Preliminary Study On The Anti-tumor Activity Of DNA Vaccine Against Prostate Cancer

Background:Prostate cancer is still a significant public health problem. Most patientseventually develop into androgen-independent PCa which is poorly responsive totraditional therapies. Immunotherapy aimed at boosting the patient's immune responseto tumor antigens represents a promising treatment option for advanced and metastaticdisease. DNA vaccines are one approach of immunotherapy that has been evaluated inmultiple preclinical models and clinical trials. The safety, specificity for the targetantigen, ease of manufacturing makes DNA vaccines particularly relevant for futuredevelopment. However, DNA vaccines have shown low efficiency of cell transfectionand immunogenicity which induce weak immune responses. Therefore, there areseveral strategies in our study taken into account for improving the DNA vaccinationefficacy, such as antigen selection, use of adjuvant and delivery systems improvement.The combination of these approaches may contribute to the optimization of the DNAvaccines for prostate cancer.Objective: In order to develop a novel anti-prostate cancer DNA vaccine, weconstructed the recombinant plasmid pVAX1-mPSCA-IRES-mIL-12which couldco-express PSCA and adjuvant IL-12in the same vector. And then we preliminarilystudied the anti-tumor activity and immune mechanism for the DNA vaccines in vitroand in vivo.Methods:1. The gene of mIL-12was amplified by PCR and cloned into downstream ofIRES gene in plasmid pVAX1-IRES to construct the plasmid vectorpVAX1-IRES-mIL-12. On this basis, the gene of mPSCA was amplified by RT-PCRfrom mouse prostate cancer cell line RM-1and cloned into upper stream of IRES geneto construct the DNA vaccine pVAX1-mPSCA-IRES-mIL-12. Then these plasmids were transfected into293T cells. The expression of the mPSCA and mIL-12gene wasidentified by ELISA and Western Blot assay respectively. Then the plasmid wasadministrated into mice by intramuscular electroporation (EP) for examining theexpression of mPSCA and IL-12gene in vivo. In this part we also have constructed theplasmid pVAX1-mPSCA as a control and examined its expression in vivo and in vitro.2. At same time, mPSCA gene was cloned into pET-42a prokaryotic expressionvector. The mPSCA protein was expressed and purified. Then the mPSCA wasidentified by SDS-PAGE and Western blot test, and its antigenicity was identified byELISA.3. C57BL/6mice were immunized by intramuscular EP using different plasmids.And then the anti-tumor activity of each plasmid was compared. Firstly, we observedand recorded the tumor growth. Meanwhile, we also studied the possible mechanism ofvaccine-induced cellular and humoral immunity. ELISA was used to detect the specificantibody titers in serum of immunized mice and the serum cytokines level; LDH assaywas used to investigate the specific CTL responses of spleen lymphocytes in vitro;ELISPOT assay was used to detect specific secreted IFN-γ for immunized mice'sspleen lymphocytes. Finally, tumor infiltrating lymphocytes (TIL) in tumor tissues wasidentified by flowcytometry. Histopathology stain (H&E) assay of several tissues wasused to find the change of immunized mice's tissues.Result:1.The plasmids pVAX1-IRES-mIL-12, pVAX1-mPSCA andpVAX1-mPSCA-IRES-mIL-12was successfully constructed and confirmed by enzymedigestion and DNA sequencing． The plasmids pVAX1-IRES-mIL-12andpVAX1-mPSCA IL-12showed successful expression mIL-12and mPSCA respectivelyin vitro and in vivo, while the plasmid pVAX1-mPSCA-IRES-mIL-12couldco-express the mPSCA and mIL-12in vitro and in vivo.2. The mPSCA prokaryotic expression vector was successfully constructed andthe fusion protein about40KD was purified．Western Blot and ELISA demonstrated theantigenicity of the purified mPSCA protein. 3. After C57BL/6mice were immunized by intramuscular EP using differentplasmids, the morphological results were record. The time of tumor forming in vaccinegroup was longer than the control groups (9.0d Vs5.8d). The new vaccine inhabitstumour growth as compared with the controls, and the tumor control rate was77.96%.The study of possible mechanism showed the high antibody titer(>12800) wasinduced in vaccine group. The antigen specific IFN-γ which released by T cells wasalso detected in vaccine immunized mice about2430.00±157.36SFU/106splenocytes.Meanwhile, cytotoxic T lymphocytes (CTLs)-mediated cytotoxicity was33.19±0.24%,26.67±0.73%and20.44±0.26%when effector-target ratio was40:1,20:1and10:1. Meanwhile, there were no histological evidences of infiltration byinflammatory cells and tissue damage in other PSCA expressing organs.Conclusion:In summary, we had made some new attempts in antigen selection, use ofadjuvant and delivery methods in this study. The recombinant plasmidpVAX1-mPSCA-IRES-mIL-12was correctly constructed. This DNA vaccine caninduce the higher cellular and humoral immune responses and inhibit tumor growthafter immunization compared with control groups, and there were no histologicalevidence of autoimmune tissue injury. This kind of novel DNA vaccines provides newexploration to overcome previous immunogenicity limitation in DNA vaccinetechnology.