Identification And Molecular Modification Of Cytotoxic T Lymphocyte Epitopes Derived From CFP21, A Secreted Protein Of Mycobact

Tuberculosis, caused by the bacterium, Mycobacterium tuberculosis (Mtb), remains a leading cause of infectious disease morbidity and mortality, which has emerged as a major opportunistic infection in individuals with HIV/AIDS. The only tuberculosis (TB) vaccine currently available is the attenuated Mycobacterium bovis strain Bacillus Calmette-Guerin (BCG), which is ineffective on preventing pulmonary TB in adults. The uncertain efficacy of the BCG vaccine for TB in adults, and the emergence of extensively drug resistant Mtb strains have further enhanced the urgency of the development of novel TB vaccines.The cellular arm of the immune response mediated by CD4+ type 1 helper T (Th1) and CD8+ cytotoxic T lymphocytes (CTL) has been determined to be a pivotal component of protective immunity against Mtb. In order to develop new vaccines against tuberculosis, attention has been directed towards the identification of antigens/epitopes recognized by CD8+ T cells secreting interferon-γ(IFN-γ). Secreted and surface-exposed cell wall proteins seem to play a pivotal role in the induction of protective cellular immunity against TB. Recently, secreted proteins encoded by different regions of difference (RDs) of the Mtb genome have been considered as attractive candidates owing to their absence in most BCG strains. Detailed knowledge of the epitopes recognized by immune responses can aid in peptide-based anti-TB vaccines development, and provide important tools for basic research.A major secreted protein, CFP21, locating in RD2 was demonstrated to be an immunodominant antigen by elevating T cell proliferate response and production of cytokines such as IFN-y. CFP21 could also induce an optimum level of cytotoxic T cell activity. These findings suggested that CFP21 could be used as an attractive candidate antigen.In the present study, to identify novel CFP21-derived HLA-A*0201/03 restricted epitopes, a series of native peptides were predicted with various prediction programs including SYFPEITHI, BIMAS and NetCTL 1.2. Recent studies suggested that modification of amino acids at the anchor position of epitopes could result in enhancing the binding affinity to HLA-I molecules. Here, we designed analogues of the native peptides by altering the native peptides with tyrosine at position 1 (1Y), leucine at position 2 (2L) and/or position 9 (9L). Based on the three prediction programs, four native peptides, p5 (SLVRIVGVV), p13 (VVATTLALV), p134 (AVADHVAAV), p189 (NIMAHVSYV) and three of their analogues, p189-1Y2L9L (YLMAHVSYL), p134-1Y2L (YLADHVAAV), p134-1Y2L9L (YLADHVAAL) with prediction scores in the top 10 detected by at least two of the programs were selected and synthesized for further study. Peptides were synthesized by standard solid phase Fmoc strategy and were purified to more than 95% purity by reverse phase high performance liquid chromatography (RP-HPLC). Their molecular weights were confirmed by electro-spray ionization mass spectrometry (ESI-MS).To evaluate the binding affinity of these peptides to HLA-A*0201/03 molecules and the stability of the peptide/HLA-A complexes in vitro, TAP-deficient T2 cells were used. In concordance with the binding affinities, these two analogues p134-1Y2L and p134-1Y2L9L showed more potent binding stabilities than the native peptide, p134. Based on the results of the T2 binding assay and peptide/HLA stability assay, among the seven peptides studied, only p134 and its two analogues were selected to investigate their ability to induce T cell response by using ELISPOT and cytotoxicity assay. Subsequent IFN-γrelease and LDH release assays by using PBMCs from HLA-A*0201+/03+ PPD+ healthy donors showed that the native peptide p134 and its two analogues p134-1Y2L and p134-1Y2L9L could induce potent T cell response, but only p134-induced CTLs could potently lyse the peptide-loaded target cells. All these results demonstrated that the amino acid sequence of 134-142 (AVADHVAAV) in CFP21 could be an immunogenic CTL epitope which could be both processed and presented endogenously via HLA-A*0201/03.In the present study, a novel HLA-A*0201/03 restricted T cell epitope, p134 (AVADHVAAV), was identified in a secreted protein of Mtb, CFP21. The potent T cell response activity and the potential to be a broad-spectrum epitope made p134 a promising epitope for vaccination and immunotherapy against TB.