The Effects Of Different Adjuvants On The Immune Activity Induced By Recombinant MUC1-MBP Fusion Protein

Mucin1(MUC1) is a tumor associated antigen, due to the abnormal expressionin tumor tissue, which is considered to be an ideal target for anti-tumorimmunotherapy. Many tumor vaccines based on the MUC1, developing rapidly inthe past years, have entered clinical trials. E. coli maltose binding protein (MBP), isusually used as a fusion protein or purification tag. Recently, our study has showedthat MBP is not only able to improve the immunogenicity of the fusion protein, butits own can also induce cellular immune response. Th1-type cellular immuneresponse and cytotoxic T lymphocyte (CTL) killing activity were importantcharacteristics of an ideal tumor vaccine, while in general, a significant Th2-typeimmune response or B cell immune response was not beneficial to the anti-tumoreffect, which is an important prompt to the development of tumor vaccines.In2001, our group firstly linked6.5MUC1gene repeat sequences and MBPgene and successfully constructed MUC1-MBP recombinant expression vector.After induced by IPTG, the transformed E. coli was able to generate stable andefficient expression of recombinant MUC1-MBP fusion protein. Generally，proteinvaccines have weak immunogenicity, and it is difficult to produce a lasting andeffective immune response, therefore we need suitable adjuvants to enhance theirimmune activity and anti-tumor effect.We will prepare MUC1-MBP fusion protein of high purity firstly and thencompare the effects of different adjuvants on the immune activity induced byrecombinant MUC1-MBP fusion protein by detecting the immune activity to explorethe optimizing adjuvants to effectively stimulate Th1-type cellular immune response,and develop a reasonable program of immunization to lay the foundation for theanti-tumor experiments. 1. Preparation and identification of MUC1-MBP fusion protein and MUC1peptidesTo prepare MUC1-MBP fusion protein, pMAL-MUC1/DH5α were massivelycultured, the expression of MUC1-MBP was induced by0.1mM IPTG. andidentified by SDS-PAGE. The engineering bacteria were broken by ultrasonication,the supernatant was collected, and MUC1-MBP protein was purified by affinitychromatography. The MUC1-MBP protein with over95%purity was obtained. Toget MUC1peptide, MUC1-GST fusion protein saved in my laboratory was digestedby thrombin, then passed through ultra filter with molecular weight cutoff (MWCO)of30kDa to remove thrombin, and further MUC1peptide concentrated by a ultrafilter tube with MWCO of3kDa. The purified MUC1-MBP fusion protein andMUC1peptide were analyzed by Western blotting. The protein concentration wasdetermined by BCA kit.2. The effects of different adjuvants on the immune activity of recombinantMUC1-MBP fusion protein in miceAccording to the different administrating drugs, ICR mice were divided intoseven groups: saline group, MUC1peptides group, MUC1combined with BCGgroup, MUC1-MBP alone group, MUC1-MBP combined with BCG group,MUC1-MBP combined with BCG-PSN group, and MUC1-MBP combined withthymosin α1group. Each group included five animals, and were injected s.c. weeklycorresponding drug on both sides of the groin for a total of two times. Mice weresacrificed at the fourth day of the last immunization. Weighed and recorded mousebody weight, spleen weight, spleen weight/body weight represented the spleenindex.20μg/ml MUC1peptide was used to stimulate spleen lymphocyte cells, andmorphology was observed under a microscope after5days, as well as the specificlymphocyte proliferation was detected by CCK-8kit. Indirect ELISA kit detectedcytokine IFN-γ and IL-4levels in lymphocyte culture supernatant and MUC1specific IgG1and IgG2a levels in mouse serum. The results showed as following: (1) Compared with the control group, the MUC1peptide alone immunizedmice did not change significantly in spleen index, MUC1-specific lymphocyteproliferation index, the level of IFN-γ and IgG2a/IgG1, indicating MUC1peptidealone can not induce Th1immune response; whereas the indexes described abovewere slightly higher in the MUC1peptide combined with BCG immunized mice,although no statistically significant, suggesting that the combination of MUC1peptide and BCG induces weak Th1immune response.(2) Compared with the control group, MUC1-MBP alone immunized mousespleen index, MUC1-specific lymphocyte proliferation index, IFN-γ level andIgG2a/IgG1increased, but no significant difference, suggesting a weak specific Th1immune response; when immunized by MUC1-MBP fusion protein combined withBCG, mouse spleen index, IFN-γ level and IgG2a/IgG1were significantly increased(P <0.01), and MUC1-specific lymphocyte proliferation index also raised(P <0.05), while IL-4levels did not change obviously, suggesting that MUC1-MBPfusion protein combined with BCG strongly induce Th1/Th2tend to Th1cellularimmunity.(3) Compared with the control group, MUC1-MBP combined with BCG-PSNgroup, IgG1and IL-4levels increased (P <0.05), spleen index, IFN-γ and IgG2alevels changed little, while the specific proliferation index and IgG2a/IgG1decreased, suggesting that MUC1-MBP combined with BCG-PSN induces Th2-typeimmune response.(4) For MUC1-MBP combined with Tα1group, mouse spleen index,MUC1-specific lymphocyte proliferation index, IFN-γ level and IgG2a/IgG1increased, but only the differences of IFN-γ level and IgG2a/IgG1had statisticallysignificant compared with the control group (P <0.05), suggesting MUC1-MBPcombined with Tα1immunization has the ability to induce Th1immune response.3. Optimization of MUC1-MBP combined with BCG-immunized programIgG2a/IgG1ratio in the serum is an indirect indicator, which can reflectTh1/Th2cell activation tendency. First, we determined the appropriate doses of BCG. MUC1-MBP fusion protein combining with BCG50mg/kg and150mg/kgevery time, respectively, immunized mice for two times in accordance with theprevious immune method. After the second immunization, we detectedMUC1-specific IgG1and IgG2a levels in mouse serum by ELISA. Then, wedetermined the immunization times. MUC1-MBP combining with BCG50mg/kgimmunized mice, once a week for a total of four times, and the immune method wasthe same as above. The serum was separated after2,3,4immunization and theMUC1-specific IgG2a/IgG1was detected by indirect ELISA. Weighted the miceevery day. The results showed as following:(1) Compared with150mg/kg of BCG，MUC1-MBP immunization combinedwith BCG50mg/kg, MUC1-specific IgG1level was decreased, the IgG2a level andIgG2a/IgG1was raised, suggesting that MUC1-MBP combined with BCG50mg/kginduces perfect Th1immune response. In addition, body weight of mice in the lattersignificantly decreased, which suggests that excessive BCG immunization is notconducive to a Th1immune response and had great side effects.(2) With immunization times increased, MUC1-specific IgG1level graduallyraised, while IgG2a reached a higher level after the second immunization and afterthat, the level had no obvious increase. As a result, the IgG2a/IgG1ratio was highestafter the second immunization. The result suggests MUC1-MBP combined withBCG50mg/kg immunization twice can induce Th1immune response perfectly.In conclusion, this study indicates that BCG as a appropriate adjuvant ofMUC1-MBP fusion protein can strongly induce Th1cell immune response in mice.Moreover we have preliminarily established MUC1-MBP combined withBCG-immunized program, which lay the foundation for the further development ofthe MUC1-MBP anti-tumor vaccine.