Background: 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.

Objective: To identify novel CTL epitopes that could induce cellular immunity against MTB infections.

Methods: 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.

Results: 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.

Conclusion: 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.

背景：结核分枝杆菌（MTB）引起的结核病（TB），尤其是耐药性MTB，给全球人类医疗保健带来了严峻挑战。细胞毒性T淋巴细胞（CTL）在针对MTB的免疫防御中起着至关重要的作用。

目的：确定可以诱导针对MTB感染的细胞免疫的新型CTL表位。

方法：通过在线算法预测并用Fmoc固相法合成来自MTB的耐药蛋白InhA的HLA-A * 0201限制性CTL表位。候选肽用于从HLA-A * 0201健康供体和HLA-2.1 / Kb小鼠的人外周血单核细胞（PBMC）诱导CTL。通过酶联免疫斑点法（ELISPOT），流式细胞仪和酶联免疫吸附法（ELISA）检测CTL的IFN-γ产生，并通过乳酸脱氢酶（LDH）分析细胞毒性。

结果：筛选出与HLA-A * 0201高亲和力的4个表位。ELISPOT和流式细胞仪分析表明，这些肽显着诱导HLA-A * 0201 + / PPD +供体的IFN-γ释放CTL，因为突变类似物具有更强的刺激作用。LDH分析显示，来自PPD +供体和免疫小鼠的CTL表现出明显的细胞毒性和低交叉反应性。ELISA分析显示，从小鼠脾脏分离的CTL释放出相对水平的IFN-γ。

结论：我们的研究确定了InhA的4种新型CTL表位，这些表位可引起有效的CTL免疫力，为开发抗药性MTB的多价肽疫苗奠定了基础。