Peptide pools

Peptides were generated by custom peptide synthesis (Biomatik) or provided as peptide pools (JPT or Miltenyi BioTec, see SI for details). Peptides were resuspended at a stock concentration of 10 mg ml⁻¹ in 10% DMSO and stored at −80°C. Peptide concentrations were adjusted in a dilution series with DMSO and an identical volume was added to B-LCL cells. The final concentrations of peptides used in cocultures are shown in Fig. 1 and fig. S1.

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.

The presence of SARS-CoV-2 specific T cells directed against the spike (S) and nucleocapsid (N) proteins were assessed with an interferon gamma release assay (IGRA) and flow cytometry four weeks after complete immunization (day 49). To specifically stimulate SARS-CoV-2 specific T cells, lithium heparin whole blood or isolated peripheral blood mononuclear cells (PBMC), were co-incubated with peptide pools (Miltenyi Biotech, Bergisch-Gladbach, Germany) consisting of 15-mer peptides with 11 amino acids overlap covering the entire sequence of the spike glycoprotein (pepS) and the complete sequence of the nucleocapsid phosphoprotein (pepN).