Development of a human respiratory tract effector cell-mediated antiviral assay using blood and airway immune cells from individuals previously hospitalised with COVID-19

Abstract Serum antibody–based neutralisation assays correlate with protective immunity to SARS-CoV-2, but cell-mediated in vitro cytotoxicity assays are lacking. Data on virus-specific respiratory tract immune responses in humans remain limited. Fifteen healthy participants who had previously been hospitalised with severe COVID-19 pneumonia were recruited. Peripheral blood mononuclear cells (PBMCs), bronchoalveolar lavage (BAL), and nasal lavage (NL) cells were phenotyped by flow cytometry. Cells were stimulated with SARS-CoV-2 S1 receptor-binding domain and nucleocapsid antigens using 15-mer peptide pools or recombinant proteins. Compartment-specific effector cell-mediated cytotoxicity of SARS-CoV-2 Delta pseudovirus by antigen-stimulated effector cells was assessed using a luciferase assay. Cytokine expression was measured by Luminex. Distinct immune profiles were observed across compartments. Macrophages and dendritic cells predominated in BAL, B cells in NL, and T cells and natural killer cells in PBMCs. BAL cells expressed significantly higher IL-1β, while PBMCs showed higher TNF-α, IL-2 and IL-6 expression. Responses did not differ between peptide and protein antigens. Antigen-stimulated PBMC, BAL, and NL cells demonstrated significantly greater pseudovirus cytotoxicity than unstimulated controls ( p < 0.05). This proof-of-concept study establishes a functional human T-cell–mediated SARS-CoV-2 cytotoxic assay in blood and alveolar compartments, providing a platform to study antiviral mucosal immunity relevant to future pandemics.