Group A3: Immunological endpoints Yacouba Cissoko Agustina Errea Mamadou Korka Diallo

PRECLINICAL STUDIES

Assesing immune response in animal model Which endpoints? Protective immune response Available Tools. challenge Humoral response Celular response Mucosal response after challenge Antigen specific

Detection of antigen especific antibodies: IgG1 and IgG2 titers * Why? Are we triggering an immune response? Other species: IgG1 correlated with response Th2 and IgG2 with Th1 Disadvantage: no correlation between B cells response and protection * How ? ELISA 10 µg/ml antigen per well Serial dilutions of sera from immunized animals from (duplicates) Anti guinea pig IgG1-HRP or Anti guinea pig IgG2- HRP Substrate: OPD - DO lecture: 492nm CTR (-): sera from non vaccinated animal CTR (+): sera from BGC vaccinated animal Titer definition: highest dilution rate yielding absorbency 3 times greater than the negative control.

Proliferation assay 1x10 6 Cells/ ml CFSE 5 µM 2x10 5 cells/ well Antigen: whole protein fusion CTR+: concavaline A CTR- : media alone spleen Single cell suspension Red blood lysis Staining cell surface markers Flow cytometry CFSE staining Harvest sample 72 hs CD4- Per CP CD8- APC IP ( live cells) CFSE techniques allows qualitative and cuantitative analysis evaluation of proliferation index Antigen Specific cellular response: proliferative response by CFSE staining and FACS

Leukocyte recruitment to lung after challenge with Mtb Why? Our boosting is able to generate immune effectors mechanism of control of the disease? Previous reports (1) : vaccinated guinea pig Determinations: Number of CD4+ cells and CD8+ Tcells Number of CD4+ CD45+ T cells and numbers of CD8+ CD45+ T cells Number of macrophages Frequency of macrophages expressing MHCII (1) Ordway D et al. Clin Vaccine Immunol 2008 Aug;15(8): Tcells in lugs Bacterial burden Activated status per gram tissue Macrophages MHC II +

Lungs ≠ times points Leukocyte recruitment to lung after challenge with Mtb Enzimatic digestion Red blood cells lysis Anti- CD4, CD8, pan T cell, MIL4, CD45. Anti-macrophages (MR-1) MHCII Singles staining and Cells without staining COMPENSATION Cells surface markers staining How? Flow cytometry Gating strategy Lymphocytes Single cell suspension

Expectations Boosted animals ( vs BCG CTR) Increased immune response at sistemic levels: Increased proliferation rates Increased levels of Antibodies with mix profile Increased capacity of development of active response against the pathogen at mucosal level: Increased and persistent levels of T cell to the lung after infection ( CD4 and CD8+ T cells) with an activation profile ( high numbers of T cells expressing CD45) Increased recruitment and activation of macrophages in response to infection.

PHASE II CLINICAL TRIAL

Primary variable to assess immune response to PFP (Ag 85A+RV2660+PPE44) CELL MEDIATED IMMUNE RESPONSE: Percentage of CD4 and CD8 T cells producing IFN-γ, TNF-α, and/or IL-2, independently or simultaneously following stimulation (peptide pools from PFV) in the different groups.

Specific immune response to PFP Vaccine (Ag85A, RV2660 and PPE44) major HLA class I related peptide Ag 85A CD8 tetramer assay. proportion of memory vs naive vs effector cells by extracellular staining for CD45RO and CCR7 HUMORAL IMMUNE RESPONSE: Assessed by Ab level in sera specific to PFP.

Brewelskloof Hospital Immunology lab Centre 1 Centre2 Centre 3 Work on frozen sample ?

Comprehensive immunomonitoring (1) ASSAYDAYPURPOSETUBE/V OL DESCRIPTION PFP Ab titer in seraD0,7,28, 37,86, 364 for groups A, B &C + 56, 112 for groups D & E Assess Humoral immunity to vaccine ½ of Dry tube /5ml Serum, 2x 0.75 ml aliquot, freeze at - 80° (field) for Ab ELISA (main lab) HLA typingD0Exploring confounding variable for CMI Ficol layer Ficol layer will be harvested for HLA typing by PCR (other lab) Cytokine titer in seraD0, D7 for all groups D37 for group D,E Assess profil of T cell response to vaccine ½ of Dry tube /5ml Serum, 2x 0.75 ml aliquot, freeze at - 80° (field) for IFN-  ELISA (main lab)

Comprehensive immunomonitoring (2) ASSAY STUDY DAY PURPOSETUBE/VOLDESCRIPTION Intracellular staining for cytokine production D0,7,28, 37,86, 364 for groups A, B &C + 56, 112 for groups D & E Assess cellular immune response to vaccine CPT /40ml Cells will be separated immediatly on the field by centrifugation in CPT, then Freeze down using linear freeze box overnight then stored in LN dryshiper /send to Main Lab/CFSE, ICS,ECS,tetram er from thawed PBMC Extra cellular staining for cell percentage Assess cellular immune response to vaccine Ag 85A - CD4 tetramer assay D0 and D364 Assess spécific CD8 response to one of the vaccine componment

Specific IgG to antigen in sera Will be mesured by ELISA, Quantitative ELISA using diluted sera of Ab will be performed:  10  g/ml antigen per well  serial dilutions of sera from study subjects 1/100 (duplicates)  Anti human IgG- HRP  substrate: OPD - reader: 492nm  negative control: diluents  positive control: will be a sample of sera from previous positive subjects We expect to have High level Ab in boosted subject signing humoral response

ELISA for INF-  in sera Quantitative ELISA with diluted INF-  standard and subjects sera :  50  l of undiluted sera per well in duplicate for each patient  Standard INF-  10 ng in 100  l PBS in first well triplicate then serial dilutions step ½ until nil (PBS)  Mouse Anti INF-  IgG- Biotin lated + Avidin Peroxydase  substrat: OPD - reader: 492nm  Standard curve will be drawn to determine function beteween dilutions and OD then apply to the sample to find quantity of IFN-g in sera of study subject. We expect to have High level of IFN-g in boosted subject signing TH1 response

PBMC separation & Thawing On CPT 2tubes of 10 ml per subject Centrifuge at 1500 rpm at 25°C for 15 min Expecting to harvest PMBC per subject per blood drawing. Resuspend in CRPMI (79%RPMI, 20% FCS) + 1%DMSO Freeze linearly (Isopropyl alcool box for 3 H at -80°C)  L. Nitrogen Thaw: washing out with RPMI; resuspending with CRPMI Expecting lost of PMBC 25% during thawing remain Use cell in different assay as needed. Field Main Lab

Extra & Intra cellular staining for cell population Stimulation of PMBC with 10  M of PFP peptide pool in presence of Befeldin A 10  g/ml. Incubate for 6 hours at 37°, 5% CO2. Control: - (non stimulated); + (stimulated/PHA). ECS with Anti CD4-FITC, Anti CD8-PE and Anti CD3 ECD, Fixe. Permeabilization, ICS of cytokine inside the producing cells with INF- , TNF- , IL2 fluorochromes labeled specific Ab. The dynamic in number of those cells will be monitored following the mentioned time points during the study. Expecting increase number of polyfunctional T cell after the boost.

HLA typing /Tetramer assay HLA typing by PCR: most common HLA A aplotype in the population to select suitable peptide for CD8 tetramer A specific CMH class 1( A*0201) tetramer of peptide p from the Ag85A,will be use to bind specific CD8 T cells. Simultaneous surface staining with Anti CD45Ro-APC, anti CCR7-PC5. To look at single peptide as inductor in the context of CMH class-1 for CD8 memory response (D0 vs D364) Expecting increase of specific CD8 memory T cell (D0 vs D364) Smith SM and al. J Immunol 2000;165;

Flow cytometry BD FACSCanto Standard System with 6-color capacities and 2-laser system (488, 633 nm) and a fully integrated fluidics cart software : BD FACSDiva™ Use for cell caracterisation, proliferation and tetramer assay.  At least events count  Good compensation  Good gate setting  Data auditing