberculosis.
Asunto(s)
Proteínas Bacterianas/inmunología , Epítopos , Hipersensibilidad Tardía/inmunología , Mycobacterium tuberculosis/inmunología , Secuencia de Aminoácidos , Animales , Escherichia coli/genética , Femenino , Cobayas , Humanos , Datos de Secuencia Molecular , Proteínas Recombinantes de Fusión/biosíntesis , Pruebas Cutáneas , Tuberculina/inmunología , Tuberculosis/diagnósticoRESUMEN
The gene of the immunogenic protein MPT64 found in culture filtrates of Mycobacterium tuberculosis H37Rv was cloned and sequenced. A comparison showed mpt64 and the gene encoding MPB64 from Mycobacterium bovis BCG Tokyo to be identical except for one silent mutation. The regions encoding the promoter and the signal peptide were also well conserved for the two sequences. Southern blot experiments on genomic mycobacterial DNA showed the presence of mpt64 in the M. tuberculosis substrains H37Rv, H37Ra, and Erdman and in the M. bovis BCG substrains Tokyo, Moreau, and Russian, whereas the M. bovis BCG substrains Glaxo, Pasteur, Canadian, Tice, and Danish 1331 and Mycobacterium leprae lack the gene. Southern blot analyses revealed differences in the restriction enzyme patterns within the M. tuberculosis substrains as well as within the M. bovis BCG substrains, indicating either different chromosomal localization of mpt64 or that mutations have occurred at different locations on the chromosomes. N-terminal and C-terminal deletion mutants were constructed for the mapping of B-cell epitopes on MPT64 with five monoclonal antibodies, C24b1, C24b2, C24b3, L24b4, and L24b5. Western blot (immunoblot) analysis revealed that the murine antibodies bind to one linear and three conformational epitopes.
Asunto(s)
Antígenos Bacterianos/inmunología , Linfocitos B/inmunología , Proteínas Bacterianas/inmunología , Epítopos/análisis , Mycobacterium tuberculosis/inmunología , Secuencia de Aminoácidos , Animales , Anticuerpos Monoclonales/inmunología , Antígenos Bacterianos/genética , Proteínas Bacterianas/análisis , Proteínas Bacterianas/genética , Secuencia de Bases , Clonación Molecular , Datos de Secuencia Molecular , Conejos , Proteínas Recombinantes/inmunologíaRESUMEN
Human mannan-binding protein (MBP) is a serum lectin that participates in the immune defence by mediating phagocytosis and activation of complement. Variant MBP alleles causing dominant low-serum concentrations have high frequencies in all populations studied, and therefore, low MBP concentrations may confer selective advantages to those individuals carrying the variant alleles. Mycobacterium leprae, the causative agent of leprosy, is an obligate intracellular parasite dependent on phagocytosis to invade host cells. The serum concentrations of MBP in 36 Ethiopian patients (median: 1688 micrograms l-1) with lepromatous or borderline lepromatous leprosy were significantly (P < 0.001) higher than in 26 healthy Ethiopian blood donors (median: 368 micrograms l-1). Only 17% of the patients vs. 58% of the donors (P = 0.0019) had the relatively low MBP concentrations usually associated with variant alleles. Functional studies revealed that M. leprae and M. tuberculosis sonicates bind MBP as strongly as pure mannan. These observations suggest a role for mycobacteria as a selective force in the positive selection of alleles causing low levels of MBP and warrant genetic studies of patients infected with these bacteria.
Asunto(s)
Proteínas Portadoras/fisiología , Lepra/sangre , Candida albicans/metabolismo , Proteínas Portadoras/sangre , Proteínas Portadoras/genética , Colectinas , Humanos , Vigor Híbrido , Inmunidad Innata , Mycobacterium leprae/metabolismo , Mycobacterium tuberculosis/metabolismoRESUMEN
Recent developments in technology have resulted in a new assistive device for profoundly deaf children: the cochlear implant. However, many variables must be considered when evaluating children for the device, one of which is parental expectations. Because expectations are sometimes unreasonable high, cochlear implant teams need to be aware of the dynamics involved in expectations and of ways to help parents come to a realistic understanding of the benefits and limitations of implants.
Asunto(s)
Cóclea/cirugía , Implantes Cocleares , Sordera/rehabilitación , Padres/psicología , Adolescente , Adulto , Niño , Preescolar , Consejo , Sordera/diagnóstico , Sordera/cirugía , Educación Especial , Femenino , Humanos , Masculino , Encuestas y CuestionariosRESUMEN
R16HBsAg is an experimental recombinant malaria vaccine consisting of 16 repeats of a four amino acid sequence (Asn-Ala-Asn-Pro or NANP) of the circumsporozoite (CS) protein of Plasmodium falciparum expressed as a fusion protein with the recombinant hepatitis B virus surface antigen (HBsAg) produced by yeast cells. Twenty male volunteers were experimentally vaccinated with the product, as well as with two doses of the commercial recombinant HBsAg vaccine Engerix B (Smith Kline Beecham Biologicals, Rixensart, Belgium) at intervals during a period of 18 months. No serious side effects were observed. Circulating antibodies to recombinant CS antigen (R32tet32) developed in all volunteers and persisted in most cases over ten months. Anti-HBs antibody production was poor initially, but a single dose of the commercial hepatitis B vaccine was sufficient to elevate these titers to high levels in all but two volunteers.