Immunotherapeutic Efficacy Of Recombinant Mycobacterium Smegmatis Expressing Ag85b-ESAT-6Fusion Protein Against Mycobacterium Tuberculosis Infection In Mice

Tuberculosis is a chronic respiratory infectious disease caused by Mycobacteriumtuberculosis. For the reasons of drug-resistant strains spreading in the world, humanimmunodediciency virus(HIV) co-infection in TB patients, population mobility increasing,and especially BCG’s poor effect on adults, the prevalent trend of TB still can not becontroled and TB continues to be the first threat of all infectious disease in the world. Newpreventive and/or therapeutic vaccines will be great helpful for the control of TBprevention.There are two major factors for TB therapy: first, after the M.tuberculosis infection,bacteria turn into a latent state, and stay in macrophages for long-term survival, whereasthere are no symptoms or just mild symptoms in TB patients who become the majorsource of new TB patients and are difficult to be detected; Secondly, drug-resistant strains,especially mutiple drugs resistant (MDR) M.tuberculosis strains remain increasing, onwhich chemotherapy drugs had a little effect. The persistent of MDR M.tuberculosisinfection can not be effectively cleared from host, which become the main infectiousstrains in TB patients. Therefore, it is important implications that develops new drugs,biological agents as well as strategies against M.tuberculosis drug-resistance strains andpersistent infection for TB control.Mycobacterium smegmatis is one of rapidly growing and non-pathogenicmycobacterium. It could effectively stimulate the host immune responses to M.tuberculosis, therefor could be used as a candidate vector for TB new vaccines. Antigen 85B (Ag85B) and early secretary target antigen6(ESAT-6) are TB early secreted antigens.DNA vaccines, subunit vaccines and recombined BCG vaccines based on them alone or asfused could induce the host immune responses to M. tuberculosis, which prove themimmunodominant antigens of M. tuberculosis.Our aim is to construct recombinant M. smegmatis expressing secreted ag85B andesat-6fusion protein, detect the immune response induced by it and estimate itsimmunotherapy effect alone or combined with drugs in M. tuberculosis infected mice.Methods and Results1. Construction and identification of recombinant M. smegmatis Ag85B-ESAT-6-rMSThe gene fragments ag85B and esat-6were amplified from H37Rv genome by PCRrespectively. After ligated by T4ligase, the fragments of ag85B and esat-6were used astemplete for new PCR to amplify the ag85B and esat-6fusion gene. A hydrophobic linkerwas designed between ag85B and esat-6; The fusion gene fragment was cloned intopMD18-T, then subcloned into pDE22shuttle expression vector and transformed into E.coli (DH5). The recombinant vector was identified by restriction enzyme digestion andsequencing analysis. Fusion gene ag85B-linker-esat-6was obtained (around1221bp)after enzyme digestion which consistent with the expected size of the fusion fragment;The sequence analysis showed that ag85B and esat-6genes were consistent with that ofGenBank published. The recombinant plasmid was named as pDE22-ag85B-esat-6.The vector pDE22-ag85B-esat-6was transformed into competent M. smegmatis.Positive clones were identified by PCR, and then transformed into E. coli (DH5) directlyand identified by enzyme digestion after plasmids were prepared. The results of PCR andenzyme digestion showed that the plasmid with ag85B and esat-6fusion gene had beentransformed into M. smegmatis. After the genetypes were identified, one positive clone ofrecombinant M. smegmatis was inoculated in liquid medium to amplify growing andinduced at42℃. The protein expression was analyzed by SDS-PAGE and furtheridentified by Western blot. The results showed that the recombinant M. smegmatis couldexpress secreted Ag85B-ESAT-6fusion protein, and the strain was named as Ag85B-ESAT-6-rMS.2. Immunological characteristics of Ag85B-ESAT-6-rMS in immunized miceMethods: Six to eight weeks old female BALB/c mice were immunizedsubcutaneously with1×107CFU Ag85B-ESAT-6-rMS or M. smegmatis(MS) in a finalvolume of100μl of PBS three times at a2weeks interval, or1×107CFU BCG once, orembedded in groin with cellulose nitrate membranes of20μg recombinant Ag85B-ESAT-6fusion protein(AE) two times at a3weeks interval. An equal volume of normal saline wasinjected in the negative control group. The peripheral blood mononuclear cells (PBMCs)were prepared from immune mice every week and the percentages of CD4+and CD8+T cellwere analyzed by fluoresecnce activated cell sorting(FACS). After6weeks fromimmunization, the mice were sacrificed by cervical dislocation after ether anesthesia, andsplenic lymphocytes were isolated for lymphocyte proliferation assay by MTS method,cytotoxic T lymphocytes response by LDH release assay and the frequency of lymphocytesproducing IFN-γ, IL-2and IL-4by ELISpot analysis.Results:①The total percentage of CD4+and CD8+T cell was significantlyincreased in Ag85B-ESAT-6-rMS immuned group compared with all the other immunedgroups including MS, AE and BCG as well as saline group (P<0.05)；②The spleniclymphocyte proliferation index is significantly higher in Ag85B-ESAT-6-rMS group thanthat of the saline, AE and MS group (P<0.05), but similar as BCG group (P>0.05)；③Cytotoxic T lymphocytes response is significantly increased in Ag85B-ESAT-6-rMSimmuned group than that of the MS and saline group (P <0.05), but no significantdifference was found between the Ag85B-ESAT-6-rMS group and AE or BCG group(P>0.05)；④The frequency of spleen lymphocytes producing IFN-γ and IL-2(spotforming cells, SFC/106) of Ag85B-ESAT-6-rMS group was significantly higher than thatof Saline, AE, MS and BCG group (P<0.05). The SFC of IL-4in Ag85B-ESAT-6-rMSgroup is higher than that of the saline and AE group (P<0.05), but no more than that ofMS group and BCG group (P>0.05). In brief, Ag85B-ESAT-6-rMS could induce strongerimmune response in mice, especially the Th1type cellular immunity. 3. Immunotherapeutic effect of Ag85B-ESAT-6-rMS in M. tuberculosis RFP-resistantstrain infected miceMethods:①Six to eight weeks old female BALB/c mice were infected with1×106CFU M. tuberculosis RFP-resistant strain via the tail vein.4weeks after infection, the micewere sacrificed by cervical dislocation after ether anesthesia, lung and spleen tissues wereisolated and taken samples for histopathologic examination after HE staining, microscopicexamination after acid-fast staining, and the rest of the organs were homogenized forbacteria loads counting by colony forming units(CFU) on plate.②The mice infected bymethods above were subcutaneously injected twice with1×107CFU MS,Ag85B-ESAT-6-rMS, or Hsp65-IL-2-rMS in upper back at a4weeks interval after6weeksinfection, or gavage administrated with4weeks Isoniazid(INH) follewed with onesubcutaneous injection of1×107CFU Ag85B-ESAT-6-rMS or Hsp65-IL-2-rMS in upperback. The INH or Rifampin(RFP) treatment were gavage administrated with drugrespectively for8weeks after6week infection. The saline was injected in infected mice asmodel control.③The mice daily activities of all groups were observed and the miceweights were recorded every week.4weeks and8weeks after therapies, the mice weresacrificed and lung and spleen tissues were isolated as above for histopathologic diagnosisafter HE staining, microscopic examination after acid-fast staining, and bacteria loadscounting by CFU on plate.④The mice survival percentages were observed after therapies.Results:①4weeks after infection, histological examination revealedgranulomatous inflammation in mice. Acid-fast bacillus could be found in lung afteracid-fast staining and assigned as strongly positive (+++) according the standards. Thebacteria counts of lung and spleen were105and104CFU respectively. The resultsshowed that the mice had infected with M. tuberculosis RFP-resistant strain.②The miceweights of saline, MS and RFP treatment group decreased significantly, but increasedsignificantly in Ag85B-ESAT-6-rMS, Hsp65-IL-2-rMS, INH+Ag85B-ESAT-6-rMS,INH+Hsp65-IL-2-rMS and INH treatment group compared with saline group respectively(P<0.05);③4weeks and8weeks after the first immunotherapy, the acid-fast stainingratings of groups were various from saline and RFP group (++++or+++), MS group (+++), Ag85B-ESAT-6-rMS and Hsp65-IL-2-rMS group (++), and INH+Ag85B-ESAT-6-rMS, INH+Hsp65-IL-2-rMS and INH group (+);④4weeks and8weeks after the firstimmunotherapy, histological examination revealed chronic inflammation. Granulomatousinflammation and pulmonary interstitial fibrosis could be found in all groups except forthe INH group, which suggested that MS and rMS could cause a certain degreepathological lesion in lung.⑤4weeks and8weeks after the first immunotherapy, Thebacteria counts (log10CFU) of mice lung and spleen were significantly decreased inAg85B-ESAT-6-rMS, Hsp65-IL-2-rMS, INH+Ag85B-ESAT-6-rMS, INH+Hsp65-IL-2-rMS and INH group than that of saline group respectively (P<0.05), which domestratedthat rMS could inhibit the growth of M. tuberculosis RFP-resistant strain in mice. ThusAg85B-ESAT-6-rMS showed immunotherapeutic effect on infected mice and the effectcould be enhanced by combined with chemotherapy.⑥The survival percentages ofINH+Ag85B-ESAT-6-rMS and INH+Hsp65-IL-2-rMS group were higher than that of rMSgroup, but no better than saline control group, which were consistent with pathologicallesion degree.4. Immunotherapeutic effect of Ag85B-ESAT-6-rMS in low dose of M. tuberculosisH37Rv strain infected miceMethods:①Six to eight weeks old female BALB/c mice were infected with1×104CFU M. tuberculosis H37Rv strain via the tail vein.6weeks and10weeks after infection,the model mice were sacrificed and lung and spleen tissues were isolated as above forhistopathologic diagnosis after HE staining, microscopic examination after acid-faststaining, and bacteria loads counting by CFU on plate.②The mice infected by methodsabove were subcutaneously injected twice with1×107CFU, Ag85B-ESAT-6-rMS in upperback at a4weeks interval after10weeks infection, or administrated with Isoniazid(INH)in drinking water for4weeks follewed with two subcutaneous injection of1×107CFUAg85B-ESAT-6-rMS in upper back at a4weeks interval, or administrated with INH andRFP in drinking water for12weeks.4weeks after finishing treatments, all group of micewere induced recurrent infection by intramuscular injection of dexamethasone.③The mice daily activities of all groups were observed and the mice weights were recordedevery week. The splenic lymophacytes were separated from all group of mice for ELISpotanalysis of the IFN-γ producing cell frequency after8,13,17,22, and26week infectionrespectively.④The mice were sacrificed and lung and spleen tissues were isolated asabove for histopathologic diagnosis after HE staining, microscopic examination afteracid-fast staining, and bacteria loads counting by CFU on plate after10,14,18,22, and26weeks infection respectively.Results:①6weeks and10weeks after infection, histological examination revealedchronic inflammation in mice. The acid-fast staining ratings of mice was positive (++).The bacteria counts of lung and spleen were104and103CFU respectively. The resultsshowed that the mice had infected with low dose of M. tuberculosis H37Rv.②The miceweights of all groups showed the increasing trendency and no difference between groups(P>0.05).③4weeks and8weeks after the first immunotherapy, the spleen lymphocytesfrequencies of producing IFN-γ in Ag85B-ESAT-6-rMS and RFP/INH+Ag85B-ESAT-6-rMS group were significantly higher than that of the untreated group (P<0.05).④4weeks and8weeks after the first immunotherapy, histological examination revealed slightto moderate chronic inflammation and no obvious pathological lesion was found in allgroups.⑤4weeks and8weeks after the first immunotherapy, The bacteria counts (log10CFU) of mice lung and spleen were significantly decreased in Ag85B-ESAT-6-rMS andRFP/INH+Ag85B-ESAT-6-rMS group compared with untreated group (P<0.05),especially in RFP/INH+Ag85B-ESAT-6-rMS group. After treated with immune inhibitorof dexamethasone, the bacteria loads were increased in Ag85B-ESAT-6-rMS andRFP/INH+Ag85B-ESAT-6-rMS group, but not significant than that of untreated group.ConclusionThis study successfully constructed a recombinant M. smegmatis of Ag85B-ESAT-6-rMS that could express secreted Ag85B and ESAT-6fusion protein. After inoculated withAg85B-ESAT-6-rMS, mice showed Th1type dominant cellular immune response. Theimmunotherapeutic effect of Ag85B-ESAT-6-rMS had been demostrated by its ability to inhibit the growth of M. tuberculosis RFP-resistant strain in infected mice, especiallyadministrated with chemotherapy. Meanwhile a certain degree of pathological lesion hadbeen observed by histological examination. The similar immunotherapeutic effect ofAg85B-ESAT-6-rMS had also been proved in low dose of M. tuberculosis H37Rv straininfected mice. In this infected mice model, the immunotherapeutic effect ofAg85B-ESAT-6-rMS to M. tuberculosis was more significant as administrated with drugsand no obvious pathological lesion was found after treatment. Thus the potentialadvantages of Ag85B-ESAT-6-rMS as therapeutic vaccines were to utility itscharacteristics of inducing and enhanceing the host immune response against M.tuberculosis, which help the host completely eliminate low load of drug-resistant andlatent bacteria hiding in tissue or cell, especially administrated with chemotherapy.