Identification Of Mycobacterium Tuberculosis Immunodominant CTL And Th1 Epitopes

BackgroudTuberculosis (TB), an infectious pulmonary disease of mankind caused by Mycobacterium tuberculosis (MTB), is one of the biggest threats to the global safe and public health. TB is the world’s most infectious and dread disease. Due to the move of population, coinfection with HIV, multidrug resistant and extensive resistant strains, TB has reemerged to become one of the biggest threats to public health. According to WHO report,1/3 of the world’s population were infected MTB, with most cases occurring in developing countries. In 2014 alone, there were an estimated 9.5 million new TB cases, causing 1.4 million deaths. China has the world’s second highest burden of MTB infection, with 45% of total population infected, resulting 100 million new cases per year and leading to 50 thousands of deads each year which means every ten minutes one person die from TB. In fact, TB has become one of the biggest threats to the global safe and public health. Lacking of quick and sensitive diagnostics lead to delay or misdiagnose of tuberculosis. Though TB has once been control by the antibiotics, the prolong cure procedures which maintain half to one year or even longer and severe side-effect have contributed to the appearance of multidrug resistant strains. WHO reported that and estimated 3.3% of new TB cases and 20% of previously treated cases have MDR-TB, a level that has changed little in recent years. An estimated 9.7% of people with MDR-TB have XDR-TB. China has the worst burden of MDR-TB with 120 thousands MDR-TB and 10 thousands XDR-TB; The treatment success rate for MDR-TB is less than 50%, and since MTB is difficult to clear up, reactivation readily happened when immunity compromise. As for XDR-TB, no standard treatment procedure is available to date. Bacillus Calmette Guerin (BCG) has been developed since 90 years ago, as the only available vaccine against TB, recently found that BCG vaccine had very unstable or variable effect on prevention against tuberculosis in adults. Especially in the area with high incidence of tuberculosis and the crowd, such as Africa, Southeast Asia etc, BCG vaccine shows slight protection. To reduce this burden, development of safe and effective anti-tuberculosis vaccine imminent is extremely urgent.Tuberculosis is a typical intracellular bacteria, T cell-induced adaptive immune responses play undeniably critical roles in protection against it. Professional antigen-presenting cells (APC) such as alveolar macrophages, macrophages, dendritic cells and B cells are capable of phagocytosis of MTB. After infected with MTB, dendritic cells (DCs) migrate to the draining lymph nodes and present MTB antigen to CD4+T and CD8+T cells. The activated lymphocytes induce immune response to promote the forming of granulomas and curb the spread of the bacteria. Identification of Immunodominant epitopes of MTB associated with protective responses would contribute significantly to a better understanding of the immunopathogenesis with respect to TB infections and provide insights for the design of more effective vaccines.Although in recent years, study on MTB antigen epitopes development made a lot of progress, several new epitopes have been reported, in general, it needs continuous development and further research. Early studies of MTB antigen epitopes focus on nonage secreted protein, with the publication of MTB genome sequence, researchers could find the target protein which can activate CD4+T cells and CD8+T cells at the molecular level. Meanwhile, some new identification methods also promote epitope discovery, such as the classic overlap method (overlapping peptide) and bioinformatics prediction-based reverse immunology. Today, approximately 800 human T-cell peptide epitopes originating from a total of 170 MTB proteins have been published in the immune epitope database. Because MTB may express approximately 4000 proteins, the identification of the entire MTB proteome is limited and need need to be further researched in the future. Furthermore, the known MTB antigen epitopes more than 20 mostly belong to a group of proteins, for instance, the early expressed virulence factors 6-kDa early secretory antigenic target (ESAT-6), 10-kDa culture filtrate antigen (CFP-10), and others. Thirdly, study on HLA restricted MTB T cell epitopes and the activation of specific T cells of many known MTB epitopes seem to be very little. At present, researchers find a large number of MTB epitopes are presented by HLA-A*02:01 molecule which is carried by European and American Caucasians. However, Africa and Southeast Asia are the most severely affected area. In addition, the number of CD4+T cell epitopes and CD8+T cell epitopes are unequal, and the former is more than the latter.In view of the fact that the significance of antigen epitopes in the immune response to tuberculosis and the important application of designing and developing anti tuberculosis vaccine、 diagnosis and evaluation of curative effect, we were committed to Screening and identification of MTB epitopes which could cover the most widely China crowd, and evaluated the anti-TB immune effect of those epitopes.Objective1. Identification of HLA-A*1101-restricted Mycobacterium tuberculosis CD8+T cell epitopes2. Identification of HLA-DRB1*0901-restricted Mycobacterium tuberculosis CD4+ T cell epitopesMethod1. Typing of HLA-A and HLA-DR allelesThe recognition and presentation of T epitopes requires the assistance of HLA molecules, so the HLA molecules play a very important role in T cell epitope. The frequencies of HLA alleles vary considerably in different regions. In order to identify the T cell epitopes covering most Chinese population, we must examine the distribution of HLA allele frequency in China and target at the most prelavent HLA alleles in HLA-A and HLA-DR loci. We collected 350 peripheral blood samples from patients with pulmonary tuberculosis in NanFang Hospital and GuangZhou Chest Hospital. The high-resolute HLA typing was conducted with sequence based technique (SBT).2. Prediction of CD8+T cell epitopes and CD4+T cell epitopes1) Prediction of HLA-A* 1101-restricted CD8+T cell epitopes① Anat Zvi, ect al defined 45 antigen proteins are suitable for vaccine candidates② Sylvie Bertholet, ect al identified 16 antigens capable of eliciting CD8+T cell responses③ 33 antigens were obtained from articles published between 2008-2013In conclusion, we finally screened a total of 94 MTB antigens2) CD4+T cell epitopesWe chose the strong immunogical antigens Rv0934c and Rv3804c. By examing the database (IEDB), we determined that there is no report on Rv0934c and HLA-DRB 1*0901 Rv3804c restricted T cell epitopes.3) Prediction of HLA-A* 1101-restricted CD8+T cell epitopesThe amino acid sequences of 94 MTB antigens were obtained from TB database; By using NetMHCⅡ algorithm on CBS database, we predicted HLA-A*1101 restricted T cell epitopes and selected 48 epitopes with the highest affinity of HLA-A* 1101 molecules.4) Prediction of HLA-DRB1*0901 restricted CD4+T cell epitopesThe amino acid sequence of 38kDa and Ag85A antigens was obtained from NCBI; By using NetMHCⅡ algorithm on CBS database, we predicted CD4+T cell epitopes and selected 18 epitopes with the highest affinity binding to HLA-DRB1*0901 molecules.3. Identification of HLA-A*1101-restricted CD8+T cell epitopes1) Binding affinity of CD8+T cell epitopes with HLA-A* 1101 molecules The binding affinity of peptides to HLA-A*1101 was assessed using the UV-induced peptide exchange assay. Briefly,50 candidate peptides were synthesized with a purity of more than 75%. Conditional HLA-A*1101 complexes containing UV-labile peptide ligands were exposed to UV light (366 nm) in the presence or absence of candidate peptides for 30 min. The efficiency of peptide exchange was assessed using an HLA class Ⅰ-specific ELISA detecting the β2m, which signals successful HLA class Ⅰ complex formation. The ELISA product is monitored via absorbance at 414 nm. The net average absorbance of each candidate peptide was normalized to that of the positive control peptide (EBNA416-424:IVTDFSVIK). The negative control peptide (NPKASLLSL) was included to confirm the specificity of the assay.2) Detection of cytokines from tuberculosis patients were stimulated by CD8+T、 CD4+T cell epitopes by ELISA PBMCs of 6 HLA-A*1101(+) patients were stimulated with 10 μg/ml individual CD8+T cell epitopes.72 hours later, The cell supernatant was collected and IFN-γ、TNF-α、 GrB were determined by ELISA Kit.3) ELISPOT detection frequency of IFN-γ-producing CD8+T cells and epitope recoginized by patients PBMCs from 30 HLA-A*1101(+) patients were seeded (2.5 × 105cells/well) in anti-IFN-y-coated ELISPOT plates in 100 ul of complete medium (RPMI+10% FBS) containing each peptide (10 μg/ml). Cells stimulated with PHA (phytohemagglutinin) were used as a positive control and cells cultured in medium alone were used as negative controls. After incubation for 24 h, Spots were analyzed by ImmunoSpot 5.0.3. Results are expressed as the mean number of spot-forming cells (SFC) per 106 cells from triplicate assays.4) 7.1 CD8+T cell epitopes induced proliferation of CD8+T cell PBMCs of 10 HLA-A*1101(+) patients,10 HLA-A*1101(-) patients and 10 HLA-A*1101(+) healthy volunteers labeled with CFSE and stimulated with CD8+T cell epitopes. On day 7, cells were analyzed by flow cytometry. The number of proliferating cells was determined by gating on the CD8+CFSEdim subset.5) Dextramer staining of MTB-specific T cellsSynthetic of HLA-A*1101 dextramer containing CD8+T epitopes p12, and HLA-A*02:01 dextramer carrying an irrelevant epitope, ENV120-128.PBMCs were obtained from patients (n=4) of HLA-A*1101 and from non HLA-A*1101 patients with pulmonary tuberculosis (n=4). Set the following three groups to label with polymersomes: ①PBMCs of HLA-A*1101 TB patients were stained with HLA-A*1101 dextramer; ②HLA-A*1101 dextramer was used for staining PBMC from non HLA-A*1101 TB patients; ③PBMCs of HLA-A*1101 TB patients were stained with HLA-A*02:01 dextramer. For each group, the cell density was 1×106/mL and stained with dextramers for 10 min at room temperature, followed by incubation with anti-CD3 and anti-CD8 fluorescence-conjugated mAbs (eBioscience, San Diego, CA) for 20 min. Then the cells were analyzed on flow cytometry.6) CD69 and CD 107 expression and ICSPBMC of HLA-A*1101(+) patients were stimulated with p12. Six hours later, the expression of CD69 and CD 107 on the cell surface was tested. T cells were also tested for intracellular secretion of IFN-γ、 TNF-α and IL-2.4. Identification of HLA-DRB1*0901-restricted CD8+T cell epitopes1) Detection of the level of IFN-γ、TNF-α、IL-4 and IL-17 excreted by PBMCPBMCs from 5 HLA-DRB1*0901(+) patients were stimulated with CD4+T cell epitopes.72 hours later, The cell supernatant was collected and the concentration of IFN-γ、TNF-α、IL-4 and IL-17 were determined by ELISA Kit.2) ELISPOT detection the frequency of IFN-y-producing CD4+T Cell PBMCs from 10 HLA-DRB1*0901(+) patients were seeded (2.5 × 105cells/well) in anti-IFN-y-coated ELISPOT plates in 100 ul of complete medium (RPMI+ 10% FBS) containing each peptide (10 μg/ml). After incubation for 24 h, spots were analyzed by ImmunoSpot 5.0.3. Results are expressed as the mean number of spot-forming cells (SFC) per 106 cells from triplicate assays.3) CD4+T cell epitopes induced proliferation of CD4+ T cellPBMCs of 10 HLA-DRB1*0901(+) patients and 10 HLA-A*0901(-) patients were labeled with CFSE and stimulated with each peptide (10 μg/ml) and incubated for 7 d. On day 7, cells were analyzed by flow cytometry. The number of proliferating cells was determined by gating on the CD4+CFSEdim subset.4) ICS detected the level of intracellular cytokines induced by CD4+T cell epitopesPBMCs of HLA-DRB1*0901(+) patients were stimulated with HLA-DRB1*0901 epitope (10 μg/ml) After incubation for 6h, Cells were labled with IFN-γ、TNF-α and IL-2 fluorescent antibody and analyzed on flow cytometry.Statistical analysesThe results of all measurement data are expressed by mean ± SE。 Data are different groups were analyzed using ANOVA followed by Tukey’s posttest correction, when variance uneven using Welch-Satterthwaite. LSD method was used to compare the variance between the two two groups, when variance uneven using Dunnett’s T3. A simplified method of ANOVA two-way factorial design was presented in this article to analysis CD4+ T and CD8+ T cell proliferation levels. Differences with a value of p< 0.05 were considered statistically significant. Data were analyzed using GraphPad Prizm 5.0 software (GraphPad Software Inc., San Diego, CA).Results1. The frequencies of HLA-A, HLA-DR alleles from the Chinese patienet with TBTo determine the requency distribution of HLA Ⅰ、Ⅱ allele, we used the SBT to examine the site high-resolution genotyping of HLA-A,HLA-DR from the TB patient in the southern China(n=350), and the data showed that the top 5 of the frequency is consistent with the information of the Chinese people from HLA allele authoritative databases AFND. The HLA-A*1101 and HLA-DRB1*0901 are the genotypes have the highest allele frequencies of the site of HLA-A and HLA-DRB1, respectively, and most of the Chinese people are inclusive.2. Prediction and confirmation of the HLA specific MTB CD8+ and CD4+ Tcells in Chinese people.1) The prediction and confirmation of HLA-A* 1101 restrictive CD8+ Tcell epitopes① Antigen selection:After screening the antigen and setting the standard, we gained Rv0079 and the other 93 antigens, which are in capable of inducing human CD8+ T cell secrete IFN-y, and be regard as vaccine targets.② HLA-A*1101 epitopes:We gained the 94 antigen protein aminoacid sequences from NCBI, and got the quantities of HLA-A* 1101 binding epitope by the way of NetMHCcons prediction technique of the CBS database as we expected. According to the scale of our experiments, we selected the 48 minimum IC50 value (the IC50 value and avidity have the negative correlation) epitope peptide, which IC50 value are< 15nM.2) The prediction and confirmation of HLA-DRB1*0901 restrictive CD4+T cell epitopes① Antigen selection:By retrieving IEDB, we found the HLA-DRB1*0901 restrictive epitopes are not reported in usual MTB antigen 38kDa (Rv3804) and Ag85A (Rv0934c), and they had been selected as the target antigen therefore.② HLA-DRB1*0901 epitopes:We gained the aminoacid sequences of Rv3804 and Rv0934c from NCBI, and got the quantities of HLA-DRB1*0901 binding epitope by the way of NetMHCIIpan prediction technique of the CBS database as we expected.18 epitope peptide IC50< 15nM had been selected.3. Screen and Immunological function verification of HLA-A*1101 restrictive T cell epitope1) We demonstrate the affinity between the predicted peptide of 48 HLA-A*1101 and HLA-A*1101 molecular by the UV-induced peptides displacement experiments. In the way of setting the affinity of positive control EBNA 416-424 (an validated epitope possess high affinity with HLA-A*1101 molecular) be 100%, we showed that the affinity of 22/48 predicted peptide and HLA-A*1101> 100%. In addition, we demonstrated that the predicted peptide are come from a same MTB antigen protein compared with the MTB proteome.2) To determine the immunocompetence of 22 HLA-A*1101 predicted peptide activating CD8+T cell and inducing the secretion of cytokines, we used different methods respectively (ELISA assay, ELISPOT assay and ICS assay) to demonstrate and screened 6 epitope peptides, which can specially induce the CD8+T cell of TB patient secrete IFN-y (experimental group vs control, P< 0.05)3) We used epitope peptides to stimulate the HLA-A*1101 positive pulmonary tuberculosis patient, negative pulmonary tuberculosis patient and the PBMC from HLA-A*1101 positive healthy volunteers respectively and ELISPOT assay to detect the IFN-y secretor CD8+T cell to demonstrate the 6 MTB specificity and HLA restrictive with HLA-A*1101 immunodominance. The result showed that all the 6 antigen epitope peptides were in capable of specifically inducing the CD8+T cell of HLA-A*1101 positive pulmonary tuberculosis patient secrete IFN-y, but were incapable of stimulating the CD8+T cells of HLA-A*1101 negative pulmonary tuberculosis patient and HLA-A*1101 positive healthy volunteers secrete IFN-y, all data are presented as the means ± SD. (P< 0.05)。4) To further determine the immunocompetence of aforementioned epitope peptides, we detected their ability about inducing CD8+T cell proliferation. We used CFSE dye-guided to mark the PBMC from HLA-A*1101 positive pulmonary tuberculosis patient, HLA-A*1101 negative pulmonary tuberculosis patient and HLA-A*1101 positive healthy volunteers, and incubated for 7 days after being stimulated by the 6 dominant antigen peptides. We used Flow cytometry to detect the proliferation activity of CD8+T cell in each group. The results clearly showed that dominant epitope peptide were in capable of specifically inducing the CD8+T cell of HLA-A*1101 positive patient to have a remarkable proliferation and were incapable of stimulating the CD8+T cells of HLA-A*1101 negative pulmonary tuberculosis patient and HLA-A*1101 positive healthy volunteers to have a remarkable proliferation (P< 0.05).5) We then synthesized the HLA-peptide polymer (dextramer) to detect the interaction among HLA-A*1101, epitope peptide p12 and T cell, and successfully detected the significance level of positive stained-dextramer CD8+T cell (controls vs experimental groups, P< 0.05) in the PB of HLA-A*11:0 positive pulmonary tuberculosis patient with the mentioned polymer marker.6) We had demonstrated that the epitope peptide p12 were in capable of inducing pulmonary tuberculosis patient CD8+T cell to secrete various cytokines at the same time by using Flow cytometry.4. Screen and Immunological function verification of HLA-DRBl*0901 restrictive T cell epitope1) To determine the immunocompetence of 14 HLA-DRB1*0901 predicted peptide activating CD4+ T cell and inducing the secretion of cytokines, we used different methods respectively (ELISA assay and ICS assay) to demonstrate and screened 6 epitope peptides, which can specially induce the CD4+ T cell of TB patient secrete IFN-γ, TNF-α and the other Th1 cytokines (experimental group vs control, P<0.05).2) We used the mentioned 6 dominant epitope peptides to stimulate the PBMC of HLA-DRB 1*0901 positive pulmonary tuberculosis patient and negative pulmonary tuberculosis patient respectively, used anti-CD4 antibody to block the CD4+T cells activity, and ELISPOT assay was used to detect the IFN-γ response. The result showed that all the 6 antigen epitope peptides were in capable of specifically inducing the PBMC of HLA-DRB1*0901 positive pulmonary tuberculosis patient secrete IFN-y, but were incapable of stimulating the PBMC of HLA-DRB1*0901 negative pulmonary tuberculosis patient secrete IFN-γ, all data are presented as the means ± SE(P< 0.05). And there IFN-γ reactions induced by 3 epitope peptides were blocked by anti-CD4 antibody (P< 0.05).3) To further determine the immunocompetence of aforementioned epitope peptides, we detected their ability about inducing CD4+T cell proliferation. We used CFSE dye-guided to mark the PBMC from HLA-DRB 1*0901 positive pulmonary tuberculosis patient, HLA-DRB 1*0901 negative pulmonary tuberculosis patient, and incubated for 7 days after being stimulated by the 3 dominant antigen peptides. We used Flow cytometry to detect the proliferation activity of CD4+T cell in each group. The results clearly showed that dominant epitope peptide were in capable of specifically inducing the CD4+T cell of HLA-DRB 1*0901 positive patient to have a remarkable proliferation and were incapable of stimulating the CD4+T cells of HLA-DRB 1*0901 negative pulmonary tuberculosis patient to have a remarkable proliferation (P< 0.05).4) We had demonstrated that the epitope peptide p12 were in capable of inducing pulmonary tuberculosis patient CD4+T cell to secrete various cytokines at the same time by using Flow cytometry.Conclusions1. We gained the allele frequency of HLA-A and DRB1 in Chinese people after having a gene-sequencing-based typing of HLA on large populations, which lays the basis of broad population coverage on detecting the MTB epitope.2. We demonstrated 6 HLA-A* 1101 restrictive CD8+T cell epitopes from different MTB antigen protein by bioinformatics prediction and the combination to experimental verification, and demonstrated the mentioned dominant epitope peptides are in capable of stimulating IFN-γ、TNF-α, GrB and the other cytokines secrete and induce CD8+T cell proliferate. We determined that p12 is a natural presented epitope with the peptide-HLA polymer marker. These results of HLA-A* 1101 restrictive epitope peptide lay the basis of further research and development for the polypeptide vaccine or diagnostic reagent of TB.3. We demonstrated 3 HLA-DRB1*0901 restrictive CD4+T cell epitopes from different MTB antigen protein by bioinformatics prediction and the combination to experimental verification, and demonstrated the mentioned dominant epitope peptides are in capable of stimulating IFN-y, TNF-a and the other cytokines secrete and induce CD4+T cell proliferate. These results of HLA-A*1101 restrictive epitope peptide lay the basis of further research and development for the polypeptide vaccine or diagnostic reagent of TB.