| [Objective] As the only vaccine against tuberculosis?TB?currently,Mycobacterium bovis Bacille Calmette-Guérin?BCG?can induce effective immune protection against severe tuberculosis such as tuberculous meningitis and miliary tuberculosis in children,but the protection period of BCG vaccine only lasts 10 to 15 years.Correspondingly,BCG provide very limit immune protection to a high-risk population of adolescents and adults.However,adolescent and adult TB remain the epidemic and deadliest infectious disease,as estimated by the World Health Organization?WHO?that there were approximately 10 million of new TB cases and 1.6 million deaths worldwide in 2018,90% of which were adolescents and adults.What's more,25% population of the world is estimated to have latent tuberculosis infection?LTBI?.Therefore,it is important to prevent and control the occurrence of TB in adolescents and adults,if the immune protection induced by BCG can be extend.Autophagy and apoptosis of macrophages can activate the antigen-presenting by lysosomal pathway and cross-presentation pathway respectively and are also important measures for the host against M.tuberculosis infection.However,M.tuberculosis hire many mechanisms to inhibit autophagy and apoptosis.Under physiological conditions,BCG vaccine cannot be targeted for autophagy in infected APCs,even if it can induce apoptosis.As BCG is a live attenuated strain derived from virulent M.bovis by repeated passage,a core issue has been raised that whether BCG vaccine inherit evasive strategies from the parental virulent strain and thus cause suboptimal effects.In order to assess the effects of the evasive mechanism on the vaccine efficacy,we aimed to find out autophagy-inhibition genes from the BCG vaccine strain in this study and confirm their effects on the efficacy of BCG vaccine against TB.[Methods] 1.Bioinformatics analysis: In order to identify antiapoptotic or autophagy-inhibition genes of BCG,we first compared DNA sequences of nuo G,pkn E,and eis of M.tuberculosis H37 Rv with the genome of M.bovis BCG Pasteur 1173P2 using BLASTn software.Then these genes were cloned from 12 world BCG strains or M.bovis and sequenced for bioinformatics analysis.2.Construction of knockout strains: In order to improve the efficacy of BCG vaccine by enhancing macrophage apoptosis or autophagy,we deleted BCG3174,BCG1782 and BCG2432c from BCG China substrain by Mycobacteria phage gene knockout system.The resulting mutant strains were named ?BCG3174,?BCG1782 and ?BCG2432c,respectively.3.Identification of autophagy and apoptosis ability of knockout strains: The THP-1 macrophage was infected by these mutant strains with MOI=5,then apoptosis or autophagy of infected macrophage were evaluated by comparing to BCG WT or blank control.In addition,the intracellular survival rate compared to BCG was evaluated,too.4.Protective effects evaluation of different mutant strains against M.tuberculosis infection: In order to compare the effect of three genes deletion on vaccine efficacy,C57BL/6 mice were used to evaluate the effect of ?BCG3174,?BCG1782 and ?BCG2432c induced immune protection against M.tuberculosis primary infection.Immune protection evaluation were according to the analysis of lung bacterial load and histopathological score compared to BCG WT and PBS control group.5.Identification of BCG2432c is an autophagy gene for BCG inhibition: Laser confocal technology was used to monitor autophagy flux change of BCG WT or ?BCG2432c infected THP-1 cells which were transfected a recombinant live lentivirus carrying autophagy marker LC3 with RFP-GFP tag.In addition,westernblot was used to analyze autophagy marker LC3 protein change.Besides,cells were pre-treated with 3-MA or rapamycin to inhibite or promote autophagy to evluate autophagy.The survival rate of ?BCG2432c in THP-1 cells were also detected.6.Identification the mechanism of BCG2432c inhibits autophagy: In order to explore the mechanism of how autophagy occurs upon infection with ?BCG2432c,the levels of intracellular ROS and ROS promoting cells autophagy were evaluated,on the other hand,cytokines released to culture medium including Th1,Th2 or Th17 were measured with time change.7.Evaluation of the inhibitory effect of BCG2432c on antigen presentation efficiency: In order to explore the influence of ?BCG2432c-induced autophagy on the antigen-presenting ability of antigen-presenting cells?APCs?,BMDMs or BMDCs was infected by BCG WT or ?BCG2432c for 1 hours and co-incubated 24 hours with Ag85B-specific CD4+T or CD8+T cells,then the concentration of IL-2 in the supernatant was detected.In addition 3-MA inhibition of autophagy was used to evluate ?BCG2432c induced special antigen presentation by autophagy.8.Evaluation of early immunogenicity: C57BL/6 mice were subcutaneous vaccinated with BCG WT or ?BCG2432c,then antigen 85 B specific effector T cells in spleen and lung were detected during 0-28 d post vaccination.In addition,the speed of antigen 85 B specific effector T cells and multifunctional T cells homing to lung were compared.9.Evaluation of immune memory found after immunization: C57BL/6 mice were subcutaneous vaccinated with BCG WT or ?BCG2432c,then 28 d,60d,90 d and120d post vaccination,the ability and speed of immunological memory found in spleen and lung were compared.10.Early and long-term protective evaluation: C57BL/6 mice were subcutaneous vaccinated with BCG WT or ?BCG2432c,then 28 d,60d,90 d and120d post vaccination,immune protection difference against M.tuberculosis infection were compared.11.Safety evaluation: SCID mice were tail vein vaccinated with BCG WT or ?BCG2432c,then survival rate,spleen and lung bacteria load were used to evluate the safety of ?BCG2432c.[Results] 1.BCG2432c is a highly conserved gene in the BCG subfamily: BCG3174,BCG1782 or BCG2432c are homologous with H37 Rv nuo G,pkn E and eis.In addition,these three genes are highly conservative and universally presented in BCG substrains and M.bovis.2.?BCG2432c enhaced autophagy in infected macrophage: The resulting mutant strains named ?BCG3174,?BCG1782 and ?BCG2432c were construced successfully.3.?BCG1782 and ?BCG2432c infected THP-1 cells showed higher apoptosis level than BCG WT group.On the other hand,BCG WT cannot induced autophagy while ?BCG3174,?BCG1782 and ?BCG2432c significant induced THP-1 cells autophagy,in which ?BCG2432c induced the highest autophagy level.The survival rate of ?BCG3174 and ?BCG1782 THP-1 cells have no difference with BCG WT,whoever,the survival rate of ?BCG2432c was significant decreased.4.Only ?BCG2432c provide more early protection than BCG WT against M.tuberculosis primary infection: Compared to BCG WT,?BCG3174 and ?BCG1782 provided the similar immune protection against M.tuberculosis primary infection while ?BCG2432c significant decreased bacterial load in the lung and histopathology from the vaccinated mice.5.BCG2432c is antophagy inhibited gene of BCG: Compared with BCG WT and control group,?BCG2432c infected macrophage appeared much more LC3 spot and co-localized with lysosome.In addition,western-blot confirmed that LC3-I changed to LC3-II in ?BCG2432c infected macrophage.The survival rate of ?BCG2432c was significant decreased compared to BCG WT.When pre-treated with rapamycin,both the survival of BCG WT and ?BCG2432c were decreased,however,when autophagy was inhibited by 3-MA,only the survival of?BCG2432c was increased.6.?BCG2432c induced autophagy was releated with the increasing of intracellular ROS: Compared with BCG WT and control group,ROS,including H2O2 and O2,in ?BCG2432c infected macrophage were significant increased at the early stage?4h and 12 h post infection?.ROS triggered ?BCG2432c infected macrophage autophagy and significant decreased the survival rate in macrophage.In addition,?BCG2432c stimulated macrophage released more TNF-? and IL-6,while other cytokines have not significant changed.7.?BCG2432c enhanced antigen presentation mediated by autophagy lysosomal pathway: ?BCG2432c infected mouse bone marrow derived dendritic cells and macrophages co-incubated with antigen Ag85 B specific CD4+ T released much more IL-2 than BCG WT group.In addition,when autophagy blocked with 3-MA,IL-2 level were decreased in ?BCG2432c group.While 3-MA stimulated cannot impact IL-2 releasing in BCG WT group.However,IL-2 released in both BCG WT and ?BCG2432c infected dendritic cells or macrophages co-incubated with antigen Ag85 B specific CD8+ T showed no difference with blank control.8.Compared to BCG WT,?BCG2432c induced much more Ag85 B specific secreting TNF-?,IFN-? and IL-2 CD4+or CD8+ T cells in spleen.What's more,?BCG2432c induced much more effector T cells early homing to lung.9.Compared to BCG WT,?BCG2432c induced much more Ag85 B specific secreting IFN-? effector immune memory cells?CD62LloCD44hi,TEM?and secreting IL-2 central immune memory cells?CD62LhiCD44hi,TCM?in spleen and lung,while the amount of these memory cells were similar 120 day post vaccinated.10.?BCG2432c induced immune against M.tuberculosis primary infection were releated with the time interval between vaccination and infection.28-60 d poat vaccination,?BCG2432c provide much more immune protection than BCG WT against M.tuberculosis primary infection.11.?BCG2432c is safety: the bacterial of spleen,lung and liver from ?BCG2432c or BCG WT infected SCID mice were similar and the surivial were no difference.[Conclusion] BCG2432c is the autophagy inhibited gene of BCG and widely presented in clinically applied BCG sub-strains of worldwide.Disruption of BCG2432c from BCG enhanced infected macrophages autophagy and promoted fusion of autophagosomes containing bacteria and lysosomes then antigen presentation were increased and activated more effector T cells homing to lung which caused mutant strain ?BCG2432c induced more immune protection to clean M.tuberculosis early infection.[Objective] Adults are the leading population affected by tuberculosis?TB?epidemic and death.Developing an effective vaccine against adult TB is urgently needed.Mycobacterium bovis Bacillus Calmette-Guérin?BCG?prime-heterologous boost strategy has been explored extensively to protect adults against primary TB infection,but the majority of experimental regimens have not improved the protection primed by the BCG vaccine.The reason attributed to the failure remains unknown.In this study,CTT3 H was chosen to develop recombinant adenovirus vector,the adjuvant DMT emulsified DNA or protein subunit vaccines in this study.Differential protective efficacy of three vaccines against primary M.tuberculosis infection was compared with BCG in vaccinated C57BL/6 mice.More importantly,the immunogenicity and protection of BCG prime-boost regimens against primary infection and late persistent infection were compared and assessed in mouse models.[Methods] 1.Construction CTT3H-based vaccines: CTT3H-based vaccines,namely DMT adjuvanted DNA vaccine?p CTT3H-DMT?and recombinant adenovirus r Ad CTT3 H were constructed.2.Protective efficacy of CTT3H-based vaccine against M.tuberculosis early primary infection: C57BL/6 mice were vaccinated with BCG or CTT3 H based vaccines,9 weeks after vaccination,mice were challenged with Mycobacterium tuberculosis H37 Rv,then 4 weeks post infection,bacterial load in the lung,spleen,and lung histopathology were used to assess vaccine-induced protection.3.Protective efficacy of BCG prime and boosted with CTT3H-based vaccines to against M.tuberculosis early primary infection: C57BL/6 mice were vaccinated with BCG,8 weeks after vaccination,mice were boosted with BCG or CTT3 H based vaccines,then at the 17 th weeks,mice were challenged with Mycobacterium tuberculosis H37 Rv,then 4 weeks post infection,bacterial load in the lung,spleen,and lung histopathology were used to assess vaccine-induced protection.4.Protective efficacy of BCG prime and boosted with CTT3H-based to against against M.tuberculosis late persistent infection: C57BL/6 mice were vaccinated with BCG,4 weeks after vaccination,mice were challenged with Mycobacterium tuberculosis H37 Rv,and then 10 weeks post boosting,bacterial load in the lung,spleen,and lung histopathology were used to assess vaccine-induced protection.5.CTT3H-based vaccines immunogenicity analysis: Protective immunogenicity of BCG or CTT3 H based boosters were compared in C57BL/6 mice models.9 weeks after vaccination,CTL response against TB10.4,antibody including Ig G,Ig G1,or Ig G2 a against CTT3 H in peripheral blood or were CTT3 H specific IFN-? secreted by splenocyte were detected.6.Immunogenicity analysis of BCG prime and boosted with CTT3H-based vaccines to against M.tuberculosis early primary infection: C57BL/6 mice were vaccinated with BCG,8 weeks after vaccination,CTL response against TB10.4,antibody including Ig G,Ig G1,or Ig G2 a against CTT3 H in peripheral blood or were CTT3 H specific IFN-? secreted by splenocyte were detected.In addition,the number of CTT3H-specific IFN-? or IL-2 secreting T cells,IL-2-expressing TCM?CD62LhiCD44hi?cells,and IFN-?-positive TEM?CD62LloCD44hi?cells in splenocytes or pneumonocyte between BCG prime-boosted mice was determined.7.Immunogenicity analysis of BCG prime and boosted with CTT3H-based vaccines to against M.tuberculosis late persistent infection: C57BL/6 mice were vaccinated with BCG,4 weeks after vaccination,mice were challenged with Mycobacterium tuberculosis H37 Rv,then 10 weeks post boosting,the number of CTT3H-specific IFN-? or IL-2 secreting T cells,IL-2-expressing TCM?CD62LhiCD44hi?cells,and IFN-?-positive TEM?CD62LloCD44hi?cells in splenocytes or pneumonocyte between BCG prime-boosted mice was determined.[Results] 1.CTT3H-based vaccines,namely DMT adjuvanted DNA vaccine?p CTT3H-DMT?,and recombinant adenovirus r Ad CTT3 H were constructed successfully.2.Similar protective efficacy against early intranasal infection was provided bydifferent CTT3H-based vaccines alone in vaccinated mice,and their protection was inferior to that of the BCG vaccine.3.CTT3H-based heterologous boosters did not enhance the protection conferred by the BCG vaccine against primary infection.4.All of these three boosters provided stronger protection against late persistent TB infection than BCG alone,regardless of vaccine types.5.CTT3 H based vaccines induced Th1 response.Among the vaccines r Ad CTT3 H induced the highest ratio of Ig G2a/Ig G1.Of all groups,the highest concentration of CTT3 H specific IFN-? was secreted by the r Ad CTT3 H group while the strongest CTL response to TB10.4 was detected in p CTT3H-DMT vaccinated mice.6.CTT3 H based vaccines pre-exposure boost BCG induced robust Th1 response.Among all the groups,r Ad CTT3 H induced the highest ratio of Ig G2a/Ig G1 and secreted highest concentration of CTT3 H specific IFN-?.All the boosting group induced stronger CTL response to TB10.4 than BCG-PBS group.CTT3 H based vaccines boosting BCG induced much more CTT3 H specific secreting IFN-? effector T cells and secreting IFN-? effector memory T cells,while CTT3 H specific secreting IL-2 effector T cells and secreting IL-2 central memory T cells have not been enhanced.7.CTT3 H based vaccines post-exposure boosting BCG induced much more CTT3 H specific secreting IFN-? or IL-2 effector T cells,IFN-? effector memory T cells or IL-2 central memory T cells.[Conclusion] BCG prime-boost strategy might be a promising measure for the prevention against late persistent TB infection by induction of IFN-?+ TEM and IL-2+ TCM cells in the lung,which can be used as alternative biomarkers for guiding the clinical practice and future development of TB vaccine for adults. |
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