1. Giulietta Saletti gsaletti@ivi.int International Vaccine Institute
Analytical methods of evaluation of antibody & T cell responses to vaccines
Giulietta Saletti
International Vaccine Institute

2. 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.

3. Cellular elements of the blood

4. Immune responses-1
T lymphocytes:
- CD4 T cells
Th1/Th2
- CD8 T cells

5. 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

6. Humoral immunity-2
Effector Functions of Antibodies

7. T cell subsets
CD4+ (helper)
CD8+ (CTL)

8. Vaccine-induced immune responses Correlate vs surrogate
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

9. Correlates of vaccine-induced immunity

10. Induction of immune responses upon vaccine administration
6 months
(?)
Long lasting
Immune responses
(Memory)
2 weeks
Serum antibodies
T cell responses
Antibody Secreting Cells
Vaccination
1 week

11. How to measure antigen-specific B cell responses
Peripheral blood
Serum antibodies
B lymphocytes
Quantitation of the secreted antibodies by
Enzyme-linked immunosorbent assay
Functional antibody assays
Neutralization (virus)
Agglutination test
Opsonophagocytosis
Bactericidal Antibody assay
Vibriocidal antibody assay
Enumeration of the
Antibody Secreting Cells (ASC)
by Enzyme-linked Immune Spot assay (ELISpot)

12. 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.

13. 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.

14. ELISA-2 Ag-specific antibody response
Substrate
Enzyme
Secondary antibody
Coloured
product
Primary antibody
Different antigens in sample

15. To detect antibody (indirect ELISA):
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
Negative controls include:
- No coating with the antigen
- No serum
- Sample replaced by buffer

16. 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
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

17. 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)

18. 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)

19. 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.

20. 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.

21. Opsonophagocytosis assay Bactericidal Antibody assay
Vibriocidal antibody assay

22. Opsonophagocytosis assay
Fcg R
CR1/CR3 +
PMN
C3b/iC3b Ab
Bacteria
Neg Ctl
pos

23. Measurement of vaccine induced B and T-cell immune responses at cellular level
The ELISpot assays
The FACS analysis 1 10
100
1000
10000
FL3-H: CD 56
FL1-H: CD3

24. Isolation of Peripheral blood mononuclear cells (PBMC)
from whole blood by Ficoll-HypaqueTM centrifugation
Plasma
Plasma
T cells
B cells
NK cells
NKT
Monocytes…
PBMC layer
Ficoll
Ficoll
Red blood cells
Red blood cells

25. 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

26. ELISPOT assay PRINCIPLE Detection of antibody-secreting cells (ASC)
Washes
Culture
Nitrocellulose
antigen
Detection
Enzyme-labeled anti-Ig
antibodies
Spot formation
Addition of precipitating substrate

27. ELISPOT Final Product

28. « Reversed » or « capture » ELISPOT
Example: detection of cytokine-secreting cells
Stimulatory agent
Cells
Culture
Washes
Nitrocellulose
mAb1 anti-cytokine X
Detection B
Biotinylated
mAb2 anti-cytokine X
Spot formation
enzyme-avidin
Addition of precipitating
substrate B E

29. 2 colour ELISPOT assay PRINCIPLE Enzyme labeled anti-Ig
Example: Detection of IgG and IgA
antibody-secreting cells (ASC)
Cells
Secreted
IgG
Secreted
IgA
Cells
Culture
Washes
Antigen
Nitrocellulose
Detection
Enzyme labeled anti-Ig
antibodies
Spot formation
Addition of precipitating substrates

30. 2 colours ELISPOT
IgA
IgG

31. Factors Influencing ELISPOT Assay

32. 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

33. 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

34. 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-5
IL-13

35. 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

36. 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.

37. The Granzyme B assay (ELISpot and FACS)
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

38. 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.

39. Fluorescence-Activated Cell Sorter FACS
Example
Staining of CD4 and CD8 T cells on human PBMCs
CD4-FITC
CD8-Percp
Data.024 10 1 2 3 4
FL1-H R2 R3 R4 R5
Surface staining
of T cell subsets
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.

40. 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.

41. 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

42. Assays of cell-mediated immune responses
Standard IVS/51Cr release assay (CRA): labor intensive, non-quantitative, 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 3H-Thymidine. 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

43. What to consider at field site
Before
Questions to be answered.
Design of experiment (both at field and bench).
Parameters to look.. How.. Availability and sensitivity vs. specificity.
During and After
Condition of samples.
Fresh samples/cells?
Aliquot samples.
Choice of antigen.

44. Reference books
Immunobiology, 7th Ed.; Kenneth Murphy, Paul Travers and Mark Walport; Garland Publishing; c2008.
Fundamental Immunology, 5th Ed.; William Paul; ISBN: ; Lippincott Williams & Wilkins; 2003.

45. THANK YOU…
International Vaccine Institute

46. Back-up slides