Download 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



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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