Characterisation of alternative expression vectors for recombinant Bacillus Calmette-Guérin as live bacterial delivery systems

BACKGROUND Bacillus Calmette-Guérin (BCG) is considered a promising live bacterial delivery system. However, several proposals for rBCG vaccines have not progressed, mainly due to the limitations of the available expression systems. OBJECTIVES To obtain a set of mycobacterial vectors using a range of promoters with different strengths based on a standard backbone, previously shown to be stable. METHODS Mycobacterial expression vectors based on the pLA71 vector as backbone, were obtained inserting different promoters (PAN, PαAg, PHsp60, PBlaF* and PL5) and the green fluorescence protein (GFP) as reporter gene, to evaluate features such as their relative strengths, and the in vitro (inside macrophages) and in vivo stability. FINDINGS The relative fluorescence observed with the different vectors showed increasing strength of the promoters: PAN was the weakest in both Mycobacterium smegmatis and BCG and PBlaF* was higher than PHsp60 in BCG. The relative fluorescence observed in a macrophage cell line showed that PBlaF* and PHsp60 were comparable. It was not possible to obtain strains transformed with the extrachromosomal expression vector containing the PL5 in either species. MAIN CONCLUSION We have obtained a set of potentially stable mycobacterial vectors with a arrange of expression levels, to be used in the development of rBCG vaccines.

Stable expression of Mycobacterium bovis antigen 85B in auxotrophic M. bovis bacillus Calmette-Guérin

BACKGROUND Bovine tuberculosis (TB) is a zoonotic disease caused by Mycobacterium bovis, responsible for causing major losses in livestock. A cost effective alternative to control the disease could be herd vaccination. The bacillus Calmette-Guérin (BCG) vaccine has a limited efficacy against bovine TB, but can improved by over-expression of protective antigens. The M. bovis antigen 85B demonstrates ability to induce protective immune response against bovine TB in animal models. However, current systems for the construction of recombinant BCG expressing multiple copies of the gene result in strains of low genetic stability that rapidly lose the plasmid in vivo. Employing antibiotic resistance as selective markers, these systems also compromise vaccine safety. We previously reported the construction of a stable BCG expression system using auxotrophic complementation as a selectable marker. OBJECTIVES The fundamental aim of this study was to construct strains of M. bovis BCG Pasteur and the auxotrophic M. bovis BCG ΔleuD expressing Ag85B and determine their stability in vivo. METHODS Employing the auxotrophic system, we constructed rBCG strains that expressed M. bovis Ag85B and compared their stability with a conventional BCG strain in mice. Stability was measured in terms of bacterial growth on the selective medium and retention of antigen expression. FINDINGS The auxotrophic complementation system was highly stable after 18 weeks, even during in vivo growth, as the selective pressure and expression of antigen were maintained comparing to the conventional vector. MAIN CONCLUSION The Ag85B continuous expression within the host may generate a stronger and long-lasting immune response compared to conventional systems.

Immunogenicity analysis of a recombinant BCG vaccine strain overexpressing Rv3478 protein / 中华微生物学和免疫学杂志

Objective To construct a recombinant Bacillus Calmette-Guerin ( BCG ) vaccine strain, rBCG::Rv3478-pMV261, expressing the Rv3478 protein of Mycobacterium tuberculosis and to inves-tigate its immunogenicity.Methods The gene fragments encoding Rv3478 antigen were amplified by PCR and then respectively cloned into pMV261 and pET-28a vectors to construct the recombinant expression plas-mids (Rv3478-pMV261 and Rv3478-pET-28a).The Rv3478-pMV261 plasmids were transformed into the BCG cells to construct the rBCG vaccine strains, while the Rv3478-pET-28a plasmids were transformed into Escherichia coli BL21 strains for the expression of Rv3478 protein.Polyclonal antibodies were induced in mice upon the immunization with Rv3478 protein.The rBCG vaccine strains overexpressing Rv3478 protein were screened out with Western blot assay.The C57BL/6 mice were divided into four groups including the PBS treated group, BCG treated group, rBCG::pMV261 ( R0) treated group and rBCG::Rv3478-pMV261 ( R3) treated group.All mice were sacrificed in 4 or 12 weeks after immunization.Enzyme-linked immunos-pot assay ( ELISPOT) , ELISA and flow cytometry analysis were performed to evaluate the induced humoral and cell-mediated immune responses in mice.Results The Rv3478 protein was successfully expressed and could induce polyclonal antibodies in mice.High levels of IFN-γand TNF-αwere detected in mice treated with R3, indicating that the immunization with R3 enhanced the cellular immunity.Moreover, the ratios of CD4+to CD8+T cells and the percentages of CD44+CD62L+T cells were increased in mice upon the immuni-zation with R3.Conclusion The recombinant BCG vaccine strain overexpressing Rv3478 protein could in-duce stronger cell-mediated immune responses in mice.It might be have a great significance as a new tuber-culosis( TB) vaccine strain against TB infection in the future.

Construction and expression of a recombinant BCG-TSOL18 vaccine of Taenia solium / 中国人兽共患病学报

We constructed a recombinant Bacillus Calmette‐Guerin(BCG)‐TSOL18 vaccine of Taenia solium and observed the expression of the TSOL18 gene in BCG .The TSOL18 gene of Taenia solium was obtained from the recombinant plasmid pGEX‐TSOL18 by digestion method and cloned into Escherichia coli (E .coli)‐mycobacterium shuttle plasmid pMV261 to con‐struct the recombinant plasmid pMV261‐TSOL18 of Taenia solium ,and the recombinant plasmid was identified by restriction enzyme digestion ,PCR and DNA sequencing .Then ,the recombinant plasmid was transformed into BCG by electroporation to construct the recombinant BCG‐TSOL18 vaccine of Taenia solium ,and the vaccine was identified by PCR .The expression of the TSOL18 gene in BCG was identified by SDS‐PAGE and Western blot .The 393 bp TSOL18 gene fragment was successfully obtained by restriction enzyme digestion .Restriction enzyme digestion ,PCR and DNA sequencing suggested that the recombi‐nant plasmid pMV261‐TSOL18 of Taenia solium was successfully constructed .PCR confirmed that the recombinant plasmid pMV261‐TSOL18 of Taenia solium was successfully transformed into BCG ,suggesting that the recombinant BCG‐TSOL18 vaccine of Taenia solium was successfully constructed .SDS‐PAGE showed that the relative molecular mass (Mr) of TSOL18 target protein was approximately 14 .7 kD .Results of western blot showed the TSOL18 target protein could be recognized by rabbit antiserum or cysticercosis swine serum .The recombinant BCG‐TSOL18 vaccine of Taenia solium was successfully con‐structed .The TSOL18 gene of Taeniasolium was successfully expressed in BCG and the expressed TSOL18 recombinant pro‐tein had specific antigenicity .This result would lay a foundation for further study of the vaccine .

Dynamic observation of immune responses induced in mice by immunization with a recombinant BCG-TSOL18 vaccine of Taenia solium / 中华地方病学杂志

Objective To dynamically observe humoral and cellular immune responses induced in mice by immunization with a recombinant BCG-TSOL18 vaccine of Taenia solium.Methods Totally 80 Kunming mice were divided into 4 groups by using random number table according to body mass, 20 mice in each group: rBCG-TSOL18 intraperitoneal injection group [mice were vaccinated with 5 × 106 colony forming units (CFU) recombinant BCG-TSOL18 vaccine of Taenia solium through intraperitoneal injection], rBCG-TSOL18 intragastric administration group(mice were vaccinated with 4 × 108 CFU recombinant BCG-TSOL18 vaccine of Taenia solium through intragastric administration), BCG control (mice were vaccinated with 5 × 106 CFU BCG through intraperitoneal injection), PBS control (mice were vaccinated with PBS through intraperitoneal injection).Zero, 2, 4, 6 and 8 weeks after immunization, eye blood was collected and serum w as separated.Levels of specific IgG and IgG2a were detected by enzyme linked immunosorbent assay (ELISA).Proliferation level of spleen lymphocytes was detected by CCK-8.Levels of interleukin-2 (IL-2) and IL-4 were determined by ELISA.Results The level of specific IgG in rBCG-TSOL18 intraperitoneal injection group and rBCG-TSOL18 intragastric administration group increased from 2 to 8 weeks, and reached the highest level by the 6th week (0.310 ± 0.022, 0.356 ± 0.026).Compared with 0 week in the same group, BCG and PBS control group of the same time periods (0.054 ± 0.005, 0.057 ± 0.006, 0.093 ± 0.014, 0.085 ± 0.010), there were statistically significant differences (all P ＜ 0.05).The level of specific IgG2a increased from 2 to 8 weeks, and reached the highest level by the 6th week (0.965 ± 0.031, 1.144 ± 0.049).Compared with 0 week in the same group, BCG and PBS control group of the same time periods (0.102 ± 0.014, 0.093 ± 0.012, 0.115 ± 0.012, 0.103 ± 0.013), there were statistically significant differences (all P ＜ 0.05).The proliferation level of spleen lymphocytes increased from 2 to 8 weeks, and reached the highest level by the 6th week (0.524 ± 0.032, 0.755 ± 0.016).Compared with 0 week in the same group, BCG and PBS control group of the same time periods (0.301 ± 0.018, 0.305 ± 0.020, 0.362 ± 0.033, 0.334 ± 0.027), there were statistically significant differences (all P ＜ 0.05).The levels of IL-2 and IL--4 in spleen lymphocyte culture supernatant increased from 2 to 8 weeks and from 4 to 6 weeks, respectively, reached the highest level by the 6th and 4th weeks, respectively [(1 665.41 ± 33.93), (1 785.11 ± 42.39)ng/L and (281.62 ± 23.79), (357.95 ± 42.57)ng/L].Compared with 0 week in the same group, BCG and PBS control group of the same time periods [(1 411.63 ± 20.54), (1 405.12 ± 21.42),(1 455.20 ± 25.03), (1 434.47 ± 17.47)ng/L and (190.17 ± 11.01), (196.96 ± 14.00), (200.51 ± 30.79), (189.64 ±40.90)ng/L], there were statistically significant differences (all P ＜ 0.05).These indexes mentioned above were statistically significantly different (all P ＜ 0.05) between rBCG-TSOL18 intragastric administration group and rBCG-TSOL18 intraperitoneal injection group.Conclusion The recombinant BCG-TSOL18 vaccine of Taenia solium might induce mice to produce humoral and cellular immune responses, and the effect of intragastric administration is better than that of intraperitoneal injection.

A Novel Recombinant BCG Vaccine Encoding Eimeria tenella Rhomboid and Chicken IL-2 Induces Protective Immunity Against Coccidiosis

A novel recombinant Bacille Calmette-Guerin (rBCG) vaccine co-expressed Eimeria tenella rhomboid and cytokine chicken IL-2 (chIL-2) was constructed, and its efficacy against E. tenella challenge was observed. The rhomboid gene of E. tenella and chIL-2 gene were subcloned into integrative expression vector pMV361, producing vaccines rBCG pMV361-rho and pMV361-rho-IL2. Animal experiment via intranasal and subcutaneous route in chickens was carried out to evaluate the immune efficacy of the vaccines. The results indicated that these rBCG vaccines could obviously alleviate cacal lesions and oocyst output. Intranasal immunization with pMV361-rho and pMV361-rho-IL2 elicited better protective immunity against E. tenella than subcutaneous immunization. Splenocytes from chickens immunized with either rBCG pMV361-rho and pMV361-rho-IL2 had increased CD4+ and CD8+ cell production. Our data indicate recombinant BCG is able to impart partial protection against E. tenella challenge and co-expression of cytokine with antigen was an effective strategy to improve vaccine immunity.

Immune response of recombinant IFN-α-2b-BCG treatment in mice bladder cancer / 中华泌尿外科杂志

Objective To study local and systemic immune response in an animal model treated with recombinant hIFN-α-2b-BCG instillation. Methods The MB49 orthotopic bladder cancer model in C57BL/6 mice was established and treated separately with rBCG, wild BCG, wild BCG combined with IFN-α-2b and PBS as the control. The changes of lymphocyte subgroups in peripheral blood were analyzed with FCM, and mTNF-α and mIL-12 in peripheral blood of mice were detected with ELISA.Immunohistochemistry was carried out to detect the local immune reaction, T cell subsets and FAS, in bladder cancer after being treated with rBCG or wBCG. Results The content of CD4+ T lymphocyte was up-regulated in the rBCG group. The CD4+/CD8+ ratio of 2. 63 was up-regulated than pretreatment, significantly different than that of wBCG group(P＜0.05). ELISA assay showed that BCG significantly up-regulated the level of mTNF-α and mIL-12 in serum of orthotopie murine bladder cancer mice. The mTNF-α 806 pg/ml, mIL-12 860 pg/ml in rBCG group, was not significantly higher than those in wBCG group and combination group. The immunocompetent cell numbers with CD3, CD4,CD8 phenotype increased significantly in the tumor tissue of BCG treated group than the control(P＜0.05). The results of CD4+ in rBCG group and the combination group, and CD8+ in rBCG group were significantly higher than that of the wBCG(P＜0.05). The expression of Fas in tumor tissues treated with intravesical BCG was increased(P＜0. 05). Conclusions The recombinant IFN-α-2b-BCG can retrieve the disproportion of systemic lymphocyte subgroups, and increases Th1-type factors and local Fas expression in orthotopic murine bladder cancer. The recombinant IFN-α-2b-BCG is effective in regulating local and systemic immune reaction in orthotopic murine bladder cancer model.

Efficiency of Recombinant Bacille Calmette-Guerin in Inducing Humoral and Cell Mediated Immunities against Human Immunodeficiency Virus Type 1 Third Variable Domain in Immunized Mice

PURPOSE: The third variable (V3) loop of the human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein has been intensively studied for AIDS vaccine development. Bacille Calmette-Guerin (BCG) is widely used to immunize against tuberculosis and has many advantages as a vaccine vehicle, such as low toxicity, adjuvant potential, low cost, and long-lasting immune-inducing capacity. This work was initiated to investigate the immunogenicity of recombinant BCG (rBCG-mV3) designed to express trimeric HIV-1 V3 loop (mV3) in rBCG-mV3-immunized animals. MATERIALS AND METHODS: HIV-1 V3-concatamer was cloned into pMV261, a BCG-expression vector, and then rBCG-mV3 was constructed by introducing the recombinant plasmid (pMV-V3). The recombinant BCG was examined with regard to its expression of V3-concatamer and the genetic stability in vivo and in vitro. The immune responses induced by recombinant BCG were tested in immunized mice and guinea pigs. RESULTS: The rBCG-mV3 expressed detectable amounts of V3-concatamer when induced by single heat-shock. The recombinant BCG was genetically stable and maintained the introduced mV3 gene for several weeks. V3-specific antibodies were clearly detected 6 weeks after inoculation. The antibody titer rapidly increased after immunization up to 10 weeks, and then maintained for over 4 weeks. IgG2a was prevalent in the V3-specific antiserum. The recombinant BCG was also effective in inducing delayed-type hypersensitivity responses in the immunized guinea pigs. rBCG-immunized mice retained substantial amounts of V3-specific T cells in the spleen, even 5 months after the first immunization. CONCLUSION: Recombinant BCG-mV3 is very efficient in inducing humoral and long-lasting cell-mediated immunity against HIV-1 V3 in the immunized animals.

Efficiency of Recombinant Bacille Calmette-Guerin in Inducing Humoral and Cell Mediated Immunities against Human Immunodeficiency Virus Type 1 Third Variable Domain in Immunized Mice

PURPOSE: The third variable (V3) loop of the human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein has been intensively studied for AIDS vaccine development. Bacille Calmette-Guerin (BCG) is widely used to immunize against tuberculosis and has many advantages as a vaccine vehicle, such as low toxicity, adjuvant potential, low cost, and long-lasting immune-inducing capacity. This work was initiated to investigate the immunogenicity of recombinant BCG (rBCG-mV3) designed to express trimeric HIV-1 V3 loop (mV3) in rBCG-mV3-immunized animals. MATERIALS AND METHODS: HIV-1 V3-concatamer was cloned into pMV261, a BCG-expression vector, and then rBCG-mV3 was constructed by introducing the recombinant plasmid (pMV-V3). The recombinant BCG was examined with regard to its expression of V3-concatamer and the genetic stability in vivo and in vitro. The immune responses induced by recombinant BCG were tested in immunized mice and guinea pigs. RESULTS: The rBCG-mV3 expressed detectable amounts of V3-concatamer when induced by single heat-shock. The recombinant BCG was genetically stable and maintained the introduced mV3 gene for several weeks. V3-specific antibodies were clearly detected 6 weeks after inoculation. The antibody titer rapidly increased after immunization up to 10 weeks, and then maintained for over 4 weeks. IgG2a was prevalent in the V3-specific antiserum. The recombinant BCG was also effective in inducing delayed-type hypersensitivity responses in the immunized guinea pigs. rBCG-immunized mice retained substantial amounts of V3-specific T cells in the spleen, even 5 months after the first immunization. CONCLUSION: Recombinant BCG-mV3 is very efficient in inducing humoral and long-lasting cell-mediated immunity against HIV-1 V3 in the immunized animals.

Study of the biological characteristics and antitumor activity of recombinant IFN-α-2B-BCG / 中华微生物学和免疫学杂志

Objective To investigate the biological characteristics of recombinant hIFN-α-2B-BCG and its direct effect to bladder tumor cells in vitro. Methods BCG and recombinant BCG(rBCG) wild-type growth and morphology were compared. After 10 generations, hIFN-α-2B content was analyzed by ELISA, of bacteria in vivo and in vitro. The effects of rBCG on bladder cancer cells EJ, MB49, were detected by elec-tron microscopy and cell inhibitory rate from MTT. Results The normal BCG and rBCG had no significant difference in growth phase. They were both positive for acid-fast stain, maintaining the characteristics of cell's connection. While rBCG slightly larger than normal BCG, no else abnormality was obvious between them. IFN-α-2B was 997.2 pg/ml in secretions, 99.3 pg. In bacteria in vivo, 990.3 pg/ml in 10th genera-tion's sections, of rBCG. Compared with 1st generation, rBCG had no significant differences in morphology and interferon expression. Normal BCG and rBCG both had, anti-proliferation directly on bladder tumor cell in vitro, the rBCG is most effective in all. After rBCG cultivated with bladder tumor cell together, tumor cell's slow proliferation, detach, quantity decreasing and death were observed under microscope. Different degeneration in degree on most of tumor cells, disorganization on organelle, aggregation on chromatin, pyc-nosis on nucleolus, and cytoplasm lysis were on tumor cell under transmission electro microscopy. MTT as-say showed rBCG inhibited the proliferation of bladder caner cell and more active than normal BCG. Conclu-sion These results suggest that the rBCG have the same characteristics in growth phase as normal BCG, and stable properties in interferon expression and morphology by generations, rBCG has more anti-tumor effects in vitro than normal BCG.

Stable expression of Leptospira interrogans antigens in auxotrophic Mycobacterium bovis BCG

Mycobacterium bovis BCG has been proposed as an effective live vector for multivalent vaccines. The development of mycobacterial genetic systems to express foreign antigens and the adjuvanticity of BCG are the basis for the potential use of this attenuated mycobacterium as a recombinant vaccine vector. Stable plasmid vectors without antibiotic resistance markers are needed for heterologous antigen expression in BCG. Our group recently described the construction of a BCG expression system using auxotrophic complementation as a selectable marker. In this work, LipL32 and LigAni antigens of Leptospira interrogans were cloned and expressed in M. bovis BCG Pasteur and in the auxotrophic M. bovis BCG ΔleuD strains under the control of the M. leprae 18kDa promoter. Stability of the plasmids during in vitro growth and after inoculation of the recombinant BCG strains in hamsters was compared. The auxotrophic complementation system was highly stable, even during in vivo growth, as the selective pressure was maintained, whereas the conventional vector was unstable in the absence of selective pressure. These results confirm the usefulness of the new expression system, which represents a huge improvement over previously described expression systems for the development of BCG into an effective vaccine vector.

Study on the changes of cytokines of splenocytes in mice by immunization with recombinant BCG-Em Ⅱ/3 vaccine against Echinococcus multilocularis / 中国地方病学杂志

Objective To investigate the changes of cytokines of splenocytes in mice immunized with recombinant BCG-Em Ⅱ/3 vaccine of Echinococcus multilocularis(Em)and consequently challenged with Em protoscoleces.Methods Balb/c mice were subcutaneously or intranasally vaccinated and challenged with Em were separated and cultured with EmAg,ConA or PHA,respectively.The supematants were gathered to measure the levels of IL-2,IFN-γ,TNF-α.and IL-4 by ELISA Kits.Results The levels of IL-2,IFN-γ,TNF-α and IL-4 in the subcutaneous group were(34.6±2.7),(34.5±2.8),(265.0 ±0.0)and(9.8±2.6)ng/L respeetively:those in the intranasal group were(32.5±2.2),(33.6±2.7),(130.0±0.0)and(10.4±27)ng/L respectively;those in the control were(25.0±1.9),(30.0±0.0),(10.0±0.0)and(12.5±2.7)ng/L,respectively:there were statistical differences between the immunized groups and control group(P＜0.01 or＜0.05);The level of TNF-α in the subcutaneous group was higher than that in the intranasal group.Conclusion Th1 response has been induced in mice vaccinated with rBCG-Em Ⅱ/3 vaccine and challenged with Em protoscoleces.

Observation on protection by immunization with mix recombinant BCG-EmⅡ/3 and BCG-Em14-3-3 vaccine of echinococcus multilocularis / 重庆医科大学学报

Objective:To investigate protection by immunization with mix recombinant BCG-EmⅡ/3 and BCG-Em14-3-3 vaccine of echinococcus multilocularis(Em) and against challenge with Em protoscoleces. Methods:BALB/c mice were vaccinated by mix recombinant BCG vaccines subcutaneously and intranasally respectively,challenged with Em protoscoleces 8w after vaccination and killed 18w after infection to count rate of reduced alveolar echinococcus weight and to examine the level of IgE,IgG and its subclasses. Blank vector,BCG and PBS served as control. Results:In the groups of immunization.The rate of reduced alveolar echinococcus weight was 45.29%～76.47%,the level of IgG,IgG2a,IgG2b and IgE increased apparently,that of IgG1 and IgG3 decreased remarkably. Conclusion:Intranasal vaccination with mix recombinant BCG-EmⅡ/3 and BCG-Em14-3-3 vaccine may be a good route,IgG,IgG2a,IgG2b and IgE may play an important role in protection induced by this vaccine.

Dynamic observation on subsets of splenocytes in mice by immunization with mixed recombinant BCG-EmⅡ/3 and BCG-Em14-3-3 vaccine of echinococcus multilocularis / 重庆医科大学学报

Objective:To dynamically observe changes of subsets of splenocytes in mice by immunization with mixed recombinant BCG-EmⅡ/3 and BCG-Em14-3-3 vaccine of Echinococcus multilocularis(Em).Methods:BALB/C mice were intranasally vacci- nated with the vaccine and killed to get spleen at 0,2,4,6,8,10,12,14,16 and 18w of immunization respectively.Splenocytes were.separated to measure subsets of CD_4~+ and CD_8~+T cells by FACsort,PBS served as control.Results:In the groups of im- munization,CD_4~+ and CD_8~+subsets increased obviously at 2～12w and 2-18w respectively,which reached the highest level at 6 and 10w respectively.Conclusion:The number of CD_4~+ subsets of splenocytes increases remarkably in the early stage (2～6week)by immunization with mixed recombinant BCG-EmⅡ/3 and BCG-Em14-3-3 vaccine.

Cytokines secretion by splenocytes of mice was promoted by recombinant BCG-EmII/3 vaccine of Echinococcus multilocularis / 基础医学与临床

Objective To dynamically observe changes of cytokines of splenocytes in mice by immunization with recombinant BCG-EmII/3 vaccine of Echinococcus multilocularis(Em).Methods BALB/C mice were intranasally vaccinated and then killed to get spleen on 0、2、4、6、8 and 10w of immunization respectively, splenocytes were isolated to culture with stimulation of EmAg or ConA,their supernatants were gathered to measure the level of IL-2,IFN-?,TNF-? and IL-4 by ELISA Kits. Results In the groups of immunization, levels of IL-2,IFN-?,TNF-? and IL-4 obviously increased on 2～6、2～6、2～10 and 8～10w respectively, reached the highest level on 4、4、8 and 10w respectively. Conclusion TH1 response was induced in mice by rBCG-EmII/3 vaccine by early immunization(2～8w).

Study of effect of recombinant human IFN-?-2b bacilli Calmette-Gu?rin in PBMCs expressing cytokines / 中华泌尿外科杂志

Objective Measure the cytokines including hIFN-?、hIL-12 and hTNF-? which secreted by PBMC stimulated with recombinant BCG, and research how recombinant BCG influence the expression of cytokines and improve the immunologic response. Methods In the experiment, Recombinant hIFN-?-2b-BCG group and wild-type BCG group stimulate peripheral blood monocytes(PBMC) with different density in vitro, which is 0.1 OD and 0.01 OD (1 OD=2.7?107CFU). In third group, we put IFN-?-2b into wild-type BCG so that we can compare it with recombinant BCG. All these components foster with PBMC(4?106/ml), then collect the supernatant in 12 hours, 24 hours, 48hours, 72 hours, 5 day, 7day, and detect hIFN-?、hTNF-?, hIL-12 by an enzyme-linked immynosorbent assay(ELISA), compare the results. Results We can learn from the result, compared with the other groups, recombinant BCG can induce higher density cytokines than wild-type BCG’s and combination group’s(P

Construction of Recombinant BCGs Overexpressing Antigen 85 Complex and Their Protective Efficacy against Mycobacterium tuberculosis Infection in a Mouse Model / 결핵및호흡기질환

Tuberculosis (TB) remains an enormous global health problem, and a new vaccine against TB more potent than the current inadequate BCG vaccine is urgently needed. We constructed three recombinant Mycobacterium bovis BCG (rBCG) strains over-expressing antigen (Ag) 85A, Ag85B, or both of M. tuberculosis using their own promoter and secretory sequence, or hsp60 promoter. SDS-PAGE analysis of rBCG proteins showed over-expression of Ag85A and Ag85B proteins in higher level than of those in their parental strain of BCG. In addition, rBCG(rBCG/B.FA) over-expressing Ag85A and Ag85B induced strong IFN-gamma production in splenocytes. However, there was no significant difference in protective efficacy between rBCG and their parental BCG strain. In this study, therefore, rBCG over-expressing Ag85A, Ag85B, or both failed to show enhanced protection against M. tuberculosis infection in a mouse model.

Changes on subsets of T cells in the spleen of mice by immunization with mixed recombinant BCG-EmⅡ/3 and BCG-Em14-3-3 vaccine against Echinococcus multilocularis / 重庆医科大学学报

Objective:To investigate changes in subsets of T cells in the spleen of mice by immunization with mix recombinant BCG-EmⅡ/3 and BCG-Em14-3-3 vaccine of Echinococcus multilocularis(Em)and against challenge with Em protoscoleces.Methods:BALB/C mice were subcutaneously and intranasally vaccinated with mix recombinant BCG vaccines respectively,then challenged with Em protoscolexes on 8w of vaccination and killed on 18w of infection to get spleen.Spleen cells were separated to measure subsets of CD4+ and CD8+T cells by FACsort,Blank vector,BCG and PBS served as control.Results:In the groups of immunization,CD4+subset increased significantly,as well as the ratio of CD4+/CD8+subset,but CD8+ subset had no change,CD4+ subset and ratio of CD4+/CD8+subset in the subcutaneous group were higher than these in the intranasal group.Conclusion:CD4+T cells subset may bear a part in protection induced in mice by mixed recombinant BCG-EmⅡ/3 and BCG-Em14-3-3 vaccine against challenge with Em protoscoleces.

Protective effect of immunization with mixed recombinant BCG-EmⅡ/3 and BCG-Em14-3-3 vaccine against Echinococcus multilocularis in mice / 第三军医大学学报

Objective To investigate the protection by immunization with mix recombinant BCG-EmⅡ/3 and BCG-Em14-3-3 vaccine of Echinococcus multilocularis(Em) and against challenge of Em protoscolexes.Methods BALB/C mice were subcutaneously or intranasally vaccinated with mixed recombinant BCG vaccines respectively.Eight weeks later,the immunized mice were challenged with Em protoscolexes.On the 18th week after vaccination,the mice were killed to count the rate of reduced alveolar echinococcus weight and to examine the levels of IgG and its subclasses,IgE;the spleen cells were separated to measure subsets of CD4+ and CD8+ T cells by FACsort.Blank vector,BCG and PBS served as control.Results In the immunized groups,the rate of reduced alveolar echinococcus weight was 45.29%-76.47%;the levels of IgG,IgG2a,IgG2b and IgE increased apparently while IgG1 and IgG3 decreased remarkably;CD4+ subset and the ratio of CD4+/CD8+ increased obviously,but CD8+ subset had no change.Conclusion Intranasal vaccination with mix recombinant BCG-EmⅡ/3 and BCG-Em14-3-3 vaccine is a good way for immunization.IgG,IgG2a,IgG2b,IgE and CD4+ T cell subset may play an important role in protecting against challenge of Em protoscolexes.

The reduced apoptosis of spleen cells in infected mice by immunization with recombinant BCG-Eg95 vaccine against Echinococcus granulosus / 中国免疫学杂志

Objective:To investigate apoptosis of spleen cells in infected mice by immunization with recombinant BCG-Eg95 vaccine of Echinococcus granulosus(Eg) and against challenge with Eg protoscoleces.Methods:BALB/c mice were vaccinated with the vaccine subcutaneously,intranasally,orally and intramuscularly respectively.The mice were then challenged with Eg protoscolexes at 8w of vaccination and sacrificed in 18w of infection to get spleen.Spleen cells were separated to measure apoptotic rate by FACsort with BCG and PBS served as control.Results:Apoptotic rate in the immunization group was lower than that in the control.Apoptotic rates in the oral or intramuscular group were significantly lower than that in the subcutaneous or intranasal group.Conclusion:Apoptosis of spleen cells in mice may be induced by infection with hydatid cyst,but is inhibited by immunization with rBCG-Eg95 vaccine.Oral or intramuscular vaccination may be the good regimen.