In vivo HA533-541-specific CD8+ T cell killing

The killing activity of HA_533-541-specific CD8+ T cells induced by the different vaccines was determined in vivo. Splenocytes from naïve BALB/c mice were loaded with cognate 5 μM HA_533-541 peptide (IYSTVASSL, JPT) or 10 μM HIV gag_199-207 control peptide (AMQMLKETI, JPT) and after 1 h the cells were loaded with CFSE (LifeTechnology) or CMTMR (LifeTechnology), respectively. Splenocytes (10⁶) were adoptively transferred into the tail vein of recipient mice 9 days or 5 - 6 weeks after the first or second immunization with SAM-H1/CNE, aMIV or sterile PBS. Mice were sacrificed 20 h after the adoptive transfer, and splenocytes were stained with live/dead yellow (LifeTechnologies) and frequencies of CFSE+ or CMTMR+ cells were measured by flow cytometry on a LSRII SORP flow cytometer (Becton Dickinson). The target cell specific lysis was calculated as % specific lysis = [1-(naïve mice/vaccinated mice)]x100 (39 (link)).