| Tuberculosis (TB) is a chronic respiratory infectious disease caused by the pathogen Mycobacterium tuberculosis (MTB). In recent years, such factors as not enough efficiency of BCG, increasing frequency of drug-resistance MTB, poverty, incidence of HIV-associated infection and increase of population movement, have made TB become the first killer to human health. BCG is currently the only vaccine against TB, but it shows little efficacy on the pulmonary TB of adults. So, developing an efficient new kind of vaccine is important to stop TB.MTB heat shock protein (HSP65) is one of the major immunoreactive proteins during the MTB infection. The study showed that the plasmid encoding HSP65 antigen could significantly reduce the bacterial loads in spleen and lungs of infected mice after intramuscular injection. It was also found that the DNA plasmid expressing HSP65 protein could eliminate the bacteria in TB mice after antibacteria chemotherapy. Cytokine IL-2 is an efficient immunological agent that can increase the antigenic responses, and switches the development of immune responses into Th1 typ which plays an important role in controling TB.AIM To construct the DNA vaccine encoding the fusion protein of MTB HSP65 with human IL-2 and its recombinant Mycobacterium smegmatis(rM.S) vaccine. Compared with BCG, to evaluate the immunological properties and protective efficacy of the two vaccines against MTB infection, in order to find a new efficient vaccine against TB.METHODS AND RESULTS1. Expression and purification of the fusion protein HSP65-IL-2The gene of HSP65 was amplified from DNA genome of Mycobacterium tuberculosis H37Rv strain by dividing it into two parts, the upper and lower fragments, using polymerase chain reaction(PCR). Inserted the PCR product into pMD18-T vector accordingly and sequenced. Amplified the human IL-2 gene from the plasmid pGEM-Teasy-IL-2 with the designed primers which we ligated a 48bp hydrophobic linker at the N termal of the upper primer of IL-2. Inserted the linker-IL-2 PCR product into pMD18-T vector and sequenced. Ligated the right sequenced HSP65 and linker-IL-2 genes into pPRO EX HTa prokaryotic expression vector and identified by restrictive enzyme digestion, the recombinant plasmid was named pPRO-hsp65-IL-2. The recombinant plasmid was transformed into E.coli DH5αstrain and induced with IPTG. The analysis of SDS-PAGE showed that there was a specific protein expression at 78kD molecular marker, and the fusion protein was further identified by Western-blot using anti-hIL-2 mAb. Purified the fusion protein by Ni-NTA purification system under native condition.2. The Construction and expression of HSP65-IL-2 fusion protein in eukaryotic expression vector.Subcloned the fused gene of HSP65-IL-2 into eukaryotic expression vector pcDNA3.1(-), the recombinant plasmid was named pcDNA-hsp65-IL-2 and identified by restrictive enzyme digestion. Transfected the recombinant plasmid into COS-7 cells transiently and P815 cells stably by Fu Gene 6 transfection reagent. The expression of HSP65-IL-2 fusion protein in the transfected cells was detected by indirect immunofluorescence technique(IIFT). The result showed that there was specific green immunofluorescence showed both in the transfected COS-7 cells and P815 cells. Six lines of transfected P815 cells stably expressing the fusion protein of HSP65-IL-2 was selected by G418.3. The immune responses of the DNA vaccine encoding the fusion protein HSP65-IL-2 in miceBALB/c mice were immunized with 100μg/mouse of plasmid pcDNA-hsp65-IL-2 DNA (1μg/μl) in separate sites in both tibialis anterior muscles three times at two weeks intervals. Also set up the BCG and saline control groups. Four weeks after the last DNA injection, the humoral and cellular immune responses induced in the mice were detected. The result showed that the DNA vaccine induced a high antibody titer, the mean is 1:16,000, which was significantly higher than that of BCG group(P<0.01), and the main antibody subtype was IgG2a, a characteristic of Th1 type of immune response. In the cellular immune responses, the index of splenocyte proliferation in DNA group was 2.2, which was higher than that of BCG group(1.9)(P<0.05). The DNA vaccine induced a greater production of IFN-γand IL-2, the amounts of them were 170.62±8.53pg/ml and 37.59±1.88pg/ml respectively, they were significantly higher than that of BCG group (120.24±6.01pg/ml and 34.25±1.71pg/ml, respectively)(P<0.05). The DNA vaccine could induce the proliferation of CD4+T cells and CD8+T in splenolymphocytes of mice significantly compared with the saline control group(P<0.05), the percentages of them were 22.8% and 11.2% respectively, however they were lower than those of BCG group(37.3% and 12.8%, respectively)(P<0.05). Using the transfected P815 cells expressing HSP65-IL-2 protein as the target cells, detected the CTL activity in immunized mice. The result showed that the DNA vaccine could induce a high antigen-specific CTL activity, the CTL activity reached 93.5±4.7% at the E:T ratio of 100:1, which was significantly higher than that of BCG group(46.7±2.2%)(P<0.5).4. The protective efficacy of the DNA vaccine encoding the fusion protein HSP65-IL-2 against MTB infection in miceThe mice were challenged i.v. with 0.2mg/100μl H37Rv after immunizatin, and 6 weeks later, seperated the lung and spleens from the mice aseptically and counted the bacteria numbers. The bacteria loads were expressed as log10CFU. The result showed that the bacteria loads in the spleen and lungs in the saline control group were 5.90±0.30log10 and 5.78±0.29log10 respectively, while the bacteria loads in the spleen and lungs in DNA group were 4.56±0.23log10 and 4.34±0.22log10 respectively. Compared with the saline control group, the DNA vaccine could significantly reduce the bacteria numbers in spleen and lungs (P<0.01) and the reduced bacteria loads were 1.34log10 and 1.44log10 respectively. This implied that the DNA vaccine could protect the mice aginst MTB infection, but the protective ability was not good as that of BCG(P<0.05), the reduced bacteria loads in BCG group were 1.70log10 and 1.88log10 respectively(P<0.05).5. The therapeutic efficacy of the DNA vaccine encoding the fusion protein HSP65-IL-2 in MTB infected miceMice were infected intravenously with 0.2mg/100μl H37Rv. Four weeks after infection, mice were treated with three doses of plasmid DNA administered intramuscularly at two weeks intervals, each mouse received 100μg plasmid DNA (1μg/μl), also set up BCG and saline control group. Six weeks after the last DNA treatment, mice were sacrificed to count the bacteria loads in spleen and lungs in mice, and the bacteria loads were expressed as log10CFU. The result showed that the bacteria loads of spleen and lungs in the saline control group were 6.50±0.33log10 and 6.38±0.32log10 respectively. While the bacteria loads in spleen and lungs in the DNA group were 6.24±0.31log10 and 6.00±0.30log10 respectively, the DNA vaccine could significantly reduce the bacteria numbers in TB mice compared with the saline group(P<0.05) and the reduced bacteria loads in spleen and lungs in DNA group were 0.26log10 and 0.38log10. This implied that the DNA vaccine had therapeutic effect on TB mice, but the efficiency was not good as that of BCG(P<0.05), while in BCG treated TB mice, the reduced bacteria loads in spleen and lungs were 0.60log10 and 0.52log10 respectively (P<0.01).6. The construction and expression of the recombinant M.smegmatis secreted expressing HSP65-IL-2 fusion proteinSubcloned the HSP65-IL-2 fused gene into the E.coli-mycobacterium shuttle plasmid pDE22. The recombinant plasmid was identified by restrictive enzyme digestion and the positive plasmid was named pDE22-hsp65-IL-2. Transformed the plasmid into M.S competent cells and selected by hygromycine. The positive rM.S was identified by PCR and the result showed that there were specific amplification of the aimed genes. Enlarged cultured rM.S and collected the supernatant which were freeze-dried to be analysized by Western-blot. The result showed that there was specific protein expressed and its size was consistent with the predicted HSP65-IL-2 fusion protein. Using IIFT to detect the fusion protein expressed in bacterium body of rM.S, the result also showed that there was specifc green immunofluorescence observed, this meant that rM.S could express and contain the goal protein.7. The immune responses of rM.S against MTB infection in miceBALB/c mice were immunized subcutaneously with 0.2mg/100μl rM.S, BCG respectively, and also set up the saline control group. One week or 4 weeks after the immunization, the humoral and cellular immune responses induced in the immunized mice was evaluated. The result showed that 1 week after immunization, rM.S could induce a high level of antibody in the mice, the titer reached to 1:16,000, which was significantly higher than that of BCG group(1:200)( P<0.01) and the main antibody subtype was IgG2a. Four weeks afer immunization, the antibody titer in rM.S group decreased significantly, and the main antibody subtype turned into IgG1. The antibody titer in BCG group was also detected, but the level of antibody was always lower and the profile of antibody subtype was in the balance of IgG1/IgG2a. In the cellular immune responses, the immunoreaction in rM.S group was also stronger at 1 week after immunization than that of 4 weeks. After 1 week of immunization, the splenolymphocyte proliferation index in rM.S group was 6.21, which was significantly higher than that of BCG group(3.33) (P<0.01) and saline control group(1.01) (P<0.01). The levels of IFN-γand IL-2 were 115.00±4.11pg/ml and 27.41±3.67pg/ml respectively, they were also higher than those of saline control group (57.42±2.87pg/ml and 9.53±1.32pg/ml, respectively) (P<0.01), and the IFN-γlevel was also higher than that of BCG group at this time point (99.37±8.87pg/ml) (P<0.05), but the IL-2 level was not different from that of BCG group (31.21±2.87pg/ml) (P>0.05). The percentages of CD4~+T cells and CD8~+ T cells in splenolymphocytes of mice induced by rM.S were 50.4% and 14.7% respectively, they were both higher than those of saline control group (20.5% and 8.7%, respectively) (P<0.01), and the percentage of CD4~+T cells were also higher than that of BCG group (43.2%) (P<0.05), but the value of CD8~+T cells were not different with that of BCG group (14.2%) (P>0.05). The activity of CTL induced by rM.S reached to 84.2±4.2%. at the E:T ratio of 100:1, which was significantly higher than that of BCG group(P<0.01) and saline control group(P<0.01).Four weeks after immunization, the splenolymphocyte proliferation index of rM.S group was 1.91, which was compared to that of BCG group(P>0.05), but higher than that of saline control group(1.15) (P<0.05), also it was lower than that of 1 week after rM.S immunization(P<0.05). The levels of IFN-γand IL-2 in rM.S group were decreased to 82.84±3.36pg/ml and 16.62±2.02pg/ml respectively, they were lower than those of 1 week after rM.S immunization and they were also lower than that of BCG group respectively(117.60±7.91pg/ml and 30.50±4.63pg/ml, respectively)(P<0.05), but still higher than that of saline control group(59.03±2.95pg/ml and 10.20±1.51pg/ml, respectively) (P<0.05). The percentage of CD4~+T cells in splenolymphocytes of mice induce by rM.S was 27.0%, which was higher than that of BCG group (P<0.05) and saline control group(20.5%) (P<0.05), but the value of CD8~+T cells was lower than that of saline control group(6.8%) (P<0.05), however equal to that of BCG group (4.8%) (P>0.05), the total percentage of CD4~+T cells and CD8~+T cells in BCG group was not different from that of saline control group(P>0.05), but in rM.S group it was still higher than that of saline control group(P<0.05), however it was lower than that of 1 week after immunization in both of rM.S and BCG groups(P<0.05). The activity of CTL in rM.S group were 62.3±3.1% at the E:T ratio of 100:1, which was higher than that of BCG group(P<0.05) and saline control group(P<0.05), but lower than that of 1 week after rM.Simmunization(P<0.05).8. The protective efficacy of rM.S vaccine against MTB infection in miceFour weeks after immunization with rM.S or BCG, mice were challenged i.v. with 0.2mg/100μl H37Rv, and 6 weeks later, seperated the lung and spleens from the mice aseptically and counted the bacteria loads, which were expressed as log10CFU. The result showed that the bacteria numbers in the spleen and lungs in rM.S immunized mice were 5.26±0.13log10 and 5.78±0.34log10, while the bacteria loads in the saline control group were 6.08±0.15log10 and 6.33±0.21log10 respectively. Compared with the saline group, the reduced bacteria loads in spleen and lungs in rM.S group were 0.82log10 and 0.55log10 (P<0.05), which implied that rM.S could protect the mice against MTB infection. In BCG group, the bacteria loads in spleen and lungs were 4.90±0.24log10 and 5.38±0.19log10 respectively, compared with saline control group, the reduced bacteria loads were significantly higher than those of rM.S group(P<0.05).9. Conclusion Both the DNA vaccine encoding HSP65-IL-2 fusion protein and the rM.S vaccine expressing the HSP65-IL-2 fusion protein could elicite great humoral and cellular immune responses. The immunoreaction induced by rM.S reached the highest after one week of immunization and it was stronger than that of BCG group, but it was also faded away quickly. Both the DNA vaccine and rM.S could significantly reduce the bacteria loads in spleen and lungs of infected mice, this meant that the two vaccines could prevent the mice against MTB infection, but the ability of protection was not better than that of BCG. In the future, we will try other methods of immunization, way of vaccination and animal models to improve the immunogenicity and protective efficacy of the two vaccines.
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