Screening And Identification Of Immune Dominant Th Epitope Of Mycobacterium Tuberculosis In Xinjiang Han Population

Objective:Screen and identify the immune dominant Th epitope of Mycobacterium tuberculosis in Xinjiang Han population,and preliminarily evaluate its immunogenicity and immune protection effect;To provide the basis for the research and design of recombinant BCG strains,the development of new epitope-based tuberculosis vaccines and tuberculosis specific diagnostic reagents.Methods:1.Predictions were carried out using bioinformatics software.The amino acid sequences of mycobacterium tuberculosis antigenic proteins Rv3874,Rv0934,Rv3875,Rv3804c,and Rv3878 were obtained using the NCBI website and analyzed for their homology with the human proteome using the EXPASY program.Four bioinformatics programs of DNAStar,SYFPEITHI,RANKPEP,and Net MHC IIpan were applied to jointly predict Th epitopes of mycobacterium tuberculosis in the Xinjiang Han population,and determine the predicted Th epitope peptides for this study.2.The SWISS-MODEL program was used to conduct the tertiary structure homology modeling of Rv3874,Rv0934,Rv3875,Rv3804c,and Rv3878.The tertiary structure positioning analysis of the predicted Th epitope peptides was performed using Py MOL program.3.Th epitope peptides were synthesized and predicted using the FMOC method.Purity analysis of the predicted epitope peptides was conducted using high performance liquid chromatography（HPLC）,and molecular mass measurement using mass spectrometry（MS）.4.CCK8 assay was used to detect the effects of the PBS group and five groups with different concentrations of polypeptide（40.0μg/ml,20.0μg/ml,10.0μg/ml,5.0μg/ml and 2.5μg/ml）on the proliferation level of lymphocytes.The optimal working concentration of Th epitope peptides for lymphocyte stimulation was investigated.5.The proliferation of peripheral blood mononuclear cells（PBMC）and CD4+T cells stimulated by the predicted Th epitope peptides was measured by CCK8 assay respectively,and the candidate Th epitope peptides were initially screened.6.The proliferation of CD4+T cells stimulated by the candidate Th epitope peptides was determined using Brd U assay.7.After the Th epitope peptides stimulating PBMC,flow cytometry was used to conduct the phenotypic analysis of CD4+T cell proliferation.8.After candidate Th epitope peptides stimulating CD4+T cells,they were cultured for 72 h,supernatant was collected,and levels of cytokines（IFN-γ,TNF-α,IL-2 and IL-10）were determined with enzyme-linked immunosorbent assay（ELISA）.9.The candidate Th epitope polypeptides were encapsulated with ELISA plate;optimal conditions for detecting serum Ig G antibodies with candidate Th epitope peptides as encapsulated antigens were determined with chessboard titration;The efficacy of candidate Th epitope peptides for detecting tuberculosis was evaluated with indirect ELISA,and Th epitope peptides with diagnostic potential were screened.Results:1.Five predicted mycobacterium tuberculosis Th epitope peptides,named P₃₉（Rv3874）,P₅₀（Rv0934）,P₄₀（Rv3875）,P₁₈₅（Rv3804c）,and P₆₂（Rv3878）,were obtained bioinformatically from the Xinjiang Han population.2.SWISS-MODEL software was used to obtain five antigenic protein tertiary prediction models with reliable results and qualities.Results of Py MOL software showed that P₃₉,P₅₀,P₄₀,P₁₈₅,and P₆₂ were located in externalβsheet and internal Loop region of the protein antigen structure.3.The purity of P₃₉,P₅₀,P₄₀,P₁₈₅,and P₆₂ was 91.79%,96.27%,96.54%,92.61%,and 96.99%,respectively;the molecular mass measurement value 1557.06,1575.30,1486.70,1410.80,and 1491.13,respectively.4.The optimal working concentration of Th epitope peptides for lymphocyte stimulation was 40μg/ml determined by CCK8 assay.5.The preliminary screening results by CCK8 assay showed that P₃₉ and P₆₂,which could stimulate CD4+T cell proliferation in the Xinjiang Han population with tuberculosis,were the candidate Th epitope peptides.6.Results of Brd U assay on the proliferation and activation of CD4+T cells stimulated by P₃₉,P₆₂,and P₃₉+P₆₂ in the Xinjiang Han tuberculosis patients showed significant differences between the PBS group,P₃₉,P₆₂ and P₃₉+P₆₂ groups（P<0.001）.7.Flow cytometry was used to conduct the phenotypic analysis of CD4+T cell proliferation after P₃₉,P₆₂ and P₃₉+P₆₂ stimulating PBMC.The results showed that the P₃₉,P₆₂ and P₃₉+P₆₂ groups were significantly different from the PBS control group（P<0.001）.8.The ELISA results showed that the levels of IFN-γ,TNF-αand IL-2 in the supernatant of CD4+T cells in the P₃₉,P₆₂ and P₃₉+P₆₂ groups were higher than those in the PBS group（P<0.001）,and the levels of IL-10 in the P₃₉+P₆₂ group were lower than those in the PBS group（P<0.001）.9.The optimum coating concentration of P₃₉,P₆₂ and P₃₉+P₆₂ was 10μg/ml,0.25μg/ml,and 10μg/ml,respectively.The optimal dilution of primary antibody serum was 1:500,1:100,and 1:50,respectively.For P₃₉,sensitivity was 75%,specificity 67.71%,and area under the receiver operating characteristic curve（AUC）0.844.For P₆₂,sensitivity was 91.66%,specificity 46.87%,and AUC 0.649.For P₃₉+P₆₂,sensitivity was 95.83%,specificity 97.91%,and AUC 0.793.Conclusion:1.In this study,a comprehensive multi-parameter prediction analysis of Rv3874,Rv0934,Rv3875,Rv3804c and Rv3878 was performed by bioinformatics,and finally a total of five predicted Th epitope peptides,namely P₃₉,P₅₀,P₄₀,P₁₈₅ and P₆₂,were screened,all of which were high purity epitope peptides after synthesis.2.The optimum working concentration of Th cell epitope peptides was determined as 40μg/ml,and P₃₉and P₆₂ were candidate immunodominant Th epitope peptides of mycobacterium tuberculosis in the Xinjiang Han population.3.This study revealed that all P₃₉,P₆₂ and P₃₉+P₆₂ possessed good immunogenicity and immunoprotective properties,and the combined use of P₃₉ and P₆₂ had a high diagnostic value in the detection of Ig G antibodies to peripheral blood tuberculosis,which could provide reference to accelerating the development of tuberculosis-specific diagnostic reagents,designing recombinant Bacillus Calmette-Geurin vaccine strains and developing new tuberculosis vaccines.