A candidate vaccine composed of live nonpathogenic Iranian Lizard Leishmania mixed with Chitin microparticles protects mice against Leishmania major infection

Background: The protective effect of immunization using Iranian Lizard Leishmania (ILL) mixed with CpG oligo-deoxynucleotides (CpG-ODN) was demonstrated in a previous study. Here, we report the effect of leishmanization using ILL mixed with chitin microparticles (CMPs) as an adjuvant against L. major infection in BALB/c mice. Methods: Briefly, 2 x 10(7) live ILL were mixed with 10 mu g CMPs (<40 mu m in size) (ILL+CMP) and were injected subcutaneously into the right footpad of BALB/c mice. Three control groups were included in the study and received ILL, chitin, and PBS respectively. Three weeks later, mice were challenged with 2 x 10(5) live L. major(EGFP) promastigotes, which were inoculated into the left footpad. The infection course was monitored using footpad swelling measurement and in vivo imaging. Eleven weeks after the challenge, all mice were sacrificed and parasite burden was measured in the spleen and the draining lymph node using three different methods including real-time PCR, flow cytometry, and direct fluorescent microscopy. In addition, cytokines levels (IFN-gamma and IL-10), and nitric oxide production were assayed in splenocytes. Results: Mice immunized with ILL+CMP had a smaller footpad diameter in comparison to control groups and notably, no lesion was developed at the inoculation site. Additionally, in vivo imaging study revealed that there was no detectable fluorescence in the ILL+CMP group footpad by the end of the tenth week. This finding was confirmed by three methods used for parasite burden assays. Moreover, higher IFN-gamma level was observed in mice immunized with ILL+CMP in comparison with other groups. On the other hand, nitric oxide concentration was higher in the ILL control group. Conclusion: ILL mixed with chitin microparticles is an effective vaccine against leishmaniasis in BALB/c mice. This vaccine is able to induce an adequate immune response to decrease the parasite burden and prevent lesion formation. Further studies are needed to evaluate long-lasting immunity, especially in experimental outbreed models.