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

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 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

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