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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. 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 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. 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 Back-up slides