Abstract
Background: Mycobacterium tuberculosis (Mtb) infects approximately one third of the world’s population, 10% of which develop disease during lifetime. Beijing genotype strains are the most predominant strains in China. The aim of this study was to screen and validate the possible HLA*0201 restricted specific T cell epitopes of latent phase Mtb strains H37Rv and Beijing genotype. Methods & Materials: MTB DNA microarray gene expression analysis was performed to screen the Mtb genes which expressed up-regulated under hypoxia. SYFPEITHI and NetCTLpan databases were used to predict the HLA-A*0201 restricted cytotoxic T lymphocyte (CTL) epitopes on Mtb, followed by peptide/HLA-A*0201 affinity and complex stability assays using the T2 cells. IFN-gammaproducing T cells were detected by enzyme-linked immunospot assay (ELISPOT) and a LDH release assay were performed to detect peptide-specific CTL activity using PBMC derived from HLAA*0201-positive human donors latently infected with Mtb (LTBI). Results: Using a whole genome microarray, we identified 130 genes of Mtb strain H37Rv and Beijing genotype whose expression was up-regulated under hypoxic conditions, 37 genes were responsible for encoding membrane protein, cell wall proteins or exported proteins. Of selected four proteins coded by upregulated genes, Six potential epitopes on Rv0350, Rv0351 and Rv0440were selected as candidate HLA-A*0201 restricted CTL epitopes by SYFPEITHI and NetCTLpan prediction. Four of these 6 study epitopes (Rv0440 p416-424, Rv0440 p362-370, Rv0351 p122-130 and Rv0350 p363-371) were showed high binding affinity and stabilization to HLA-A*0201 molecules by T2 binding studies. Synthesis of multi-epitope peptides Rv0351-A-T (containing Rv0351 p122-130, APDRE and Trojan peptide) and Rv0350-A-T (containing Rv0350 p363-371, APDRE and Trojan peptide) via ufrinsensitive linker VRKR. Cytotoxic activity showed that significant lysis of T2cellspulsedwithRv0351-A-TorRv0350-A-Twas induced by peptide-specific T cells derived fromHLA-A*0201-positive LTBI. IFNgamma was released by PBMC in vitro from HLA-A*0201positive LTBI when challenged with the peptides by an ELISPOT assay. Conclusion: Our data suggest that Rv0351p122-130 and Rv0350p363-371 are HLA-A*0201-restricted CTL epitopes and could be useful in the design of an Mtb vaccine for LTBI. Acknowledgments: This investigation was funded by a grant from Key Projects in the National Science & Technology Program (2009ZX10004-305).
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