Tehran University of Medical Sciences Iranian Journal of Parasitology 1735-7020 11 2 2016 12 21 144 158 Vahid NASIRI Department of Parasitology, Faculty of Medical Sciences, Tarbiat Modares University, Tehran, Iran Abdolhossein DALIMI Department of Parasitology, Faculty of Medical Sciences, Tarbiat Modares University, Tehran, Iran Fatemeh GHAFFARIFAR Department of Parasitology, Faculty of Medical Sciences, Tarbiat Modares University, Tehran, Iran Azam BOLHASSANI Department of Hepatitis & AIDS, Pasteur Institute of Iran, Tehran, Iran 2016 07 02 2016 07 02 Background: The aim of present study was to evaluate the protective efficacy of live recombinant L. tarentolae expressing KMP11-NTGP96-GFP fusion as candidates for live engineered recombinant vaccine against visceral leishmaniasis in BALB/c mice. Methods: KMP-11 and NT-GP96 genes cloned into the pJET1.2/blunt cloning vector and then into pEGFP-N1 expression vector. The KMP-11, NT-GP96 and GFP fused in pEGFP-N1 and subcloned into Leishmanian pLEXSY-neo vector. Finally this construct was transferred to L. tarentolae by electroporation. Tranfection was confirmed by SDS-PAGE, WESTERN blot, flowcytometry and RT-PCR. Protective efficacy of this construct was evaluated as a vaccine candidate against visceral leishmaniasis. Parasite burden, humoral and cellular immune responses were assessed before and at 4 weeks after challenge. Results: KMP- NT-Gp96-GFP Fusion was cloned successfully into pLEXSY -neo vector and this construct successfully transferred to L. tarentolae. Finding indicated that immunization with L. tarentolae tarentolae-KMP11-NTGP96-GFP provides significant protection against visceral leishmaniasis and was able to induce an increased expression of IFN-γ and IgG2a. Following challenge, a reduced parasite load in the spleen of the KMP11-NTGP96-GFP immunized group was detected. Conclusion: The present study is the first to use a combination of a Leishmania antigen with an immunologic antigen in live recombinant L. tarentolae and results suggest that L. tarentolae-KMP11-NTGP96-GFP could be considered as a potential tool in vaccination against visceral leishmaniasis and this vaccination strategy could provide a potent rout for future vaccine development.  https://ijpa.tums.ac.ir/index.php/ijpa/article/view/1054 https://ijpa.tums.ac.ir/index.php/ijpa/article/download/1054/687