Identification of Novel Cytotoxic T Lymphocyte Epitopes of Drug- Resistance Related Protein InhA from Mycobacterium tuberculosis.

Abstract

Tuberculosis (TB) caused by Mycobacterium tuberculosis (MTB), especially the drug-resistant MTB, poses serious challenges to human healthcare worldwide. Cytotoxic T lymphocytes (CTLs) play a vital role in immune defense against MTB. To identify novel CTL epitopes that could induce cellular immunity against MTB infections. The HLA-A*0201 restricted CTL epitopes of the drug-resistant protein InhA from MTB were predicted by online algorisms and synthesized by the Fmoc solid phase method. The candidate peptides were used to induce CTLs from human peripheral blood mononuclear cells (PBMCs) of HLA-A*0201 healthy donors and the HLA-2.1/Kb mice. IFN-γ productions of CTLs were detected by enzyme linked immunospot assay (ELISPOT), flow cytometry and enzyme-linked immunosorbent assay (ELISA), and cytotoxicity was analyzed by lactate dehydrogenase (LDH) assay. A group of 4 epitopes were screened out with high affinities to HLA-A*0201. ELISPOT and flow cytometry analysis indicated these peptides significantly induced that IFN-γ release of CTLs from the HLA-A*0201+/PPD+ donors, as the mutant analogues had more potent stimulation effects. LDH assay showed that CTLs from PPD+ donors and the immunized mice exhibited significant cytotoxicity and low cross-reactivity. ELISA analysis revealed comparative levels of IFN-γ were released by CTLs isolated from the mice spleen. Our study has identified 4 novel CTL epitopes of InhA that could elicit potent CTL immunity, establishing a foundation for the development of multivalent peptide vaccines against the drug-resistant MTB.