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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. - PowerPoint PPT Presentation
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Group A3:Immunological
endpointsYacouba CissokoAgustina 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
1x106 Cells/ ml
CFSE 5 µM
2x105 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 stainingHarvest 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):1248-58.
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)ASSAY DAY PURPOSE TUBE/
VOLDESCRIPTION
PFP Ab titer in sera D0,7,28, 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 typing D0 Exploring confounding variable for CMI
Ficol layer
Ficol layer will be harvested for HLA typing by PCR (other lab)
Cytokine titer in sera D0, D7 for all groupsD37 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)
ASSAYSTUDY DAY
PURPOSE TUBE/VOL DESCRIPTION
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,tetramer 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:
10g/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 :
50l of undiluted sera per well in duplicate for each patient
Standard INF- 10 ng in 100l 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 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.
Fie
ldM
ain
Lab
Extra & Intra cellular staining for cell population
Stimulation of PMBC 500.103 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 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
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