Immunization against Leishmania major infection in BALB/c mice using a subunit-based DNA vaccine derived from TSA, LmSTI1, KMP11, and LACK predominant antigens

Abstract

Objectives: To design a multivalent DNA vaccine encoding the most immunogenic regions of the Leishmania major antigens including TSA (Thiol-specific antioxidant protein), LmSTI1 (Leishmania major stress-inducible protein1), LACK (Leishmania homologue of receptors for activated C Kinase), and KMP11 (kinetoplastid membrane protein-11) on BALB/c mice.

Materials and methods: The chimeric construct was generated including the most immunogenic epitopes containing a combination of domains and oligopeptides of the aforementioned genes. The construct was cloned into pcDNA 3.1 plasmid and named "pleish-dom." Following intramuscular injection of mice, the capability of the vector pleish-dom alone and with pIL-12 (expressing murine IL-12) to raise protective cytokines and parasite burden was evaluated in the BALB/c mice as a susceptible animal model against L. major.

Results: The immunized mice with pleish-dom/pIL-12 showed the highest and the lowest levels of interferon-gamma (IFN-γ) and interleukin-10 (IL-10), as well as the lowest leishmanin skin test (LST) reactions, were found through 8 weeks post-infection.

Conclusion: Although the obtained DNA vaccine from the immunogenic chimeric protein of L. major antigens was able to induce a high level of IFN-γ, it partially protected mice against L. major. However co-administration with pIL-12 led to shift immune response to Th1 phenotype, granuloma formation, and lowered parasite burden, and consequently distinct protection was found.

Keywords: BALB/c mice; Cytokines; DNA; Epitopes; Leishmania major; Vaccines.

Figure 1

Antigen structures with the number of protein sequence are shown in rectangles and domains within it. Experimental and predicted epitopes have been displayed in green and pink color, respectively above the rectangle. Selected Domains/oligopeptides in any antigen have been marked with red dash lines

Figure 2

A schematic representation of the construct. A. Selected fragments of each protein with the number of nucleotides have been shown as linear successive domains separated by flexible linkers. The predicted and experimental validated peptides have been listed under each fragment. B. The sequence of the chimeric construct with linkers and His-tag

Figure 3

GFP expression of pEGFP-leish-dom in HEK293 cells. A: A merged picture of HEK293 cells after 48 hr transfection; B: G418-treated cells after two weeks incubation with an expanding colony

Figure 4

Western blotting analysis. Lane 1: The stable transfected Hek293 cells by pleish-dom with the desired protein band (46 kDa); Lane 2: protein marker

Figure 5

Lesion sizes were measured in experimental groups of animals for ten weeks post-infection. Lesion development was ceased at weeks 4 and 6 post-infection in pleish-dom/pIL-12 and pleish-dom groups, respectively. There was a significant difference (P<0.05)between the pleish-dom/pIL-12 group and control groups

Figure 6

Spleen parasite burden in experimental groups. Mice were inoculated 3 times with pleish-dom alone and with pIL-12 as vaccinated groups and with pCDNA plasmid and PBS as control groups with a 2-week interval. Mice were challenged with 106 metacyclic promastigotes 4 weeks post last time of immunization. Spleen parasite burdens were evaluated 8 weeks post-challenge. Vaccination with pleish-dom/pIL-12 was able to prevent visceralization of the parasites in almost all the immunized mice except one mouse with a little parasite burden

Figure 7

Cytokine assessment in the supernatant of spleen cell cultures. Extracted spleen cells were stimulated with soluble Leishmania antigen (25 μg/mL) for 48 hr and 72 hr for IL-10 and IFN-γ, respectively. The levels of cytokines were determined using ELISA. Tukey’s test followed by one way ANOVA was used to perform statistical analysis (* P≤0.05); A. increased level of the IFN-γ level in vaccinated groups before challenge; B. Higher level of IL-10 in mice vaccinated with pleish-dom alone before challenge; C. 4-fold increasing of IFN-γ level in mice vaccinated with pleish-dom/pIL-12 than before challenge; D. elevated level of IL-10 in control groups at 8 weeks post-challenge compared to before challenge

Figure 8

Microscopic examination: Site of infection; diffuse macrophages with plenty of internalized amastigotes infiltrated with a few polymorphs and lymphocytes without or rare granuloma formation in the site of infection of control groups(A) and many granulomas with entrapped amastigotes in vaccinated groups (B). Spleen; visceral Leishmania infection in control groups’ mice altered the histological structure of the spleen, led to the loss of cell populations in the spleen, and disorganized architecture with indistinct border between red and white pulps (C) versus regular architecture in spleen of mice vaccinated with pleish-dom/pIL-12(D). Liver; immature granuloma formation with diffused amastigotes out of granuloma in liver sections of control groups (E) and the parasitized multinucleated granulomas seen in the liver sections of mice vaccinated with pleish-dom (F)