Identification of the murine firefly luciferase-specific CD8 T-cell epitopes

Adeno-associated virus serotype 9-mediated pulmonary transgene expression: effect of mouse strain, animal gender and lung inflammation

“…The use of BALB/c strain in our study was based on the fact that transgenes are more immunogenic in BALB/c mice. In fact, it has been reported that three of the most widely used transgenes lacZ, 16 enhanced green fluorescent protein 30 and firefly luciferase 31 are more immunogenic in BALB/c mice than in C57BL/6 mice. Previous studies reporting long-term expression in the lungs after AAV2/9-mediated delivery were performed in C57BL6 per mice 8,14 using human a1-antitrypsin and nuclear-localized lacZ as marker genes.…”

A combination of intradermal jet-injection and electroporation overcomes in vivodose restriction of DNA vaccines

“…Splenocytes were purified by Ficoll-Paque separation (GE Healthcare, Stockholm, Sweden) and IFN-γ ELISpot and IFN-γ/IL-2 FluoroSpot assays (Mabtech, Nacka Strand, Sweden) were performed according to the manufacturer’s protocol and as previously described [37]. 1 × 10 5 viable cells were plated per well and stimulated with a peptide pool covering Gag p24B (15mers with ten amino acids overlap) or the H2-K d restricted AMQMLKETI (Gag) and GFQSMYTFV (luciferase) [38] peptides. The final concentration of peptides was 5 μg/peptide/ml.…”

Evaluation of immunogen delivery by DNA immunization using non-invasive bioluminescence imaging

“…A total of 1 × 10 5 splenocytes per well were plated and stimulated for 20 h with antigens: recombinant luciferase (Promega, #E1701) at 6 μg/ml and 12 μg/ml and luciferase-derived peptide GFQSMYTFV (GL Biochemical) at 5 μg/ml and 10 μg/ml of RPMI. 17 The costimulatory anti-CD28 antibody was added to the cells during the incubation. Bound cytokines were detected with fluorescein isothiocyanate (FITC)-labeled antibody (R4-6A2) and biotynylated antibody (5H4) followed by anti-FITC antibody conjugated to a green fluorochrome and streptavidin conjugated to a red fluorochrome.…”

“…Next, we compared superficial and deep injections in their ability to induce the luciferase-specific immune response. To assess cellular responses, we measured the ability of splenocytes of immunized mice to secrete type 1 cytokines (IFN-γ or IL-2) when stimulated with luciferase-derived peptide corresponding to its CTL epitope recognized in mice 17 or with the recombinant luciferase to monitor CD4+ T-cell response. Superficial immunization induced a 3-fold higher IFN-γ secretion compared with the at this time point (9.5 × 10 6 photons/sec difference; p = 0.085, t-test).…”