Download Antibody Secreting Cells

Analytical methods of evaluation
of antibody & T cell responses to
vaccines
Giulietta Saletti
[email protected]
International Vaccine Institute
Distribution of
lymphoid tissues
Lymphocytes arise from stem cells in
bone marrow, and differentiate in the
central lymphoid organs (yellow), B cells in
bone marrow and T cells in the thymus.
They migrate from these tissues and are
carried in the bloodstream to the
peripheral or secondary lymphoid organs
(blue), the lymph nodes, the spleen, and
lymphoid tissues associated with mucosa,
like the gut-associated tonsils, Peyer's
patches, and appendix.
The peripheral lymphoid organs are the
sites of lymphocyte activation by antigen,
and lymphocytes recirculate between the
blood and these organs until they
encounter antigen.
Cellular elements of the blood
Immune responses-1
T lymphocytes
- CD4 T
cells
Th
Th
- CD8 T
cells
Humoral immunity-1
Antibodies are variable proteins
produced by B lymphocytes in
response to an infection
Once activated, naïve B cells
become effector plasma cells whose
secrete large amounts of antibody.
hey reside within the secondary
lymphoid tissue or the bone marrow
A subset of B cells will become
memory cells which can quickly be
activated and produce high affinity
antibodies of isotypes other than
IgM
A secondary immune response is
more rapid and characterized by
high levels of IgG
Humoral immunity-2
Effector Functions of Antibodies
T cell subsets
CD4+ (helper)
CD8+ (CTL)
Vaccine-induced immune responses
Correlate vs surrogate
Correlate
A specific immune response to a vaccine that is closely related to protection
against infection, disease, or other defined end-point
Surrogate
A quantified specific immune response to a vaccine that is not itself protective
But that substitute for the true (perhaps unkonwn) correlate
Correlates of vaccine-induced immunity
Induction of immune responses upon
vaccine administration
Antibody Secreting Cells
Vaccination
1 week
Serum antibodies
T cell responses
2 weeks
Long lasting
Immune responses
(Memory)
6 months
(?)
How to measure antigen-specific B cell
responses
Peripheral blood
Serum antibodies
Quantitation of the secreted antibodies by
Enzyme-linked immunosorbent assay
Functional antibody assays
• Neutralization (virus)
•Agglutination test
• Opsonophagocytosis
• Bactericidal Antibody assay
• Vibriocidal antibody assay
B lymphocytes
Enumeration of the
Antibody Secreting Cells (ASC)
by Enzyme-linked Immune Spot assay (ELISpot)
ELISA
(enzyme-linked immunosorbent assay)
What the assay tells you :
The ELISA can be used both qualitatively and quantitatively to
measure antigen-antibody binding.
Depending on what variation you use, it will detect antigen or
antibody in body fluids or tissue culture supernatants.
2- ELISA
What you need to do the assay :
Purified antigen (if you want to detect or quantify antibody).
Purified antibody (if you want to detect or quantify antigen).
Standard solutions (positive and negative controls).
Sample to be tested.
Microtiter dishes: plastic trays with small wells in which the assay is
done.
 Wash fluid (buffer).
 Enzyme-labeled antibody and enzyme substrate.
 ELISA reader (spectrophotometer) for quantitative measurements.
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

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ELISA-2
Ag-specific antibody response
Substrate
Secondary
antibody
Coloured
product
Primary
antibody
Different antigens in sample
To detect antibody (indirect ELISA):
Negative controls include:
- No coating with the antigen
- No serum
- Sample replaced by buffer
1- Coating with a purified antigen
2- Wash off unbound antigens
3- Add blocking agent to avoid
non-specific binding
4- Add serum samples
5- Wash off unbound antibodies
6- Add anti-Ig binding to Fc
region of specific antibody. This
anti-Ig is covalently linked to an
enzyme (HRP, AP)
7- Wash off unbound antibodies
8- Add chromogenic substrate that
the enzyme will convert to a
colored product
To detect antigen (sandwitch ELISA):

Coat a plate with an antibody for
the protein you want to find

Wash, block, and wash the plate

Put on the substrate with the protein
in question

Wash, block, and wash the plate

Put on another antibody (covalent
linked to an enzyme) for the protein
that binds at a different epitope

Wash the plate

Add the chromogenic substrate

Read the plate
How to measure functional antibodies
(correlate/surrogate of protection)
Antibody Neutralization assay (Viruses and Bacteria)
Agglutination test (Viruses and bacteria)
Opsonophagocytosis assay (Bacteria)
Bactericidal Antibody assay (Bacteria)
Vibriocidal Antibody assay (Bacteria)
Agglutination tests-principle
The reaction of an antibody with the antigen can be detected by
agglutination (clumping) of the antigen.
The general term agglutinin is used to describe antibodies that
agglutinate particulate antigens.
Antigens may be:
- On a cell (direct agglutination)
- Attached to latex spheres
(indirect or passive agglutination)
Hemagglutination tests
Agglutination tests are used to measure the level of
antibodies to particulate antigens. When the antigen is an
erythrocyte the term hemagglutination is used.
In this test, serial dilutions are
made of a sample to be tested for
antibody and then a fixed number of
red blood cells or bacteria or other
such particulate antigen is added.
The maximum dilution that gives visible agglutination is called the
titer. The results are reported as the reciprocal of the maximal
dilution that gives visible agglutination.
Viral hemagglutination inhibition test
Viral hemagglutination involves agglutination of red blood cells
by viruses
Viral hemagglutination inhibition tests the antibodies' ability to
prevent viruses from agglutinating RBCs.
Opsonophagocytosis assay
Bactericidal Antibody assay
Vibriocidal antibody assay
Opsonophagocytosis assay
Fcg R
CR1/CR3
Bacteria
Ab
+
C3b/iC3b
PMN
Neg Ctl
pos
Measurement of vaccine induced B
and T-cell immune responses at
cellular level
The ELISpot assays
10000
FL1-H: CD3
The FACS analysis
1000
100
10
1
1
10
100
1000
FL3-H: CD 56
10000
Isolation of Peripheral blood mononuclear cells (PBMC)
from whole blood by Ficoll-HypaqueTM centrifugation
Plasma
T cells
PBMC layer B cells
Ficoll
Red blood cells
NK cells
NKT
Monocytes…
ELISPOT assay
What the assay tells you:
The ELISPOT is used to count the number of cells producing:
 Antibodies (Abs): Antibody secreting cells (ASC)
 Cytokines secreted by T cells in culture (T cells Elispot)
 Release of Perforin or granzymeB: surrogate for cytotoxic T
cells
ELISPOT assay PRINCIPLE
Detection of antibody-secreting cells (ASC)
Cells
Culture
Washes
antigen
Nitrocellulose
Detection
Spot formation
Addition of precipitating substrate
Enzyme-labeled anti-Ig
antibodies
ELISPOT Final Product
« Reversed » or « capture » ELISPOT
Example: detection of cytokine-secreting cells
Stimulatory agent
Cells
Culture
Washes
mAb1 anti-cytokine X
Nitrocellulose
Detection
Spot formation
Biotinylated
mAb2 anti-cytokine X
enzyme-avidin
Addition of precipitating
substrate
B
B
B
2 colour ELISPOT assay PRINCIPLE
Example: Detection of IgG and IgA
antibody-secreting cells (ASC)
Culture
Cells
Secreted
Cells
Secreted
IgG
IgA
Washes
Antigen
Nitrocellulose
Detection
Spot formation
Addition of precipitating substrates
Enzyme labeled anti-Ig
antibodies
2 colours ELISPOT
IgA
IgG
Factors Influencing ELISPOT
Assay
I. Responding cells
1. Anticoagulant & Processing time
2. Isolation
3. Cryopreservation & Thawing
Cell Populations
I. Responding cells
II. APC
Cell Counter
1. Automated
2. Manual
ELISpot assay
1. Coating Antibody
2. Detecting Antibody
3. Substrate
Quality of spots
Spots Reader
1. Manual
2. Automated
Results
Criteria of positivity
Storage of data source
Antigens
1. Recombinant constructs
2. Proteins & lysates
3. Peptides
Broad applicability
The ELISPOT assay and its reverse (capture)
variants can be applied to any system in order to
detect any secreted antigenic substance
(antibodies, cytokines, hormones, metabolites
etc..)
at the single cell level
Application of ELISPOT
 To investigate specific immune responses in various
diseases including infections, cancer, allergies and
autoimmune diseases
 Development and monitoring of new vaccines and
vaccine candidates
Application of ELISPOT
 Humoral responses (Antibody Secreting Cells)
- Vaccine monitoring
 Cell-mediated Immune responses
Cytokines
- Vaccine efficacy (HIV IFN-γ)
- Diagnostic (M.Tuberculosis-IFN-γ)
- Quality of immune responses (Th1/Th2)
Perforin and Granzyme B
- Cytotoxic T Cell responses
IL-13
IL-5
Measurement of vaccine induced T cell
(CD4 and CD8) responses
T cells
• Secreted Cytokines: ELISA, ELISpot and Luminex
• Intracellular cytokine staining (ICS): FACS
• Phenotype of the antigen-specific T cells: FACS
• Cytotoxic T lymphocytes: Radioactive, ELISpot, FACS
The above assays require an in-vitro activation of the cells
with vaccine antigen
Cytotoxic T Lymphocytes (CTL) Assay
1. The
51Cr-release
assay
Effector Cytotoxic T Lymphocytes (CTL) bind targets
(infected cells) bearing virus peptide on Class I MHC and
signal the targets to undergo apoptosis.
If the targets are labeled with 51Chromium before the CTL
are added, the amount of 51Cr released into the supernatant is
proportional to the number of targets killed.
2. A non-radioactive alternative for monitoring
cell-mediated cytotoxicity:
The Granzyme B assay (ELISpot and FACS)
Granzyme B is secreted by cytolytic
effector cells that target cells through
transmembrane pores formed by
another granule protein, perforin.
In the target cell, Granzyme B, a
neutral serine protease, induces
apoptosis by cleaving and activating
members of the caspase family.
The detection of Granzyme B
secreting cells in ELISPOT assays
correlates with cytolytic
responses measured by the classic
radioactive chromium 51Cr-release
assay
Fluorescence-Activated Cell Sorter
FACS
Flow cytometry can be used to count the number of cells (cell
suspension) having specific molecules on their membrane or (with
fixation and permeabilization) in their cytosoplasm.
Possibility to detect up to 10 different parameters on the same cell
How it works?
Fluorochrome-labeled antibody is used to bind specific molecules.
When illuminated by a laser emitting UV light, the fluorochrome
emits visible light in a specific wavelength that can be detected by
a photomultiplier tube.
Fluorescence-Activated Cell Sorter
FACS
Example
Staining of CD4 and CD8 T cells on human PBMCs
Data.024
CD8-Percp
R3
R4
R5
10 0
R2
10 1
10 2
10 3
10 4
FL1-H
Morphology
1: Prepare PBMC cell suspension
2: Staining of the cells with Fluorochrome-conjugated antibodies
against the molecule expressed on the target cells
-Surface staining: incubation of the antibodies with cells
-Intracellular staining: Cell fixation and permeabilization followed
by staining with specific antibodies
3: Run cells through flow cytometer.
CD4-FITC
Surface staining
of T cell subsets
Interpretation of flow cytometry data
Cell Morphology
SSC

Its relative size (Forward Scatter—FSC): Related to
cell surface area

Its relative granularity or internal complexity (Side
Scatter—SSC)

Fluorescent labeling of cell surface or intracellular
structures using fluorescent antibodies allows
investigation of cell structure and function.
Application of flow cytometry for monitoring
Immune responses
Cytokines-supernatant (MBA/ELISA)
CTL-Lysis (Cr-realese) or granzyme/perforin (ELISA, ELISpot, FACS)
B cells: Antibodies: Supernatant (ELISA-MBA), Antibody secreting cells (ELISpot),
frequencies of antigen specific memory cells
Assays of cell-mediated immune responses
 Standard IVS/51Cr release assay (CRA): labor intensive, nonquantitative, difficult to perform in large scale trials, difficult to
transfer to developing countries, difficult on cryopreserved samples.
 Lymphoproliferative assay (LPA): mainly used to detect CD4 T cell
responses after Ag stimulation in vitro with incorporation of 3HThymidine. Non-quantitative assay, can be performed using
cryopreserved cells.
 ELISPOT assay: quantitative; easier to perform and to transfer; can be
performed with cryopreserved samples; affected by operator gating
bias.
 ICS assay: quantitative; high sensitivity, identification of CD4 & CD8
responses in same sample, affected by operator gating bias. Multiple
parameters (up to 12) can be detected simultaneously
What to consider at field site
 Before
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Questions to be answered.
Design of experiment (both at field and
bench).
Parameters to look.. How.. Availability and
sensitivity vs. specificity.
 During and After
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Condition of samples.
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Fresh samples/cells?
Aliquot samples.
Choice of antigen.
Reference books
 Immunobiology, 7th Ed.;
Kenneth Murphy, Paul Travers and Mark
Walport; Garland Publishing; c2008.
 Fundamental Immunology, 5th Ed.; William
Paul; ISBN: 0781735149; Lippincott
Williams & Wilkins; 2003.
THANK YOU…
International Vaccine Institute
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