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ABSTRACT: Tuberculosis remains the leading cause of human death. Currently, Bacillus Calmette-Guérin (BCG) is the only available vaccine against tuberculosis but its efficacy is highly variable. Thus, developing new tuberculosis vaccines becomes an urgent task. In this study, we evaluated in BALB/c mice the humoral and cellular immune responses of recombinant BCG expressing the antigen ESAT-6 from Mycobacterium tuberculosis.
Escherichia coli-BCG shuttle plasmid named pDE22-esat-6 was constructed by inserting the BamHI/EcoRI digested esat-6 gene PCR product into the similarly digested parental plasmid pDE22. BCG cells were transformed with pDE22-esat-6, which was named recombinant BCG (rBCG). BALB/c mice were immunized subcutaneously on the back with 100 microl normal saline containing 10(6) CFU of BCG or rBCG. They were sacrificed after 4 weeks to detect their humoral and cellular responses.
There was no any significant differences in the growth characteristics between the conventional BCG and rBCG. In immunized mice, the IgG antibody titres of rBCG group were as high as 1:8000, which was significantly higher than that in BCG group (1:1400, P < 0.05). The elicited IFN-gamma level of rBCG group was (1993 +/- 106) pg/ml, which was also significantly higher than that in BCG group ((1463 +/- 105) pg/ml, P < 0.05). The splenocyte proliferation index of rBCG group reached 4.34 +/- 0.31, which was higher than that of BCG group (3.79 +/- 0.24, P < 0.05).
rBCG secreted expressing antigen ESAT-6 stimulated stronger humoral and cellular immune responses than BCG did, and, therefore may be the better vaccine against mycobacterium tuberculosis.
Chinese medical journal 07/2007; 120(14):1220-5. · 0.86 Impact Factor
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ABSTRACT: The live vaccine Mycobacterium bovis bacillus Calmette-Guérin (BCG) provides variable efficacy against adult pulmonary tuberculosis (TB). Recombinant BCG, expressing either immunodominant antigens or Th1 cytokines, is a promising strategy for developing a new TB vaccine. However, not much is known about whether the introduction of cytokine and specific antigen genes concurrently into the BCG strain could improve the immunogenicity of BCG. In this study, a recombinant BCG strain (rBCG) expressing the fusion protein human interleukin (IL)-2 and ESAT-6 (early secreted antigenic target-6 kDa) antigen of Mycobacterium tuberculosis was constructed. Six weeks after BALB/c mice (H-2d) were immunized with 106 colony forming units (CFUs) BCG or rBCG, splenocyte proliferation was determined with MTT [3-(4,5-dimethylthiazolyl-2)-2, 5-diphenyltetrazolium bromide] assay, IL-4 and interferon (IFN)-gamma produced by splenocytes were tested by enzyme linked immunosorbent assay (ELISA,) and the cytotoxicity of splenocytes from immunized mice to P815 cells (H-2d) expressing ESAT-6 protein was measured using CytoTox 96 Non-Radioactive Cytotoxicity Assay. Compared with native BCG-vaccinated mice, rBCG induced stronger Th1 responses that were confirmed by high lymphoproliferative responses and IFN-gamma production to culture filtrate protein (CFP) or ESAT-6 protein. Moreover, rBCG induced significant enhanced CTL responses against P815-ESAT-6 cells. Results from rBCG-immunized mice demonstrated that introducing the il-2 and esat-6 genes into BCG could enhance Th1 type immune responses to ESAT-6. Further investigation is needed by introducing other Th1 cytokines and antigens into BCG to optimize the protective efficacy against TB.
Acta Biochimica et Biophysica Sinica 11/2006; 38(10):683-90. · 1.38 Impact Factor
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Chinese medical journal 06/2005; 118(9):762-5. · 0.86 Impact Factor
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ABSTRACT: To investigate the effects of rBCG vaccination containing foreign antigen Der p2 in the form of lipoprotein on murine immune response.
6 to 8 weeks old and newborn BALB/c mice were vaccined intraperitoneally with 10(6) CFU rBCG or BCG. At the same time, the control group was injected with saline. Six weeks later, all animals were injected with Der p2 (20 microg). After two weeks later, the concentrations of IL-4 and IFN-gamma in the serum and splenocyte culture supernatant (STLCS) were determined by ELISA, and Th subgroups were determined by double fluorescent staining and flow cytometry.
After vaccination, the serum and STLCS from both rBCG-immunized and BCG-immunized group of adult and newborn BALB/c mice had significantly higher level of IFN-gamma and lower level of IL-4 than those from control groups. Besides, there was the larger percentage of CD4 (+) IFN-gamma (+) cells in spleen from rBCG-vaccined and BCG-vaccined mice than that from control group. However, the percentage of CD4 (+) IL-4 (+) cells in spleen cells from rBCG-vaccined and BCG-vaccined group was lower than that from control group. Moreover, the level of IFN-gamma in STLCS from rBCG-immunized was significantly higher, compared with that from BCG-immunized mice. At the same time, the percentage of CD4 (+) IFN-gamma (+) cells in spleen from rBCG-vaccined mice was larger than that from BCG-vaccined group.
Both rBCG and BCG could stimulate Th1 predominant immune response, when injected intraperitoneally into adult or newborn BALB/c mice, The Der p2 expressed on the cell wall of BCG can work as the component of BCG and be recognized by the immune system of mice, therefore stimulates Der p2-specific Th1 predominant immune response. These data indicate that recombinant BCG-expressing antigens can be used as the antigen-specific vaccines against allergic diseases by regulating the balance of Th1/Th2.
Xi bao yu fen zi mian yi xue za zhi = Chinese journal of cellular and molecular immunology 06/2005; 21(3):287-9.
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ABSTRACT: To construct the E. coli.-BCG (Bacille Calmette-Guerin) shuttle vector expressing Mycobacterium tuberculosis secreted protein Ag85B-ESAT-6 on the surface of Mycobacterium vaccae.
The gene fragment containing 19 000 antigen (19-ss) were amplified by polymerase chain reaction (PCR) from the Mycobacterium tuberculosis H(37)Ra. We cloned the 19ss gene into the E. coli.-BCG shuttle vector pOLYG and named the pCW, which can shuttle and express exogenous antigen gene on cell wall of Mycobacterium. Then Mycobacterium tuberculosis secret protein Ag85B and ESAT-6 gene were cloned into the vector and determined by indirect immunofluorescence.
The sequence of 19-ss gene was identified with Genbank reported by sequencing. The constructed E. coli.-BCG shuttle vector using 19ss gene had the function of shuttle between E. coli. and Mycobacteria. By indirect immunofluorescence technique the secreted protein Ag85B-ESAT-6 can be fused and expressed on surface of Mycobacterium vaccae.
The E. coli.-BCG shuttle vector is constructed successfully which could express exogenous antigen gene as a chimeric exported membrane.
Zhonghua jie he he hu xi za zhi = Zhonghua jiehe he huxi zazhi = Chinese journal of tuberculosis and respiratory diseases 05/2004; 27(4):249-52.
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ABSTRACT: To investigate the fused expression of secreted protein Ag85B-ESAT6 of Mycobacterium tuberculosis, and to provide a promising preventive subunit vaccine against tuberculosis.
The gene encoding Ag85B and ESAT6 protein was amplified by PCR from genome of Mycobacterium tuberculosis H(37)Rv strain, and inserted into cloning vector P(GEM)-T-easy. After sequence analysis, and digestion by restriction endonuclease, Ag85B-ESAT6 was cloned into corresponding sites of the expression vector P(PRO) EXHT, and the recombinant plasmid was transformed into expressive strain E. coli DH5 alpha, induced with IPTG and fusion protein was purified by Ni-NTA purification system. The specific antibody titer in the sera of BALB/c mouse immunized with two fusion protein was detected by ELISA.
The sequences of Ag85B and ESAT6 by PCR amplification were identical to those reported by GenBank. The recombinant plasmid fused expression protein of Ag85B-ESAT6 with relative molecular mass (Mr) of 37,000, which was confirmed by Western-blot analysis with specific monoclonal antibody against 6 x HismAb. The fused expression protein was insoluble. It could be purified by Ni-NTA purification system. The specific antibody titer in the sera of BALB/c mouse immunized with fusion Ag85B-ESAT6 was 1:1000 and that of mouse immunized with fusion protein ESAT6-Ag85B was 1:5000.
Secreted protein Ag85B-ESAT6 of Mycobacterium tuberculosis was successfully fused expressed in E. Coli DH5 alpha. It may become a new type of vaccine against tuberculosis.
Zhonghua jie he he hu xi za zhi = Zhonghua jiehe he huxi zazhi = Chinese journal of tuberculosis and respiratory diseases 03/2004; 27(2):89-92.
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ABSTRACT: To investigate the effects of plasmid containing mouse IL-12 and human IL-18 genes on the humoral immune response of mice immunized by CFP10 gene of Mycobacterium tuberculosis (MTB) H(37)R(v) strain.
Human IL-18 cDNA was amplified from RNA of PBMCs by RT-PCR and cloned into the pGEM-Teasy vector. After sequencing it was subcloned into the the sites of BamH I and EcoR I digestion of pcDNA3.1. BALB/c mice were injected intramuscularly by eukaryotic expression plasmid pcmIL12 and pcIL18, together with MTB CFP10 DNA vaccine, respectively. The same immunization repeated three times at intervals of two weeks. Mouse sera were collected at two weeks after the each injection. The titer of anti-CFP10 antibody was detected by ELISA.
IL-18 cDNA was amplified successfully from RNA of human PBMCs by RT-PCR and the result of sequencing was correct. The IL-18 gene was correctly inserted into the vector pcDNA3.1 by being confirmed with BamH I and EcoR I digestion analysis, positive plasmid was called pcIL18. After being immunized with pcCFP10 three times, the end point titer of anti-CFP10 was 1:4 000, while the titer obtained by being immunized with pcIL18 + pcCFP10 was 1:8 000, but yet, after being immunized with pcmIL12+pcCFP10, the end point titer of anti-CFP10 antibody was only 1:200.
Combination of IL-18 gene with MTB CFP10 DNA vaccine can enhance the humoral immune responses to pcCFP10, whereas the immunization with IL-12 gene plus pcCFP10 made humoral immune response markedly descent. As for whether IL-18 gene plus MTB CFP10 DNA vaccine can induce markedly the cellular mediated immune response to CFP10 or not remains to be further investigated.
Xi bao yu fen zi mian yi xue za zhi = Chinese journal of cellular and molecular immunology 06/2003; 19(3):260-2.
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ABSTRACT: To construct the E.coli-BCG shuttle vector carrying and expressing dust mite antigen gene Der p2 on cell wall of mycobacterium vaccae.
The gene fragment encoding 19 kDa antigen and the upstream control element (19-ss) was amplified by PCR from the mycobacteria tuberculosis H37Rv.Subsequently, the 19-ss gene was cloned into the E.coli-BCG shuttle vector pOLYG, which can schlepped and expressed exogenous antigen gene on cell wall of mycobacteria and containing the Der p2 gene. The expression of Der p2 gene in mycobacterium vaccae determined by indirect immunofluorescence staining.
Sequencing proved that the cloned sequence of 19-ss gene was correct. The constructed E.coli-BCG shuttle vector (pCW) containing 19-ss gene had function of shuttle between E.coli and mycobacteria, and mediated the expression of antibiotic resistance gene. Indirect immunofluorescence staining indicated that the Der p2 gene was expressed in the form of lipoprotein on surface of the mycobacterium vaccae.
E.coli-BCG shuttle vector has been constructed successfully, which can express exogenous antigen gene as a chimeric protein on cell wall.
Xi bao yu fen zi mian yi xue za zhi = Chinese journal of cellular and molecular immunology 04/2003; 19(2):132-5.
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ABSTRACT: To explore protective efficacy of pTB30m and pTB30s encoding Ag85B protein against infection with M. tuberculosis H (37)R (v).
BALB/c mice were infected intravenously with 5x10(5) CFU of M. tuberculosisH (37)R (v) six weeks after the last vaccination of pTB30m and pTB30s. At the same time, normal BALB/c mice were injected intravenously with 5x10(6) T cells separated from immunized BALB/c mouse spleen through nylon wool column, and challenges were injected with 10(5) CFU of M. tuberculosis immediately intravenously. After 4 weeks, the CFU in spleens of infected mice were counted respectively.
Vaccination with pTB30m and pTB30s produced significant protection against M. tuberculosis proliferation in the mouse spleens following challenge. As compared with the saline-injected mice, CFU in spleens of the mice vaccinated with pTB30m or pTB30s were reduced significantly, being 0.645(log(10) CFU, P<0.01) and 0.839(log(10) CFU, P<0.001), respectively. In contrast, bacterial load in the mice spleens vaccinated with empty plasmid only had little reduction. After adoptive immunization by T cells from the mice vaccinated with pTB30m and pTB30s, the mice might induce partial protection against M. tuberculosis proliferation in the mouse spleens following challenge.
Protective efficacy of the mice immunized with pTB30s against challenge with M. tuberculosis virulent strain was better than that immunized with pTB30m.Thus, pTB30s is a promising DNA vaccine with respect to the prevention and treatment of tuberculosis.
Xi bao yu fen zi mian yi xue za zhi = Chinese journal of cellular and molecular immunology 02/2003; 19(1):90-2.
