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.
Related
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- Knowledge and misconceptions about antibiotic use and antibiotic resistance among secondary school students in Riyadh, Saudi Arabia: implications for school- based antibiotic resistance education
- Beliefs and Behaviours related to Antibiotic Use and Antimicrobial Resistance in Poland: General Population Survey
BibTeX
@article{taheri2026generation,
title = {{Generation of a recombinant, antibiotic resistance gene-free Leishmania tarentolae strain as a live vaccine platform}},
author = {Tahereh Taheri and Elaheh Davarpanah and Ana Maria Murta Santi and Elham Gholami and Negar Seyed and Gerald F. Späth and Sima Rafati},
journal = {Scientific Reports},
year = {2026},
month = oct,
doi = {10.1038/s41598-026-73756-5},
url = {https://www.nature.com/articles/s41598-026-73756-5},
note = {CC BY 4.0},
}
RIS
TY - JOUR
TI - Generation of a recombinant, antibiotic resistance gene-free Leishmania tarentolae strain as a live vaccine platform
AU - Tahereh Taheri
AU - Elaheh Davarpanah
AU - Ana Maria Murta Santi
AU - Elham Gholami
AU - Negar Seyed
AU - Gerald F. Späth
AU - Sima Rafati
PY - 2026
DA - 2026/10/01
JO - Scientific Reports
DO - 10.1038/s41598-026-73756-5
UR - https://www.nature.com/articles/s41598-026-73756-5
AB - 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.
SN - 2045-2322
ER -