Supplementary Figure 1

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Gillian Jordan
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1 Supplementary Figure 1 FMO control (no HLA-DR antibody) FMO control (no HLA-DR antibody) Supplementary Figure 1. Gating strategy for HLA-DR+ T-cells. A gate was drawn around lymphocytes based on cell size (FSC-A/SSC-A) and then a second gate was drawn around single cells (FSC-A/FSC-H). Live singlet cells were then gated as CD3+CD19. CD3+ T-cells were then individually gated as either CD4+ or CD8+. A fluorescence minus one control tube (FMO) containing all stains except HLA- DR was used to determine the position of the HLA-DR+ gate on either CD4+ or CD8+ T-cells. 1

2 Supplementary Figure 2 Supplementary Figure 2. Distribution of immune response variables in case and control infants at D0 and D28. Infants are stratified according to TB case (red) or control (grey) status. Immune responses at D0 (panels A, C, E) or D28 (panels B, D, F) are shown. The log frequency of live cells expressing cell surface phenotype markers is shown. SFC/million PBMC is the number of IFN- spot forming cells per million PBMC (only results from infants with a PHA response greater than 1000 SFC/million are shown). 2

3 Supplementary Table 1. Spearman rank correlations between activated T cells and CMV response D0 Correlations HLA-DR+ CD4+ CD8+HLADR+ ELISpot CMV CD4+ HLA-DR+ CD4+ R value P value N CD4+ R value P value N CD8+HLADR+ R value P value N CMV ELISpot R value P value N D28 Correlations HLA-DR+ CD4+ R value P value N CD4+ R value P value N CD8+HLADR+ R value P value N CMV ELISpot R value P value N Significant correlations highlighted in bold 3

4 Supplementary Table 2. Estimated odds ratio for the association between risk of TB disease and HLA-DR+ CD4+ by treatment group MVA85A or Placebo Estimated odds ratio for the association between risk of TB disease and A.CD4 by treatment group MVA85A or Placebo Day Model N Cases Est OR LL UL P value D0 CD4+ HLA-DR D0 CD4+ HLA-DR MVA85A or Placebo D0 CD4+ HLA-DR *MVA85A or placebo^ D28 CD4+ HLA-DR <0.001 D28 CD4+ HLA-DR <0.001 MVA85A or placebo D28 CD4+ HLA-DR * MVA85A or placebo ^ ^Est OR is for the control group. 4

5 Supplementary Table 3. Estimated Odds-Ratio (OR) of TB disease from a Conditional Logistic Regression of Day 28 immunological variable for MVA85A and placebo arms Quantitative Est OR* 95% CI P value Est 95% CI P variable MVA85 MVA85A MVA85 OR* Placebo value A A Placeb Placeb o o CD3+ T cell , , CD4+ T cell , , CD4+HLADR+ T , , cell CD4+CD25+CD , , T cell CD8+ T cell , , HLA-DR+ CD , , T cell CD14+CD , , monocyte CD14+CD , , monocyte CD19+ B cell , , BCG MGIA , , A ELISpot , ,

6 BCG ELISpot , , PPD ELISpot , , TB10.3/ , , ELISpot EBV ELISpot , , CMV ELISpot , , FLU ELISpot , , GAM.DEL , , (putative) NK.16NEG , , (putative) NK.16POS (putative) , , CD14+CD16+ /CD , , CD14+CD16-0 0, , /CD3+ Ag85A IgG , ,

7 Supplementary Table 4. Conditional Logistic Regression with Z-transformed variables Estimated Odds-Ratio (OR) of TB disease from a Conditional Logistic Regression of Day 0 immunological variable Quantitative variable N Cases Est OR* 95% CI P value FDR value AUROC CD3+ T cell , CD4+ T cell , CD4+HLADR+ T cell , CD4+CD25+CD127- T , cell CD8+ T cell , HLA-DR+ CD8+ T cell , CD14+CD , monocyte CD14+CD , monocyte CD19+ B cell , BCG MGIA A ELISpot , BCG ELISpot , PPD ELISpot , TB10.3/10.4 ELISpot ,

8 EBV ELISpot , CMV ELISpot , FLU ELISpot , GAM.DEL (putative) , NK.16NEG (putative) , NK.16POS (putative) , CD14+CD16+ /CD , CD14+CD16- /CD , Exploratory Variable Ag85A IgG , Estimated Odds-Ratio (OR) of TB disease from a Conditional Logistic Regression of Day 28 immunological variable CD3+ T cell , CD4+ T cell , HLA-DR+ CD4+ T cell , CD4+CD25+CD127- T , cell CD8+ T cell , HLA-DR+ CD8+ T cell , CD14+CD , monocyte CD14+CD ,

9 monocyte CD19+ B cell , BCG MGIA A ELISpot , BCG ELISpot , PPD ELISpot , TB10.3/10.4 ELISpot , EBV ELISpot , CMV ELISpot , FLU ELISpot , GAM.DEL (putative) , NK.16NEG (putative) , NK.16POS (putative) , CD14+CD16+ /CD , CD14+CD16- /CD , Exploratory Variable Ag85A IgG , NA = model did not converge; results not available *represents the odds ratio for a 1 unit change in the immune response 9

10 Supplementary Table 5. Conditional Logistic Regression based on tertile of immune response Conditional Logistic Regression based on tertile of immune response D0 D28 Range N OR 95% CI p Range N OR 95% CI p HLADR+ CD4+ T-cells Low 2.34, Ref , Medium 7.20, , , , High 10.54, , , , BCG ELISpot Low 0.30, Ref , Medium 1.51, , , , High 1.82, , , , CD4+ T-cells Low 0.01, Ref , Ref Medium 1.02, , , , High 1.99, , , , Ag85A-specific IgG Low 0.01, Ref , Ref Medium 1.02, , , , High 1.99, , , ,

11 Table 6: Assay identification for C case-control correlates analysis: Preliminary selection Study Group / Pilot Study Sample type Assay Stimulation condition Group 2 Fresh PBMC ELISPOT UNS, BCG, Ag85A Group 3 Fresh PBMC PBMC ICS UNS, BCG, Ag85A Group 4 Whole blood Whole blood ICS UNS, BCG, Ag85A Pilot 1 Frozen PBMC Assay 1: ELISPOT (2 antigens) Pilot 1 Frozen PBMC Assay 2: Illumina HT-12 Gene expression analysis (GEX) (5 conditions) Pilot 1 Frozen PBMC Assay 3: Mycobacterial growth inhibition assay (MGIA) (1 mycobacterial strain) Pilot 1 Frozen PBMC Assay 4: Flow surface staining Live/dead, CD3, CD4, CD8, γδ, CD19, CD14 (6 populations) Pilot 1 Supernatant from stimulated frozen PBMC Assay 5: Luminex (42 plex) MGIA supernatants Assay 6: Luminex (42 plex) GEX supernatants Pilot 2 Frozen PBMC ELISPOT (duplicate wells) (3 antigens) Pilot 2 Frozen PBMC ELISPOT (triplicate wells) (3 antigens) Pilot 2 Frozen PBMC Illumina HT-12 Gene expression analysis (GEX) (5 conditions) Pilot 2 Frozen PBMC Mycobacterial growth inhibition assay (MGIA) (2 strains of mycobacteria) Pilot 2 Frozen PBMC Flow surface staining Live/dead, CD3, UNS, BCG, Ag85A UNS, BCG, Ag85A Reliability (externally validated assay or ICC) Sample available Detection of antigen specific immune response Correlation between assays (Spearmans correlation p<0.01) externally validated No Yes ELISPOT/WB- ICS externally No Yes validated externally No Yes ELISPOT/WBvalidated ICS passed Yes Yes ELISPOT/ GEX Ag85A passed Yes Yes ELISPOT/ GEX Ag85A BCG Pasteur passed Yes - - UNS passed Yes - - UNS, BCG, Ag85A UNS, BCG, PPD, Ag85A UNS, BCG, PPD, Ag85A UNS, BCG, Ag85A BCG Pasteur, MTB H37rV Externally validated Yes Yes Some subsets of analytes and GEX/MGIA NB. Supernatants collected from MGIA and GEX experiments passed Yes Yes ELISPOT/ GEX Ag85A / Ki67 proliferation passed Yes Yes ELISPOT/ GEX Ag85A / Ki67 proliferation passed Yes Yes ELISPOT/ GEX Ag85A passed Yes - MGIA and CD14+CD16+ monocytes on cell surface flow UNS passed Yes - MGIA and CD14+CD16+ monocytes on 11

12 CD4, CD8, γδ, CD19, CD14, CD16, CD25, CD127, CTLA4 (90 cell populations) Pilot 2 Frozen PBMC Assay 7: Ki67 proliferation assay (4 parameters 2 antigens CD4 and CD8) Pilot 3 Pilot 3 Frozen PBMC (short rest) Frozen PBMC (long rest) ELISPOT (8 antigens) ELISPOT (8 antigens) UNS, PPD, Ag85A UNS, BCG, PPD, Ag85A, Flu+CMV+EBV, tetanus toxoid, TB10.3/4 UNS, BCG, PPD, Ag85A, Flu+CMV+EBV, tetanus toxoid, TB10.3/4 cell surface flow Not done Yes Yes ELISPOT/ Ki67 proliferation passed Yes Yes - passed Yes Yes - The maximum volume of blood collected from each infant at each time point was 8ml. PBMC were isolated and cryopreserved in liquid nitrogen in 1-3 separate vials. Based on pilot studies conducted with non-case samples from the TB20 study the median expected recovery of viable cells from 1-2 thawed vials is 17 million (range 2-32 million). 12

13 Supplementary Table 7: Assay identification for C case-control correlates analysis: Intermediate selection of key assays/variables Assay Antigen/Cell population/cytokine Notes 1 MGIA BCG Log growth in sample tube/log growth in control tube (possible measure of vaccine response) 2 ELISPOT 85A SFC/million PBMC (measure of vaccine response) 3 ELISPOT BCG SFC/million PBMC (possible measure of vaccine response) 4 ELISPOT PPD SFC/million PBMC (possible measure of vaccine response) 5 ELISPOT 10.3/10.4 SFC/million PBMC 6 ELISPOT CMV SFC/million PBMC 7 ELISPOT EBV SFC/million PBMC 8 ELISPOT Flu SFC/million PBMC 9 Flow Cytometry T cells % of Live CD3+ lymphocytes 10 Flow Cytometry CD4+ T cells % of Live CD3+CD4+CD8- lymphocytes 11 Flow Cytometry CD8+ T cells % of Live CD3+CD8+CD4- lymphocytes 12 Flow Cytometry B cells % of Live CD3-CD19+ lymphocytes 13 Flow Cytometry Monocytes % of Live CD3-HLA-DR+CD14+ CD16-14 Flow Cytometry Inflammatory monocytes % of Live CD3-HLA-DR+CD14+ CD Flow Cytometry Regulatory T cells % of Live CD3+CD4+CD8- CD25+CD127- lymphocytes 16 Flow Cytometry Monocytes/lymphocytes % of Live CD3-HLA-DR+CD14+ CD16-/ % of Live CD3+CD4+CD8- lymphocytes 17 Flow Cytometry Inflammatory monocytes/lymphocytes % of Live CD3-HLA-DR+CD14+ CD16+/ % of Live CD3+CD4+CD8- lymphocytes 18 Flow Cytometry other -possible gamma delta T % of Live CD3+CD4-CD8- lymphocytes cells 19 Flow Cytometry Other possible natural killer % of Live CD3-CD19-CD14- lymphocytes cells 1 20 Other possible natural killer cells 2 % of Live CD3-CD19-CD14- CD16+lymphocytes 21 Flow Cytometry Activated CD8+ T cells % of Live CD3+CD8+HLA-DR+CD4- lymphocytes 22 Flow Cytometry Activated CD4+ T cells % of Live CD3+CD8-HLA-DR+CD4+ lymphocytes 13

14 Supplementary Table 8: Description of outliers Day SUBJID Variable Value assay type % live cellsinvestigation Retain yes/no E.BCG elispot viability low (61% and PHAno 6 elispot excluded, 5 retained E.BCG elispot viability low (51%) and PHno 6 flow retained, non-excluded log(e.bcg) elispot 50.8 PHA<1000 no ed and 1 excluded log(e.bcg) elispot 57.4 PHA<500 no 19 outliers 12 retained log(e.bcg) elispot 63.7 HIGH BACKGROUND EXCLno 7 excluded log(e.ppd) elispot 63.7 HIGH BACKGROUND EXCLno E.PPD elispot viability low (62%) but PHyes log(e.ppd) elispot 62.8 ok low viability yes X85A elispot 74.6 ok yes X85A elispot 78.6 ok yes X85A 55 elispot 84.4 DAY 28 and DAY -7 WRONG yesway AROUND NOW CORRECTED IN FILE C.MON flow ok requested Iman to look yes at raw flow fliles CML flow ok requested Iman to look yes at raw flow fliles I.MON flow ok requested Iman to look yes at raw flow fliles I.MON flow ok requested Iman to look yes at raw flow fliles IML flow ok requested Iman to look yes at raw flow fliles log(iml) flow ok requested Iman to look yes at raw flow fliles M.BCG MGIA replicates ok, viabilty ok. But no control tubes low on week 10 and I do have a concern regarding the robustness of MGIA log(m.bcg) MGIA 80.9 batch 10 incorrectly entered yesdata NOW CORRECTED log(e.bcg) elispot 53.6 PHA<500 no log(e.bcg) elispot 61.4 HIGH BACKGROUND EXCLUDE no ELISPOT RESULTS ALSO DAY -7 AND DAY log(e.bcg) elispot 65.7 batch 10 incorrectly entered yesdata NOW CORRECTED log(e.bcg) elispot 76.2 ok yes X85A 140 elispot 52.3 ok low viability yes X85A elispot 76.3 ok yes X85A elispot 78.3 ok yes X85A 110 elispot 79.9 ok yes X85A elispot 80 ok yes X85A 150 elispot 81.1 ok yes X85A elispot 81.7 ok yes X85A elispot 83.4 ok yes X85A elispot 84.9 ok yes X85A elispot 85.7 ok yes NK.16NEG 37.8 flow 64.2 Iman checking - machine blocked no sample run twice. Exclude A.CD4 43 flow 53.8 ok low viability yes A.CD flow 61.2 ok low viability yes C.MON 29.9 flow 80 ok yes CML flow 80 ok yes GAM.DEL 7.72 flow 53.8 ok low viability yes GAM.DEL 9.77 flow 73.4 ok yes I.MON flow 61.2 ok low viability yes I.MON 7.48 flow 65.2 batch 10 incorrectly entered yesdata NOW CORRECTED I.MON 7.12 flow 80 ok yes I.MON 10.5 flow 80.6 ok yes I.MON 7.01 flow 84.2 ok yes IML flow 61.2 ok low viability yes IML flow 65.2 batch 10 incorrectly entered yesdata NOW CORRECTED IML flow 80 ok yes IML flow 80.6 ok yes IML flow 84.2 ok yes NK.16NEG 17.2 flow 52.3 ok low viability yes NK.16NEG 12.3 flow 53.8 ok low viability yes NK.16NEG flow 61.1 ok low viability yes NK.16NEG 12.4 flow 64.3 ok low viability yes NK.16NEG 12.2 flow 65.3 batch 10 incorrectly entered yesdata NOW CORRECTED NK.16NEG 15.1 flow 67.8 ok low viability yes NK.16NEG 16.8 flow 67.8 ok low viability yes NK.16NEG 12.9 flow 71.6 batch 10 incorrectly entered yesdata NOW CORRECTED NK.16NEG 14.5 flow 73.9 batch 10 incorrectly entered yesdata NOW CORRECTED NK.16NEG 20.4 flow 76.4 batch 10 incorrectly entered yesdata NOW CORRECTED NK.16NEG 17.1 flow 76.5 batch 10 incorrectly entered yesdata NOW CORRECTED NK.16NEG 13 flow 77.2 incorrect ID number now corrected yes NK.16NEG 13.8 flow 77.5 batch 10 incorrect NOW CORRECTED yes NK.16NEG 23.7 flow 78.1 batch 10 incorrectly entered yesdata NOW CORRECTED NK.16NEG 18.6 flow 78.5 batch 10 incorrectly entered yesdata NOW CORRECTED NK.16NEG 25.5 flow 79 batch 10 incorrectly entered yesdata NOW CORRECTED NK.16NEG 24.9 flow 80 batch 10 incorrectly entered yesdata NOW CORRECTED NK.16NEG 24.7 flow 80 batch 10 incorrectly entered yesdata NOW CORRECTED NK.16NEG 14.1 flow 80.7 batch 10 incorrectly entered yesdata NOW CORRECTED NK.16NEG 15.7 flow 81.5 batch 10 incorrectly entered yesdata NOW CORRECTED NK.16NEG 16 flow 82 batch 10 incorrectly entered yesdata NOW CORRECTED NK.16NEG 21.9 flow 82.7 batch 10 incorrectly entered yesdata NOW CORRECTED NK.16NEG 16.4 flow 84.4 batch 10 incorrectly entered yesdata NOW CORRECTED NK.16POS 21.3 flow 77.8 ok yes T.LIVE 50 flow 50 ok low viability yes T.LIVE 52.3 flow 52.3 ok low viability yes T.LIVE 52.9 flow 52.9 ok low viability yes T.LIVE 53.6 flow 53.6 ok low viability yes T.LIVE 53.8 flow 53.8 ok low viability yes

CD25-PE (BD Biosciences) and labeled with anti-pe-microbeads (Miltenyi Biotec) for depletion of CD25 +

Supplements Supplemental Materials and Methods Depletion of CD25 + T-cells from PBMC. Fresh or HD precultured PBMC were stained with the conjugate CD25-PE (BD Biosciences) and labeled with anti-pe-microbeads