Figure 2
Response to Dband Kbbinding NS1 peptides. (A) Splenocytes were cultured with NS1 peptides for 48 h and IFN-γ production was measured using ELISPOT assays. Peptides NS1(141/152/166/222/403) showed specific IFN-γ in only 3 or 4 mice out of the 6 mice used in these experiments. Peptide NS1(14) was only positive in one mouse, whereas peptide NS1(125) was positive for IFN-γ production in 5 out of 6 mice. (*p < 0.05 Mann–Whitney test NS1(125) vs control). Data are presented as the mean number ± SD of IFN-γ-producing cells in response to stimuli in 6 mice. (B) CTL lines were generated by in vitro NS1 peptide stimulation (effector: E) and tested against RMA-S cells (target: T) pulsed with the NS1 peptide used for in vitro stimulation or with the control peptide gp(33). Peptides NS1(125/152/235/403) generated specific CTL responses, whereas no CTLs were generated using NS1(141/166(I)/222) peptides. Data presented are representative of at least 4 mice for each peptide (*p < 0.05 unpaired Student t test NS1 peptide vs control gp(33) peptide). (C) CTL lines specific for NS1(125/152/235/or 403) peptide were also tested against MC57 cells as target (E:T = 50:1) pulsed with relevant NS1 peptide as positive control, mock BHK lysate as negative control, or BEI-BTV-1, 4 or 8 to assess epitope processing. CTLs specific for NS1(152) and NS1(403), but not NS1(125) peptide, were capable of recognising target cells pulsed with BTV from different serotypes (p < 0.05 unpaired Student t test: mock lysate vs NS1 peptide, BTV-1, 4 or 8). CTLs specific for NS1(235) peptide were able to recognise target cells pulsed with BTV-8. However due to the limited number of CTL obtained in these experiments, we were not able to confirm the expression of this epitope in BTV-1 or 4. All CTL data presented are representative of at least 4 mice for each peptide. §: not done.