Maryam Aghaei - Skin Diseases and Leishmaniasis Research Centre, Isfahan University of Medical Sciences, Isfahan, Iran
Hossein Khanahmad - Department of Genetics and molecular biology, School of Medicine, Isfahan University of Medical Sciences, Isfahan, Iran
Akram Jalali - Research Center for Molecular Medicine, Hamadan University of Medical Sciences, Hamadan, Iran
Shahrzad Aghaei - Department of Molecular Medicine, School of Advanced Technologies, Shahrekord University of Medical Sciences, Shahrekord, Iran
Manizheh Narimani - Department of Parasitology and Mycology, School of Medicine, Isfahan University of Medical Sciences, Isfahan, Iran
Sayed Mohsen Hosseini - Department of Biostatistics & Epidemiology, School of Public Health, Isfahan University of Medical Sciences, Isfahan, Iran
Fatemeh Namdar - Department of Parasitology and Mycology, School of Medicine, Isfahan University of Medical Sciences, Isfahan, Iran
Hossein Hejazi - Skin Diseases and Leishmaniasis Research Centre, Isfahan University of Medical Sciences, Isfahan, Iran

Objective(s): Leishmaniasis is a complex infection against which no confirmed vaccine has been reported so far. Transgenic expression of proteins involved in macrophage apoptosis-like BAX through the parasite itself accelerates infected macrophage apoptosis and prevents Leishmania differentiation. So, in the present research, the impact of the transgenic Leishmania major including mLLO-BAX-SMAC proapoptotic proteins was assayed in macrophage apoptosis acceleration. Materials and Methods: The coding sequence mLLO-Bax-Smac was designed and integrated into the pLexyNeo۲ plasmid. The designed sequence was inserted under the ۱۸srRNA locus into the L. major genome using homologous recombination. Then, mLLO-BAX-SMAC expression was studied using the Western blot, and the transgenic parasite pathogenesis was investigated compared with wild-type L. major in vitro and also in vivo. Results: Western blot and PCR results approved mLLO-BAX-SMAC expression and proper integration of the mLLO-Bax-Smac fragment under the ۱۸srRNA locus of L. major, respectively. The flow cytometry results revealed faster apoptosis of transgenic Leishmania-infected macrophages compared with wild-type parasite-infected macrophages. Also, the mild lesion with the less parasitic burden of the spleen was observed only in transgenic Leishmania-infected mice. The delayed progression of leishmaniasis was obtained in transgenic strain-injected mice after challenging with wild-type Leishmania. Conclusion: This study recommended transgenic L. major including mLLO-BAX-SMAC construct as a pilot model for providing a protective vaccine against leishmaniasis. 

Homologous recombination, Integration, Leishmaniasis, Transfection, Vaccine