1. Group B3 (BCG replacement strategy) Define the immunogenicity endpoints to be used for the preclinical studies and the clinical trial Claude MERIC, Narges MIANDEHI 1

2. This definition is based on information or the immunological correlates of protection proposed by group B1 to be used for groups B4 and B5 2  Which immunogenicity assays (T and/or B cell assays) should be utilized for the preclinical studies and the clinical trial ;  A detailed description of the assays selected, including the limitations and advantages of the assays ;  The results expected.

3. Introduction Correlates of protection against TB infection  CMI response Th1 (CD4+, IFN-g, IL-2 and TNF-a) Importance of polyfunctional T cells Th17 (CD4+, IL-17) may be important for protective immunity CTL (CD8+, IFN-g and TNF-a) cytotoxic activity  Activated macrophages (the major source of TNF-a) able to kill phagocytosed Mtb Antibodies are not considered to be critical  Would be measured by ELISA or OPA with macrophages (?) 3

4. Immunological endpoints Focus on cell mediated immunity as Th1 and CD8+ responses correlate with protection CD4+ cell response to BCG proteins (PPD) and to each overexpressed antigen  Polyfunctional IFN-g, IL-2, TNF-a expression  IL-17 expression by CD4+ T cells => Th17 response CD8+ to each overexpressed antigen  Polyfunctional IFN-g, IL-2, TNF-a expression 4

5. Preclinical studies Mouse  Cytokine (IFN-g and others) production after restimulation of mouse lymphocytes with antigen or with peptide pools Detection with a multiplex assay (Luminex)  Intracellular cytokine detection by flow cytometry following stimulation of mouse lymphocytes with antigen or peptide pools CD4+ and CD8+, IFN-g, IL-2, TNF-a, IL-17  Functional CTL assay (?) Effectors: stimulated CD8+ cells Targets: Autologous TB infected macrophages Endpoint: Bacterial killing (bacterial culture) Guinea pig  Protection against challenge only 5

6. Phase I clinical study Screening (volunteers negative for TB infection at enrolment)  TST Classical and modified test using selected antigens  IGRA Kits and modified test using selected antigens Immunogenicity  Intracellular cytokine detection on stimulated whole blood CD4+ and CD8+, IFN-g, IL-2, TNF-a, IL-17 PBMC control for some subjects to check for coherence (for artefacts with whole blood)  Measurements on D0, D7, D14, D28, D42, D84, D182  ELISPOT: nd  Tetramers: nd 6

7. Screening  Tuberculin test - in vivo In use for about 100 years Measures a delayed-type hypersensibility response Uses tuberculin PPD, a crude mixture of antigens, many of them shared by M. tuberculosis, bovis and BCG Produces a white or rose-colored induration which peaks after 48-72h and lasts up to 1 month Infiltration of CD4+ T cells and activated macrophages Inflammatory reaction  Interferon-  release assay (IGRA) - ex vivo IFN-  production by circulating T cells 16-20h stimulation by Mtb-specific antigens (not in BCG, M bovis, M avium)  Peptides from proteins encoded in region of difference (RD): CPF-10, ESAT-6, TB7.7 (only QuantiFERON) Measurement of IGN-g by ELISPOT or whole-blood ELISA Responding cells likely to be effector memory, recently induced by Mtb Cannot distinguish LTBI from TB 7

8. TST and IGRA T-SPOT (Oxford Immunotec)  Type of ELISPOT assay  Counts the number of effector T cells produce IFN-  8

9. TST and IGRA QuantiFERON (Cellestis)  ELISA  Measures IFN-  in whole blood following stimulation with TB antigens  Test Principle (source: Cellestis web site) The QuantiFERON-TB Gold In-Tube assay is an in vitro diagnostic laboratory test that aids in the detection of infection with Mycobacterium tuberculosis. It uses human whole blood, with patented assay technology based on the measurement of Interferon-gamma (IFN-  ) secreted from stimulated T-cells previously exposed to Mycobacterium tuberculosis. The QuantiFERON-TB Gold In-Tube assay is a straightforward laboratory test that involves 4 simple steps:  Collection of blood into QuantiFERON-TB Gold In-Tube Blood Collection Tubes.  Overnight incubation at 37°C. TB infected patients' blood cells will produce IFN- .  Detection of released IFN-  in harvested plasma using a quick and easy ELISA.  Analysis of data using the QuantiFERON-TB Gold In-Tube Analysis Software. 9

10. Intracellular cytokine staining Mouse  Lymphocytes washed from the spleen  Stimulation using overexpressed antigens and peptides pools from them, PPD, PHA positive control  Addition of Brefeldin A (to prevent excretion of newly made cytokines)  Permeabilization solution  Fluorescence conjugated antibodies to CD3, CD4, CD8, IFN-g, TNF-a, IL-2, IL-17  Fixation  Analysis using a flow cytometer 10

11. Intracellular cytokine staining Whole blood method (Humans) Principle and reagents  Stimulation of lymphocytes in whole blood in the presence of anti-CD49d and anti-CD28 costimulatory antibodies to enhance the specific response (1ug/ml each)  To be done immediately after blood collection Blood stored at 2-8°C until stimulation 250 μl needed to acquire 40,000 events (sufficient to detect events <1% of the considered cell type) Stimulation for approx. 12hr at 37°C Brefeldin added at 7hr  Lysis of red cells using FACS lysing solution  Fixation  Frozen until labeling and analysis using a flow cytometer Pros and cons  Need for small volume of blood assay: 250 ul  Freeze the fixed cells: limits variability due to viability of frozen cells  Detection of only effector/memory cells (short stimulation)  More tricky to detect multiple markers than PBMC  Adapted to rural, developing country trial setting *Hanekom et al, 2004, JIM 291:185; Duramad et al, 2004, Cancer Epi Biom & Prev 13:1452 11

12. Intracellular cytokine staining PBMC assay (Humans) Principle and reagents  Purification of PBMC from blood  Blood layered on Ficoll-Paque based on density gradiant  Centrifuge  4 phases are formed (top to bottom) plasma and other constituents a layer of mono-nuclear cells called buffy coat (PBMC/MNC) Ficoll-Paque erythrocytes & granulocytes (pellet)  PBMC are frozen at -70°C (critical step)  Viability checked upon thawing  Stimulation with antigen  Labeling and analysis by flow cytometry Pros and cons  Use of purified cell preparation for stimulation  Need for larger volume of blood (for purification) than whole blood stimulation  Freeze of PBMC in clinical trial Due to workload: critical step 12

13. Validation of the assays for human testing Steps to be validated  Whole blood stimulation  Cell freezing/thawing (PBMC)  Staining  Flow cytometry General principles of assay validation: to be adapted to the specific assays  Specificity Discrimination of the signal from different channels  Linearity / range For % of cells with a defined phenotype (gating?)  Accuracy Comparison with other method (ELISPOT?)  Precision Repeatability Intermediate precision ( days, analysts, equipment, etc)  Reproducibility Inter-laboratory trial: not needed  Detection limit  Robustness 13

14. Expected results from immunology analysis Stimulation of T lymphocytes with the phenotype described as correlating with a protective response against TB  Antigen-specific CD4, CD8, and Th17 responses Polyfunctional T cells of long duration specific to BCG and to overexpressed antigens  Responses to BCG antigens as strong as after BCG vaccination Significant response to the overexpressed antigens  Higher response than in Mtb infected individuals 14

15. BACKUP 15

16. TST and IGRA QuantiFERON (Cellestis) source: Cellestis web site 16

17. Flow cytometry - Equipment 17 Institut Pasteur, eLearning

18. Flow cytometry – Data analysis 18 Forward scatter (FSC): proportional to particle size Side scatter (SSC): granularity, structural complexity Institut Pasteur, eLearning

19. 19 Figures below show Peyer's patch cells obtained from a C57BL/6 mouse and stained with FITC anti-B220 and PE anti-CD3 or with FITC anti-B220 and PE anti-CD19. The purpose of this example is to illustrate the information that can be generated from a sample using two antibodies simultaneously. Using scatter measurements, a gate has been drawn to include all lymphocytes, thus excluding dead cells and debris. Gating enables the number of cells positive for CD3, CD19 and/or B220 to be expressed as a percentage of lymphocytes only. CD3, CD19 and B220 staining parameters found on gated cells are shown in the appropriately labeled dot plot. In each quadrant of dot plots (a, b, c, d) are indicated the percentage of positive cells in the gated population. In the right dot plot, quadrant b contains the B220+/CD19+ cells (B lymphocytes) (73.4% of the cell population analyzed), quadrant d contains the B220-/CD19- cells (mostly T lymphocytes). In middle dot plot, quadrant a contains the B220- /CD13+ cells (T lymphocytes) (19.5% of the cell population analyzed), quadrant c contains the B220+/CD3- cells (B lymphocytes) (76.9% of the cell population analyzed), and quadrant b shows the absence of the B220+/CD3+ cells; the low percentage of double positive cells (1.1% of the cell population analyzed) may rely on the presence of clusters containing one B220+ cell and CD3++ cell. Institut Pasteur, eLearning