Acute and Persistent Mycobacterium tuberculosis Infections Depend on the Thiol Peroxidase TPX

The macrophage is the natural niche of Mycobacterium tuberculosis infection. In order to combat oxidative and nitrosative stresses and persist in macrophages successfully, M. tuberculosis is endowed with a very efficient antioxidant complex. Amongst these antioxidant enzymes, TpX is the only one in M. tuberculosis with sequence homology to thiol peroxidase. Previous reports have demonstrated that the M. tuberculosis TpX protein functions as a peroxidase in vitro. It is the dominant antioxidant which protects M. tuberculosis against oxidative and nitrosative stresses. The level of the protein increases in oxidative stress. To determine the roles of tpx gene in M. tuberculosis survival and virulence in vivo, we constructed an M. tuberculosis strain lacking the gene. The characteristics of the mutant were examined in an in vitro stationary phase model, in response to stresses; in murine bone marrow derived macrophages and in an acute and an immune resistant model of murine tuberculosis. The tpx mutant became sensitive to H2O2 and NO compared to the wild type strain. Enzymatic analysis using bacterial extracts from the WT and the tpx mutant demonstrated that the mutant contains reduced peroxidase activity. As a result of this, the mutant failed to grow and survive in macrophages. The growth deficiency in macrophages became more pronounced after interferon-γ activation. In contrast, its growth was significantly restored in the macrophages of inducible nitric oxide synthase (iNOS or NOS2) knockout mice. Moreover, the tpx mutant was impaired in its ability to initiate an acute infection and to maintain a persistent infection. Its virulence was attenuated. Our results demonstrated that tpx is required for M. tuberculosis to deal with oxidative and nitrosative stresses, to survive in macrophages and to establish acute and persistent infections in animal tuberculosis models

Preclinical Development of an In Vivo BCG Challenge Model for Testing Candidate TB Vaccine Efficacy

There is an urgent need for an immunological correlate of protection against tuberculosis (TB) with which to evaluate candidate TB vaccines in clinical trials. Development of a human challenge model of Mycobacterium tuberculosis (M.tb) could facilitate the detection of such correlate(s). Here we propose a novel in vivo Bacille Calmette-Guérin (BCG) challenge model using BCG immunization as a surrogate for M.tb infection. Culture and quantitative PCR methods have been developed to quantify BCG in the skin, using the mouse ear as a surrogate for human skin. Candidate TB vaccines have been evaluated for their ability to protect against a BCG skin challenge, using this model, and the results indicate that protection against a BCG skin challenge is predictive of BCG vaccine efficacy against aerosol M.tb challenge. Translation of these findings to a human BCG challenge model could enable more rapid assessment and down selection of candidate TB vaccines and ultimately the identification of an immune correlate of protection

Comparative cutaneous testing with purified protein derivative and the antigen complex A60 in vaccinated subjects and tuberculosis patients.

Some 840 bacille Calmette-Guérin (BCG)-vaccinated healthy controls and tuberculosis patients from two Chinese hospitals were submitted to comparative skin tests with purified protein derivative of tuberculin (PPD; as reference) and with the antigen complex A60 from Mycobacterium bovis BCG. In a first trial, including 581 persons (185 healthy juveniles, 180 healthy adults and 216 tuberculosis patients), a limited dose of A60 (1 microgram) was used. Performance of the A60 test was similar to that of 5 I.U. PPD for controls (cut-off values = 5 mm induration diameter), but lower than that seen for tuberculosis patients (10 mm cut-off values). A second survey was conducted on 259 persons (109 recently revaccinated healthy persons, considered as tuberculin-negative in the first trial, and 150 tuberculosis patients), using a higher dose of A60 (2 micrograms) and the same dose of PPD (5 I.U.). Similar results were obtained with the two tests in all cases, thus supporting the possibility of PPD replacement by A60 in cutaneous testing. The pattern of induration diameter distribution in healthy subjects who took part in the first testing round (64% positively rate) was displaced to the inactivity side (with a peak at 5 to 9-mm diameter), in comparison with the second round (90% positivity rate and peak at 10-14 mm). This indicates a progressive fading of cellular immunity reactions after BCG vaccination. In tuberculosis patients, no correlation was found among the following three parameters: positivity at cutaneous testing (with PPD or A60), titer of anti-A60 mycobacterial immunoglobulins in blood (IgG titer higher than cut-off line) and presence of mycobacteria in sputum