RESUMEN
It is estimated that one-third of the world's population is infected with Mycobacterium tuberculosis. Infection typically remains latent, but it can reactivate to cause clinical disease. The only vaccine, Mycobacterium bovis bacillus Calmette-Guérin (BCG), is largely ineffective, and ways to enhance its efficacy are being developed. Of note, the candidate booster vaccines currently under clinical development have been designed to improve BCG efficacy but not prevent reactivation of latent infection. Here, we demonstrate that administering a multistage vaccine that we term H56 in the adjuvant IC31 as a boost to vaccination with BCG delays and reduces clinical disease in cynomolgus macaques challenged with M. tuberculosis and prevents reactivation of latent infection. H56 contains Ag85B and ESAT-6, which are two of the M. tuberculosis antigens secreted in the acute phase of infection, and the nutrient stress-induced antigen Rv2660c. Boosting with H56/IC31 resulted in efficient containment of M. tuberculosis infection and reduced rates of clinical disease, as measured by clinical parameters, inflammatory markers, and improved survival of the animals compared with BCG alone. Boosted animals showed reduced pulmonary pathology and extrapulmonary dissemination, and protection correlated with a strong recall response against ESAT-6 and Rv2660c. Importantly, BCG/H56-vaccinated monkeys did not reactivate latent infection after treatment with anti-TNF antibody. Our results indicate that H56/IC31 boosting is able to control late-stage infection with M. tuberculosis and contain latent tuberculosis, providing a rationale for the clinical development of H56.
Asunto(s)
Vacuna BCG/administración & dosificación , Macaca fascicularis/inmunología , Oligodesoxirribonucleótidos/administración & dosificación , Oligopéptidos/administración & dosificación , Vacunas contra la Tuberculosis/administración & dosificación , Tuberculosis/prevención & control , Adyuvantes Inmunológicos/administración & dosificación , Animales , Antígenos Bacterianos/genética , Modelos Animales de Enfermedad , Combinación de Medicamentos , Genes Bacterianos , Humanos , Inmunización Secundaria , Interferón gamma/biosíntesis , Tuberculosis Latente/inmunología , Tuberculosis Latente/prevención & control , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/inmunología , Linfocitos T/inmunología , Tuberculosis/inmunologíaRESUMEN
Region of difference (RD1) genes are present in virulent Mycobacterium tuberculosis but not the vaccine strain M. bovis bacille Calmette-Guérin (BCG). The deletion of RD1 from M. tuberculosis produces an attenuation strikingly like that of BCG, which suggests the use of RD1 mutant strains for improvement of the tuberculosis (TB) vaccine. We performed long-term murine infection with M. tuberculosis H37Rv: Delta RD1 and BCG. Mice infected with H37Rv: Delta RD1 gained less weight than did BCG-infected control mice, and, after >1 year, their lungs harbored many more bacteria and displayed significant levels of inflammation. This difference in virulence has important implications for the pursuit of strains lacking RD1 in the development of the TB vaccine.
Asunto(s)
Eliminación de Gen , Genes Bacterianos , Mycobacterium bovis/patogenicidad , Mycobacterium tuberculosis/patogenicidad , Tuberculosis Pulmonar/patología , Animales , Vacuna BCG , Humanos , Pulmón/microbiología , Pulmón/patología , Ratones , Ratones Endogámicos C57BL , Mycobacterium tuberculosis/genética , Vacunas contra la Tuberculosis , Tuberculosis Pulmonar/microbiología , Vacunas Atenuadas , VirulenciaRESUMEN
The RD1 genomic region is present in virulent strains of Mycobacterium tuberculosis (MTB), missing from the vaccine strain M. bovis BCG, and its importance to virulence has been established experimentally. Based on in silico analysis, it has been suggested that RD1 may encode a novel secretion system, but the mechanism by which this region affects virulence is unknown. Here we examined mutants disrupted in five individual RD1 genes. Both in vitro and in vivo, each mutant displayed an attenuated phenotype very similar to a mutant missing the entire RD1 region. Genetic complementation of individual genes restored virulence. Attenuated mutants could multiply within THP-1 cells, but they were unable to spread to uninfected macrophages. We also examined export of two immunodominant RD1 proteins, CFP-10 and ESAT-6. Export of these proteins was greatly reduced or abolished in each attenuated mutant. Again, genetic complementation restored a wild-type phenotype. Our results indicate that RD1 genes work together to form a single virulence determinant, and argue that RD1 encodes a novel specialized secretion system that is required for pathogenesis of MTB.
Asunto(s)
Antígenos Bacterianos/genética , Proteínas Bacterianas/genética , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/patogenicidad , Animales , Vacuna BCG/genética , Secuencia de Bases , Línea Celular , Mapeo Cromosómico , Cartilla de ADN , Genes Bacterianos/genética , Prueba de Complementación Genética , Vectores Genéticos , Humanos , Ratones , Ratones Endogámicos C57BL , Mutagénesis , Fenotipo , Eliminación de Secuencia , Virulencia/genéticaRESUMEN
Unlike many pathogens that are overtly harmful to their hosts, Mycobacterium tuberculosis can persist for years within humans in a clinically latent state. Latency is often linked to hypoxic conditions within the host. Among M. tuberculosis genes induced by hypoxia is a putative transcription factor, Rv3133c/DosR. We performed targeted disruption of this locus followed by transcriptome analysis of wild-type and mutant bacilli. Nearly all the genes powerfully regulated by hypoxia require Rv3133c/DosR for their induction. Computer analysis identified a consensus motif, a variant of which is located upstream of nearly all M. tuberculosis genes rapidly induced by hypoxia. Further, Rv3133c/DosR binds to the two copies of this motif upstream of the hypoxic response gene alpha-crystallin. Mutations within the binding sites abolish both Rv3133c/DosR binding as well as hypoxic induction of a downstream reporter gene. Also, mutation experiments with Rv3133c/DosR confirmed sequence-based predictions that the C-terminus is responsible for DNA binding and that the aspartate at position 54 is essential for function. Together, these results demonstrate that Rv3133c/DosR is a transcription factor of the two-component response regulator class, and that it is the primary mediator of a hypoxic signal within M. tuberculosis.
Asunto(s)
Proteínas Bacterianas/metabolismo , Regulación Bacteriana de la Expresión Génica , Hipoxia/metabolismo , Mycobacterium tuberculosis/metabolismo , Factores de Transcripción/metabolismo , Ácido Aspártico/metabolismo , Proteínas Bacterianas/genética , Marcación de Gen , Genes Reporteros , Humanos , Mycobacterium tuberculosis/genética , Análisis de Secuencia por Matrices de Oligonucleótidos , Oxígeno/metabolismo , Unión Proteica , Secuencias Reguladoras de Ácidos Nucleicos , Factores de Transcripción/genética , Tuberculosis/metabolismoRESUMEN
The tuberculosis (TB) vaccine bacille Calmette-Guérin (BCG) is a live attenuated organism, but the mutation responsible for its attenuation has never been defined. Recent genetic studies identified a single DNA region of difference, RD1, which is absent in all BCG strains and present in all Mycobacterium tuberculosis (MTB) strains. The 9 open-reading frames predicted within this 9.5-kb region are of unknown function, although they include the TB-specific immunodominant antigens ESAT-6 and CFP-10. In this study, RD1 was deleted from MTB strain H37Rv, and virulence of H37Rv:DeltaRD1 was assessed after infections of the human macrophage-like cell line THP-1, human peripheral blood monocyte-derived macrophages, and C57BL/6 mice. In each of these systems, the H37Rv:DeltaRD1 strain was strikingly less virulent than MTB and was very similar to BCG controls. Therefore, it was concluded that genes within or controlled by RD1 are essential for MTB virulence and that loss of RD1 was important in BCG attenuation.