SARS-CoV-2 T Cell Reactivity Assay

An interferon gamma response assay (IGRA) was used to approximate T cell reactivity to SARS-CoV-2 [34 (link)]. A total of 5 mL of blood was collected in a lithium heparin tube and processed within 4 h of collection. 1 mL of blood was decanted into blood monitor tubes and relevant stimulants were added: unstimulated/null (negative control); lyosphere (anti-CD3 & R848, positive control); and peptide pools (Miltenyi Biotec, Bergisch Gladbach, Germany) consisting of spikes (S, S1, and S+; 0.5 μg/mL), membranes, and nucleocapsid (0.5 μg/mL) and a recombinant spike protein trimer (amino-acids 14-1213, 1 μg/mL). Tubes were incubated for 24 h at 37 °C then centrifuged at 2000–3000 g for 15 min. Interferon gamma (IFN-ɣ) production was measured by ELISA (Qiagen, Manchester, UK). Qiagen QuantiFERON^® Monitor ELISA kit was used to measure IFN-ɣ levels. Results are reported as IU/mL in response to stimulus, subtracting the ‘Unstimulated/Null’ value from the ‘Stimulated’ value. The reference range for IFN-ɣ response to spike peptides (S, S1, S+) was 0.11 IU/mL. This range was established using receiver operator characteristic curves for 30 healthy controls pre- and post-vaccination.