| BackgroundTuberculosis(TB)is a global infectious disease caused by Mycobacterium tuberculosis(Mtb)mainly through the respiratory tract.Bacille Calmette-Guerin(BCG)is the only licenced vaccine for TB.While,the protective efficiency of BCG is insufficient in adult which may be involved in the lack of some immunodominant antigens,such as the region difference(RD1).Therefore,the development of new vaccines is essential for the prevention of TB.6 k Da early secretory antigenic target(ESAT-6)is the difference antigen between Mtb and BCG.It was reported that ESAT-6 could induce protective immune response against Mtb infection,which was widely used in TB vaccine research.As far,four vaccines containing ESAT-6 and other antigens have entered clinical trial.In addition,ESAT-6 as a virulence factor involved in the pathogenicity of Mtb and regulated the immune response of the host infected by Mtb.Therefore,ESAT-6 is one of the potential candidate antigens in TB vaccine research.While,we found that ESAT-6 induced immune response was insufficient.It was reported that c-di-AMP,a bacterial signaling molecule,not only regulates the physiological process in bacteria,but also regulates host innate immune response.Besides,c-di-AMP as a mucosal adjuvant enhanced the immune response induced by antigen.Therefore,this study aims to explore the immunological characteristics of ESAT-6 and evaluate the protective effect of ESAT-6 subunit vaccines with c-di-AMP as an adjuvant against Mtb infection.It is expected to provide theoretical and experimental basis for the development of ESAT-6 for TB mucosal vaccine.ObjectiveExploring the role of Mtb ESAT-6 homologue Ms ESAT-6 in the regulation of immune response induced by fast-growing Mycobacterium smegmatis(Ms).Exploring the regulatory effect of recombinant Mtb ESAT-6 protein on macrophage innate immunity.Clarifing the immunological characteristics of subunit vaccine based on Mtb ESAT-6 with c-di-AMP as adjuvant immunized through mucosal route.Evaluating the protective efficiency of ESAT-6-based subunit vaccine against Mtb infection.Methods and Results(1)Immunological characteristics of Ms ESAT-6 knockout strain1)Ms ESAT-6 is the homologue of Mtb ESAT-6 in Ms strain.The similarity of the two amino acid sequences is 72%.The Ms ESAT-6 knockout strain Ms?E6(?E6)was successfully constructed by CRISPR/Cas9,and the complementary strain?E6 C was constructed.It was found that knockout of ESAT-6 had a certain effect on bacterial growth,but did not affect the acid-fast staining of bacteria.2)Establishing a model of Ms infection of macrophages in vitro.It was found that ESAT-6 played a role in the production of inflammatory factors,inducing inflammasome activation and autophagy after Ms infected macrophages,but the impact level is limited.3)Intracellular survival results showed that the replication of the ESAT-6 knockout strain was reduced in macrophages,indicating that ESAT-6 was related to the survival of Ms in macrophages.(2)The role of ESAT-6 in regulating the innate immunity of macrophages1)Establishing a macrophage stimulation model in vitro,and found that c-di-AMP promoted the type I IFN response induced by Mtb recombinant ESAT-6 protein.ESAT-6 and c-di-AMP triggered autophagy initiation in the early stage of treatment,and long-term treatment of both two was mainly manifested as the inhibition of autophagy degradation.This process was regulated by m TOR but did not depend on IL-17.2)ESAT-6 induced the inflammatory factor response of macrophages,and c-di-AMP promoted the inflammatory factor response induced by ESAT-6.3)Mtb intracellular survival results showed that ESAT-6 and c-di-AMP synergistically restricted the replication of Mtb in the early stage of infection.As the infection prolonged,the inhibitory effect on the growth of Mtb faded away.(3)Immunological characteristics of ESAT-6-based subunit vaccine1)Mice were inoculated with ESAT-6,ESAT-6+c-di-AMP through nasal mucosa route.The humoral immune response assay results showed that ESAT-6 induced the systemic and mucosal specific antibody,c-di-AMP as a mucosal adjuvant enhanced the humoral immune response induced by ESAT-6.2)Cellular immune response assay results showed that c-di-AMP as an adjuvant promoted the systemic Th1/Th2/Th17 and inflammatory factor responses induced by ESAT-6.And c-di-AMP adjuvant promoted the local mucosa Th17 and inflammatory factor responses.3)The immune cell subpopulation analysis results in lung showed that ESAT-6,ESAT-6+c-di-AMP vaccination reduced the ratio of CD8~+T cells and macrophages.Meanwhile,c-di-AMP as a mucosal adjuvant significantly increased the differentiation of ILC1 and NK cells induced by ESAT-6.(4)Immune protection of ESAT-6-based subunit vaccine against Mtb infection1)After Mtb infection,ESAT-6 immunized mice exhibited enhanced mucosal immune response,and c-di-AMP promoted the mucosal immune response induced by ESAT-6.2)After Mtb infection,mice immunized with ESAT-6 and ESAT-6+c-di-AMP showed reduced Th1 type(IFN-?),Th2(IL-10),Th17(IL-17)and inflammatory factors(IL-1?),IL-6,TNF-?)production.3)ESAT-6,ESAT-6+c-di-AMP inoculated through mucosa route could provide protection against Mtb infection,and c-di-AMP as an adjuvant improved the immune protection of ESAT-6 to a certain extent.ConclusionMs ESAT-6 played a certain role in the host innate immunity induced by Ms infection and affected the intracellular survival of bacteria,overall the effect of Ms ESAT-6 was limited.Mtb ESAT-6 and the immunomodulator c-di-AMP synergistically activated type I IFN,regulated autophagy,induced inflammatory response,and promoted the elimination of Mtb infection in macrophages.EAT-6 induced humoral immune response vaccinated via mucosal route,and c-di-AMP as an adjuvant promoted the mucosal immune response and Th1/Th2/Th17 type cellular immune responses induced by ESAT-6.ESAT-6 immunized via mucosal route could provide protection against Mtb infection,and c-di-AMP improved the immune protection efficiency of ESAT-6 to a certain extent. |
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