IFN-γ ELISPOT assay.

The IFN-γ ELISPOT plus kit (Mabtech, Nacka, Sweden) was used according to the instructions of the manufacturer. Fresh peripheral blood mononuclear cells (PBMC) were stimulated with linear peptide pools matching DNA-SMI-Gag_p37 and MVA-CMDR-Gag_p55 (described below; JPT, Berlin, Germany) (Fig. 2). Frequencies of antigen-specific SFC were measured with an automated ELISPOT assay reader (ImmunoSpot CTL, Bonn, Germany). Responses were considered positive when the number of SFC was at least four times the medium control and >55 SFC/10⁶ PBMC (33). Mapping of individual peptide responses was performed based on the peptide pool matrix ELISPOT assay results and testing cryopreserved PBMC. Responses of stimulated PBMC with an SFC count higher than 2-fold the unstimulated medium control and ≥50 SCF/10⁶ PBMC cells were considered positive. Samples with a medium control of >60 SFC/PBMC were excluded from analyses.