Expression and immunogenicity of recombinant Mycobacterium bovis Bacillus Calmette-Guérin strains secreting the antigen ESAT-6 from Mycobacterium tuberculosis in mice

Background Tuberculosis remains the leading cause of human death. Currently, Bacillus Calmette-Guerin （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.Methods 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 μl normal saline containing 106 CFU of BCG or rBCG. They were sacrificed after 4 weeks to detect their humoral and cellular responses. Results 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-γ, 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）. Conclusion 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.

Repeated inoculations of Mycobacterium bovis Bacille Calmette-Guérin (BCG) are needed to induce a strong humoral immune response against antigens expressed by the bacteria

The cellular immune response elicited by Mycobacterium bovis Bacille Calmette-Guérin (BCG) has been carefully investigated, but the humoral immune response has been partially neglected. BALB/c mice were immunized with BCG strain used to immunize humans. Anti-BCG antibodies, as assayed by ELISA, began to appear in the sera after the third week of immunization and plateaued three weeks after the 8th immunization. The total immunoglobulins (Igs) were purified by caprylic acid method from pooled serum collected after the 8th immunization. Anti-BCG antigen antibodies were detected in the total Igs preparation as well as in IgG, IgM, IgA, IgG1, IgG2a, and IgG2b, but not in the IgG3. Distinct BCG proteins were recognized the IgGs in Western blot analysis. Opsonization of BCG bacilli by the purified Igs potentiated internalization of the bacteria by murine Raw 264.7 macrophages. The intracellular BCG elimination coincided with the induction of NO production, which was more pronounced in cells infected with opsonized BCG compared to those infected with the non-opsonized bacteria. Coincidently, the production of NO was also higher in macrophages infected with opsonized BCG (maximal NO production at 48 h of incubation). The obtained results demonstrate that repeated inoculations of BCG effectively activate the humoral immune response, justifying the use of BCG as a live recombinant vaccine vector to insert genes encoding virulence factors controlled by antibodies.

Antitumor effects of human interferon-alpha 2b secreted by recombinant bacillus Calmette-Guérin vaccine on bladder cancer cells

Objective:Our objective was to construct a recombinant bacillus Calmette-Guérin vaccine(rBCG) that secretes human interferon-alpha 2b(IFNα-2b) and to study its immunogenicity and in vitro antitumor activity against human bladder cancer cell lines T24 and T5637.Methods:The signal sequence BCG Ag85B and the gene IFNα-2b were amplified from the genome of BCG and human peripheral blood,respectively,by polymerase chain reaction(PCR).The two genes were cloned in Escherichia coli-BCG shuttle-vector pMV261 to obtain a new recombinant plasmid pMV261-Ag85B-IFNα-2b.BCG was transformed with the recombinant plasmid by electroporation and designated rBCG-IFNα-2b.Mononuclear cells were isolated from human peripheral blood(PBMCs) and stimulated with rBCG-IFNα-2b or wild type BCG for 3 d,and then cultured with human bladder cancer cell lines T24 and T5637.Their cytotoxicities were measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide(MTT) assay.Results:BCG was successfully transformed with the recombinant plasmid pMV261-Ag85B-IFNα-2b by electroporation and the recombinant BCG(rBCG-IFNα-2b) was capable of synthesizing and secreting cytokine IFNα-2b.PBMC proliferation was enhanced significantly by rBCG-IFNα-2b,and the cytotoxicity of PBMCs stimulated by rBCG-IFNα-2b to T24 and T5627 was significantly stronger in comparison to wild type BCG.Conclusions:A recombinant BCG,secreting human IFNα-2b(rBCG-IFNα-2b),was constructed successfully and was superior to control wild type BCG in inducing immune responses and enhancing cytotoxicity to human bladder cancer cell lines T24 and T5637.This suggests that rBCG-IFNα-2b could be a promising agent for bladder cancer patients in terms of possible reductions in both clinical dosage and side effects of BCG immunotherapy.

全球结核病疫苗研究进展

本文旨在对全球结核病疫苗研究进展进行系统综述,描述国际上目前进入临床试验不同阶段的新型疫苗,包括重组卡介苗、亚单位疫苗、治疗性疫苗等,分析我国结核病疫苗研究现状,介绍国际研究发展趋势,如人类疫苗计划、全细胞疫苗、多阶段疫苗等,并对存在的问题和挑战进行讨论,展望未来发展趋势。

抗结核重组BCG疫苗研究进展

结核病仍是严重威胁人类健康的传染病之一,卡介苗(mycobacterium bovis bacillus calmette-guérin,BCG)是唯一被批准用于结核病预防的疫苗,但近年来的研究发现其免疫保护效果极不稳定,构建重组BCG成为目前新型抗结核疫苗研究的重要方向之一。本文概括性地介绍了抗结核重组BCG疫苗研究的必要性、抗结核重组BCG的研究策略与研究进展以及存在的问题和未来展望。

BCG Infection after Bladder Cancer Treatment—3 Clinical Case Reports

Immunotherapy with Bacillus Calmette-Guérin (BCG) to treat non-muscle invasive bladder cancer has become an effective and superior alternative to chemotherapy. Intravesical treatment with BCG appears to be relatively safe;however, occasionally BCG infection complicates such treatment. In the present work we describe three patients in whom BCG infection occurred after intravesical BCG therapy. All patients had positive urine culture forMycobacterium tuberculosis complex, using AccuProbe culture identification and then Genotype Mycobacterium MTBC test identified Mycobacterium bovis BCG. The diagnosis is difficult and microbiologic study is usually negative, so high index of suspicion is essential.