IFN-γ ELISpot assay

The frequency of IFN-γ–producing T cells was measured by ex vivo ELISpot-forming cell assay after antigen-specific stimulation. PBMCs were resuspended in R10, stimulated with a set of peptides designed to cover the 209 FSPs encoded by the vaccine, and arranged into 16 peptide pools (P1 to P16). Cells were plated at 2 × 10⁵ cells per well in ELISpot plates (Human IFN-γ ELISpot^PLUS kit, Mabtech) and incubated for 18 to 20 hours in the presence of the peptides in a 37°C (±1°C), humidified CO₂ incubator. At the end of incubation, the ELISpot assay was developed according to the manufacturer’s instructions. Spontaneous cytokine production (background) was assessed by incubating PBMCs with the medium only plus the peptide diluent DMSO (negative control) (Sigma-Aldrich), whereas CEFX [a pool of known peptide epitopes for a range of human leukocyte antigen (HLA) subtypes and different infectious agents, namely, Clostridium tetani, coxsackievirus B4, Haemophilus influenzae, Helicobacter pylori, human Ad5, human herpesvirus 1, human herpesvirus 2, human herpesvirus3, human herpesvirus 4, human herpesvirus 5, human herpesvirus 6, human papillomavirus, JC polyomavirus, measles virus, rubella virus, Toxoplasma gondii, and vaccinia virus (JPT Peptide Technologies)] was used as positive control. Results are expressed as SFC/10⁶ PBMCs in stimulating cultures after subtracting the DMSO background. The cutoff values for a positive response were calculated on 20 healthy donors. A response was considered positive if (i) the number of SFC/10⁶ cells was greater than 48 and (ii) higher than three times the background DMSO value. A subject is defined as a responder if reactivity to at least 1 of the 16 FSP peptide pools is induced after vaccination. Induction of an immune response is defined as a change from negative at baseline (after pembrolizumab) to positive at any of the time points collected after vaccination. In case of detection of a positive reactivity against one of the peptide pools at baseline, the vaccine is expected to determine an increase (of at least 80%) of the preexisting reactivity to at least 1 of the 16 FSP peptide pools, and in this case, such patients will be considered as responders.