Group B3 (BCG replacement strategy)
Define the immunogenicity endpoints to
be used for the preclinical studies and
the clinical trial
Claude MERIC, Narges MIANDEHI
1
This definition is based on information or the
immunological correlates of protection proposed
by group B1 to be used for groups B4 and B5
 Which immunogenicity assays (T and/or B cell assays)
should be utilized for the preclinical studies and the clinical
trial;
 A detailed description of the assays selected, including the
limitations and advantages of the assays;
 The results expected.
2
Introduction

Correlates of protection against TB infection

CMI response

Th1 (CD4+, IFN-g, IL-2 and TNF-a)
•

Th17 (CD4+, IL-17)
•

Importance of polyfunctional T cells
may be important for protective immunity
CTL (CD8+, IFN-g and TNF-a)
•
cytotoxic activity
 Activated macrophages (the major source of TNF-a)
•

able to kill phagocytosed Mtb
Antibodies are not considered to be critical

Would be measured by ELISA or OPA with macrophages (?)
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Immunological endpoints


Focus on cell mediated immunity as Th1 and CD8+
responses correlate with protection
CD4+ cell response to BCG proteins (PPD) and to
each overexpressed antigen



Polyfunctional IFN-g, IL-2, TNF-a expression
IL-17 expression by CD4+ T cells => Th17 response
CD8+ to each overexpressed antigen

Polyfunctional IFN-g, IL-2, TNF-a expression
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Preclinical studies

Mouse

Cytokine (IFN-g and others) production after restimulation of mouse
lymphocytes with antigen or with peptide pools


Intracellular cytokine detection by flow cytometry following stimulation of
mouse lymphocytes with antigen or peptide pools


CD4+ and CD8+, IFN-g, IL-2, TNF-a, IL-17
Functional CTL assay (?)




Detection with a multiplex assay (Luminex)
Effectors: stimulated CD8+ cells
Targets: Autologous TB infected macrophages
Endpoint: Bacterial killing (bacterial culture)
Guinea pig

Protection against challenge only
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Phase I clinical study

Screening (volunteers negative for TB infection at enrolment)

TST


IGRA


Classical and modified test using selected antigens
Kits and modified test using selected antigens
Immunogenicity

Intracellular cytokine detection on stimulated whole blood





CD4+ and CD8+, IFN-g, IL-2, TNF-a, IL-17
PBMC control for some subjects to check for coherence (for artefacts with whole
blood)
Measurements on D0, D7, D14, D28, D42, D84, D182
ELISPOT: nd
Tetramers: nd
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Screening

Tuberculin test - in vivo







In use for about 100 years
Measures a delayed-type hypersensibility response
Uses tuberculin PPD, a crude mixture of antigens, many of them shared by
M. tuberculosis, bovis and BCG
Produces a white or rose-colored induration which peaks after 48-72h and
lasts up to 1 month
Infiltration of CD4+ T cells and activated macrophages
Inflammatory reaction
Interferon-g release assay (IGRA) - ex vivo


IFN-g production by circulating T cells
16-20h stimulation by Mtb-specific antigens (not in BCG, M bovis, M avium)




Peptides from proteins encoded in region of difference (RD): CPF-10, ESAT-6,
TB7.7 (only QuantiFERON)
Measurement of IGN-g by ELISPOT or whole-blood ELISA
Responding cells likely to be effector memory, recently induced by Mtb
Cannot distinguish LTBI from TB
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TST and IGRA

T-SPOT (Oxford Immunotec)

Type of ELISPOT assay
 Counts the number of effector T cells produce IFN-g
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TST and IGRA

QuantiFERON (Cellestis)



ELISA
Measures IFN-g in whole blood following stimulation with TB antigens
Test Principle (source: Cellestis web site)

The QuantiFERON-TB Gold In-Tube assay is an in vitro diagnostic
laboratory test that aids in the detection of infection with Mycobacterium
tuberculosis. It uses human whole blood, with patented assay technology
based on the measurement of Interferon-gamma (IFN-g) secreted from
stimulated T-cells previously exposed to Mycobacterium tuberculosis. The
QuantiFERON-TB Gold In-Tube assay is a straightforward laboratory test
that involves 4 simple steps:




Collection of blood into QuantiFERON-TB Gold In-Tube Blood Collection
Tubes.
Overnight incubation at 37°C. TB infected patients' blood cells will produce IFNg.
Detection of released IFN-g in harvested plasma using a quick and easy ELISA.
Analysis of data using the QuantiFERON-TB Gold In-Tube Analysis Software.
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Intracellular cytokine staining

Mouse
Lymphocytes washed from the spleen
 Stimulation using overexpressed antigens and peptides pools
from them, PPD, PHA positive control
 Addition of Brefeldin A (to prevent excretion of newly made
cytokines)
 Permeabilization solution
 Fluorescence conjugated antibodies to CD3, CD4, CD8, IFN-g,
TNF-a, IL-2, IL-17
 Fixation
 Analysis using a flow cytometer

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Intracellular cytokine staining
Whole blood method (Humans)

Principle and reagents

Stimulation of lymphocytes in whole blood in the presence of anti-CD49d and anti-CD28
costimulatory antibodies to enhance the specific response (1ug/ml each)
 To be done immediately after blood collection




Blood stored at 2-8°C until stimulation
250 μl needed to acquire 40,000 events (sufficient to detect events <1% of the considered cell type)
Stimulation for approx. 12hr at 37°C
Brefeldin added at 7hr

Lysis of red cells using FACS lysing solution
 Fixation
 Frozen until labeling and analysis using a flow cytometer

Pros and cons





Need for small volume of blood assay: 250 ul
Freeze the fixed cells: limits variability due to viability of frozen cells
Detection of only effector/memory cells (short stimulation)
More tricky to detect multiple markers than PBMC
Adapted to rural, developing country trial setting
*Hanekom et al, 2004, JIM 291:185; Duramad et al, 2004, Cancer Epi Biom & Prev 13:1452
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Intracellular cytokine staining
PBMC assay (Humans)

Principle and reagents




Purification of PBMC from blood
Blood layered on Ficoll-Paque based on density gradiant
Centrifuge
4 phases are formed (top to bottom)









plasma and other constituents
a layer of mono-nuclear cells called buffy coat (PBMC/MNC)
Ficoll-Paque
erythrocytes & granulocytes (pellet)
PBMC are frozen at -70°C (critical step)
Viability checked upon thawing
Stimulation with antigen
Labeling and analysis by flow cytometry
Pros and cons



Use of purified cell preparation for stimulation
Need for larger volume of blood (for purification) than whole blood stimulation
Freeze of PBMC in clinical trial Due to workload: critical step
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Validation of the assays for human testing

Steps to be validated





Whole blood stimulation
Cell freezing/thawing (PBMC)
Staining
Flow cytometry
General principles of assay validation: to be adapted to the specific assays

Specificity


Linearity / range




Repeatability
Intermediate precision (days, analysts, equipment, etc)
Reproducibility


Comparison with other method (ELISPOT?)
Precision


For % of cells with a defined phenotype (gating?)
Accuracy


Discrimination of the signal from different channels
Inter-laboratory trial: not needed
Detection limit
Robustness
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Expected results from immunology analysis

Stimulation of T lymphocytes with the phenotype described as
correlating with a protective response against TB


Polyfunctional T cells of long duration specific to BCG and to
overexpressed antigens


Antigen-specific CD4, CD8, and Th17 responses
Responses to BCG antigens as strong as after BCG vaccination
Significant response to the overexpressed antigens

Higher response than in Mtb infected individuals
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BACKUP
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TST and IGRA

QuantiFERON (Cellestis)
source: Cellestis web site
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Flow cytometry - Equipment
Institut Pasteur, eLearning
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Flow cytometry – Data analysis
Forward scatter (FSC): proportional to particle size
Side scatter (SSC): granularity, structural complexity
Institut Pasteur, eLearning
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Figures below show Peyer's patch cells obtained from a C57BL/6 mouse and stained with FITC anti-B220 and PE anti-CD3
or with FITC anti-B220 and PE anti-CD19. The purpose of this example is to illustrate the information that can be generated
from a sample using two antibodies simultaneously. Using scatter measurements, a gate has been drawn to include all
lymphocytes, thus excluding dead cells and debris. Gating enables the number of cells positive for CD3, CD19 and/or B220
to be expressed as a percentage of lymphocytes only. CD3, CD19 and B220 staining parameters found on gated cells are
shown in the appropriately labeled dot plot. In each quadrant of dot plots (a, b, c, d) are indicated the percentage of positive
cells in the gated population.
In the right dot plot, quadrant b contains the B220+/CD19+ cells (B lymphocytes) (73.4% of the cell population analyzed),
quadrant d contains the B220-/CD19- cells (mostly T lymphocytes). In middle dot plot, quadrant a contains the B220/CD13+ cells (T lymphocytes) (19.5% of the cell population analyzed), quadrant c contains the B220+/CD3- cells (B
lymphocytes) (76.9% of the cell population analyzed), and quadrant b shows the absence of the B220+/CD3+ cells; the low
percentage of double positive cells (1.1% of the cell population analyzed) may rely on the presence of clusters containing
one B220+ cell and CD3++ cell.
Institut Pasteur, eLearning
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