Figure 5.

B8R_70–78 peptide of VACV is mutated in ECTV and does not elicit CD8⁺ T cell responses. B7.2^tg mice (n = 6) were primed with a mixture of peptides using TriVax vaccination protocol and boosted 14 days later with maturated BMDCs that were pulsed with the indicated peptides as described in “Materials and methods” to elicit peptide-specific CD8⁺ T cell responses. Ten days after boost, splenocytes were harvested, and peptide-reactive CD8⁺ T cell responses were monitored. (A) Representative contour plots (n = 6) depicting D1R_808-817, B8R_70–78 and C4R_70–78 specific CD8⁺ T cell responses that were detected with the indicated pB7.2 tetramers, each of which was labelled with two different fluorochromes. Numbers in oval gates correspond to the frequency of antigen specific CD8⁺ T cells. (B) Frequencies (% of CD8⁺) of B8R_70–78, C4R_70–78 or D1R_808-817 specific CD8⁺ T cells were enumerated with pB7.2 tetramer staining in infected spleocytes. Data are mean ± sem; n as in (A). (C) Peptide binding to conditional pB7.2 monomer was measured by the ability of the exchange peptide to stabilize HLA-I. Soluble AARG-J-TLAM/B7.2 monomer was incubated with the peptides indicated at the concentrations shown and subjected to a 60-min UV radiation. No UV (un-exchanged) AARG-J-TLAM/B7.2 monomer was used as a positive control and monomer without any peptide served as negative/background control. Peptide exchange-stabilized pB7.2 was monitored by ELISA by detecting human β2-m bound to MHC-I tethered to the ELISA plates using the pan-HLA-I mAb W6/32. Data are mean ± sem of exchange performed in triplicate for each peptide concentration, and representative of three independent experiments. (D) Dose dependent IFN-γ response elicited by TriVax primed and peptide-pulsed BMDC-boosted mouse splenocytes that was measured by epitope titration in an ELISpot assay. Data represent the mean of triplicate wells; representative of two independent experiments. n = 3 mice in each experiment.