Protective Antigens ESAT-6 And CFP-10 Of Mycobacterium Tuberculosis Expressed In Recombinant Salmonella Vehicle And Mechanisms Of Their Mucosal Immune Responses

Tuberculosis (TB) has a long history. It was presented before the beginning of recorded history. TB, together with acquired immune deficiency syndrome(AIDS) and malaria, remains today one of the leading infectious diseases. TB causes an estimated of at least 200 millions deaths now, and it was declared a global emergency by the World Health Organization. In 1882, Robert Koch identified Mycobacterium tuberculosis (MTB) as the causative agent of TB. More than one third of people over the world have infected MTB, and there has more than 20 million patients. There were an estimated 8.8 million new TB cases per year. A total of 1.6 million people died of TB, including 195000 patients infected with HIV. The reported efficacy of the currently used Bacillus Calmette-Guérin (BCG) vaccine against TB is highly variable, ranging from 50% against pulmonary tuberculosis to 80% against disseminated tuberculosis. The alarming increase in the incidence of TB , due to emergence of TB strains resistant to all major chemotherapeutic drugs and to co-infection with human immunodeficiency virus (HIV), has emphasized the need to develop immunological tools for TB control.Adaptive immunity against TB plays an immportant role in clearing infectious MTB. BCG vaccine protected efficiently against leprosy as well as childhood manifestations of TB. However, the protective efficacy against adult pulmonary TB was limited. Therefore, the new vaccine against TB should include protective efficacy against pulmonary TB. Comparative genomics identified up to 100 coding sequences absent from BCG but present in MTB. Some of these coding sequences encode potential antigens that could improve immunogenicity if reintroduced into BCG. RD1(Regions of Difference 1) is one of the regions absent from BCG. Among the missing genes are the coding sequences esxB and esxA, which respectively encode CFP-10 (Culture filter protein 10, CFP-10) and ESAT-6 (Earlier secreted antigen target 6, ESAT-6), low-molecular-weight proteins that induce potent Th1 responses. Both M.bovis and some clinical pathogenic MTB share the two proteins. ESAT-6 and CFP-10 protein elicit protection against TB in animal models and become two of the most potential candidates in diagnosis and vaccine research. The purpose of this study were to: (1) express ESAT-6 and CFP-10 antigens using attenuated Salmonella and/or chimeric flagellin vector; (2) analyze the mucosal immune responses and their mechanisms induced by recombinant ESAT-6 and CFP-10 proteins through intranasally immunization; (3) study the immune responses initiated by chimeric flagellin fliC/esat co-immunized with BCG.1. Cloning, expression and immunogenic analysis of Mycobacterium tuberculosis ESAT-6 and CFP-10 proteinTo analyze immunogenic characteristics of ESAT-6 and CFP-10 proteins of Mycobacterium tuberculosis. ESAT-6 and CFP-10 coding genes were cloned and identified by PCR. Prokaryotic exprssion plasmids pBCX-esat and pET-cfp were constructed harboring the coding gene respectively and transformed into E.coli BL21(DE3). Recombinant bacteria were identified and induced by IPTG. SDS-PAGE results showed that both ESAT-6 and CFP-10 proteins were expressed and the sizes of them were 34kD and 19kD, respectively. Western-blotting results showed that fusion protein CFP-10 could react with the specific mAb 6E8. And, fusion protein ESAT-6 can bind to the serum of TB patients in ELISA assay. C57BL/6 mice were immunized intraperitoneally with ESAT-6 and CFP-10 fusion proteins respectively, which were emulsified by Freund's adjuvant. At the 7th day of second immunization in three-week intervels, splenocytes were collected from the immunized mice. IFN-γ-producing and IL-4-producing CD4~+ T cells were detected by intracellular cytokine staining assay. The results showed that both ESAT-6 and CFP-10 proteins can induce higher level of IFN-γcompared with that of IL-4. Furthermore, both ESAT-6 and CFP-10 proteins can significantly up-regulate CD69 molecule expression on CD3~+ T cells. Overall, both ESAT-6 and CFP-10 fusion proteins are capable of up-regulating CD69 molecule of CD3~+ T cells and inducing Th1 immune responses.2. Immune responses specific for ESAT-6 and CFP-10 antigen expressed by recombinant Salmonella typhimuriumTo determine mucosal immune responses induced by recombinant Salmonella harboring ESAT-6 and CFP-10 antigens, prokaryotic exprssion plasmids pYA33-esat and pYA33-cfp carrying the ESAT-6 and CFP-10 coding genes were constructed firstly and eletro-transformed into an attenuated strain X4550 of Salmonella typhimurium, the recombinant bacteria were named as X4550(33-esat) and X4550(33-cfp), respectively. C57BL/6 mice were immunized intranasally with 1×108 cfu recombinant bacteria in 18d intervals. Cells from spleen, lung, mesenteric lymph node (MLN) and Peyer's patches (PP) were collected from mice after second immunization, and the specific IFN-γ-secreting cells and IL-4-secreting cells were detected using ESAT-6 and CFP-10 proteins as stimuli. The results showed that the immune responses specific for ESAT-6 and CFP-10 proteins could be detected by the ELISPOT assay. In lung and PP cells, immune responses against ESAT-6 and CFP-10 proteins both were biased toward Th1 response, the frequency of IFN-γ-secreting cells was much higher than that of IL-4 -secreting cells. However, in splenocytes and MLN cells, the antigen specific immune responses acted as Th1 and Th2 balance, the frequency of IFN-γ-secreting cells was close to that of IL-4-secreting cells. Also, costimulatory molecules CD80 and CD86 from splenocytes, MLN, PP and bronchoalveolar lavage fluid (BAF) cells were up-regulated after the second immunization. Furthermore, IgG and IgA antibodies from serum, BAF and fecal extracts were detected by ELISA respectively, and the levels of antibodies in immunized group had significantly increased compared with those of the negative control. These results suggested that intranasal immunization of recombinant Salmonella not only can induce cellular immune responses, but also can elicit humoral and mucosal immune responses. It provided the bases for the development of mocusal vaccine against infectious disease of respiratory tract.3. Immunologic characteristics of ESAT-6 antigen expessed by Salmonella chimeric flagellin To understand the mucosal immune responses against ESAT-6 in the Salmonella chimeric flagellin system, which act as a vector to deliver ESAT-6 antigen of Mycobacterium tuberculosis. Chimeric flagellin gene fliC/esat were constructed by overlap PCR technique. The ESAT-6 coding gene was inserted to the hypervariable region of Salmonella flagellin gene fliC. Prokaryotic exprssion plasmid pET-fliC/esat carrying the chimeric fragment was constructed firstly and eletro-transformed into the strain LB5000 of Salmonella typhimurium, the recombinant bacteria were named as LB5000(fliC/esat). And then, the transduction was conducted between LB5000(fliC/esat) and Salmonella dublin strain SL5928 using bacteriophage P22HT int. The positive transductants were further identified by PCR, motolity observation and serological agglutination assay. In order to determine the invasion and adhesion capacities of SL5928(f/e). The colon carcinoma cells HT-29 were infected with SL5928(f/e). The results showed that SL5928(f/e) had favourable invasion and adhesion. It was detected TLR-5 mRNA from BMDC, F4/80+ MФand enterocytes infected with SL5928(f/e) by RT-PCR. All three type cells expressed TLR-5 mRNA at 3h post infection and the quantity of TLR-5 mRNA up-regulated at 6h. Furthermore, C57BL/6 mice were immunized intranasally with 1×107 cfu SL5928(f/e) in 18d intervals. Cells from spleen, lung, MLN, PP and nasopharynx associated lymph node (NALT) were harvested from mice after second immunization, and the specific IFN-γ-secreting cells and IL-4 secreting cells were detected using ESAT-6 protein or peptide of ESAT-6(1-20aa) as stimuli. The results showed that in lung, PP cells and NALT cells, immune responses against ESAT-6 protein or ESAT-6 peptide (1-20aa) both were biased toward Th1 response, the frequency of IFN-γ-secreting cells was markedly higher than that of IL-4 -secreting cells. However, in spleen and MLN cells, the frequency of IFN-γ-secreting cells was close to that of IL-4-secreting cells. Then, the percentage of specific lysis of SL5928(f/e) was higher than that of X4550(33-esat). Finally, the levels of IgG and IgA antibodies from serum and BAF in immunized group were significantly higher compared with those of the control group. These results suggested that Salmonella chimeric flagellin delivery system was effective after intranasal immunization. The cellular and mucosal immune responses specific for ESAT-6 can be induced by tranductant SL5928(f/e). This system could develop a new pathway for TB vaccine.4. Immune responses initiated by intranasal co-immunization of chimeric flagellin fliC/esat and BCGTo define the role of ESAT-6 chimeric flagellin in TB immunology, prokaryotic expression plasmids pET-fliC/esat and pET-fliC were transformed into E.coli and induced by IPTG. SDS-PAGE results showed the ESAT-6 chimeric flagellin and fliC protein both are soluble. The sizes of the two proteins were 64kD and 57kD respectively. Western-blotting analysis showed that chimeric flagellin and fliC protein both could be bind to serum of Salmonella typhymurium. BMDC maturation was triggered by both ESAT-6 chimeric flagellin and fliC protein. In contrast, ESAT-6 protein alone didn't activate BMDC. There were up-regulated the expression level of CD40, CD80, CD86 and CD54 costimulatory molecules on F4/80+ MФafter the stimulation of ESAT-6, ESAT-6 chimeric flagellin and fliC protein, respectively. IL-12p70 was detected in the superantants of BMDC and F4/80+ MФ. The results showed that chimeric flagellin was able to induce higher level of IL-12p70 than that of ESAT-6 protein. However, there weren't any IL-12p70 from F4/80+ MФafter the three proteins stimulating. In order to further demonstrate the adjuvant activity of flagellin, C57BL/6 mice were immunized intravenously with ESAT-6 chimeric flagellin. The results showed that ESAT-6 chimeric flagellin could enhance the immunogenic activity of ESAT-6 protein. Furthermore, mice were co-immunized with BCG and ESAT-6 chimeric flagellin. This immune strategy increased the level of immune responses and biased toward Th1 response in lung, PP, NALT and spleen. Percentage of CD8+CD69+ cells of co-immunization group were higher than that of BCG or ESAT-6 chimeric protein group. Overall, flagellin can present adjuvant activity for ESAT-6 protein, and the novel co-immunization of Chimeric flagellin and BCG could enhance the specific Th1 immune responses.