Construction Of Recombinant Mycobacterium Smegmatis Expressing Ag85B And Its Preliminary Study Of Immunotherapy On Mycobacterium Tuberculosis-infected Mice

Tuberculosis(TB),caused by Mycobacterium tuberculosis(Mtb),is a chronic infectious disease that spreads widely in the world.Bacillus Calmette-Guerin(BCG)is a live attenuated vaccine,which provides various protection against TB in adults.Although drug chemotherapy is still one of the main approaches to treat TB,long course of treatment and inevitable side effects are still series problems.The therapeutic vaccine inducing immune response contributes to clearance of latent TB infection.Therefore,the development of novel TB therapeutic vaccine will contribute to the prevention and treatment of TB.Ag85B is one of the TB vaccine candidate antigens widely studied.Subunit vaccine and DNA vaccine based on Ag85B have immunotherapeutic potential against Mtb infection in animal.Mycobacterium smegmatis(Ms)acts as an immune adjuvant and is widely used as vaccine vector.In this study,rMs-Ag85B strain expressing Ag85B in cytoplasm and rMs-s Ag85B strain secreting Ag85B were constructed,respectively.The biological and immunological characteristics were investigated,and immunotherapeutic effects of these rMs vaccines were evaluated in Mtb-infected mice.The results showed that the rMs-Ag85B expressing Ag85B in cytoplasm and rMs-s Ag85B secreting Ag85B were successfully constructed.The growth of these two rMs strains slowed in 7H9 complete medium and sauton medium under static culture conditions rather than shaking culture conditions.Compared with Ms,the biofilm formation of rMs-Ag85B was significantly reduced.In order to investigate the immunological characteristics of rMs strain,BALB/c mice were immunized subcutaneously with rMs-Ag85B and rMs-s Ag85B twice at 2-week interval.Mice were sacrificed at 6-week post the last vaccination for immune responses.The results showed that rMs-Ag85B and rMs-s Ag85B subcutaneous immunization could induce higher humoral immune response.Compared with Ms-immunized mice,rMs-Ag85B immunization could increase the secretion level of Th1/Th2 type cytokines,and promote secretion of inflammatory cytokines of TNF-αand IL-6 in splenocytes.While rMs-s Ag85B could only induce an increased Th1 cellular immune response and an increasing trend for the secretions of TNF-αand IL-6.The transcription levels of Th1/Th2/Th17 cytokines of rMs-Ag85B and rMs-s Ag85B groups in lung showed a decreasing trend in comparison with Ms group,and the transcription levels of TNF-αand IL-6 significantly decreased.Compared with Ms group,rMs-Ag85B immunization could increase the transcription level of IL-1βand i NOS in lung,but there were no significantly difference in rMs-s Ag85B group.Finally,we evaluated the immunotherapeutic effect of rMs-Ag85B and rMs-s Ag85B vaccine in Mtb H37Ra intranasal infected mice.The results showed that compared with Ms,both these two rMs strains induced high levels of humoral immune response in Mtb-infected mice,and the antibody level induced by rMs-Ag85B was higher than that of rMs-s Ag85B.rMs-s Ag85B immunotherapy increased the proportion of CD4~+and CD8~+T cells in spleen of Mtb-infected mice.rMs-Ag85B immunotherapy increased the proportion of CD4~+T cells secreting IFN-γin Mtb-infected mice.In addition,two rMs strains immunotherapy increased the frequency of splenocytes secreting IFN-γof Mtb-infected mice.rMs-s Ag85B immunotherapy significantly increased the secretion level of Ag85B-specific Th1/Th2cytokines,TNF-αand IL-6 inflammatory cytokines in splenocytes of Mtb-infected mice.rMs immunotherapy promoted the accumulation of T cells in lung tissue of Mtb-infected mice to a certain extent,but the transcriptional levels of inflammatory cytokines induced by rMs immunotherapy in lung of Mtb-infected mice were significantly different.rMs immunotherapy alleviated pulmonary pathological damage and reduced bacterial loads in lungs caused by Mtb infection.Moreover,rMs-s Ag85B immunotherapy reduced bacterial loads in spleen of Mtb-infected mice.In conclusion,the growth of the rMs-Ag85B and rMs-s Ag85B strains were slowed under static culture conditions,and the biofilm formation of rMs-Ag85B strain was reduced.rMs-Ag85B and rMs-s Ag85B immunotherapy could induce higher humoral immune response,Th1/Th2 cellular immune response and inflammatory response in mice.rMs could alleviate the pathological damage caused by Mtb infection to a certain extent,and provides immunotherapeutic effect against Mtb infection.Therefore,rMs-Ag85B and rMs-s Ag85B can be used for further study of therapeutic vaccines against Mtb.