Laboratory evaluation 23OCT2023 (2).pdf

Laboratory evaluation of humoral and cellular immune response Gwenn Waerlop, 2023 TABLE OF CONTENTS Part 1 Part 2 - Why measure immune responses? - - How to measure immunity in vivo? - How to measure humoral immunity in vitro? - How to measure cell-mediated immunity (CMI) in vitro? - W...

Laboratory evaluation of humoral and cellular immune response Gwenn Waerlop, 2023 TABLE OF CONTENTS Part 1 Part 2 - Why measure immune responses? - - How to measure immunity in vivo? - How to measure humoral immunity in vitro? - How to measure cell-mediated immunity (CMI) in vitro? - When to validate immunogenicity testing? - General concepts of assay validation Exercise on study endpoints and immunogenicity testing I. Why measure immune responses? • To study the magnitude and quality of the immune response induced by natural infection or vaccination • To study correlates of protection (CoP) against disease • CoP is an immune response that is statistically correlated with protection. • CoP can facilitate demonstrating vaccine efficacy and support healthcare workers to manage vaccination of (immunocompromised) individuals, especially if a ‘protective threshold’ is available. • To identify and characterise functional subsets of immune cells • To evaluate the effect of adjuvantation on immune responses, where relevant II. How to measure immunity in vivo? • • Assessment of protective immunity • Administer vaccine and challenge with pathogen • Evaluate disease susceptibility following vaccination Transfer protective immunity • Serotherapy: transfer of passive immunity by administering convalescent plasma/antibodies • Adoptive cell transfer: transfer of (treated) autologous or heterologous lymphoid cells (subsets) • Testing for allergic responses • The tuberculin test (Mantoux test) III. How to measure humoral immunity in vitro? • Main actors of humoral immunity • Antibodies: Neutralizing versus non-neutralizing • B cells III. How to measure humoral immunity in vitro? • Detection and quantification of antibodies • Ligand Binding Assays • Functional assays • Haemagglutination inhibition - HI • Single radial haemolysis - SRH • (Micro)neutralisation of viral infection - MN • Opsonophagocytosis • Detection and quantification of memory B cells • Antibody Dependent Cell Cytotoxicity (ADCC) Ligand binding assays Since 1900: ELISA (enzyme-linked immuno assay) ELISA: colorimetric read-out OPTICAL DENSITY (OD) Ligand binding assays STD CONCENTRATION Many variants available based on this principle: - Fluorescence immunoassays - Chemiluminescent immuno assays (CLIA) Advantages: - Multiplexing (e.g. Luminex or Cytometric bead array (CBA)) - easy to standardise due to the availability of reference material or standards Haemagglutination-inhibition (HI) assay • Based on the haemagglutinating property of some enveloped viruses such as influenza • Incubation of Ag (virus) + RBC (sheep, chicken/turkey, horse) + serum • Golden standard for influenza antigenic characterization and Correlate of Protection (CoP) • Since 1940 • Very difficult to standardise https://www.cdc.gov/flu/about/professionals/antigenic.htm “teardrop” Single radial haemolysis (SRH) assay Since 1975 Agarose gel containing • sheep red blood cells coupled with antigen(s) (from eg rubella, influenza,…) • guinea pig complement Wells are punched in the agarose and filled with test sera. Antibodies in the test sera (if present) and complement lyse the antigen-coated red blood cells, resulting in a clear, hemolytic zone around the well. The area of this zone of hemolysis (measured in mm 2) is directly proportional to the amount of antibody present in the test serum. Very difficult to standardize. Virus neutralization (VN) assay (1) Based on cytopathic effect or or 1. Add serum 2. Add virus (pseudoparticles) Virus neutralization (VN) assay (2) Based on evidence of viral infection Virus or viral pseudoparticles can be used + TMPRSS2 E.g. SARS-CoV-2 3. Add cells No neutralizing Ab + TMPRSS2 Neutralizing Ab 4. Wash 5. Readout Neutralization titer: reciprocal of the highest dilution at which the virus infection is blocked (Micro)neutralization versus pseudovirus neutralisation assay (PNA) • • • • Requires “live” virus Wild type or recombinant flu virus BSL-2, BSL-3, BSL-4 Outcome is determined by virus entry and virus replication • • • • • • No “live” virus Can be created with HA gene sequence BSL-2 Outcome is determined by selected pseudoparticle High throughput ELISA format and equipment Opsonophagocytic killing (OPK) assay - PBMC + bacteria + complement + serum - Serial dilutions of serum are added in drops on agar plates - After incubation, bacterial colony forming units (cfu) are counted - Eg Streptococcus pneumoniae Detection memory B cells – B cell Elispot • • • • PBMC are stimulated in vitro with a polyclonal B cell activator, eg CpG, pokeweed mitogen (PWM) Activated B cells are plated on a plate coated with an antigen of interest After overnight incubation, cells are washed away Bound Ig (total Ig or antigen-specific) is revealed using labeled reagent Antibody dependent cell cytotoxicity (ADCC) Detection by - Lysis target cell - Or activation of NK cells: expression of degranulation marker CD107a - Specificity is determined by the involved IgG antibody - Another major Fc effector function is ADCP: Antibody-Dependent Cellular Phagocytosis (macrophages) IV. How to measure cell-mediated immunity (CMI) in vitro? - Antigen-specific lymphoproliferation - ELISpot assay - Flow cytometry - - Intracellular cytokine staining (ICS) - Cytokine secretion assays - Tetramer staining New approaches to immunogenicity analysis - RNA sequencing - Multispectral imaging flow cytometry Lymphoproliferation assay Measurement of the capability of lymphocytes to proliferation as a response to antigenic stimulation. General principle: Cells collected after vaccination or infection are stimulated with a recall antigen. Cells will be activated and start to proliferate more than when kept in culture without stimulus (control condition). 1. 3H-Thymidine assay (gold standard) 2. CFSE staining 3. Ki-67 expression Lastovicka et al. Hum Immunology. 2016 Dec;77(12):1215-1222. A.k.a. thymidine incorporation assay A radioactive nucleoside (e.g. thymidine), such as titrated thymidine (3H-thymidine or tritium), is incorporated into new strands of chromosomal DNA during mitotic cell division. 5 days overnight Harvesting of cells and read-out Lymphoproliferation assay – 3H-thymidine assay Radioactivity is measured in stimulated and unstimulated cultures. Data is expressed as cpm (counts per minute). Interpretation of the data: D cpm = [cpm in stimulated culture] – [cpm in non-stimulated culture] Stimulation Index or SI = [cpm in stimulated culture]:[cpm in non-stimulated culture] It is a sensitive method but requires radioactive labels and does not allow to discern individual lymphocyte subpopulations Lastovicka et al. Hum Immunology. 2016 Dec;77(12):1215-1222. Lymphoproliferation assay – CFSE staining Carboxyfluorescein succinimidyl ester (fluorescent cell staining dye) is added at culture start (‘first generation cells’) and binds to cytoplasmic structures. It will be passed on to progeny cells, resulting in a halving of the fluorescence of daughter cells. Read-out is performed by flow cytometry. This method gives information about the number of divisions of each cell and about pre-cursor cell frequency (how many parental cells responded to antigenic stimulus). No differentiation is possible between the different mitosis phases and and CFSE is toxic to the cells (and diminishes the proliferative response of PBMC). Lastovicka et al. Hum Immunology. 2016 Dec;77(12):1215-1222. Lymphoproliferation assay – Ki-67 expression Nuclear protein used as a marker of proliferation. Not detectable during G0 phase (quiescent cells), appears as of G1 phase, resulting in a high assay sensitivity. Read-out with by flow cytometry. Intracellular staining does require cell permeabilization. Anti-Ki-67 fluorochrome-labeled antibodies can be combined with others, facilitating the identification and quantification of cell phenotypes. Lastovicka et al. Hum Immunology. 2016 Dec;77(12):1215-1222. Enzyme-linked ImmunoSpot (ELISpot) Read-out: spot-forming unit (SFU) per million PBMC e.g. IFNɣ ELISpot Unstimulated Stimulated (e.g. HBsAg) Flow cytometry - allows individual cells to be identified by their cell surface and intracellular antigens (and to be sorted) - Providing information on cell size, granularity and target protein composition - Up to 50 parameters can be simultaneously detected - BIG DATA! Intracellular cytokine staining (ICS) Detection of antigen-specific T cells - Quantification - Characterisation Intracellular cytokine staining (ICS) Complex set of data - Single cytokines - Boolean combination - Polypositivity can be important Cytokine secretion assay anti-IFN- 1 Y 2 Y anti-CD45 16 hr Stimulation with antigen add catch reagent 4 3 Y anti-IFN- Y anti-CD3/CD4/CD8 Y + VZAg blank Cytokine secretion assay CD14-PI Gating on lymphocytes CD14-PI FSC FSC IFN -g IFN-g IFN- secreting T cells CD3 I F N-g CD3 IFN- g IFN- g IFN- secreting CD4+ T cells CD4 CD4 Tetramer staining Tetramer staining Tetramer assays are used for single-cell phenotyping and enumeration, and have an important advantage over other methods, such as ELISPOT and ICS, by enabling - the recovery and further study of sorted cells based on fluorescent tetramer binding in flow cytometry. - the identification of immunogenic epitopes (e.g. peptide libraries) New approaches to immunogenicity analysis: RNA-seq RNA sequencing (RNA-Seq) is used for transcriptome profiling. RNA-seq data analyses are applied in: (1) accurate mapping of millions of short sequencing reads to a reference genome; (2) quantifying expression levels of genes, transcripts, and exons; (3) differential analysis of gene expression among different biological conditions; (4) biological interpretation of differentially expressed genes cDNA: complementary DNA, NGS: next-generation sequencing New approaches to immunogenicity analysis: RNA-seq In bulk RNAseq, we measure the average expression of genes from multiple cells. • Gene expression profiles between various conditions/treatments/timepoints • limiting when attempting to understand gene expression patterns within the cell In single-cell RNAseq, we measure the expression of genes for each single analysed cell. • scRNA allows us to understand cellular differences in expression • directly applicable to the studies of cell heterogeneity, cell population and subpopulation identification. • This comes with even more bioinformatic challenges. New approaches to immunogenicity analysis: MIF-C Multispectral Imaging Flow Cytometry (MIF-C) Imaging flow cytometry extends the power of traditional fluorescent cytometry by adding morphological information. Applications: • Co-localization of two proteins • Binding of two cells • Changes in morphological changes • Immune synapse formation • Nuclear translocation V. When to validate immunogenicity testing In a clinical research trial, a clinical endpoint generally refers to occurrence of a disease, symptom, sign or laboratory finding that constitutes one of the target outcomes of the trial. The primary endpoint of a clinical trial is the endpoint for which subjects are randomized and for which the trial is powered. Secondary endpoints are endpoints that are analyzed post hoc, for which the trial may not be powered nor randomized. Pre-Clinical Phase I (Safety) QUALIFICATION SET-UP REPORT Phase II (Safety/ Immunogenicity) Phase III (Efficacy) VALIDATION (if primary endpoint) Assay validation Validating the analytical method ensures that the data are reliable by addressing certain key questions, including: • Does the method measure the intended analyte? For example, does anything interfere with the measurement, and is the method specific or selective for the analyte? • What is the variability associated with these measurements? For example, what are the accuracy and precision of the method? Repeatability and reproducibility, Linearity • What is the range of measurements that provide reliable data? For example, what is the sensitivity of the method (e.g., what is the lower limit of quantitation (LLOQ) of the method, and what is the upper limit of quantitation of the method (ULOQ)?) • How do sample collection, handling, and storage affect the reliability of the data from the bioanalytical method? For example, what steps need to be followed while collecting samples? Do the samples need to be frozen during shipping? What temperatures are required to store the samples, and how long can the samples be stored? Stability, robustness https://www.fda.gov/files/drugs/published/Bioanalytical-Method-Validation-Guidance-for-Industry.pdf General concepts of assay validation POC Assay type to be defined. Search for reagents Pre-Clinical ….. ASSAY SET-UP Technical details. Search for reagents Transfer to Clinical setting ….. ASSAY OPTIMISATION ASSAY QUALIFICATION ASSAY VALIDATION Standard Calibrator is fixed Uncontrolled Standard Calibrator is fixed – Controlled SOP is final Design of Experiment (DoE) • • • • • • Calibration Selectivity Carry-Over Matrix effect MRD …. Design of Experiment (DoE) • • • • • • • • • Accuracy LLOQ-ULOQ LOB-LOD Reproducibility Specificity Linearity Interference Robustness Stability LIFE CYCLE MANAGEMENT “The Real World” • • • • • • Accuracy LLOQ-ULOQ LOB-LOD Reproducibility Specificity Linearity • • • • • Transfer Change Control Equipment Pre-analytical Cross-validation POC = proof of concept LLOQ = lower limit of quantification ULOQ = upper limit of quantification LOB = Limit of blank LOD = limit of detection Assay validation: characterizing the assay ULOQ LLOQ Analytical range LOB LOD ULP LLP LLL ULL https://www.fda.gov/files/drugs/published/Bioanalytical-Method-Validation-Guidance-for-Industry.pdf Any questions? Tip: try to phrase in your own words the purpose of the lab test ‘this test is applied for…’ GWENN WAERLOP Lab Manager CEVAC Lab [email protected]