Peptivator

Overlapping (15 oligomers with 11 amino acids overlap) peptide pools spanning the entire S protein (315 peptides, PepMix; JPT) were used for the detection of WT SARS-CoV-2–specific T cell responses. To further study T cell responses to WT, B.1.1.7, and B.1.351 VOC, commercially available PepTivator Prot_S B.1.1.7 mutant pool was used. PepTivator Prot_S B.1.1.7 mutant pool covers selective mutated regions in the S protein of B.1.1.7 through 34 peptides that include deletion 69, deletion 70, deletion 144, N501Y, A570D, D614G, P681H, T716I, S982A, and D1118H. Similarly, PepTivator Prot_S B.1.351 mutant pool was used to assess cellular immune responses against B.1.351. PepTivator Prot_S B.1.351 mutant pool covers selective mutated regions in the S protein of B.1.351 through 30 peptides that include D80A, D215G, 242 deletion, 243 deletion, 244 deletion, K417N, E484K, N501Y, D614G, and A701V. For both the B.1.1.7 and B.1.351 peptide pools, a specific PepTivator WT reference pool consisting of 34 or 30 homologous peptides from the SARS-CoV-2 Wuhan strain (GenBank MN908947.3) was included as a control, respectively. All PepTivator (Miltenyi Biotec) peptide pools consist of mainly 15-nucleotide oligomers with 11 amino acids overlap.

PBMC-s were thawed in X-Vivo 15 cell medium (Lonza). After washing, mix of diluted costimulants [anti-CD28 (BD) and anti-49d (BD)], pure CD40 antibody (for blocking CD40-CD154 binding; Miltenyi Biotec) and also CMV pp65 peptide pool (1 μg of each peptide per ml, PepTivator, Miltenyi Biotec) were added to each sample. As a negative control, cells were incubated only with costimulants and CD40. PBMCs were stimulated for 16-24h in CO2 enriched thermostat at 37°C.

Murine IFN-γ-secreting CD8⁺ T cells were performed by ELISPOT assays as previously described (27 (link)). Ninety six well filtration plates were coated with rat anti-mouse IFN-γ (R4-6A2; BD) overnight at 10 µg/mL. CD8⁺ T cells were isolated from the spleens of naïve or aAVC-treated mice by using CD8 MACS beads (Miltenyi Biotec). To prepare for antigen-presenting cells (APCs), splenic DCs from naïve mice were isolated using CD11c MACS beads. Additionally, CD8⁺ T cells (5 × 10⁵/well) were co-cultured with DCs (1 × 10⁵/well) pulsed with or without PepTivator (Miltenyi Biotec) for 24 h. After the culture, the plates were incubated with biotinylated anti-mouse IFN-γ (XMG1.2; BD) (2 µg/mL) for 2 h. Finally, IFN-γ-secreting spots were developed with streptavidin-HRP (BD) and stable DAB substrate (Research Genetics). IFN-γ SFCs were quantified using microscopy.

SARS-CoV-2 specific T-cell responses were determined by interferon gamma (IFN-γ) enzyme-linked immunospot (ELISpot) assay using synthetic peptide pools of the SARS-CoV-2 spike (S1/S2) and membrane (M) protein (600 pmol/mL of each peptide; PepTivator^®, Miltenyi Biotec, Bergisch Gladbach, Germany). The peptide pools mainly consisted of 15-mer sequences with 11 overlapping amino acids. In total, 250,000 mononuclear cells isolated from patient blood were incubated with the respective peptides and added onto anti-interferon-gamma-coated strip assay plates (Merck Millipore Ltd., Tullagreen, Ireland). After 19 h of incubation, substrate solution (Oxford Immunotec, Oxford, UK) was added and spots were analyzed by an ELISpot reader (AID Fluorospot, Autoimmun Diagnostika GmbH, Strassberg, Germany). Mean values of duplicate samples were considered and a total of at least three spots after subtraction of unstimulated (background) spots (spot increment) was defined as positive [17 (link)].

IFN-γ ELISpot assays were performed on mouse splenocytes using a commercial kit (cat.no. ab64029, AbCam) following the manufacturer’s recommendations. Cells were stimulated with 2 μg/mL anti-CD28 (Clone 37.51: BD) and 15-mer sequences with 11 amino acids overlap peptides from influenza A (H1N1) HA (Peptivator; Miltenyi Biotech). The spots were counted using the AID iSpot reader (AID) and ELISpot Reader software V 7.0.