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

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

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

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

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

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

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

Distribution of reported single nucleotide polymorphisms (SNPs) throughout the <i>pncA</i> gene.

<p>Dashed lines indicate the open reading frame for the <i>pncA</i> gene.</p

Forest plot for the summary estimates of pyrazinamide prevalence by WHO region and presence or risk of MDR-TB.

<p>Abbreviations: CI, confidence interval; DST, drug susceptibility test; MDR-TB, multi-drug resistant tuberculosis; N/A, not applicable; WHO, world health organization. MDR-TB was defined as an isolate being resistant to RIF and INH. High risk of MDR-TB was defined as an isolate being resistant to at least one anti-TB drug. *Any TB was defined as the inclusion of patients independent of drug resistance profile.</p

Flow diagram describing article selection.

<p>Flow diagram describing article selection.</p