Investigation On Mycobacterial Lipoprotein Z Specific Immune Profiles And Immuno-protection Against Tuberculosis

Tuberculosis（TB）is an infectious disease caused by Mycobacterium tuberculosis（M.tb）and is mainly infected through respiratory tract.One fourth of the world population were estimated to be infected with M.tb,while in 2018,China was ranking the 2^nd in WHO’s list of 30 high TB burden countries.Therefore,it is still indispensable to investigate new tools for TB diagnosis and prevention as well as immune protection against TB.Due to prevalent distribution and high immunogenicity,mycobacterial lipoproteins are considered to play important roles in mediating direct host-pathogen interaction.Some of lipoproteins are subjected to promising biomarkers in TB diagnosis and novel candidate antigens for TB vaccine development.According to our previous study,Lipoprotein Z（shortened as LppZ,encoded by Rv3006）was identified to induce strong cellular immune responses among TB patients.In this study,we used purified LppZ protein from prokaryotic expression system not only to systematically explore the significance of LppZ in TB diagnosis and prevention,but also to dissect the relevant mechanisms.This thesis mainly includes three parts as shown below:Part 1:Diagnostic significance of Mycobacterial LppZPeripheral blood samples were collected,including 125 TB patients,92 latent tuberculosis infection（LTBI）individuals and 165 healthy controls（HC）.Using ELISPOT and home-made ELISA,we detected LppZ-specific cellular and humoral immune response levels.The results showed that compared to HC group,LppZ-specific IFN-γreleasing levels were significantly higher in TB patients,displaying similar promising diagnostic value to ESAT-6 and CFP-10 in discriminating TB according to ROC analysis.Meanwhile,LppZ-specific IgA levels were also abundantly higher in TB and LTBI than HC.Interestingly,LppZ-specific IgA level gradually decreased following anti-TB treatment,which indicated that LppZ-specific IgA could become an adjunctive biomarker in screening M.tb-infection.Therefore LppZ is capable of inducing strong cellular and humoral immune responses upon M.tb infection and is a novel M.tb antigen with strong immunogenicity.Part 2:Mycobacterial LppZ immunization exerts immuno-protection against M.tb infectionUpon LppZ immunization in a mouse model,antigen-specific antibody levels were detected in serum,while ELISPOT and ICS were conducted to determine antigen-specific T cell responses in the spleens.The results showed that LppZ could induce strong antigen-specific adaptive immune responses,including higher LppZ-specific Ig G levels,abundantly increased secretion of Th1-type cytokines（IFN-γ、IL-2 or TNF-α）by CD4⁺T and IFN-γsecretion by CD8⁺T cells.Futhermore,antigen-specific polyfunctional T cell responses were also enhanced upon LppZ immunization.In murine H37Rv challenge experients,it was indicated that compared with ADJ group,LppZ immunized mice showed significantly lower clony forming units（CFUs）in both spleens and lungs and comparable to those from BCG immunized group.In the meantime,alleviated lung damage was observed in LppZ immunized mice.The results above demonstrated that LppZ could play immuno-protective roles against TB,showing great promise to become a candidate antigen for a novel TB subunit vaccine.Part 3:Mechanism Exploration of Immuno-protection by Mycobacterial LppZ against TB InfectionCombining in-vivo and in-vitro strategies,we systematically studied the mechanisms of LppZ-induced immuno-protection upon M.tb infection.Using an Air pouch model,it was found that more neutrophils and macrophages were infiltrated into the pouches at the early stage after LppZ treatment.When RAW264.7 cell lines or peritoneal macrophages from C57BL/6 mice were stimulated with LppZ protein in-vitro,it was showed that LppZ stimulation could enhance the production of ROS and pro-inflammatory cytokines（including TNF-α,IL-6 and IL-12）,induce non-canonical autophagy（showing higher LC3-Ⅱand p62 expression levels with unchanged Beclin-1）and inhibit intracellular OXPHOS level（displaying decreased maximal respiration and spare capacity）,resulting in increased capacity of eliminating BCG bacilli.In addition,upon LppZ stimulation,the expression levels of CD80,CD86 and MHC-Ⅱmolecule were upregulated time-and dose-dependently in the macrophages.Subsequently,LppZ-activated macrophages could induce Th1-and Th17-type cytokines production（such as IFN-γ,IL-2,IL-6,TNF-αand IL-17）by antigen-specific CD4⁺T cells,suggesting the enhancement of antigen presenting capacity of macrophages after LppZ treatment.Last but not least,in-vitro virus challenge assay in LppZ-pretreated macrophages was conducted.It was shown that LppZ treatment could magnificently inhibit intracellular VSV virus duplication whereas scarcely affect HSV duplication.Taken all together,LppZ can induce strong immune responses and is a novel M.tb antigen with great immunogenicity.LppZ immunization in mice leads to immuno-protection against M.tb infection.Results from mechanism exploration indicate that LppZ-induced activation in macrophages is dependent on TLR2 and TLR4.LppZ triggers increased cytokine secretion,enhanced BCG bacilli elimination capacity and upregulated antigen presenting capacity in macrophages.Furthermore,we resport here for the first time that LppZ-treated macrophages could resist other pathogen infection,suggesting that LppZ might induce trained immunity in the macrophages.Therefore,mycobacterial LppZ induces both innate and adaptive immune responses,displaying potential values in both TB diagnosis and new TB vaccine development.