Whole blood IFN-Υ responses to individual and pooled PE/PPE peptides.

<p>Peptide pools eliciting positive responses were further analysed by assessing the contribution of each constituent peptide at 3, 5 or 9 weeks post-infection<b>.</b> Representative data are shown for peptides corresponding to (A) One pool derived from PE8, and (B) One pool derived from PPE15. The solid lines represent the temporal response to a peptide pool, while individual symbols correspond to individual peptides. (C) and (D) BLASTP homology searches were performed with peptides which elicited positive responses, and closest matches are shown in a CLUSTALW alignment.</p

IFN-γ responses of short-term T cell lines to potentially cross-reactive PPE peptides.

<p>Short-term T cell lines were raised by primary stimulation of bovine PBMC with individual PE or PPE peptides in the presence of IL-2. Following initial expansion with (A) PE31.10, (B) PPE18.14 or (C) PPE60.14, cells were re-stimulated with selected peptides, predicted to be immunologically cross-reactive. IFN-γ production was then measured using the ELISA-based Bovigam assay. The amino acid sequence of the initial stimulation peptide is shown in alignment with restimulation peptides, with amino acid substitutions indicated. Responses are shown as ΔOD₄₅₀ values, and the dashed line indicates the positivity cut-off value of 0.1.</p

Comparison of proportion responders in human and bovine hosts with active TB.

<p>Responses to 28 PE/PPE peptide pools were assessed in both cattle and humans with active TB, by Bovigam or ELISPOT assays. Individual cattle samples were scored as positive if ΔOD₄₅₀ values were ≥0.1, and human samples were scored as positive if ≥20 SFC were counted per 10⁶ PBMC. Linear regression analysis produced the line shown. A Pearson correlation test revealed a statistically significant correlation between the proportion human responders and the proportion bovine responders; r(26)  = 0.539, 2-tailed p value  = 0.0031.</p

Temporal profile of bovine IFN-γ responses to PE/PPE peptide pools.

<p>IFN-γ responses were measured following stimulation of whole bovine blood with PE/PPE peptides and control antigens before, and at multiple time points following, experimental infection of cattle with <i>M. bovis</i> AF2122/97. (A) Responses to controls antigens; avian PPD (PPD-A), bovine PPD (PPD-B) and an EsxA/B peptide cocktail. (B) to (D) Representative responses to sets of PE/PPE peptide pools. Open grey symbols, PE pools; Solid black symbols, PPE pools; ▪, ▴, ▾, ♦, •, pools A-E, respectively (B) IFN-γ responses to PE18/PPE26 peptide pools. (C) IFN-γ responses to PE31/PPE60 peptide pools. (D) IFN-γ responses to PE8/PPE15 peptide pools. Mean responses for a single representative animal are shown.</p

Immune responses to PE/PPE proteins in humans with active and latent infection.

<p>PBMC from <i>M. tuberculosis</i>-sensitized individuals were stimulated with PE/PPE peptide pools, and IFN-γ producing cells were enumerated by ELISPOT. (A) Proportion responders to all PE/PPE pools analyzed. Grey bars, individuals with latent infection; Black bars, individuals with active infection. (B) Number of IFN-γ producing cells measured after stimulation with PE/PPE peptide pools. White symbols, individuals with latent infection; Black symbols, individuals with active infection. No statistically significant differences were observed between responses in individuals with latent and active infection for any of the peptide pools analyzed (as determined by Mann Whitney U test). Results for a representative set of pools shown, horizontal lines indicate median responses.</p

Bovine and human responses to selected PE/PPE peptide pools.

<p><b>a.</b> Individual cattle samples were scored as positive if ΔOD₄₅₀ values were ≥0.1, and human samples were scored as positive if ≥20 SFC were counted per 10⁶ PBMC.</p

PE/PPE proteins selected for this study.

<p><b>a.</b> C, PE/PPEs encoded by complete <i>esx</i> gene cluster; P, PE/PPE encoded by partial <i>esx</i> cluster; I, PE/PPE encoded by isolated <i>pe/ppe</i> pair (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0040890#pone.0040890.s001" target="_blank">Figure S1</a>).</p><p><b>b.</b> Single peptide for Rv3018A/PE27A mixed with PE13 set.</p><p><b>c.</b> Pool does not include all possible overlapping peptides, some were excluded due to 100% identity.</p

Supervised classification algorithms results.

<p>Sensitivity, Specificity and Misclassification Error Rates are shown for training and test sets. 1000 repeated trials have been performed (as described in the Methods) for each classification algorithm. (A) results for the <i>single cytokine dataset</i>. (B): results for the <i>memory cytokine dataset</i>. (C): results for the <i>multiple cytokine dataset</i>.</p

Methodology roadmap.

<p>The left side of the diagram (gray boxes) summarizes all of the datasets generated in this study. We have generated experimental datasets from blood samples and lung necropsies of non-human primates (NHPs), and datasets generated by computational model simulations (<i>in silico</i> data). The right side of the diagram (yellow boxes) represents the analyses performed on each dataset. Each dataset is displayed by a different shape. The blue arrows point to the type of analysis performed on each dataset. The circled numbers represent the chronological order of operations (referred to as steps in the text). Details on which Fig or table in the manuscript contain a dataset or analysis are given in each box.</p

Computational model calibration: LUNG.

<p>NHP experimental data on CFU/granuloma (<a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1004804#pcbi.1004804.s012" target="_blank">S1 Table</a>) are plotted here versus the i<i>n silico</i> datasets of CFU/granuloma (lung compartment) from i<i>n silico</i> repository of 10,000 granulomas coupled to the blood and LN dynamics). Although the <i>in silico</i> dataset has time courses up to 600 days, the x-axis always shows a time span of infection up to 200 days to match the NHP blood data. The y-axis represents CFU/granuloma (A-B). (A) <i>In silico</i> dataset of time courses of CFU/granuloma generated in the lung compartment (black circles, with the black solid line representing the median trajectory) compared to experimental data on NHP CFU/granuloma (with the solid red line representing the median, and the dotted red lines representing the min and max values in the NHP data). The median trajectories for both the NHP and <i>in silico</i> data are calculated including the sterilized granulomas, while the min trajectories excluded the sterilized granulomas. (B) Mtb trajectories (total [solid thick], extracellular [solid with empty circles], intracellular [dotted] and non-replicating [solid thin]) in a representative granuloma (containment) compared to the NHP CFU/granuloma experimental data (red circles). (C) Snapshots of 4 different granulomas. The top row of Panel C is for H and E staining of two NHP granulomas. The left granuloma is from NHP 22810, CFU~40. The right granuloma is from NHP 17211, CFU~1240. Both granulomas are ~ 2mm in diameter (see <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1004804#pcbi.1004804.s012" target="_blank">S1 Table</a> for details). The bottom row of Panel C is for <i>in silico</i> granulomas, matching lesion size and CFU/granuloma of the NHP images. Cell types displayed are the following: macrophages (resting-green, activated-blue, infected-orange, chronically infected-red), effector lymphocytes (pro-inflammatory IFN-γ producing T cells-Tγ in pink, cytotoxic T cell-Tc in purple, regulatory T cell-Treg in light blue), extracellular bacteria (olive green), vascular sources (grey and necrotic spots (white)).</p