Fig. 3

IFNγ T-cell and Ab responses induced by peGn, pscDEC-eGn and pscCD11c-eGn in lambs. a Schematic representation of the immunization protocol. The 2-month-old lambs were immunized at weeks 0, 4 and 8 with 400 µg of peGn, pscDEC-eGn, pscCD11c-eGn and 400 µg of pGM-CSF as adjuvant. Control lambs received pGM-CSF only. The plasmids were injected intradermally and the injection was immediately followed by SEP (532 V/cm). Sera were collected and the IFNγ T-cell responses were assessed by ELISPOT at the indicated time points. The syringe drawing is from Servier Medical Art which provides open source illustrations. b PBMCs (2.5 × 10⁵) were plated in Multiscreen plates coated with a capture anti-ovine IFNγ mAb (duplicated wells) and re-stimulated for 18 h with overlapping eGn peptides covering the N- and C-terminal parts (5 µg/ml), or with an irrelevant peptide (5 µg/ml). IFNγ spot counts were considered eGn specific when both duplicated well values from cells stimulated with eGn peptides were strictly superior to the duplicated well values from cells stimulated with the irrelevant peptide. The mean spot numbers in stimulated wells minus the ones stimulated with irrelevant peptides are shown (net mean values). Each symbol in the graphs represents an individual animal and the mean is indicated. P values between the two groups were determined according to the Mann–Whitney test (*p < 0.05; **p < 0.01). c The serum of the lambs immunized with peGn, pscDEC-eGn, pscCD11c-eGn and of control lambs were assayed for the detection of eGn-specific IgG using indirect ELISA. A lysate from HEK293 cells transfected with peGn was used as coating (see Materials and methods section) and the individual sera were tested in a 1:100 dilution. The OD signals at 450 nm from the sera collected at weeks 0, 4, 8 and 12 are shown in 3 C (1 repeated experiment). Each lamb serum is shown as a distinct symbol and the mean OD value is represented as an horizontal bar. P values were determined using the two-way ANOVA with Bonferroni’s correction to evaluate the statistical significance of the OD value differences between vaccinated groups (*p < 0.05; **p < 0.01; ****p < 0.0001) and the significance of the differences between the OD values measured at the different weeks versus at day 0 was evaluated for each vaccinated group using paired t-test and the groups with statistically significant differences are indicated by a box