| Background:Tuberculosis (TB) caused by Mycobacterium tuberculosis (M.tb) remains one of the top three infectious disease killers. World Health Organization (WHO) estimated that there were about 960 million new cases and 150 million individuals died from TB around the world in 2013. About 1/3 population was infected with TB, and in which 90-98% was latent TB infection (LTBI) and 2-10% developed into active TB. Drug-resistant TB and co-infections of M.tb with human immunodeficiency virus (HIV) or hepatitis C virus (HCV) further threaten to undermine tuberculosis control. In recent years, although we have made some achievements on the researches of TB diagnosis, prevention and pathogenic mechanism, but there were remained many deficiencies and further researches were urgently needed. Current laboratory methods for tuberculosis diagnosis mainly rely on X-ray, acid-fast staining, tests of tuberculin skin test (TST), PCR, sputum culture etc; However these methods are often with low sensitivity or long detection period. In recent years, interferon-gamma release assay (IRGA) has been developed and used for diagnosis of both active TB and LTBI with a high sensitivity and specificity. In the IRGA, only 6-kD early secreted antigenic target (ESAT6) and 10-kD culture filtrate protein (CFP10) polypetides were used as stimulated antigens, therefore, a lot of MTB antigen epitopes are missed for diagnosis. On the research of vaccine, Bacillus Calmette-Guerin (BCG), the only available TB vaccine, decreases TB in children but gives little protection against TB in adults. Some researches have proved that recombinant BCG is the best method to improve vaccine, but the difficulty was to find suitable target antigen. Screening dominant target antigen will be helpful for not only diagnosis but also vaccine. Macrophages are the main M.tb target cells of host. M.tb can evade the killing of macrophages of the host immune system. So, deeply understanding the interaction between M.tb with macrophage will be important to explore the pathogenic mechanism of the long period parasitical M.tb in macrophage.Comparative genomic studies have helped in revealing some regions of the genome known as regions of differernce (RDs) that are present in Mycobacterium tuberculosis complex or part of Mycobacterium bovis but absent in Mycobacterium bovis Bacillus Calmette Guerin (BCG). In recent years, more and more researches have revealed that some RD genes which expressing functional proteins have important roles with strong immunogencity or mediating immune evasion.Objective:1. Screening the dominant immunogenic antigens from RD regions and further evaluating their diagnostic purposes;2. Constructing recombinant BCG vaccine (rBCG::Rv2645) which Rv2645 gene inserted into BCG and evaluate its immunogenicity;3. Screening RD genes which mediate evading the killing of macrophage, and further clarifying bacterial escape mechanism.Method:In order to screen the dominant immunogenic antigens located in RD10-14, different pET-28-RD recombinant plasmids were constructed, and RDs protein from E. coli were expressed and purified. We examined the IFN-y releasing with individual RD proteins stimulated splenocytes from the M.tb immunized mice with enzyme-linked immunospot (ELISPOT) assay. We selected the highest levels of antigen-specific IFN-y among the tested RD proteins. We further evaluated the diagnostic potential of a novel M.tb-specific Rv2645 from DNA segment region of RD13 of M.tb and investigated T-cell recognition during natural infection in humans and experiment mice.Recombinant BCG (rBCG::Rv2645) was constructed and its immunogenicity and protective effects with BCG in Balb/c mice and rhesus monkeys were compared and evaluated.We further identify which RD proteins which can resist the killing of macrophage through measurement phagocytosis of macrophage, colony counting and lactic dehydrogenase (LDH) releasing test. We clarify the immune evasion mechanism and the interaction between RD protein and macrophages.Results:In this study, we found Rv2645, a novel early secreted RD13 protein, evoked the highest level of antigen specific IFN-y among the tested RD proteins (RD10-14). Rv2645-specific IFN-y levels were much higher in the peripheral blood mononuclear cells (PBMCs) of TB patients than that in healthy donors (HDs) (including Bacille Calmette-Guerin (BCG)-vaccinated donors). In the differential diagnosis of TB and HDs, the sensitivity and specificity of Rv2645 were 90.0% and 98.2%, and Rv2645 had a high overall agreement (98.0%) with the results from the clinical T-SPOT.TB with CFP10 and ESAT6 peptides. The combination of Rv2645 and CFP10-ESAT6 was better than the individual protein CFP10-ESAT6 or Rv2645, with increased sensitivity from about 88% to 96.0%, and with a similar specificity about 98.2%, respectively. Rv2654 also induced M.tb-specific skin-test responses can be used to distinguish TB infection and BCG vaccinated population. At the same time, we have constructed rBCG::Rv2645 candidate vaccine successfully. In the Balb/c and rhesus challenge experiments, we found rBCG::Rv2645 could evake stronger immunogenicity and protective capacity compared with the traditional BCG vaccine. Furthermore, we found an early secreted protein Rv1768 (PEPGRS31) from RD14 region could evade the killing of macrophage. Rv1768 binds to Annexin A2 of macrophage and reduced the calcium ion concentration in RAW264.7 macrophages, which might result in the inhibition of phagolysosome maturation.Conclusion:Our research suggest that Rv2654 protein is a strongly recognized T-cell antigen that is highly specific for TB and has potential as a novel cell-mediated immunity-based TB diagnostic agent and used for vaccine design. The rBCG::Rv2645 is a good vaccine cadidate which has stronger immunogenicity and protective effects compared with BCG. Furthermore, our study provide a new insight of mechanism that Rv1768 antigen might result in phagosome maturation arrest in macrophage and mediate M.tb escape from macrophage. |
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