Siinfekl peptide

The in vitro cytotoxicity assay used was derived from the VITAL assay, originally described by Hermans et al. [34 (link)]. Female C57BL/6 mice aged 6–8 weeks were assigned to each treatment in groups of 9 per assay. Vaccines were administered subcutaneously into the left flank on days 0 (1D), 0 and 1 (2D) or 0, 1 and 2 (3D), consisting of 100 μg VLP and 25 μg CpGs in CPBS, 3 mice per vaccination regimen. Lymph nodes were resected on day 7, strained, washed and RBCs were lysed. CD8⁺ cells were sorted using CD8α (Ly-2) microbeads (Miltenyi Biotec, Germany) on an AutoMACS Pro cell separator (Miltenyi Biotec, Germany) by positive selection. Target cells were prepared as previously described, with the exception that a population pulsed with synthetic SIINFEKL peptide (JPT Peptide Technologies, Germany) was used. Sorted CD8⁺ T cells were co-cultured with target cells at an effector:target ratio of 0.1:1 for 4 h at 37 °C + 5% CO₂. Cells were stained with near-IR live/dead stain and fixed with 2% PFA. Target populations were detected on a Gallios flow cytometer, and analysed using Kaluza version 1.2. The gating strategy is provided in Additional file 1: Figure S1.

Mice harboring memory OT-1 cells, primed with MCMV-SIINFEKL or RAE-1γMCMV-SIINFEKL, were sacrificed and splenocytes isolated using a standard protocol. 2x10⁶ cells were then incubated for 6h with different concentrations of SIINFEKL peptide (JPT PeptideTechnologies GmbH) in RPMI 1640 (PAN-Biotech) supplemented with 10%FCS (PAN-Biotech), Brefeldin A (Invitrogen), Monensin (Invitrogen), and CD107 (Invitrogen) at 37°C.
For the in vitro killer assay, mice harboring memory OT-1 cells (CD45.1) were sacrificed and splenocytes isolated using a standard protocol. Splenocytes were pooled from 4-5 mice/group. CD8 T cells were purified by negative selection using magnetic beads (Miltenyi Biotec), and OT-1 (CD45.1) cells were stained with CD45.1 antibody and sorted using FACSAria II (BD) using high-speed sorting into RPMI supplemented with 20% FCS. Sorted OT-1 (CD45.1) cells were co-incubated with E.G7-OVA (CD45.2) cells for 4h in 2:1, 1:1, and 0.5:1 effector to target ratios at 37℃. After co-incubations, target cells were identified as CD45.1 negative, and viability was determined using Fixable Viability Dye (eBioscience). OT-1 cytotoxicity was calculated using following formula: [(% FVD⁺CD45.1^- cell-specific lysis − % FVD⁺CD45.1^- cell spontaneous lysis)/(100 − % FVD⁺CD45.1^- cell spontaneous lysis)] × 100 as described in (19 (link)).