Immunogenicity refers to the ability of a substance, such as an antigen or a vaccine, to provoke an immune response in an organism. The recognition of the substance as foreign triggers an immune response, which involves the production of antibodies and the activation of immune cells to fight against or neutralize the perceived threat. Immunogenicity can be either desired, in case of vaccination for instance, or unwanted when an immune reaction develops against a therapeutic antibody or a replacement therapy. In both settings, immunogenicity data will help compare a compound to a placebo or standard-of-care, different populations of patients, different doses, and dose regimens. To do so, one can rely on various technologies that include multiplex immunoassays, multiparametric flow cytometryELISpot, for analysis at the protein level, and qPCR or RNAseq for analysis at the transcript level. Herein, we will focus on the comparison of platforms that can measure protein biomarkers.

While all these assays require cell-culture aseptic conditions for stimulation, they can each provide specific and/or complementary information on the immunogenicity of a compound, based on different read-outs such as the production of cytokinic or cytotoxic mediators, cell proliferation, and activation/exhaustion status…).

Cytokines, or more globally, immune mediators, can be measured in different ways. On one hand, profiling of soluble mediators can be done in biofluids, namely PBMC supernatants, based on immunoassays that can be run on multiple technological platforms such as Meso Scale Discovery® (MSD) ECL (ElectroChemiLuminescence), Luminex™, Ella™ Automated Immunoassay System, HD-X ™ analyzer or classical ELISA. These immunoassays provide information on the levels of soluble biomarkers (cytokines, chemokines, growth factors…) secreted by stimulated cells, but not on which type of cells is producing which biomarker.

On the other hand, both flow cytometry panels and ELISpot assay allow quantification of the cells that are capable of secreting cytokines, with only multiparametric flow cytometry being able to provide information on the antigen-reactive cell type [1,2]. Cell-surface markers used to characterize the responding cell subsets (e.g., CD4, CD8 T cells), their differentiation (e.g. Effector, Naïve, effector memory, central memory) and activation status (e.g. expression of CD137, CD154, CD25, CD69….) and even their specificity (e.g. Multimer/Tetramer staining), can be combined with intracellular markers. These will more deeply depict the functional profile of antigen-reactive cells, i.e. their capacity to proliferate (expression of Ki-67, dilution of fluorescent labeled cells), produce cytokines (e.g. IFN-g, TNF-a, IL-2, IL-4, IL-17A) or lyse target cells (e.g. Granzyme B, perforin, CD107a/b).

While polychromatic flow cytometry is a powerful tool for measuring multiple markers at the single-cell level, its sensitivity is limited by the number of events that can be acquired and the staining background. Conversely, ELISpot offers higher sensitivity than flow cytometry, making it the assay of choice for antigens known to be poorly immunogenic, such as chronic diseases.

Ultimately, choosing the right technology involves a good knowledge of the disease, the immunopathological processes, the mechanism of action of the therapeutic compound, that is the context-of-use of the biomarkers to be analyzed. Being able to implement most of these methodologies in a high-quality environment, we can comprehensively explore the immunogenicity of your substance and help you understand its efficacy, safety, and potential for triggering immune responses in individuals.

  1. Tischer S, Dieks D, Sukdolak C, Bunse C, Figueiredo C, Immenschuh S, Borchers S, Stripecke R, Maecker-Kolhoff B, Blasczyk R, Eiz-Vesper B. Evaluation of suitable target antigens and immunoassays for high-accuracy immune monitoring of cytomegalovirus and Epstein-Barr virus-specific T cells as targets of interest in immunotherapeutic approaches. J Immunol Methods. 2014 Jun;408:101-13. doi: 10.1016/j.jim.2014.05.011. Epub 2014 May 28. PMID: 24877879.
  2. Villemonteix J, Cohen L, Guihot A, Guérin V, Moulin C, Caseris M, Carol A, Bonacorsi S, Carcelain G. Comparison between enzyme-linked immunospot assay and intracellular cytokine flow cytometry assays for the evaluation of T cell response to SARS-CoV-2 after symptomatic COVID-19. Immun Inflamm Dis. 2022 Oct;10(10):e617. doi: 10.1002/iid3.617. PMID: 36169252; PMCID: PMC9449588.

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