Fig 5. Impact of distinct immunization protocols on the NO production elicited early and late after L. chagasi-challenge.

NO levels (μM) were determined in supernatants from PBMCs cultures maintained upon vaccine-soluble antigen (VSA) or soluble Leishmania chagasi antigen (SLcA) stimuli in vitro. Data were analyzed early (90 days—T₉₀) and late (885 days—T₈₈₅) after experimental L. chagasi-challenge. The groups are represented as follows: C (“Control”; white bars); “Sal” (Lutzomyia longipalpis salivary glands; light gray bars); “LbSal” (antigen of L. braziliensis plus Lutzomyia longipalpis salivary glands; dark gray bars); and “LbSapSal” (L. braziliensis antigen plus saponin and Lutzomyia longipalpis salivary glands; black bars). Top panels: The x-axis displays the different experimental groups (“Control”, “Sal”, “LbSal” and “LbSapSal”) according to the in vitro stimuli (control culture [CC], VSA or SLcA). The y-axis represents the nitrite levels [μM]. Data are presented as mean values ± standard deviations. The connecting lines represent significant difference (P <0.05) between the CC, VSA or SLcA-stimulated cultures. The symbols C, Sal and LbSal indicate significant differences in comparison to the “Control”, “Sal” or “LbSal” groups, respectively. Bottom panels: Correlation between NO levels and spleen parasite load (# amastigotes/20ng of total DNA) at T₈₈₅ considering CC (bottom left panel) or the presence of a stimulus (VSA: bottom middle panel; or SLcA: bottom right panel) in all groups. The groups are distinguishable by colors as follows: as follows: “C” (white circles); “Sal” (ligh gray circles); “LbSal” (dark gray circles) and “LbSapSal” (black circles). The quadrants represented in the bottom panels delimit the low and high NO producers (y-axis) and the low and high spleen parasite load (x axis).