Parasites of the genus Leishmania are intracellular protozoa which are transmitted to their mammalian host by the bite of infected sand flies and cause a group of diseases known as Leishmaniasis. Despite attempting different vaccination strategies, no human vaccine is yet available against this disease. There is increasing evidence that presence of a small number of live parasites is necessary to maintain durable immunity, and the only way to meet this requirement is by using attenuated live vaccines . The main obstacle about attenuated live strains is the risk of reversion of the organism to its virulent state. Another approach to reach this strategy is to use nonpathogenic Leishmania such as L. tarentolae. This parasite is lizard parasite and has never been found associated with any leishmaniasis in humans and is considered as non-pathogenic to humans. Previous studies have shown that L. tarentolae can be used as a live vaccine against L. donovani and elicit a protective Th1 immune response . Recently, by comparative genomic analysis and expression profiles of well-characterized virulence factors such as GP63, CPB, LPG3, Amastin and A2 between pathogenic Leishmania species (e.g. L. major, L. infantum and L. braziliensis) and non pathogenic L. tarentolae revealed that only A2 is absent at the level of DNA . A recombinant L. tarentolae expressing the A2 protein was generated and its potential as a live vaccine against L. infantum infection in BALB/c mice was examined . The A2 expressing recombinant parasites showed higher macrophage infectivity in comparison to L. tarentolae used as a control. Immunization (i.v. and i.p.) of BALB/c mice with recombinant L. tarentolae A2 elicited a strong protective immunity against virulent L. infantum challenge, manifested by a dramatic decrease in parasite burdens in the liver and the spleen of immunized mice. IFN-g production upon antigen stimulation indicated that protection is associated with a Th1 cell-mediated immunity accompanied by reduced levels of IL-5 production (the Th2 type response). Interestingly, although IFN-g production is also induced in groups of mice immunized with wild type L. tarentolae, cytokine levels are increased in the group immunized with the recombinant L. tarentolae A2 and especially when the vaccine regimen is administered via the i.p. route . In continuation of these promising results, we are expanded this idea against L. major infection as a novel vaccine regimen by including two immunogenic parasite proteins (cysteine proteinases A and B, CPA/CPB).