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