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Updated: May 28, 2026
Whole Blood Assay with Dual Co-Stimulation for Antigen-Specific Analysis of Host Immunity to Fungal and Viral Pathogens
Published on: September 20, 2024
Comprehensive longitudinal profiling identifies differential frequency, epitope specificity, and effector function of
Susana Patricia Amaya Hernandez1, Kamilla Kjærgaard Munk1, Konstantin Danilov1
1Department of Health Technology, Section of Experimental and Translational Immunology, Technical University of Denmark, Kongens Lyngby, 2800, Denmark.
Background:
CD8+ T-cells are crucial for controlling and resolving SARS-CoV-2 infection, yet their epitope specificity and relationship to COVID-19 disease severity remain incompletely understood.
Methods:
We performed comprehensive longitudinal profiling of antigen-specific CD8+ T-cell populations using DNA-barcoded peptide-HLA multimers, analysing 553 SARS-CoV-2 epitopes across globally prevalent HLA alleles in patients with mild and severe COVID-19. Functional and phenotypic characterisation was performed using multidimensional single-cell analysis and detailed cytokine profiling. The impact of post-infection COVID-19 vaccination on T-cell memory was also assessed.
Findings:
Severe and mild COVID-19 were associated with robust yet distinct patterns of CD8+ T-cell activation. In the acute phase, severe disease was characterised by a broader T-cell repertoire (139 unique epitopes) with a median frequency of 1.4% (IQR 0.2-5.0) and a high-frequency of immunodominant epitope-specific T-cells that exhibited reduced cytotoxic profile. In contrast, patients with mild COVID-19 mounted responses against a more limited set of epitopes (98 unique epitopes), partially overlapping with those observed in severe disease, with a median T-cell frequency of 0.7% (IQR 0-1.9) and displayed a stronger cytotoxic phenotype and functional state. Over time, the memory T-cell compartment contracted to a restricted subset of immunodominant epitopes in the two patient groups and COVID-19 vaccination further enhanced frequencies of spike-specific T-cells independent of prior disease severity.
Interpretation:
These findings delineate the epitope-specific frequency, function, and persistence of antigen-specific T-cell populations during SARS-CoV-2 infection, highlighting how differential activation, rather than magnitude alone, shapes immune outcomes across disease severities and other viral infections.
Funding:
This work was supported by the Independent Research Fund Denmark (DFF-Sapere Aude, 2066-00044B), the EU Horizon Europe REACT project (101057129), the European Research Council (ERC) Starting Grant MIMIC (101045517), and the Danish National Research Foundation (DNRF170).
