Generation of a recombinant, antibiotic resistance gene-free Leishmania tarentolae strain as a live vaccine platform

Abstract Live vaccines based on non-pathogenic microorganisms are among the most effective vaccine platforms. L. tarentolae , a non-pathogenic protozoan for humans, is a valuable expression system capable of producing eukaryotic proteins while preserving their native structure and post-translational modifications. Leishmaniasis, a sand fly–borne disease with diverse clinical forms, remains a significant health concern. Sand fly salivary proteins such as PpSP15 and PsSP9 have been identified as promising protective vaccine candidates. However, the common use of antibiotic resistance genes as selection markers in recombinant strains raises safety concerns for human applications. In this study, an antibiotic marker–free system was developed by removing the neomycin resistance gene (Neo r ) from the pLEXSY-Neo r vector to construct pLEXSY-noNeo r . The PpSP15-T2A-PsSP9 fragment was cloned into this modified vector, and CRISPR-Cas9 technology was used to integrate the genes into the SSU locus of L. tarentolae . Successful gene insertion and expression were confirmed at DNA, RNA, and protein levels. Importantly, mutant parasites showed infectivity comparable to wild-type strains. These findings demonstrate the feasibility of generating antibiotic resistance–free transgenic L. tarentolae , supporting its potential as a safe live vaccine platform.