Restructuring Shame Scale Mycobacteria Inducing Immune Response In Mice, Protection And Immune Treatment Of Infected Mice

BACKGROUUND:M. smegmatis is the rapidly growing non-pathogenic mycobacteria withconversion of high-efficiency. It propagates one generation every1-3hours,10times higher growth rate than the Bacillus Calmette-Guerin (BCG). It isnon-pathogenic and commensal to humans, mice and guinea pigs with its rapidclearance by the host. The level of exogenous protein expressed in M. smegmatisis5-10times as that in MTB, and secreted protein production of unit of time inM. smegmatis is50-100times higher than that in MTB. Based on the aboveadvantages, the M. smegmatis can be developed into a vaccine vector to replaceBCG. Heparin-binding hemagglutinin (HBHA) has a clear effect of treatment onMTB, and interleukin12(IL-12) can augment both innate and cellular immunityin many ways against intracellular pathogens. Here, we constructed arecombinant M. smegmatis (rMS) strain expressing a fusion protein of HBHAand human IL-12. It is expected that the recombinant M. smegmatis could combine the advantages of the target antigen and live vector, improving theHBHA-induced body to produce protective immune response, and enhancingthe killing effect of body macrophages in vivo MTB.AIM：To screen new vaccine candidates for the prevention and treatment oftuberculosis, construct a recombinant M. smegmatis (rMS) strain expressing afusion protein of HBHA and human IL-12using molecular biology techniquesand detect of rMS–induced cellular responses and protective and therapeuticefficacy against M. tuberculosis in mice.METHODS AND RESULTS：1. Construction and identification of recombinant M. smegmatisAccording to the HBHA and hIL12gene sequences of MTB and human beingin GenBank, primers of the two genes were designed. Shuttle plasmidpSMT3-HBHA-hIL12was constructed using molecular biology techniques, thefusion protein expression was also identified by western-blot andImmunofluorescence detection, and the recombinant M. smegmatis strainshowed no significant changes in proliferation characteristics by optical densityat600nm.2. The immune response and protective effects of the rMS vaccineIn this experiment, groups of mice were vaccinated by the homologousprime-boost method. A number of mice in each group were used for analysis oflymphocyte proliferation index, IFN-γ, IL-2, IL-12and CD4+and CD8+T cellphenotypes in PBMCs. The other mice in each group were retained for an Mtbvirulent strain H37Rv infection experiment. Administration of this novel recombinant M. smegmatis enhanced Th1-type cellular responses in mice byproducing higher amounts of IFN-γ and IL-2, and reduce bacterial burden andalleviate histopathological damage in lungs. The recombinant M. smegmatis canbe considered as a TB vaccine candidate in TB vaccines．3. Therapeutic effects of the rMS vaccine in mice infected with MTBMice infected with the M. tuberculosis H37Rv strain were treated with rMS,and the therapeutic effects were determined by the bacteria load in the lung andlung histopathology. The decrease in bacterial burden in mice that received rMScorrelated with a decrease in lung consolidation and number of pathologicallesions when compared to the saline treated group. However, treatment with therMS vaccine was significantly less effective than treatment with INH+RFP.CONCLUSIONS：The recombinant M. smegmatis could effectively control the growth of M.tuberculosis in the lung and reduce lung bacterial load in established infectionmice. It may a preventive vaccine candidate or a useful vaccine candidatecombined treatment with the therapeutic drugs.