Lymphoproliferation responses and production of IFN-γ after Soluble Leishmania Antigen-specific stimulation. Panel A: Soluble Leishmania Antigen-specific lymphoproliferation. This assay detects the ability of the specific T cells to proliferate after being exposed for 5 days to Soluble Leishmania Antigens (SLA). The cells were pulsed during the last 24 h with 10 μM 5-bromo-2-deoxyuridine (BrdU). The lymphoproliferation index is the ratio of the mean optical density obtained for the SLA stimulated samples compared to the mean optical density obtained for the non-stimulated samples using peroxydase-labelled anti-BrdU antibodies. This figure presents the results before challenge on week 58 (W58), which was also one year after the last vaccination, and on weeks 90 and 105. Panel B: ELISpot detection of IFN-γ secreting lymphocytes. This assay detects the ability of lymphocytes to secrete IFN-γ after 72 h specific stimulation with Soluble Leishmania Antigens (SLA) in coated wells with canine IFN-γ capture antibody. The clones of cells secreting IFN-γ (or spots) were detected using specific biotinylated antibodies and incubation with Streptavidin-AP and the BCIP/NBT Chromogen. The data presented here are the number of spots per 2 × 105 cells after stimulation with SLA minus the equivalent value obtained with the negative control using medium alone. This figure presents the results before challenge on week 58 (W58), which was also one year after the last vaccination, and on weeks 90 and 105.