Immunological endpoints for preclinical studies
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Transcript Immunological endpoints for preclinical studies
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
Mucosal response after challenge
challenge
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:
* How ?
no correlation between B cells response and protection
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.
Antigen Specific cellular response: proliferative
response by CFSE staining and FACS
• CFSE techniques allows qualitative and cuantitative analysis evaluation of
proliferation index
Harvest sample
spleen
CFSE staining
Proliferation assay
72 hs
1x106 Cells/ ml
•CD4- Per CP
•CD8- APC
CFSE 5 µM
•IP ( live cells)
•Single cell
suspension
•Red blood
lysis
Staining cell surface
markers
2x105 cells/ well
Antigen: whole protein
fusion
CTR+: concavaline A
CTR- : media alone
Flow cytometry
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
Tcells in lugs
Macrophages MHC II +
Bacterial
burden
Activated status
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
per gram
tissue
(1) Ordway D et al. Clin Vaccine Immunol 2008 Aug;15(8):1248-58.
Leukocyte recruitment to lung after
challenge with Mtb
How? Flow cytometry
Cells surface markers
staining
Lungs
Single cell
suspension
≠ times points
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
Gating strategy
Lymphocytes
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.
Work on frozen
sample ?
Brewelskloof
Hospital Immunology
lab
Centre 1
Centre 3
Centre2
Comprehensive immunomonitoring (1)
ASSAY
DAY
PURPOSE
TUBE/V
OL
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 Serum, 2x 0.75 ml
tube /5ml aliquot, freeze at 80° (field) for Ab
ELISA (main lab)
HLA typing
D0
Exploring
Ficol
confounding
layer
variable for CMI
Cytokine titer in sera D0, D7 for Assess profil of ½ of Dry
all groups T cell response tube /5ml
D37 for
to vaccine
group D,E
DESCRIPTION
Ficol layer will be
harvested for HLA
typing by PCR
(other lab)
Serum, 2x 0.75 ml
aliquot, freeze at 80° (field) for IFN-g
ELISA (main lab)
Comprehensive immunomonitoring (2)
ASSAY
STUDY
DAY
D0,7,28,
Intracellular staining 37,86,
for cytokine
364 for
production
groups
A, B &C
+ 56, 112
Extra cellular
for
staining for cell
groups
percentage
D&E
Ag 85A - CD4
tetramer assay
D0 and
D364
PURPOSE
TUBE/VOL DESCRIPTION
Assess cellular
immune
response to
vaccine
Assess cellular
immune
response to
vaccine
Assess spécific
CD8 response
to one of the
vaccine
componment
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
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
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-g 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
Main Lab
Field
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.
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.
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 p4856 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