The Identification And Evaluation Of 16 Novel Antigens From Mycobacterium Tuberculosis

Tuberculosis(TB)is a chronic and communicable disease which caused by Mycobacterium tuberculosis(MTB)complex infection and is a serious hazard to human health in the world.There have been one third of the world's population infected with MTB,in which there are 10%developing tuberculosis in the future.There were 10.4 million new TB cases and 1.4 million TB deaths in 2015 in the world.In recent years the incidence and burden of TB has increased with the occurance of multidrug-resistant TB and coinfection with HIV/AIDs.Although the number of TB deaths fell by 22%from 2000 to 2015,TB remained the first of the 10 grave causes of death wordwide.BCG,a live attenuated vaccine,derived from continuous culture of Mycobacterium bovis,is currently the only TB vaccine widely used in human beings.Although BCG has some effect on preventing children from severe disseminated diseases such as tubercular meningitis,and hematogenous disseminated tuberculosis,There was a great difference in the protection rate of BCG for adult tuberculosis according to the reports from 0%to 80%.Studies found that among different BCG vaccine strains there is significant different protection with the loss of the protective antigens or epitopes in BCG strains.Therefore it is urgent to find new immunodominant antigens and epitopes used to construct recombinant BCG or develop novel enhanced immune vaccine for BCG vaccination,to provide new key and effective means for better control of tuberculosis.In this study,we screened new protein antigens in H37Rv genome and predicted the human T cell epitopes of the new antigens by means of genomics and bioinformatics.Then we synthesised the epitopes with high score predicted by TEpredict and IEDB.Peripheral blood mononuclear cells(PBMCs)collected from the blood of TB patients,patients with no TB and healthy donors were stimulated by the peptides to conduct interferon gamma release assay(IGRA)for selecting the immunodominant antigens and epitopes of MTB.BALB/c mice were immunized with subcutaneous injection the T cell epitopes with higher positive reaction after emulsified with adjuvants DDA and poly(I:C).The cytokines IFN-y,IL-2,IL-4,and IL-10 in supernatant fraction produced by splenocyte after isolating from the immunized mice and stimulated with the same epitopes were tested together with IgG/IgG1/IgG2a in the sera.The immunogenicity of these epitopes was evaluated.The ratio of CD3+CD4+ T cell and CD3+CD8+T cell were also determined with flow cytometry.B cell epitopes predicted by seppa2.0 also were synthesised and serologically evaluated with the sera from TB patients and healthy donors.Rv2351c is an antigen from RD7 of Mycobacterium tuberculosis prepared by our lab.And we also conduct the IGRA assay and animal experiment with Rv2351c recombinant protein.As a result,2726 T cell epitopes and B cell epitopes of 273 protein antigens in H37Rv genome were predicted,of which we selected and synthesised 265 T cell epitopes from 13 MTB antigens and 22 B cell epitopes from 3 TB antigens.We have identified 30 human T cell epitopes from 13 TB antigen:Rv3793,Rv0585c,Rv1490,Rv0446c,Rv1547,Rv2201,Rv3573,Rv0197,Rv0674,Rv 1566c,Rv1798,Rv2318 and Rv2941.The sensitivity and the specificity of these 30 T cell epitopes were from 0 to 30%and from 96%to 100%.The sensitivity of these antigens increased and ranged from 1.9%to 50%with combined the epitopes.The sensitivity and specificity of Rv2351c were 61.7%and 91.45%respectively.In animal experiments,11 peptides could induce different levels of cellular immune response and humoral immune response.P1(Rv3793,P24(Rv0585c),P26(Rv0585c),P55(Rv1490)and P120(Rv0446c)could induce a mixed Thl/Th2 cellular immune response with the production of IFN-y,IL-2,IL-4,and IL-10 when P123(Rv0446c),P136(Rv2201),P166(Rv0197),P309(Rv1798)and P318(Rv2318)could induce Th1-type cellular immune response with the only production of IFN-? and IL-2.Increased number of CD3+CD4+ T cell was observed with mice immunized with P1(Rv3793),P26(Rv0585c),P120(Rv0446c)and P121(Rv0446c).Increased number of CD3+CD8+ T cell was observed in mice immunized with P123(Rv0446c),P166(Rv0197),P309(Rv1798),P318(Rv2318)and Rv2351c.Increased number of CD3+CD4+ T cell and Increased number of CD3+CD8+ T cell was observed in mice immunized with P24(Rv0585c),P55(Rv1490)and P166(Rv0197).The titer of IgG were from 1:44 to 1:5079 in mice immunized with the 11 T cell epitopes.The titer of Rv2351 c-specific IgG was up to 1:2.2 × 106.The sensitivity and the specificity of the 9 B-cell epitope polypeptides in Rv0674 were from 54.55%to 79.55%and 71.25%to 95%respectively.The area under the ROC curve was more than 0.8 except P203 and P204.The sensitivity and the specificity of the 7 B-cell epitope polypeptides in Rv1411c were from 46.5%to 88.6%and 75%to 92.5%respectively.The sensitivity and the specificity of the 6 B-cell epitope polypeptides in Rv1477 were from 60.23%to 76.14%and 86.25%to 95%respectively.The area under the ROC curve was more than 0.8 except P220.In this study,30 new human T cell epitopes from 14 new TB antigens were successfully identified to prove their certain antigenicity,in which 11 epitopes could induce cellular immune response and humoral immune response in BALB/c mice to prove their certain immunogenicity.We have also evaluated the serodiagnosis value of 22 B cell epitopes from 3 TB antigens.And the 22 B cell epitopes have the potential in TB diagnosis.In all,we have identified 16 new TB antigens.And these discoveries have provide the experimental evidences for developing the specific diagnostic reagents and new TB vaccine.