Mycobacterium tuberculosis sigE mutant ST28 used as a vaccine induces protective immunity in the guinea pig model

With more than 9 million new infections and 1.5 million deaths claimed every year, tuberculosis remains one of the major scourges of humankind. The only vaccine available against this disease, the attenuated strain Mycobacterium bovis, BCG is effective against severe forms of the disease in infants, but scarcely effective in protecting adults from the pulmonary form of the disease, thus not stopping transmission. Consequently, the development of an effective anti-tuberculosis vaccine is a major goal for improving global health. The most common concept is that a more effective vaccine should include a first immunization with a live vaccine followed by the administration of an acellular boosting vaccine. In this approach, the live vaccine might be either BCG or a different, more efficient attenuated strain. Recently, we showed that a Mycobacterium tuberculosis mutant missing the gene encoding for the extracellular function sigma factor SigE, is strongly attenuated and is able to induce a more effective protection from M.\ua0tuberculosis infection compared to BCG in mice. We now further characterize the protective potential of this novel strain in the guinea pig model of tuberculosis. In the guinea pig, it had limited growth but induced a Th1 immune response and was able to significantly reduce the number of colony forming units as well as prolong survival. Taken together these data provide evidence for the use of the M.\ua0tuberculosis sigE mutant as the basis for further development as a vaccine against infection

Evaluation of peripheral blood markers as early endpoint criteria in Guinea Pigs (Cavia porcellus) when testing tuberculosis vaccine candidates

The guinea pig model of tuberculosis is used extensively to assess the efficacy of novel tuberculosis vaccines. There are established parameters to determine vaccine efficacy in this model, but the science community currently lacks established biomarkers for early detection and monitoring of experimental disease in guinea pigs. To define a set of biomarkers that could be used as benchmarks for disease progression and early endpoint criteria, we assessed serum biochemical and hematology parameters in 2 groups of guinea pigs-one vaccinated with the attenuated Mycobacterium bovis vaccine strain (BCG) and one sham-vaccinated with saline-and then experimentally infected with a virulent strain of Mycobacterium tuberculosis. After infection, WBC showed the strongest differences between saline-inoculated and vaccinated animals, with more subtle changes in other serum biochemical parameters, including ALT and ALP. Therefore, this study provides a starting point for evaluating the utility of blood values as possible early endpoint criteria in the guinea pig model of tuberculosis

Virulence of Mycobacterium tuberculosis after acquisition of Isoniazid resistance: individual nature of katG mutants and the possible role of AhpC

In the last decade, there were 10 million new tuberculosis cases per year globally. Around 9.5% of these cases were caused by isoniazid resistant (INHr) Mycobacterium tuberculosis (Mtb) strains. Although isoniazid resistance in Mtb is multigenic, mutations in the catalaseperoxidase (katG) gene predominate among the INHr strains. The effect of these drug-resistance- conferring mutations on Mtb fitness and virulence is variable. Here, we assessed differences in bacterial growth, immune response and pathology induced by Mtb strains harboring mutations at the N-terminus of the katG gene. We studied one laboratory and one clinically isolated Mtb clonal pair from different genetic lineages. The INHr strain in each pair had one and two katG mutations with significantly reduced levels of the enzyme and peroxidase activity. Both strains share the V1A mutation, while the double mutant clinical INHr had also the novel E3V katG mutation. Four groups of C57BL/6 mice were infected with one of the Mtb strains previously described. We observed a strong reduction in virulence (reduced bacterial growth), lower induction of proinflammatory cytokines and significantly reduced pathology scores in mice infected with the clinical INHr strain compared to the infection caused by its INHs progenitor strain. On the other hand, there was a subtle reduction of bacteria growth without differences in the pathology scores in mice infected with the laboratory INHr strain. Our results also showed distinct alkyl-hydroperoxidase C (AhpC) levels in the katG mutant strains, which could explain the difference in the virulence profile observed. The difference in the AhpC levels between clonal strains was not related to a genetic defect in the gene or its promoter. Cumulatively, our results indicate that the virulence, pathology and fitness of INHr strains could be negatively affected by multiple mutations in katG, lack of the peroxidase activity and reduced AhpC levels

AhpC leves from soluble cellular fractions of <i>Mtb</i> cultures.

<p>Western blot using anti-AhpC_<i>Mtb</i> [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0166807#pone.0166807.ref017" target="_blank">17</a>] from <b>c</b>ulture supernatants and cytosolic fraction of the four strains used in the study grown in GAS media (1 and 2 represent the two culture replicates of each strain used). CFP of H37Rv from BEI was used as a positive control. *indicates the lane for the ladder.</p

KatG levels and peroxidase activity in the clonal <i>Mtb</i> strains.

<p>A and B). Western blot using anti-KatG (from mouse clone IT-57) from culture supernatants and cytosol fractions of <i>Mtb</i> clonal strains cultured in GAS media respectively (1 and 2 represent the two culture replicates of each strain used). Recombinant KatG was used as a positive control. C). Standard curve for peroxidase activity using 3,3′,5,5′-Tetramethylbenzidine (TMB) as substrate and recombinant KatG. D). KatG peroxidase activity (derived from the standard curve, C) from laboratory and clinical <i>Mtb</i> pairs using recombinant KatG as reference. Non-paired <i>t</i>-test without assuming consistent standard deviation, *<i>p</i><0.05.</p

Dynamics and comparison of cytokine levels in mouse lung homogenates.

<p>A. Comparison of pro-inflammatory cytokines levels throughout the infection with pairs of clinical and laboratory clonal <i>Mtb</i> strains (t-test, *p<0.05). Limit of detection (LD) for IFN-γ: 0.5pg/mL, TNF-α: 0.9 pg/mL, and IL-6: 1.4 pg/mL.) Pair comparison between mice infected B). IL-10 (LD: 16.8 pg/mL) and IL-2 (LD: 0.1 pg/mL) levels in mice infected with laboratory and clinical <i>Mtb</i> pairs. Bars represent the mean values of cytokine concentration for five mice and the error bars represent the standard deviation.</p

Comparison of bacterial growth rates in BACTEC media using MGIT^™320.

<p>Time to positivity (TTP) of each strain was determined by seeding BACTEC tubes with 10-fold serial dilutions. TTP assessed in the MGIT^™320.</p

Comparison of bacterial growth in C57BL/6 mice after low dose aerosol infection.

<p>A) and C). Lung CFU count of mice infected with Laboratory and Clinical clonal strain pairs respectively. B) and D). Spleen CFU counts of Laboratory and Clinical clonal strain pairs respectively. Non-paired <i>t</i>-test without assuming consistent standard deviation (SD).*p<0.05, **p<0.001. Each time point represents the mean values for five mice infected with each of the four strains with the respective standard deviation shown in the error bars.</p