C-Terminal Domain Deletion Enhances the Protective Activity of cpa/cpb Loaded Solid Lipid Nanoparticles against Leishmania major in BALB/c Mice

Cutaneous leishmaniasis (CL) is the most common form of leishmaniasis with an annual incidence of approximately 2 million cases and is endemic in 88 countries, including Iran. CL's continued spread, along with rather ineffectual treatments and drug-resistant variants emergence has increased the need for advanced preventive strategies. We studied Type II cysteine proteinase (CPA) and Type I (CPB) with its C-terminal extension (CTE) as cocktail DNA vaccine against murine and canine leishmaniasis. However, adjuvants' success in enhancing immune responses to selected antigens led us to refocus our vaccine development programs. Herein, we discuss cationic solid lipid nanoparticles' (cSLN) ability to improve vaccine-induced protective efficacy against CL and subsequent lesion size and parasite load reduction in BALB/c mice. For this work, we evaluated five different conventional as well as novel parasite detection techniques, i.e., footpad imaging, footpad flowcytometry and lymph node flowcytometry for disease progression assessments. Vaccination with cSLN-cpa/cpb-CTE formulation showed highest parasite inhibition at 3-month post vaccination. Immunized mice showed reduced IL-5 level and significant IFN-ã increase, compared to control groups. We think our study represents a potential future and a major step forward in vaccine development against leishmaniasis

cSLN formulations and their characteristics.

<p>Nanoparticles were formulated from cetyl palmitate, cholesterol and DOTAP hydrochloride. Results represent mean±SD of three independent SLN preparations.</p

Average parasite inhibition percent (PI%) by different methods.

<p>Average PI% measured in terms of the parasitic load decrease in the footpad (FP) and/or lymph node (LN) of vaccinated animals compared to the control groups via different methods. (“NS” represent as “not significant” and “*” represents significant difference between the mentioned groups).</p

Cytokine production by lymph node (LN) cells from <i>L. major</i>-infected mice.

<p>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 <i>Leishmania</i> 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.</p

<i>L. major</i> burden of popliteal lymph nodes.

<p>(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 <i>L. major</i> 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 (*<i>p</i><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 (<i>p</i><0.05).</p

Antigen-specific IgG1 and IgG2a responses.

<p>Before and at week 9^th 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.</p

The ratio of IgG2a/IgG2a production.

<p>The ratio presentation at 9^th week after challenge in the different groups determined against rCPA. This ratio was significantly (*<i>p</i><0.05) higher in the animals immunized with S_pa/b^-CTE (G3).</p

Flowcytometry analysis of the footpads.

<p>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 (*<i>p</i><0.05).</p

Assessement of footpad swelling in vaccinated mice.

<p>Schematic presentation of the mean footpad measurements via caliper based method in mm (left axis) with standard error over the course of the <i>Leishmania</i> infection following pcDNA-<i>cps</i> immunization. BALB/c mice, thirteen animals in each group, were immunized subcutaneously with cSLN-pcDNA-<i>cpa/cpb</i> (G 1); pcDNA-<i>cpa/cpb</i> (G 2); cSLN-pcDNA-<i>cpa/cpb^-CTE</i> (G 3), pcDNA-<i>cpa/cpb^-CTE</i> (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 <i>L. major</i> 5days stationary phase promastigotes (3×10⁶ cells/mouse). Weekly measurements of footpad thickness represent the mean score ± SD in each group (<i>n</i> = 10). From the 9^th week, a statistically significant difference (*<i>p</i><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).</p