Journal: BMC Infectious Diseases

Article Title: A new tool for tuberculosis vaccine screening: Ex vivo Mycobacterial Growth Inhibition Assay indicates BCG-mediated protection in a murine model of tuberculosis

doi: 10.1186/s12879-016-1751-4

Figure Lengend Snippet: Ex vivo MGIA comparing growth inhibition conferred by BCG SSI and BCG Pasteur Aeras. a 1 × 10 6 splenocytes or 2 × 10 6 splenocytes from mice immunized with BCG SSI (grey circles) or given saline (open squares) were co-cultured with 3800, 675, or 90 CFU of BCG SSI. b 1 × 10 6 splenocytes from mice immunized with BCG Pasteur Aeras (grey circles) or given saline (open squares) were co-cultured with 2000, 500, or 100 CFU of BCG Pasteur Aeras. Splenocytes were obtained from a total of 5 immunized and 5 control animals ( a ) or a total of 6 immunized and 6 control animals ( b ). Aliquots from each spleen were cultured with different numbers of mycobacteria as indicated, and are represented by individual data points. Error bars represent the median +/- interquartile range. Statistical significance was tested using the unpaired t test function in GraphPad Prism

Article Snippet: We found that protection was conferred by both BCG strains, with a significant reduction of bacterial burden in the lung by 0.73 log10 CFU (BCG SSI) or 0.79 log10 CFU (BCG Pasteur Aeras), and in the spleen by 0.93 log10 CFU (BCG SSI) or 1.06 log10 CFU (BCG Pasteur Aeras), compared to control animals.

Techniques: Ex Vivo, Inhibition, Saline, Cell Culture, Control

Journal: BMC Infectious Diseases

Article Title: A new tool for tuberculosis vaccine screening: Ex vivo Mycobacterial Growth Inhibition Assay indicates BCG-mediated protection in a murine model of tuberculosis

doi: 10.1186/s12879-016-1751-4

Figure Lengend Snippet: Optimisation of the ex vivo MGIA using BCG Pasteur Aeras. a 1 × 10 6 , 3 × 10 6 , and 5 × 10 6 splenocytes (1 M, 3 M, and 5 M, respectively) from mice immunized with BCG Pasteur Aeras (grey circles) or given saline (open squares) were co-cultured with 2000 CFU ( a ), 500 CFU ( b ) or 100 CFU ( c ) of BCG Pasteur Aeras. Splenocytes were obtained from a total of 6 immunized and 6 control animals. Aliquots from each spleen were cultured with different numbers of mycobacteria as indicated, and are represented by individual data points. Error bars represent the median +/- interquartile range. Statistical significance was tested using the unpaired t test function in GraphPad Prism. d Analysis of variation of the data shown in a - c . *Sample sizes (alpha 0.05, power 0.8) were calculated using the mean values of BCG immunized and naïve groups and using the standard deviation of the BCG group (standard deviation was greater in the BCG groups than in the saline groups). **Optimal conditions for vaccine candidate testing determined as delta >0.5 log CFU, CoV < 50 % and required sample size < 10 per group. The analysis was carried out using STATA software

Article Snippet: We found that protection was conferred by both BCG strains, with a significant reduction of bacterial burden in the lung by 0.73 log10 CFU (BCG SSI) or 0.79 log10 CFU (BCG Pasteur Aeras), and in the spleen by 0.93 log10 CFU (BCG SSI) or 1.06 log10 CFU (BCG Pasteur Aeras), compared to control animals.

Techniques: Ex Vivo, Saline, Cell Culture, Control, Standard Deviation, Software

Journal: BMC Infectious Diseases

Article Title: A new tool for tuberculosis vaccine screening: Ex vivo Mycobacterial Growth Inhibition Assay indicates BCG-mediated protection in a murine model of tuberculosis

doi: 10.1186/s12879-016-1751-4

Figure Lengend Snippet: Protection conferred to infection with M. tuberculosis by BCG SSI and BCG Pasteur Aeras. Groups of 6 C57BL/6 mice were infected 6 weeks after immunization with BCG SSI (grey squares) or BCG Pateur Aeras (grey circles) via the intranasal route with 700 CFU M. tuberculosis Erdman. Control animals received saline at the time of immunization (open squares). CFUs per organ were determined 4 weeks after infection in lungs ( a ) and spleens ( b ). Each data point represents an individual animal. Error bars represent the median +/- interquartile range. Statistical significance was determined by one-way ANOVA with Holm-Sidak correction for multiple comparisons using GraphPad Prism

Article Snippet: We found that protection was conferred by both BCG strains, with a significant reduction of bacterial burden in the lung by 0.73 log10 CFU (BCG SSI) or 0.79 log10 CFU (BCG Pasteur Aeras), and in the spleen by 0.93 log10 CFU (BCG SSI) or 1.06 log10 CFU (BCG Pasteur Aeras), compared to control animals.

Techniques: Infection, Control, Saline

Journal: BMC Infectious Diseases

Article Title: A new tool for tuberculosis vaccine screening: Ex vivo Mycobacterial Growth Inhibition Assay indicates BCG-mediated protection in a murine model of tuberculosis

doi: 10.1186/s12879-016-1751-4

Figure Lengend Snippet: Ex vivo MGIA using M. smegmatis distinguishes between immunized and control animals. 5 × 10 6 splenocytes from mice immunized with BCG Pasteur Aeras (grey circles) or given saline (open squares) were co-cultured with 50 CFU of M. smegmatis 5 weeks after immunization. Splenocytes were obtained from 6 immunized and 6 control animals, each represented by an individual data point. Error bars represent the median +/- interquartile range. Statistical significance was tested using the unpaired t test function in GraphPad Prism

Article Snippet: We found that protection was conferred by both BCG strains, with a significant reduction of bacterial burden in the lung by 0.73 log10 CFU (BCG SSI) or 0.79 log10 CFU (BCG Pasteur Aeras), and in the spleen by 0.93 log10 CFU (BCG SSI) or 1.06 log10 CFU (BCG Pasteur Aeras), compared to control animals.

Techniques: Ex Vivo, Control, Saline, Cell Culture

Journal: BMC Infectious Diseases

Article Title: A new tool for tuberculosis vaccine screening: Ex vivo Mycobacterial Growth Inhibition Assay indicates BCG-mediated protection in a murine model of tuberculosis

doi: 10.1186/s12879-016-1751-4

Figure Lengend Snippet: Ex vivo MGIA reflects importance of IFNγ for growth control of mycobacteria. 1 × 10 6 , 2 × 10 6 , and 3 × 10 6 splenocytes from IFNγ-deficient (filled diamonds) or wild type mice (grey triangles) were co-cultured with 750 CFU ( a ) or 100 CFU ( b ) of BCG Pasteur Aeras. Splenocytes were obtained from a total of 5 IFNγ-deficient and 5 wild type animals. Aliquots from each spleen were cultured with different numbers of mycobacteria as indicated, and are represented by individual data points. Error bars represent the median +/- interquartile range. Statistical significance was tested using the unpaired t test function in GraphPad Prism

Article Snippet: We found that protection was conferred by both BCG strains, with a significant reduction of bacterial burden in the lung by 0.73 log10 CFU (BCG SSI) or 0.79 log10 CFU (BCG Pasteur Aeras), and in the spleen by 0.93 log10 CFU (BCG SSI) or 1.06 log10 CFU (BCG Pasteur Aeras), compared to control animals.

Techniques: Ex Vivo, Control, Cell Culture

Journal: Nature Communications

Article Title: Early mucosal responses following a randomised controlled human inhaled infection with attenuated Mycobacterium bovis BCG

doi: 10.1038/s41467-025-60285-4

Figure Lengend Snippet: The first volunteers were screened on 19 th March 2019 and enrolment commenced on 29 th April 2019. The final volunteer was enrolled on 12 th February 2020, and the last volunteer visit was 30 th July 2020. Volunteers were randomised to receive either 1 × 10 7 CFU aerosol BCG Danish (Group A) or aerosol saline (Group B) with a bronchoscopy at either Day 2 (D2; Group 1) or Day 7 (D7; Group 2) following inhalation. Volunteers were blinded to intervention. Reasons for exclusion during screening included blood dyscrasias, respiratory disease or poor baseline lung function, IGRA positivity.

Article Snippet: 1 ml BAL or WB were stimulated overnight with BCG Pasteur (1.2 x 10 6 CFU/ml; Aeras), and otherwise as previously described .

Techniques: Aerosol, Saline

Journal: Nature Communications

Article Title: Early mucosal responses following a randomised controlled human inhaled infection with attenuated Mycobacterium bovis BCG

doi: 10.1038/s41467-025-60285-4

Figure Lengend Snippet: BAL post-BCG or saline inhalation, or blood post-BCG inhalation. Stained with Panel 1. A Eosinophils were defined as live singlets, Lin- (CD19, CD56, CD3), CD16- Siglec8+ and shown as % CD3- cells. BAL D7 control v BCG p = 0.03, BCG D2 v D7 p = 0.0007; after correction BCG D2 v D7 p = 0.01. Blood baseline to D2 p = 0.03, D56 p = 0.045. B Neutrophils were defined as live singlets, Lin- (CD19, CD56, CD3), CD16+ , CD66b+ . Shown as % CD3- cells. BAL D2 control v BCG p = 0.007, BCG D2 v D7 p = 0.009. Blood baseline to D7 p = 0.03, D14 p = 0.07, D28 p = 0.003, D56 p = 0.003. After Dunns correction D7 p = 0.02, D28 p = 0.03, D56 p = 0.02. C Antigen presenting cells (APCs) refer to macrophages, monocytes and dendritic cells and were defined as live singlets, CD19-CD3-CD56-CD66b- CD45+ and shown as % leucocytes, defined as live singlets, CD19-. BAL D7 control v BCG p = 0.003, BCG D2 v D7 p = 0.0007; after correction BCG D2 v D7 p = 0.002. Blood baseline to D14 p = 0.03, D28 p = 0.006. After Dunns correction D28 p = 0.01. D Natural Killer (NK) cells defined as CD19-, CD3-, CD56+ to identify the total NK cell population as % CD3- cells; or E CD16+ NK cells as % total NK cell population. BAL NK cells BCG D2 v D7 p < 0.0001, after correction p = 0.0009. BAL CD16+ NK cells D7 control v BCG p = 0.007, BCG D2 v D7 p = 0.02. After correction D7 control v BCG p = 0.01. Blood baseline to D2 p = 0.04. Differences in cell frequency between BAL groups were calculated using a two-sided Mann-Whitney and corrected with Dunns; and in the blood, differences between baseline samples and corresponding timepoints following BCG inhalation were calculated using a paired two-sided Wilcoxon signed rank test against baseline only and corrected with Dunns. Statistically significant differences are presented in the figure, red asterix indicates significant differences after Dunn’s correction. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. See Supplementary Note and source data for sample sizes per group. Source data are provided as a Source Data file. BAL: Black dots indicate BCG, grey squares saline control. Blood: Solid dots indicate Group 1 (D2 bronchoscopy) volunteers and circles represent Group 2 (D7 bronchoscopy) volunteers. Box and whisker plots show median, IQR and min/max.

Article Snippet: 1 ml BAL or WB were stimulated overnight with BCG Pasteur (1.2 x 10 6 CFU/ml; Aeras), and otherwise as previously described .

Techniques: Saline, Staining, Control, MANN-WHITNEY, Whisker Assay

Journal: Nature Communications

Article Title: Early mucosal responses following a randomised controlled human inhaled infection with attenuated Mycobacterium bovis BCG

doi: 10.1038/s41467-025-60285-4

Figure Lengend Snippet: BAL post-BCG or saline inhalation or blood post-BCG inhalation stained with Panel 2 (γδ T cells and CD3+ CD56+ cells, whole blood) and Panel 3 (MAITs, iNKTs, CD161 + T cells, PBMCs). As Panel 3 was third priority, there were only sufficient BAL cells for three D2 BCG-infection and four D7 BCG-infection BAL samples to be stained for detection of MAIT, iNKT and CD161+ T cells. A MAITs defined as live singlets, CD19-, CD14-, CD3+ 5-OP-RU MR1 tetramer + , 6-FP control MR1 tetramer- (NIH Tetramer Core facility); and shown as % CD14-CD19- cells. B CD161+ T cells defined as live singlets, CD14- CD19- CD3 + CD161+ and shown as % CD14-CD19- cells. C γδ T cells were live singlets, CD19- CD14- CD3+ γδpan+ cells combined with live singlets, CD19- CD14- CD3 + γδ2+ cells. Expressed as % CD14- CD19- cells. BAL: BCG D2 v D7 p = 0.02; after correction p = 0.04. Blood: baseline to D7 p = 0.002. D iNKTs defined as live singlets, CD19- CD14- CD3+ , Vα24+ Vβ11+ CD56+ CD3+ cells and shown as % CD14-CD19- cells. Blood: baseline to D2 p = 0.03. E NKT-like CD3+ CD56+ cells defined as live singlets, CD19- CD14- CD3+ CD56+ cells and shown as % CD14- CD19- cells. BAL: BCG D2 v D7 p = 0.0001; after correction BCG D2 v D7 p = 0.0006. MAITs ( F ), CD161 + T cells ( G ) and iNKT cells ( H ) were further defined by CD4 CD8 subsets; median % parent. DN: double negative (CD4-CD8-) ( F – H BAL on upper panels and blood on lower panels). Differences in cell frequency between BAL groups were calculated using a two-sided Mann-Whitney and corrected with Dunn’s; and in the blood, differences between baseline samples and corresponding timepoints following BCG inhalation were calculated using a two-sided paired Wilcoxon signed rank test against baseline only and corrected with Dunn’s. Statistically significant differences are presented in the figure, red asterix indicates significant differences after Dunn’s correction. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. See Supplementary Note and source data for sample sizes per group. Source data are provided as a Source Data file. BAL: Black dots indicate BCG, grey squares saline control. Blood: Solid dots indicate Group 1 (D2 bronchoscopy) volunteers and circles represent Group 2 (D7 bronchoscopy) volunteers. Box and whisker plots show median, IQR and min/max.

Article Snippet: 1 ml BAL or WB were stimulated overnight with BCG Pasteur (1.2 x 10 6 CFU/ml; Aeras), and otherwise as previously described .

Techniques: Saline, Staining, Infection, Control, MANN-WHITNEY, Whisker Assay

Journal: Nature Communications

Article Title: Early mucosal responses following a randomised controlled human inhaled infection with attenuated Mycobacterium bovis BCG

doi: 10.1038/s41467-025-60285-4

Figure Lengend Snippet: A BAL (upper panels) and whole blood (lower panels) cells (baseline, D2, D7, D14, D28, D56), Panel 2 staining. T cells (live singlets, CD19-, CD14-, CD3+ ); % Leucocytes (singlets, CD19-). (i) Total T cells; (ii) CD4 + T cells; (iii) CD8+ T cells; (iv) CD4-CD8- T cells (double negative, DN); (v) CD4+ CD8+ T cells (double positive, DP). (i) Total T cells: BAL BCG D2 v D7 p < 0.0001, after correction p = 0.0003; Blood baseline to D2 p = 0.003, after correction p = 0.03. (ii) CD4 + T cells: BAL BCG D2 v D7 p = 0.0016, after correction p = 0.003. (iii) CD8+ T cells: BAL D7 control v BCG p = 0.02, after correction p = 0.03; BCG D2 v D7 p = 0.02. (iv) DN T cells: BAL D7 control v BCG p = 0.04. (iv) CD4+ CD8+ cells: BAL D2 control v BCG p = 0.01; D7 control v BCG p = 0.009 after correction p = 0.02. BCG D2 v D7 p = 0.002, after correction p = 0.01. B BAL, Panel 2 or 4 staining. Antigen (BCG)-specific IFN-γ+ T-cells in (i) Total T cells; (ii) CD4+; (iii) CD8+; (iv) DN; or (v) CD4+ CD8+ T cells. Shown as % parent. (i+ii) Total and CD4 + T cells: BCG D2 v D7 p = 0.02 with correction p = 0.03. (v) CD4+ CD8+ T cells: D7 control v BCG p = 0.02, BCG D2 v D7 p = 0.03. C, D BCG-stimulated whole blood cells. Panel 2 or 4 staining. C Antigen (BCG)-specific IFN-γ+ (i), TNF-α+ (ii) or IL-2+ (iii) CD4+ T cells or CD8+ T cells. Shown as % parent. C (i) IFN-γ+ CD4+ T cells: D7 p = 0.03, D28 p = 0.03. IFN-γ+ CD8+ T cells: D28 p = 0.03. (iii) IL2+ CD4+ T cells: D28 p = 0.03, D56 p = 0.0002. IL2+ CD8+ T cells: D2 p = 0.03. D TNF-α+ BCG-reactive monocytes. D7 p = 0.004, D14 p = 0.02, D56 p = 0.003. E Antigen (PPD)-specific IFN-γ ELISpot responses in fresh PBMCs. SFC = spot-forming cells. Different timepoints after BCG: Wilcoxon paired test, D7 p < 0.0001, D14 p = 0.0003, D28 p = 0.01, D56 p = 0.04; after correction, D7 p = 0.0008, D14 p = 0.003. BCG v control: Mann-Whitney, D2 p = 0.049, D7 p = 0.0006, D14 p = 0.04, D56 p = 0.01; after correction, D7 p = 0.001, D56 p = 0.04. Statistically significant differences are presented in the figure. BAL: Two-sided Mann-Whitney; blood: two-sided paired Wilcoxon against baseline. Red asterix indicates significant differences after correction (Dunns). * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. See Supplementary Note and source data (provided as a Source Data file) for sample sizes. BAL: Black dots BCG, grey squares saline control. Blood: Solid dots Group 1 (D2 bronchoscopy), circles Group 2 (D7 bronchoscopy). Box and whisker plots show median, IQR and min/max, crosses show mean.

Article Snippet: 1 ml BAL or WB were stimulated overnight with BCG Pasteur (1.2 x 10 6 CFU/ml; Aeras), and otherwise as previously described .

Techniques: Staining, Control, Enzyme-linked Immunospot, MANN-WHITNEY, Saline, Whisker Assay