Additional file 1: Figure S1. of Selection strategy of phage-displayed immunogens based on an in vitro evaluation of the Th1 response of PBMCs and their potential use as a vaccine against Leishmania infantum infection

Parasitological and immunological evaluations obtained in the infected and/or vaccinated animals in the second vaccine experiment. BALB/c mice (n = 16, per group) were vaccinated subcutaneously in their left hind footpad with Wild-type (WT), Random, B1 or D11 phage clones. Additional mice received only saline (n = 16). Three doses were administered at 14-day intervals. 30 days after the last vaccine, animals (n = 8, per group) were infected in the right hind footpad with 1 × 107 stationary promastigotes of L. infantum. Before and 60 days after challenge, the anti-phage and anti-parasite IgG2a and IgG1 isotype antibody levels were obtained, and the ratios between IgG2a and IgG1 results were calculated and are shown before (a) and after (b) infection. In addition, spleen cells were collected to evaluate the cytokine response, when they were incubated in complete RPMI 1640 medium (negative control) or in vitro stimulated with SLA (25 μg/ml) or with the respective clone (1 × 1010 phages), for 48 h at 37 °C in 5% CO2. IFN-γ, IL-12, GM-CSF, IL-4 and IL-10 levels were then measured by ELISA in the culture supernatants before (c) or after (d) infection. 6 days after challenge, the parasite burden was determined in the liver, spleen, draining lymph nodes and bone marrow of the animals, by a limiting dilution assay (e). Using the cell supernatants employed to evaluate cytokines, the nitrite production was also evaluated in this time (f). +indicates a statistically significant difference in relation to the B1 and D11 phages groups (P < 0.0001). ***indicates a statistically significant difference in relation to the saline, WT and Random groups (P < 0.0001). (TIFF 139 kb