Rapid Cytokine Release Assays for Analysis of Severe Acute Respiratory Syndrome Coronavirus 2-Specific T Cells in Whole Blood

We developed a simple and sensitive whole-blood stimulation assay to detect severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-specific T cells in previously infected or vaccinated individuals. Virus-specific T-cell responses persisted significantly longer than SARS-CoV-2-specific immunoglobulin G responses. Background Waning of immunoglobulin G (IgG) antibodies against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) complicates the diagnosis of past infection. The durability of T-cell memory against SARS-CoV-2 remains unclear, and most current T-cell protocols are unsuited for large-scale automation. Methods Whole-blood samples from 31 patients with verified past coronavirus disease 2019 (COVID-19) and 46 controls, of whom 40 received COVID-19 vaccine, were stimulated with peptides spanning the nucleocapsid (NC) or spike 1 (S1) regions of SARS-CoV-2 and analyzed for interferon gamma in supernatant plasma. Diagnostic accuracy of these assays was evaluated against serum anti-NC and anti-receptor-binding domain S1-IgG. Results Induction of interferon gamma in whole blood by NC or S1 peptides diagnosed past COVID-19 with high accuracy (area under the receiver operating characteristic curve, 0.93 and 0.95, respectively). In accordance with previous studies, NC-IgG levels rapidly waned with only 5 of 17 patients (29%) remaining seropositive >180 days after infection. By contrast, NC peptide-induced T-cell memory responses remained in 13 of 17 study participants (76%) >180 days after infection (P = .01 for comparison with NC-IgG; McNemar test). After 2 vaccine doses, all 18 donors exhibited S1-specific T-cell memory. Conclusions Cytokine release assays for the monitoring of T-cell memory in whole blood may be useful for evaluating complications following unverified past COVID-19 and for long-term assessment of vaccine-induced T-cell immunity.