| Tuberculosis is one of the major infectious diseases that threatening the human health all over the world. There are nearly one third of the world’s people infected with Mycobacterium tuberculosis(M.tb), if not treated, about 5%-10% people with latent tuberculosis infection(LTBI) will develop into active tuberculosis in their lifetime. People with LTBI has become an important source of tuberculosis patients. So far, LTBI lacks of unified criteria and method for the diagnosis, the existing diagnostic methods can not distinguish the active tuberculosis infection from LTBI, and LTBI also lacks of effective prevention and intervention measures. Therefore, the breakthrough of LTBI early diagnosis technology and the development of new vaccines become the important challenges in the current control of tuberculosis. With the deepening of the research on LTBI, it has been found that some LTBI associated proteins may provide immune protective effect to prevent LTBI from developing into active tuberculosis. These LTBI associated proteins may become new diagnostic markers and potential vaccine candidates for LTBI, and provide new ideas for the diagnosis and treatment of LTBI. M.tb Rv2629 protein and Rv1736 c protein are hypoxia associated proteins during LTBI. Although the studies have showed that both of them can stimulate the whole blood from TB patients to release a variety of cytokines in vitro, but the immunological characteristics of these two proteins are not yet clear, and the possible roles of these proteins in anti-tuberculosis immunity needs further study.In this study, we predicted and analyzed the epitopes of M.tb Rv2629 protein and Rv1736 c protein, identified their HLA-A2 restricted CTL epitopes, and evaluated their immunogenicity, which laid the foundation for the development of immune diagnostic reagents and new vaccines for LTBI.1. Prediction of epitopes of Rv2629 protein and Rv1736 c proteinThe amino acid sequences of Rv2629 protein and Rv1736 c protein were analyzed by protean software of DNAStar software package. Their B cell and T cell epitopes were predicted using multi-parameters including the secondary structure, hydrophilicity, antigenicity, surface probability, peptide backbone flexibility. The results showed that Rv2629 protein and Rv1736 c protein had many sections with high antigen index, in which the B cell epitopes of Rv2629 protein might be located in 22-27, 39-51, 103-105, 112-119, 161-177, 199-215, 226-236, 242-252, 345-354, 364-374 amino acid residues and the potential B cell epitopes of Rv1736 c protein may located in 4-11, 35-42, 82-105, 112-124, 151-167, 191-199, 244-255, 268-273, 331-338, 347-361, 447-457, 526-532, 637-652 amino acid residues. Two kinds of online softwares, SYFPEITHI and RANKPEP, were used to predict the Th cell epitopes. The results showed that Rv2629 protein might have 20 potential Th epitopes, while Rv1736 c protein might have 40 potential Th epitopes. Their CTL epitopes were predicted using SYFPEITHI, BIMAS and Net CTL online softwares. After a preliminary prediction, some wild-type candidate epitopes with higher scores were selected for amino acid substitutions. Finally, fifteen CTL epitopes including three kinds of phenotypes(HLA-A2, HLA-A3 and HLA-B7) were obtained in Rv2629 protein, in which there were twelve HLA-A2 restricted CTL epitopes. While 23 CTL epitopes including three phenotypes mentioned above were predicted in Rv1736 c protein, in which there were 13 HLA-A2 restricted CTL epitopes. After prediction, the candidate T cell epitopes of Rv2629 accounted for 76.1% of its total predicted epitopes, while the candidate T cell epitopes of Rv1736 c accounted for 82.9% of its total predicted epitopes. These two proteins are T cell epitope dominant antigens.2. The syntheses of HLA-A2 restricted CTL candidate epitope peptides in M.tb Rv2629 and Rv1736 c proteinsTwenty-five HLA-A2 restricted CTL candidate epitope peptides were synthesized by standard solid phase synthesis method and purified to more than 95% purity by high performance liquid chromatography(HPLC).3. Determination of affinity and stability of HLA-A2 restricted candidate CTL epitopes in M.tb Rv2629 and Rv1736 c proteins binding with HLA-A2 moleculeThe candidate epitopes were incubated with T2 cells, then HLA-A2 molecules on the surface of T2 cells were labeled by FITC-labeled HLA-A2 antibody. The mean fluorescence intensity(MFI) were detected by flow cytometry, then the fluorescence index(FI) was calculated, FI>1.5 indicated high affinity, 1.0<FI≤1.5 indicated medium affinity, FI≤1.0 indicated low affinity. The candidate epitopes with high affinity were incubated with T2 cells again. After incubation, HLA-A2 antibody was added separately at 0, 2, 4, 6 hours. The MFI was detected by flow cytometry, then the required time was determined when the MFI at t=0 decreased to 50%(DC50). DC50≥6h indicated high stability, 4h≤DC50<6h indicated medium stability, DC50<4h indicated low stability. The results of affinity experiment showed that the FI value of 7 candidate epitope peptides(Rv2629-p190-2L, Rv2629-p190-1Y, Rv2629-p190-1Y2 L, Rv2629-p274, Rv2629-p315, Rv1736c-p611-1Y and Rv1736c-p576-1Y) were greater than 1.5, which showed higher affinity with HLA-A2 molecules. The stability of these seven candidate epitopes were detected in subsequent experiment. The result showed that Rv2629-p190-1Y had higher affinity, but low stability. The affinity and stability of Rv1736c-p611-1Y and Rv1736c-p576-1Y binding with HLA-A2 molecule were in the medium level. Rv2629-p190-2L, Rv2629-p190-1Y2 L, Rv2629-p274 and Rv2629-p315 binding with HLA-A2 molecule had higher affinity and stability, and the immune activities of these four peptides were further detected in vitro.4. Detection of immune activities of candidate CTL epitope peptides of Rv2629 in vitroPeripheral blood mononuclear cells(PBMCs) were isolated from healthy donors, LTBI donors and tuberculosis patients, and stimulated by four candidate epitope peptides with high affinity and stability and recombinant human interleukin 2(rh IL-2) once a week. At 3 days after each stimulation, half amount of culture medium was replaced and the culture supernatant was preserved. The interferon-gamma(IFN-γ) level secreted was detected by ELISA method. After three times of stimulation, the newly induced CTLs were used as effect cells, while T2 cells loading with peptides were used as target cells. The numbers of CTLs secreting IFN-γwere detected by ELISPOT assay. The specific cytotoxic killing activity of CTLs was detected by LDH assay. The results of ELISA showed that the IFN-γ levels in culture supernatants of PBMCs from healthy donors, LTBI donors and tuberculosis patients increased in different degrees with the increase of stimulation times. But the secretion level and growth rate of IFN-γin tuberculosis patients were significantly higher than those in the other two groups. The results of ELISPOT assay showed that candidate epitopes Rv2629-p190-2L, Rv2629-p190-1Y2 L, Rv2629-p315 and Rv2629-p274 could stimulate the PBMCs from 5 TB patients generating CTLs which produced IFN-γ, while in healthy donors and LTBI donors could not be detected significantly increased specific spots. There was statistically significant difference among these three groups, p<0.01. The results of cytotoxic killing experiment showed that the killing activity of CTLs induced by the candidate epitope peptides increased with the increase of effector cell and target cell ratio(E/T). When the E/T was 100∶1, the killing activity of CTLs induced by Rv2629-p190-2L was highest and the killing rate reached 53.91%. The killing effects of CTLs induced by Rv2629-p190-1Y2 L and Rv2629-p315 on target cells were similar and in the middle level, their killing rates were 27.11% and 23.91%, respectively. The CTLs induced by Rv2629-p274 also had certain killing effect, but the killing rate was relatively lower, only 13.75%. After cross stimulation test, the CTLs induced Rv2629-p190-2L and Rv2629-p190-1Y2 L modified could specificly recognise and kill the T2 cells loading with their native peptide(Rv2629-p190). The anti-HLA-A2 monoclonal antibody was added into effector cell and target cell reaction system, and the cytotoxic activity was blocked, indicating that the specific cytotoxic killing effect was HLA-A2 restricted. Therefore, Rv2629-p190-2L, Rv2629-p190-1Y2 L, Rv2629-p315 and Rv2629-p274 peptides are HLA-A2 restricted CTL epitopes.In summary, four nonapeptide fragments of M.tb Rv2629 protein(Rv2629-p190-2L, Rv2629-p190-1Y2 L, Rv2629-p274 and Rv2629-p315) were identified as HLA-A2 restricted CTL epitopes, and confirmed that they had higher immunogenicity in TB patients. These four epitope peptides have the potential to become candidate targets for tuberculosis immune treatment and epitope vaccine development. In the future, we will further study their effect on the treatment and prevention of tuberculosis in vivo to provide experimental basis for the development of novel epitope vaccine. |
