Preliminary Screening And Identification Of The Candidates As New Tuberculosis Vaccines And Diagnostic Biomarker

TB (tuberculosis, TB) remains a severe public health problem globally. TB is a disease of great antiquity that is the leading cause of death in the single bacterial infections. Different methods can be used to diagnose tuberculosis, such as sputum smear, sputum culture, PCR, ELISPOT, TST, chest X-ray and so on. However, time consuming and high false positive rate are the limitations of the above detection methods. One of the most significant limitations is the misdiagnosis concerning patients infected by M.tb when Mycobacterium tuberculosis negative. A new diagnostic method is needed that can quickly and accurately identify whether a patient is suffering from tuberculosis, in order to provide the basis for the late-stage clinical treatment. Nowadays, ESAT-6, CFP10 and TB7.7 act as antigens in ELISPOT test, and 38kDa (Rv0934) is the antigen in serological test. At present, the only prophylactic tuberculosis vaccine is BCG. It has a protective effect against childhood tuberculosis, whereas it has variable protection against pulmonary disease young people and adults. New and effective TB vaccine is urgently needed.The aim of this study is to find out candidates for potential tuberculosis vaccine components and diagnostics biomarker by using a strategy of screening secreted and membrane proteins.Bioinformatics software is used to study the proteome of M. tuberculosis H37Rv strain and predict target proteins.Then the candidate proteins were regrouped and screened through the Western Blot and ELISPOT method one by one. According to the results of the second round cellular immunity,5 proteins were selected to immunize C57BL/6 mice for testing their immunogenicity effect in vivo. Results were as follows:After predicting,240 open reading frame encoding membrane proteins and 242 open reading frame encoding secreted proteins were enrolled.467 expression clones were constructed by the Gateway technology and one step cloning.219 membrane proteins and 141 secreted proteins were expressed and purified. The ion trap mass spectrometry track (LTQ-Orbitrap Velos) was used to confirm the proteins’ amino acid sequences.360 proteins’sequences were confirmed.After three rounds (serum from 229 active TB patients) of Western Blot screening with sera of tuberculosis patients,18 membrane proteins and 26 secreted proteins’mean reaction intensities are higher than positive control Rv0934. In order to verify the results, three groups of serum were selected in this study. The three groups are from healthy subjects who have no BCG or have a BCG scar or active TB patients respectively. At last, 5 proteins among those perform are differently in the three groups.After first round of ELISPOT, the reactivity mean ratio of 17 proteins (8 membrane proteins and 9 secreted proteins) were above 50% compared with ESAT-6. According to the primary results of screening, we expand the sample size to verify 17 proteins by testing 15 ESAT-6-positive patients and 6 ESAT-6-negative patients. There are 5 proteins which can achieve the 40% of ESAT-6 strength. Interestingly, there are 3 proteins that may be complementary to ESAT-6/CFP-10. When the three protein and ESAT-6 act as a panel, may increase the rate of matching active tuberculosis detection.S43 protein can stimulate mouse CD4+, CD8+T lymphocytes to produce IFN-y and CD8+T lymphocytes to produce IL-2. S4 protein can stimulate mouse CD8+T lymphocytes to produce IFN-γ. S62, P2, M1 protein can stimulate mouse CD8+T lymphocytes to produce IL-2. When P2, M1, S43, S4, S71 act.as lymphocyte antigen, the phenomenon of CD4+T, CD8+T lymphocyte to proliferate can be observed. However compared with the PBS control group, there was no statistical significance. Furthermore,10 out of 360 proteins were confirmed by Western Blot and ELISPOT experiment. They would be well worth for further study.In conclusion, screening secreted and membrane proteomics are utilized to find out candidates of potential tuberculosis vaccine components and diagnostics biomarker for the first time. Due to the complexity of Mycobacterium tuberculosis, the proteomics research discovering whether a new tuberculosis vaccine candidate or diagnostic biomarker with far-reaching implications. This method can effectively discover new TB antigen, and improve diagnosis and prevention of tuberculosis now. Therefore, this study of proteomics has good prospects for development. This strategy could be taken as routine work in other organisms’ new antigens.