Therapeutic Effects Of Granulysin/Interleukin-12 Recombinant M. Smegmatis In A Murine Model Of M.Tuberculosis Infection

Background:1.Tuberculosis (TB) is primarily a pulmonary disease and remains the leading cause of death from an infectious disease worldwide. Factors that have recently appeared put more burdens on the treatment of TB including co infection with HIV, prolonged treatments and the emergence of Mycobacterium tuberculosis (Mtb) strains that are resistant to the front-line drugs.2. Vaccines have traditionally been designed to prevent the disease. But therapeutic vaccine can induce immune responses for therapy of the diseases. Up to now, the researches of tuberculosis therapeutic vaccines mainly focus on gene vaccine ,the killed mycobacterium vaccae vaccine and alive vaccine. 3. The Th1 response has the most important role in anti-mycobacterial immunity in humans and mice. Mycobacteria primarily infect host macrophages,which represent the first line of cellular defense against microbial invasion. IL-12 is crucial to the generation of antigen-specific lymphocytes that are able to produce IFN-γ.4. Granulysin is a cytolytic granule protein of natural killer (NK) cells and cytotoxic T lymphocytes (CTL) with a broad range of antimicrobial and tumoricidal activities. Serum levels of granulysin are related to host cellularimmunity. Granulysin directly killed extracellular Mtb, altering the membrane integrity of the bacillus, and, in combination with perforin, decreased the viability of intracellular Mtb.Objective:To construct a GLS/IL-12 recombinant Mycobacterium smegmatis (recombinant MS)strain that can targetedly deliver the eukaryotic coexpression plasmid pZM03 encoding human granulysin and murine single chain IL-12 into macrophage. To explore the effect and immunology mechanism in launching specific immune response against Mtb in vitro and in vivo. At last, to evaluate therapeutic effects of recombinant MS with anti-tuberculosis chemotherapy in murine model of Mtb infection. Methods:1. The coding sequences of the granulysin, IL-12 and OriM were subcloned into the HindIII/BamHI, NotI/KpnI and NheI MCS sites of the eukaryotic coexpression plasmid pBudCE4.1 respectively to form the plasmid pZM03 and then transfected RAW264.7 cells. The expression of these genes was detected by RT-PCR, Westernblot, ELISA and immunocytochemistry.2. The shuttle-plasmid pZM03 was transformed into ATCC607 and identified by PCR. The targeted delivery property of the recombinant was demonstrated by co-incubating the strains with murine macrophage RAW264.7. The expression of GLS and IL-12 was detected by RT-PCR, immunocytochemistry and ELISA.3. BALB/c mice were intranasally immunized 3 times with pZM03, BCG, ATCC607, recombinant MS, inactivated recombinant MS and normal saline respectively. The mice were sacrificed in 4th week after the first immunization. The levels of IL-12, IFN-γ, IgG2a, lL-4 in serum, SIgA in BALF(bronchoalveolar lavage fluid)and IFN-γreleased from spleen lymphocytes stimulated with PPD were detected with ELISA. The expression of the two genes was detected by RT-PCR and immunocytochemistry. The proliferation of spleen lymphocytes stimulated with PPD was detected with MTT assay. The pathology analysis and acid-fast staining are also done at the same time. 4. The virulent strain Mtb H37Rv was passed through mice and cultured on Lowenstein-Jensen media for 4 weeks, then harvested and diluted to 5×104CFU/50μl, in saline containing 0.05% Tween80. Mice were challenged intranasal (i.n.)with live Mtb (H37Rv).5. BALB/c mice infected with Mtb were treated with normal saline, MS, pZM03 and recombinant MS respectively. The numbers of viable bacteria in the lung and spleen were counted. The levels of IL12, IFN-γin serum and IFN-γ, TNF-αreleased from spleen lymphocytes stimulated with PPD were detected with ELISA. The expression of GLS in tissue was detected with immunohistochemistry. Lungs and spleens were prepared for pathological analysis.6. BALB/c mice infected with Mtb were treated 12 weeks with normal saline, recombinant MS, anti-tuberculosis chemotherapy(INH+PZA), recombinant MS + INH+PZA, and Vaccae+ INH+PZA respectively. To evaluate the therapeutic effects of GLS/IL-12 recombinant MS by pathological analysis and changes of the Th1 response in mice. Results:1. The eukaryotic coexpression plasmid pZM03 carrying the coding sequences of GLS, murine IL-12 and OriM was successfully constructed. The expression of GLS and IL-12 in transfected RAW264.7 cells was successfully detected by RT-PCR, ELISA and immunocytochemistry.2. The recombinant MS was successfully constructed. The expression of GlS and IL-12 was detected by RT-PCR in murine macrophage RAW264. 7 infected with the recombinant bacteria.3. When BALB/c mice intranasally immunized with recombinant MS were sacrificed in the 4th week, the levels of IL-12, IFN-γ, IgG2a in serum, SIgA in BALF and IFN-γreleased from spleen lymphocytes stimulated with PPD were markedly higher than that of the control groups. The expression of GLS and IL-12 was detected by RT-PCR and immunocytochemistry. The pathology analysis showed no obviously pathological changes at the same time.4. Mycobacteria were found in the lungs and spleens by acid-fast staining in all mice infected with Mtb H37Rv. The bacterial detected from the lungs and spleens. The lesions on the lungs were excessive and extensive, lveoli and interalveolar septae were effaced by infiltrates of macrophages and lymphocytes, but the necrosis was not found.5. Twelve weeks after treatment initiation with recombinant MS, a significant reduction in the number of Mtb organisms was achieved in this group. The lesions in the lungs were slight, limited, and a few epithelioid macrophages and foamy cytoplasm aggregated there. The Th1 response of the recombinant Mtb group is higher than that of the other groups. The expression of GLS in lungs and spleens were found.6. Twelve weeks after treatment initiation with recombinant MS, anti-tuberculosis chemotherapy(INH+PZA), recombinant MS + INH+PZA, Vaccae+ INH+PZA, and normal saline respectively, the changes in histological and the reduction of pulmonary and splenic bacterial loads of anti-tuberculosis chemotherapy group have an advantage over the recombinant MS group, the Th1 response of the recombinant MS group is higher than in INH+PZA groups. The therapeutic effect of combining INH+PZA chemotherapy and recombinant MS immunotherapy against tuberculosis is superior to chemotherapy alone and anti-tuberculosis chemotherapy in combination with Vaccae. Conclusions:1. Human GLS and murine IL-12 can be coexpressed in murine macrophages.2. It is feasible to clone and deliver foreign therapeutic genes into target cells at a lower cost and on a larger scale by using mycobacterium smegmatis as an attenuated organism vector.3. The recombinant MS has excellently targeted delivery property,and can inspire specific cellular immunity of Th1 and mucosal immunization against the Mtb.4. The recombinant MS had immunotherapeutic effects, which were associated with a switch to Th1 response and the antibacterial activity of GLS. The recombinant MS had a synergism to anti-tuberculosis chemotherapy.