Granzyme A Is Expressed in Mouse Lungs during <i>Mycobacterium tuberculosis</i> Infection but Does Not Contribute to Protection <i>In Vivo</i>

<div><p>Granzyme A, a serine protease expressed in the granules of cytotoxic T and Natural Killer cells, is involved in the generation of pro-inflammatory cytokines by macrophages. Granzyme A has been described to induce in macrophages <i>in vitro</i> the activation of pro-inflammatory pathways that impair intracellular mycobacterial replication. In the present study, we explored the physiological relevance of Granzyme A in the control of pulmonary <i>Mycobacterium tuberculosis</i> infection <i>in vivo</i>. Our results show that, even though Granzyme A is expressed by cytotoxic cells from mouse lungs during pulmonary infection, its deficiency in knockout mice does not have an effect in the control of <i>M</i>. <i>tuberculosis</i> infection. In addition our findings indicate that absence of Granzyme A does not affect the protection conferred by the live-attenuated <i>M</i>. <i>tuberculosis</i> vaccine MTBVAC. Altogether, our findings are in apparent contradiction with previously published <i>in vitro</i> results and suggest that Granzyme A does not have a crucial role <i>in vivo</i> in the protective response to tuberculosis.</p></div

GZMA-/- mice are not less protected by MTBVAC vaccination.

<p>Groups of nine C57BL/6 WT or GZMA-/- mice were vaccinated subcutaneously with 10⁶ CFU of MTBVAC (WT VAC or GZMA-/- VAC), or non-vaccinated. <b>A</b>, at two months post-vaccination, mice were inoculated intranasally with a low-dose challenge of H37Rv, and four weeks later lung bacterial burden was determined. Data from one experiment are represented in the graph. One-way ANOVA test with Bonferroni post analysis was performed to calculate statistical significance. * p<0.05; ** p<0.01; *** p<0.001. <b>B</b>, cells were stimulated with PPD as described in materials and methods section, and CD4+IFNγ+ cells frequency in lungs was determined by flow cytometry. Data from one experiment are represented in the graph as mean± SEM.</p

Granzyme A expression is increased in lungs after <i>M</i>. <i>tuberculosis</i> infection.

<p><b>A</b>, lung cells from WT or GZMA-/- mice were intracellularly stained with anti-GZMA. A representative dot-plot showing GZMA staining is shown. GZMA-positive cells are contained in the red-circled region. <b>B</b>, <b>C</b>, <b>D</b>, Groups of five C57BL/6 wild-type mice were inoculated intranasally with a low-dose H37Rv challenge. Four weeks later, mice were sacrificed and GZMA expression analyzed in lung cellular populations. <b>B</b>, total GZMA-expressing cells. <b>C</b>, GZMA-expressing cell populations. Graph shows the relative proportions in percentage of the GZMA-positive cell populations. Right panels show representative CD4, CD8 and NK1.1 staining dot-plots and histogram gated from a GZMA-positive region. D, graphs show frequency (upper panels) and absolute number (lower panels) of CD4, CD8 and NK cells positive for GZMA staining, comparing non-infected and infected mice. A representative of two independent experiments is shown in the Figure. Data in the graphs are represented as mean ± SEM. Unpaired t-student analysis was performed to calculate statistical significance. * p<0.05; ** p<0.01; *** p<0.001.</p

Attenuated <em>Mycobacterium tuberculosis</em> SO2 Vaccine Candidate Is Unable to Induce Cell Death

<div><p>It has been proposed that <em>Mycobacterium tuberculosis</em> virulent strains inhibit apoptosis and trigger cell death by necrosis of host macrophages to evade innate immunity, while non-virulent strains induce typical apoptosis activating a protective host response. As part of the characterization of a novel tuberculosis vaccine candidate, the <em>M. tuberculosis phoP</em> mutant SO2, we sought to evaluate its potential to induce host cell death. The parental <em>M. tuberculosis</em> MT103 strain and the current vaccine against tuberculosis Bacillus Calmette-Guérin (BCG) were used as comparators in mouse models <em>in vitro</em> and <em>in vivo</em>. Our data reveal that attenuated SO2 was unable to induce apoptotic events neither in mouse macrophages <em>in vitro</em> nor during lung infection <em>in vivo</em>. In contrast, virulent MT103 triggers typical apoptotic events with phosphatidylserine exposure, caspase-3 activation and nuclear condensation and fragmentation. BCG strain behaved like SO2 and did not induce apoptosis. A clonogenic survival assay confirmed that viability of BCG- or SO2-infected macrophages was unaffected. Our results discard apoptosis as the protective mechanism induced by SO2 vaccine and provide evidence for positive correlation between classical apoptosis induction and virulent strains, suggesting apoptosis as a possible virulence determinant during <em>M. tuberculosis</em> infection.</p> </div

Virulent MT103 strain, but not the attenuated SO2 and BCG strains, replicates in vivo, causes lung pathology and induces apoptosis in mouse lungs.

<p>Groups of five C57BL/6 mice were intratracheally infected with a low dose (100 bacteria/mouse) of MT103, BCG or SO2 strains as described in Materials and Methods. Lungs were harvested and CFU counted at 21 days post-infection (A, left panel). Lung histopathology (hematoxilin/eosin) representative images (10x magnification) of mock-treated (A, right panel, image 1) or MT103-, BCG- or SO2-infected mice (A, right panel, images 2, 3, 4, respectively) at 3 weeks post inoculation. Representative images of active caspase 3 immunohistochemical and Ziehl-Neelsen staining of mock-treated or MT103-, BCG- or SO2-infected lungs at three weeks post inoculation (B): primary antibody control of MT103-infected lung section incubated only with secondary antibody (10x magnification) (image 1); active caspase-3 staining of MT103-infected lung section (10x magnification) (image 2); active caspase-3 staining of MT103-infected lung section (100x magnification) (image 3); active caspase-3 staining of MT103-infected lung section (600x magnification) (image 4); Ziehl-Neelsen staining of MT103-infected lung section (600x magnification) (image 5); active caspase-3 staining of mock-treated lung section (10x, 100x and 600x magnification, images 6, 9 and 12, respectively); active caspase-3 staining of BCG-infected lung section (10x, 100x and 600x magnification, images 7, 10 and 13, respectively); active caspase-3 staining of SO2-infected lung section (10x, 100x and 600x magnification, images 8, 11 and 14, respectively).</p

Attenuated SO2 strain does not induce PS translocation in primary mouse macrophages.

<p>Mouse bone marrow-derived primary macrophages (BMDM) were mock-treated or infected at low MOI of 1∶1 (A) or 10∶1 (B) with the indicated strains. After 4 hours of infection, cells were washed and incubated with complete medium. At the indicated times post infection, both detached and adhered cells were pooled and PS exposure (annexin-V-FITC) and 7-AAD staining were analyzed by flow cytometry as described in Materials and Methods (A, B). Numbers in the dot-plot diagrams correspond to the percentage (%) of cells in each quadrant. A representative experiment is shown in the left panels. Data in the graph (figure B, right panel) is represented as mean±S.E.M. of at least three independent experiments. Statistical analyses were done with one-way ANOVA with Tukeýs post-test comparing every strain with MT103. Upper symbols  =  statistical analyses of Ann+AAD+ cells; lower symbols =  statistical analyses of Ann+AAD- cells. ns =  not statistically significant; *, **, ***  =  statistically significant; * p<0,05; ** p<0,01; *** p<0,001.</p

Statistical Analysis Plan

Statistical analysis plan for the MTBVAC 201 clinical trial. A phase Ib, randomised, double-blind, dose-escalation clinical trial of the safety, reactogenicity and immunogenicity of MTBVAC compared to BCG Vaccine SSI, in newborns living in a tuberculosis endemic region with a safety arm in adults

Cllinical Trial Protocol

Clinical trial protocol for MTBVAC 201 trial

Correlations of disease along the infection phase.

<p>Thorax radiology, bacterial burden, and hilar LN pathology correlate significantly with lung PA. Loss of total body weight (wasting), and C-reactive protein (CRP) levels and decreasing mean corpuscular volume (MCV) and mean corpuscular hemoglobin (MCH) as measures of systemic inflammation, are highly significant correlates of disease in this high dose challenge model for TB vaccine evaluation. Parameters are plotted per individual (with colouring as indicated in the legend to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0005264#pone-0005264-g002" target="_blank">Figure 2</a> on group colouring) against lung PA for CXR scores at autopsy (A), CFU counts from lung homogenates (B), hilar LN involvement (C), disseminated extra-thoracic lesions (D), and against total pathology (the sum of lung, hilar LN and extra-thoracic PA scores) for relative change in body weight (E), change in CRP (F), change in MCV (G) and in MCH (H). Spearman's rho (R_s) as correlation factor and p-value are indicated. (AU for arbitrary units.)</p

Immune correlations of disease.

<p>Maximal PPD-specific IFNγ levels post-vaccination and maximal Ag85A-specific IFNγ post-infection show significant inverse correlations with TB disease by total gross lesion PA scores. Maximal antigen-specific IFNγ response levels are plotted per individual against total pathology scores (with colouring as indicated in the legend to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0005264#pone-0005264-g002" target="_blank">figure 2</a> on group colouring) and post-vaccination and post-infection for PPD (A and B, respectively) and Ag85A (C and D, respectively), and for ESAT6-CFP10 fusion protein post-infection only (E).</p