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Group A3: Immunological endpoints

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. Antigen specific. Celular response. Humoral response.

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Group A3: Immunological endpoints

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

  2. PRECLINICAL STUDIES

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

  4. 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.

  5. Harvest sample CFSE staining Proliferation assay Staining cell surface markers 72 hs spleen • CD4- Per CP • CD8- APC • IP ( live cells) 1x106 Cells/ ml CFSE 5 µM • Single cell suspension • Red blood lysis Flow cytometry 2x105 cells/ well Antigen: whole protein fusion CTR+: concavaline A CTR- : media alone Antigen Specific cellular response: proliferative response by CFSE staining and FACS • CFSE techniques allows qualitative and cuantitative analysis evaluation of proliferation index

  6. 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 Macrophages MHC II + Bacterial burden Tcells in lugs Activated status per gram tissue (1) Ordway D et al. Clin Vaccine Immunol 2008 Aug;15(8):1248-58.

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

  8. 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.

  9. PHASE II CLINICAL TRIAL

  10. 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.

  11. 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.

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

  13. Comprehensiveimmunomonitoring (1)

  14. Comprehensive immunomonitoring (2)

  15. Specific IgG to antigen in sera • Will be mesured by ELISA, • Quantitative ELISA using diluted sera of Ab will be performed: • 10mg/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

  16. ELISA for INF-g in sera • Quantitative ELISA with diluted INF-g standard and subjects sera : • 50ml of undiluted sera per well in duplicate for each patient • Standard INF-g 10 ng in 100ml PBS in first well triplicate then serial dilutions step ½ until nil (PBS) • Mouse Anti INF-gIgG- Biotin lated + AvidinPeroxydase • 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

  17. PBMC separation & Thawing Field Main Lab On CPT 2tubes of 10 ml per subject Centrifuge at 1500 rpm at 25°C for 15 min Expecting to harvest 30.106 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 22.5. 106. Use cell in different assay as needed.

  18. Extra & Intra cellular staining for cell population Stimulation of PMBC 500.103 with 10 mM of PFP peptide pool in presence of Befeldin A 10 mg/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-g, TNF-a, 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.

  19. 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 p48-56 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;7088-95

  20. 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 100 000 events count • Good compensation • Good gate setting • Data auditing

  21. THANKS!

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