Evaluation of the long-term immune responses following leishmanization using a live- Lizard Leishmania mixed with chitin

Background: Leishmanization using non-pathogenic to human Leishmania spp. is considered a reliable approach to immunize subjects against Leishmania infection. Objectives: : Here, we evaluated the long-term immune responses (14 weeks) after immunization with either live- or killed-Iranian Lizard Leishmania (ILL) mixed with chitin microparticles (CMPs) against L. major infection in BALB/c mice. Methods: : In total, nine groups of mice were included in the study. To evaluate short-term immunity, mice were immunized with live-ILL and three weeks later were challenged with L. major EGFP . To evaluate the long-term immunity, mice were immunized with either live- or killed-ILL, and 14 weeks after immunization were challenged with L. major EGFP . A group of healthy mice who received no injection was also included in the study. Eight weeks after the challenge with L. major EGFP all subjects were sacrificed and the parasite burden (quantitative real-time PCR), cytokines levels (IFN-γ, IL-4, and IL-10), Leishmania -specific antibody concentration, and total levels of IgG1 and IgG2a were measured. In addition, nitric oxide concentration, and arginase activity were evaluated. Results: : In mice that were immunized using live-ILL+CMP, the induced proactive immune response lasted at least 14 weeks since, when they were challenged with L. major EGFP at the 14 th -week post-immunization, no open lesion was formed during 8 weeks follow-up, and the footpad swelling was significantly lower than controls. As well, they showed a significant reduction in the parasite burden in splenocytes, in comparison to the control groups including the group that received killed-ILL+CMP. The observed protection was associated with a higher IFN-γ and a lower IL-10 production by splenocytes. Additionally, the results demonstrated that arginase activity was decreased in the ILL+CpG group compared to other groups. Conclusion: The long-term response against L. major infection induced by Live-ILL+CMP was more competent than the response elicited by killed-ILL+CMP to protect mice against infection with L. major EGFP .