Analysis Of Immune Responses Induced By Mucosal Immunization Of BCG And Chimeric Flagellin Expressed ESAT-6

Tuberculosis (TB) is a chronic infectious disease caused by Mycobacterium tuberculosis (M.tb) complex, affecting as many as one-third of humans alive, and killing about 3 million people each year, mostly in developing countries. Vaccination is the most cost-effective strategy for control and eventual elimination of tuberculosis. The current vaccine is Bacille Calmette and Guérin (BCG), which is effective in preventing the most severe disseminated forms of disease in children and newborns. However its efficacy against active TB in adults is limited. In recent years, looking for a new and more effective vaccine or an improved TB vaccine is urgently needed.Much of the published reports assign a protective role for secreted antigens of M.tb. Among the secreted proteins, the 6-kDa early secreted antigenic target (ESAT-6) is an important protective antigen. ESAT-6 is present in many virulent mycobacterial strains, but absent in BCG and most of the environmental mycobacteria. ESAT-6 has been described as a potent T-cell antigen secreted by M.tb, activating cell-mediated immunity and inducing IFN-γrelease. It is suggested used as a diagnostic reagent with the ability to discriminate infection with M.tb from previous BCG vaccination. Thus, in this study Salmonella flagellin was used as a vector delivering ESAT-6 antigen, combining with BCG by intranasal immunization, analyzing the immunologic characteristics of the secreted antigen and the immunologic strategy.1. Kinetics of immune responses induced by BCG co-immunized intranasally with fliC/esat protein The fliC/esat protein was inducible expression and purified from the E.coli express system, and Western-blotting analysis showed that fliC/esat protein could be reacted to specific sera of Salmonella and Ms mAb to ESAT-6, respectively. Bone marrow dendritic cells (BMDC) could be activated by fliC/esat protein, and the expression levels of theirs CD80, CD86, MHC-Ⅰ, MHC-Ⅱsurface molecules were up-regulated after fliC/esat protein stimulation. BCG co-immunized intranasally with fliC/esat protein in C57BL/6 mice model, increased the IgA titer of sera and bronchoalveolar lavage fluid (BAF), as well as the secretion of IFN-γforming cells and IL-4 forming cells, and induced Th-1 immune responses. The dynamic observation on the changes during 2-14 weeks after immunization showed that the level of mucosal and cellular responses maintain 8-10 weeks post inoculation. It indicated that BCG priming plus fliC/esat protein boost would be a new vaccination strategy for the prevention of TB.2. Immune responses triggered by BCG co-immunized intranasally with Salmonella chimeric flagellinPhage P22HT int was used for mediating transduction between LB5000(fliC/esat) and Salmonella dublin strain SL5928. And the transductan SL5928(fliC/esat) was identified by PCR, motolity and serum agglutination. The flagellin of the transductan SL5928(fliC/esat) was purified, and Western-blotting analysis showed that SL5928(flic/esat) flagellin could be binded to specific sera of Salmonella and Ms mAb to ESAT-6, respectively. We also detected TLR-5 mRNA from BMDC stimulated with SL5928(fliC/esat) flagellin by RT-PCR, and found that the level of BMDC TLR-5 mRNA was inhanced. Then, C57BL/6 mice were immunized intranasally with SL5928(flic/esat) flagellin and BCG. The results showed that the level of IgA antibodies in immunized group had significant increased compared with that of immunized with BCG or SL5928(flic/esat) flagellin only, as well as the level of IFN-γand IL-4 secreted by lymphocytes. All these results suggest that SL5928(flic/esat) flagellin would be a potential TB subunit vaccine candidate and provides the bases for research of mucosal TB vaccines.