ABSTRACT
Objective: To determine the effects of toll-like receptor 2 (TLR-2) agonist, Pam3CSK4, on cellular and humoral immune response against recombinant Mycobacterium bovis bacille Calmette-Guérin (rBCG) expressing the C-terminus of merozoite surface protein-1 of Plasmodium falciparum. Methods: Six groups of mice (n=6 per group) received intraperitoneal phosphate buffered saline T80 (PBS-T80), BCG or rBCG in the presence or absence of Pam3CSK4. Enzyme-linked immunosorbent assay was carried out to measure serum total IgG, IgG1, IgG2a, and IgG2b production. Spleens were also harvested and splenocytes were co-cultured with rBCG antigen for in vitro determination of IL-4 and IFN-γ via enzyme-linked immunosorbent assay. Results: The production of total IgG and the isotype IgG1, IgG2a and IgG2b was significantly higher in rBCG-immunised mice than in the BCG and PBS-T80-immunised mice, and Pam3CSK4 further enhanced their productions. A similar pattern was also observed in IFN-γ production. Moreover, there was no significant difference in IL-4 production in all groups either in the presence or absence of Pam3CSK4. Conclusions: We present evidence of the adjuvant effects of TLR-2 agonist in enhancing the production of total IgG, IgG1, IgG2a, IgG2b, as well as IFN-γ in response to rBCG. However, the presence or absence of Pam3CSK4 had no effect on IL-4 production.
ABSTRACT
Objective: To determine the effects of toll-like receptor 2 (TLR-2) agonist, Pam3CSK4, on cellular and humoral immune response against recombinant Mycobacterium bovis bacille Calmette-Guérin (rBCG) expressing the C-terminus of merozoite surface protein-1 of Plasmodium falciparum. Methods: Six groups of mice (n=6 per group) received intraperitoneal phosphate buffered saline T80 (PBS-T80), BCG or rBCG in the presence or absence of Pam3CSK4. Enzyme-linked immunosorbent assay was carried out to measure serum total IgG, IgG1, IgG2a, and IgG2b production. Spleens were also harvested and splenocytes were co-cultured with rBCG antigen for in vitro determination of IL-4 and IFN-γ via enzyme-linked immunosorbent assay. Results: The production of total IgG and the isotype IgG1, IgG2a and IgG2b was significantly higher in rBCG-immunised mice than in the BCG and PBS-T80-immunised mice, and Pam3CSK4 further enhanced their productions. A similar pattern was also observed in IFN-γ production. Moreover, there was no significant difference in IL-4 production in all groups either in the presence or absence of Pam3CSK4. Conclusions: We present evidence of the adjuvant effects of TLR-2 agonist in enhancing the production of total IgG, IgG1, IgG2a, IgG2b, as well as IFN-γ in response to rBCG. However, the presence or absence of Pam3CSK4 had no effect on IL-4 production.
ABSTRACT
Objective@#The inhibition function on EB virus positive tumors with rBCG (recombinant BCG) was researched.@*Methods@#After cancer models of EB virus positive tumor cells(GT39)were established in C57BL/6 mice, the mice survival conditions, tumor weight and tumor formation time were analyzed, flow cytometry was used to research the proliferation of CD4+ T. Inhibition of rBCG to cancer was researched, HE staining of the mice tumor tissue was used to detect and analyze lymphocyte infiltration. Single factor analysis method of variance (One-way ANOVA) was processed for rBCG′s inhibition evaluation by SPSS 11.0 statistical software.@*Results@#Compared with the control group, rBCG group significantly slowed tumor growth, tumor formation time was delayed, rBCG significantly prolonged survival time of the mice.@*Conclusions@#rBCG had inhibition to EB virus positive tumors in mice.
ABSTRACT
Objective:To study the antitumor activity and immunological mechanisms of rBCG including GM-CSF and EB virus LMP2A gene fusion.Methods: Animal models of EB virus-positive tumors was built.The formation time of tumors in mice,survival time,tumor weight was analyzed to detect rBCG anti-tumor activity;ELISA method was used to detect the specific antibodies which was produced in the mice stimulated by rBCG,specific CTL killing effect was detected by lactic dehydrogenase assay,ELISPOT was used to assay the secretion of IFN-γand flow cytometry, HE staining of tumor tissue was used to detected lymphocyte infiltration in mice immunized with recombinant BCG.Statistical methods were used for rBCG immunization effect preliminary analysis and evaluation.Results:Comparing to other control,tumor formation time was significantly delayed and tumor growth was slow, survival time of mice prolonged .ELISA test results showed that the rBCG immunization groups of mice could produce specific IgG antibodies of GM-CSF and LMP2A.Specific CTL activity was detected in mice immunized with rBCG.IFN-γsecretion was detected by ELISPOT method, flow cytometry and morphological observation detected tumor tissue infiltration of lymphocytes growth in mice immunized with rBCG.Conclusion:The rBCG induced a humoral and cellular immune responses and induced C57BL/6 mice to produce a strong anti-tumor effect and the EB virus-positive tumor cells was significantly inhibited.
ABSTRACT
Objective To identify the expression of a fusion gene GCA formed from GM-CSF gene and LMP2A gene of Epstein-Barr virus (EBV) in a recombinant BCG (rBCG) and to study its immunoge-nicity.Methods The rBCG was constructed to express the fusion gene GCA and the expressed products were detected by Western blot assay .ELISA was performed to measure specific antibody titers in serum sam-ples from mice immunized with rBCG .Lactate dehydrogenase assay was used to analyze the cellular immuni-ty of mice.A mouse model of EBV-positive gastric carcinoma was established to evaluate the therapeutic effects of rBCG.Results The target proteins of GM-CSF and LMP2A were successfully expressed in rBCG . The specific antibodies were detected in rBCG immunized mice as indicated by ELISA .The maximum anti-body titer reached 1 ∶27 900 [(326.5±7.8) pg/ml] as injection with rBCG 5×108/mouse.The rBCG in-duced cytotoxicity of cytotoxic lymphocytes (CTLs) to EBV-positive gastric carcinoma cells (GT39) (with a killing rate of 89.6%±6.8%) was significantly higher than that of control group (P<0.05) The sizes of tumor in PBS control group [(1964.0±548.7) mm3] and BCG group [(1268.65±72.4) mm3] were big-ger than those in rBCG group [(168.64±78.80) mm3].Conclusion The rBCG expressing GM-CSF and LMP2A fusion gene was successfully constructed .The rBCG could induce humoral and cellular immune re-sponses in mice and inhibit the growth of tumor .
ABSTRACT
Objective To construct a recombinant bacillus Calmette-Guérin(BCG) vaccines based on different tandem repeats of MUC1 and GM-CSF, rBCG-MVNTR1/4/8-CSF, and to observe the ability of three recombinant BCG vaccines in the inhibition of breast cancer. Methods After MUC1 variable-number tandem repeats (MVNTR1/4/8) were cloned in a stepwise manner, the E. coli-Mycobacteria shuttle expression vector pDE22-MVNTR1/4/8-CSF were constructed by fusing MVNTR1/4/8 and GM-CSF, and then used to transform competent BCG by electroporation after identification by restriction endonuclease digestion analysis and DNA sequencing. A novel breast cancer vaccines, rBCG-MVNTR1/4/8-CSF was constructed. The expression of fused MVNTR1/4/8-CSF protiens was analyzed by SDS-PAGE and Western blot. The ability of rBCG vaccines inhibiting the growth of breast cancer was observed in hu-PBL-SCID mice. The specific T cell responses in mice were assessed by immunohistochemistry. Results The expression of recombinant MVNTR1/4/8-CSF fusion proteins were detected by SDS-PAGE and Western Blot in rBCG-MVNTR1/4/8-CSF vaccines, respectively. Tumor incidence in mice prophylactic immunized with rBCG-MVNTR4-CSF (37.5%) or rBCG-MVNTR8-CSF (25%) significantly decreased compared with PBS and BCG-pDE22 control ( 100% ) at 42 days after tumor implantation ( P ＜ 0. 05 ). MCF-7 tumor growth inhibition in rBCG- MVNTR4/8-CSF-immunized mice was more significant than that in controls ( P ＜0. 01 ).The inhibition effect of three rBCG vaccines on breast rumor growth appeared to rise with increase of numbers of the tandem repeats of MUC1. Survival rate was 75% of mice in the rBCG-MVNTR4-CSF-treated group and 87. 5% of mice in the rBCG-MVNTR8-CSF-treated groups at 70 days after tumor implantation; however,survival rate was only 12. 5% in control group( P ＜0. 05). The CD4-positive and CD8-positive lymphocytes were detected only in rBCG-MVNTR4/8-CSF-immunized mice. Conclusions rBCG-MVNTR4/8-CSF immunization inhibits breast cancer growth in mice.
ABSTRACT
Objective To elucidate potential roles of tyrosine kinase 2 (Tyk2) in the generation and maintenance of Ag-specific CD8+ T cells. Methods We followed the fate of OVA-specific CD8 + T cells in Tyk2-deficient ( Tyk2 -/- ) mice after infection with recombinant OVA-expressing BCG ( rBCGOVA ). Because the immunostimulatory BCG-derived peptides recognized by CD8 + T cells have not been defined, and the OVA is definite peptide for specific CD8 + T cells that has been accepted widely, therefore we examine the kinetics of the OVA-Ag-specific CD8 + T cell response after rBCG-OVA infection in mice.Tyk2-/- and wild type(Tyk2+/+ ) mice were inoculated with rBCG-OVA by intra-trachea( i. t. ), after the examination of bacterial growth in the lung and spleen, the population of CD8 + T cells were detected by FACS analysis, the epitope-specific CD8 + T cells were followed with tetrameric H-2Kb molecule folding with OVA257 264 peptide, and the kinetics of Ag-specific CD8 + Tc1 cells were detected by intracellular IFN-γ production in response to OVA257-264 peptide by cytokine FACS analysis. Results After rBCG-OVA challenge,the bacteria number in spleen and lung of Tyk2 -/- mice were significantly larger than those in Tyk2 +/+ mice on days 14, 21 and 49. Almost as same as that in Tyk2+/+ mice, the size of epitope-specific CD8+ T cella with OVA257-264/Kb-tetramer-positive and the CD8 +Tc1 (T eytotoxic 1 )cells positive for intracellular IFN-γ could proliferate to its peak on day 21, then contract and maintain to the memory phase in spleen and lung of Tyk2-/- mice, but the population of CD8+ T cells in spleen and lung of Tyk2 -/- mice were significantly smaller than those in Tyk2+/+ mice on days 21 and 49, the number of epitope-specific CD8+ T cells in spleen and lung of Tyk2 -/- mice were significantly decreased and the frequency of CD8 + Tc1 cells in spleen and lung of Tyk2 -/- mice significantly reduced on day 21,49 and 70 after rBCG-OVA infection. So correspond with the larger number of bacteria in Tyk2-/- mice than those in Tyk2 +/+ mice, the expansion of OVA257-264-specific CD8 + T cells and CD8+ Tc1 response were attenuated in Tyk2 -/- mice following rBCG-OVA infection. Conclusion These results suggest that the lack of Tyk2 signaling impairs the proliferation and difference of effector CD8 + T cell to rBCG-OVA infection and at least, is partly responsible for the susceptible to the rBCG-OVA infection.
ABSTRACT
Objective:To detect the effects of recombinant BCG-Em Ⅱ/3 vaccine on apoptosis of splenocytes in mice challenged with protoscoleces of Echinococcus multilocularis.Methods:BALB/C mice were immunized with rBCG-Em Ⅱ/3 vaccine by subcutaneous injection or intranasal inoculation.After 8 weeks of immunization,all the mice were challenged with 50 protoscoleces of Echinococcus multilocularis by intraperitoneal injection.On the 18th week after infection,mice were killed to separate splenocytes and stimulated with ConA for 16～18h,then apoptosis of splenocytes was detected by Annexin V-FITC staining methods.Results:Apoptotic rate of splenocytes in all groups with or without ConA stimulation was obviously lower than that of PBS group(P