ESAT-6 and TB10.4; gene expression, immunogenicity and post-exposure vaccine efficacy.

<p>CB6F1 mice were infected, treated with antibiotics as described above and vaccinated with ESAT-6/CAF01 (n=7), TB10.4/CAF01 (n=7) or CAF01 alone (n=8) at week 10, 13 and 16 post infection (p.i.). (A) At week 37 and 49 p.i. mice were sacrificed, lungs harvested for assessment of protection by culturing lung homogenates at 37° C and enumerating the number of bacteria (expressed as log₁₀). Log₁₀ CFUs are shown for CAF01 only control animals (open triangles), TB10.4 (open squares) and ESAT-6 (open circles) vaccinated animals. Data are shown as median ± interquartile range and represent 6-8 individual animals. Evaluations of ESAT-6 and TB10.4 have been done in several experiments with similar outcome (the overall dataset is depicted in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0080579#pone-0080579-g006" target="_blank">Figure 6</a>). (B) <i>M.tb</i>. RNA was extracted from lung lobes of CB6F1 mice and used for real-time PCR to assess the gene expression level of the <i>acr, esxA, esxH</i> and <i>fhpB</i>. Data shown as relative transcript numbers (normalized data) and depicts the mean ± standard deviation (n=4). The dotted line denotes the level of detection at 100 relative RNA copies per bacterial genome. (C) Lymphocytes were extracted at week 17 and 49 p.i. from perfused lungs of six pooled mice, vaccinated with TB10.4 or ESAT-6. The cells were re-stimulated with their respective vaccine antigen and stained for CD4, IFN-γ, IL-2 and TNF-α for ICS analysis. Data is displayed as the total frequency of cytokine producing CD4 T cells (producing IFN-γ, IL-2, TNF-α or any combination of the three). The pie-charts represent the proportion of the subpopulation of responding CD4 T cells color-coded according to their cytokine profile.