Novel Biomarkers Distinguishing Active Tuberculosis from Latent Infection Identified by Gene Expression Profile of Peripheral Blood Mononuclear Cells

BACKGROUND: Humans infected with Mycobacterium tuberculosis (MTB) can delete the pathogen or otherwise become latent infection or active disease. However, the factors influencing the pathogen clearance and disease progression from latent infection are poorly understood. This study attempted to use a genome-wide transcriptome approach to identify immune factors associated with MTB infection and novel biomarkers that can distinguish active disease from latent infection. METHODOLOGY/PRINCIPAL FINDINGS: Using microarray analysis, we comprehensively determined the transcriptional difference in purified protein derivative (PPD) stimulated peripheral blood mononuclear cells (PBMCs) in 12 individuals divided into three groups: TB patients (TB), latent TB infection individuals (LTBI) and healthy controls (HC) (n = 4 per group). A transcriptional profiling of 506 differentially expressed genes could correctly group study individuals into three clusters. Moreover, 55- and 229-transcript signatures for tuberculosis infection (TB&LTBI) and active disease (TB) were identified, respectively. The validation study by quantitative real-time PCR (qPCR) performed in 83 individuals confirmed the expression patterns of 81% of the microarray identified genes. Decision tree analysis indicated that three genes of CXCL10, ATP10A and TLR6 could differentiate TB from LTBI subjects. Additional validation was performed to assess the diagnostic ability of the three biomarkers within 36 subjects, which yielded a sensitivity of 71% and specificity of 89%. CONCLUSIONS/SIGNIFICANCE: The transcription profiles of PBMCs induced by PPD identified distinctive gene expression patterns associated with different infectious status and provided new insights into human immune responses to MTB. Furthermore, this study indicated that a combination of CXCL10, ATP10A and TLR6 could be used as novel biomarkers for the discrimination of TB from LTBI

Preliminary results in the immunodiagnosis of tuberculosis in children based on T cell responses to ESAT-6 and PPD antigens

The aim of this work was to study the difference in interferon gamma (IFN-gamma) production by T lymphocytes after early secretory antigen target 6 (ESAT-6) or purified protein derivate (PPD) stimulation in whole blood culture supernatants from children with suspected tuberculosis (TB) disease (n = 21), latent TB infection (n = 16) and negative controls (NC) (n = 22) from an endemic area in Brazil. The concentration of IFN-gamma (pg/ml) was measured by enzyme linked immunosorbent assay and the differences in the IFN-gamma levels for each group were compared and evaluated using an unpaired Student's t-test; p values < 0.05 were considered significant. Measurement of IFN-gamma levels after ESAT-6 stimulation raised the possibility of early diagnosis in the latent TB group (p = 0.0030). Nevertheless, the same group showed similar responses to the NC group (p > 0.05) after PPD stimulation. The IFN-gamma assay using ESAT-6 as an antigenic stimulus has the potential to be used as a tool for the immunodiagnosis of early TB in children

<i>M.tuberculosis</i> Mutants Lacking Oxygenated Mycolates Show Increased Immunogenicity and Protective Efficacy as Compared to <i>M. bovis</i> BCG Vaccine in an Experimental Mouse Model

<div><p>The existing vaccine against tuberculosis (<i>M. bovis</i> BCG) exerts some protection against the extrapulmonary forms of the disease, particularly in young children, but is not very effective against the pulmonary form of TB, which often results from the reactivation of a latent <i>M. tuberculosis</i> (<i>M.tb</i>)infection. Among the new approaches in TB vaccine development, live attenuated <i>M.tb</i> mutants are a promising new avenue. Here we report on the vaccine potential of two highly attenuated <i>M.tb</i> mutants, MGM1991 and <i>M</i>.<i>tb</i>hma::hyg (HMA), lacking all oxygenated mycolates in their cell wall. In C57BL/6 mice, stronger Th1 (IFN-γ, IL-2 and TNF-α) and IL-17 responses could be induced following subcutaneous vaccination with either of the two mutants, than following vaccination with <i>M. bovis</i> BCG. Significantly more mycobacteria specific IFN-γ producing CD4⁺ and particularly CD8⁺ T cells could be detected by intracellular cytokine staining in mice vaccinated with the <i>M.tb</i> mutants. Finally, vaccination with either of the two mutants conferred stronger protection against intratracheal <i>M.tb</i> challenge than vaccination with BCG, as indicated by reduced bacterial replication in lungs at 4 to 12 weeks after challenge. Protection against <i>M. tb</i> dissemination, as indicated by reduced bacterial numbers in spleen, was comparable for both mutants to protection conferred by BCG. </p> </div

Increased B and T Cell Responses in M. bovis Bacille Calmette-Guérin Vaccinated Pigs Co-Immunized with Plasmid DNA Encoding a Prototype Tuberculosis Antigen.

The only tuberculosis vaccine currently available, bacille Calmette-Guérin (BCG) is a poor inducer of CD8(+) T cells, which are particularly important for the control of latent tuberculosis and protection against reactivation. As the induction of strong CD8(+) T cell responses is a hallmark of DNA vaccines, a combination of BCG with plasmid DNA encoding a prototype TB antigen (Ag85A) was tested. As an alternative animal model, pigs were primed with BCG mixed with empty vector or codon-optimized pAg85A by the intradermal route and boosted with plasmid delivered by intramuscular electroporation. Control pigs received unformulated BCG. The BCG-pAg85A combination stimulated robust and sustained Ag85A specific antibody, lymphoproliferative, IL-6, IL-10 and IFN-γ responses. IgG1/IgG2 antibody isotype ratio reflected the Th1 helper type biased response. T lymphocyte responses against purified protein derivative of tuberculin (PPD) were induced in all (BCG) vaccinated animals, but responses were much stronger in BCG-pAg85A vaccinated pigs. Finally, Ag85A-specific IFN-γ producing CD8(+) T cells were detected by intracellular cytokine staining and a synthetic peptide, spanning Ag85A131-150 and encompassing two regions with strong predicted SLA-1*0401/SLA-1*0801 binding affinity, was promiscuously recognized by 6/6 animals vaccinated with the BCG-pAg85A combination. Our study provides a proof of concept in a large mammalian species, for a new Th1 and CD8(+) targeting tuberculosis vaccine, based on BCG-plasmid DNA co-administration