Table 1.
cSLN formulations and their characteristics.
Figure 1.
Assessement of footpad swelling in vaccinated mice.
Schematic presentation of the mean footpad measurements via caliper based method in mm (left axis) with standard error over the course of the Leishmania infection following pcDNA-cps immunization. BALB/c mice, thirteen animals in each group, were immunized subcutaneously with cSLN-pcDNA-cpa/cpb (G 1); pcDNA-cpa/cpb (G 2); cSLN-pcDNA-cpa/cpb-CTE (G 3), pcDNA-cpa/cpb-CTE (G 4); cSLN (G 5), cSLN-pcDNA (G 6) and PBS (G 7). Mice were immunized again 3 weeks later with the same schedule. Three weeks after the booster immunization, mice were challenged in the left footpad with L. major 5days stationary phase promastigotes (3×106 cells/mouse). Weekly measurements of footpad thickness represent the mean score ± SD in each group (n = 10). From the 9th week, a statistically significant difference (*p<0.05) was seen between G3 and the control groups. This difference continued up to the end of study course. (“G”represents “group” in all the graphs).
Figure 2.
Fluorescence imaging of the footpads.
Fluorescence imaging was performed at 9th week after challenge Four mice from each group were selected randomly for this experiment, but one representative mouse of each group was chosen for the photographs, taken by fluorescence imaging system. Footpads from each group were averaged for each data point to demonstrate sum green pixel count in the fluorescence image at this time point. The mean of the sum green pixel count with standard error from footpad images plotted for each group. The vaccinated mice in group 3 showed significantly (*p<0.05, n = 4) lower footpad size and sum green pixel count from the other groups.
Figure 3.
Flowcytometry analysis of the footpads.
The bars represent the average percentage of GFP positive footpad cells ± SD of three mice per each group GFP fluorescence expression was significantly lower in the footpads of group 3 (*p<0.05).
Figure 4.
L. major burden of popliteal lymph nodes.
(A) microtitration analysis: a limiting dilution analysis was performed 9 weeks after infection on the cells isolated from popliteal LN of 3 individual mice from each group and cultured in triplicate in Schneider's medium for 15 days at 26°C in serial 10-fold dilution. The wells were assessed microscopically for L. major growth, and the number of viable parasites was determined from the well with the highest dilution. The bar represents the average score±SD of three mice per group. Results are representative of at least two independent experiments and revealed a significant (*p<0.05) decrease in G1, G3 of the vaccinated mice compared to the control groups. (B) flowcytometry analysis. 9 weeks post-infection, intracellular fluoresence of the lymph node cells (pool of lymph nodes of three mice from each group) was quantified by Partec PASIII flow cytometer. “*” reveals significantly decrease in group 1 and 3 compared to unvaccinated control groups (p<0.05).
Table 2.
Average parasite inhibition percent (PI%) by different methods.
Figure 5.
Cytokine production by lymph node (LN) cells from L. major-infected mice.
Single-cell suspensions were prepared from the LN of three mice in the designated groups, before (n = 13) and 9 weeks after infection (n = 10). Cells cultured in triplicate for 5 days in the presence of recombinant antigens (10 µg/ml), soluble Leishmania antigen (SLA, 4 µg/well), Con A (as positive control) and RPMI (as negative control). Culture supernatants were assayed for levels of IFN-γ, before challenge (A), after challenge (B) and IL-5 (C) production by ELISA. There were no difference in Con A-induced cytokine production, among the groups. Each bar represents the mean ± SD for three mice per group (n = 3). Results are representative of two independent experiments, each performed in triplicate.
Figure 6.
The ratio of IFN-γ/IL-5 production.
The ratio presentation at week 9th after challenge in the lymph node cells stimulated by SLA and respective rCPs. This ratio was significantly (*p<0.05) higher in the animals immunized with Spa/b-CTE (G3).
Figure 7.
Antigen-specific IgG1 and IgG2a responses.
Before and at week 9th after challenge, blood samples were collected and pooled sera (diluted 1∶50) per each group was prepared. Antibody responses stimulated by rCPA (A, C) and rCPB or rCPB-CTE (B, D) and SLA (E) showed the profile of CP-specific antibodies, induced before (n = 13) and after (n = 10) challenge. The data represent means±SD. Results are representative of two independent experiments, each performed in triplicate.
Figure 8.
The ratio of IgG2a/IgG2a production.
The ratio presentation at 9th week after challenge in the different groups determined against rCPA. This ratio was significantly (*p<0.05) higher in the animals immunized with Spa/b-CTE (G3).