Preliminary Study On The Anti-tumor Activity Of A Novel Replicable Double-plasmid DNA Vaccine Against Tumors

Background：Following the application of proplylatic vaccines and with the deepdevelopment of immunology, molecular biology and cell biology, thearaputic vaccineshave achieved great progress in recent years. Through reboosting the patient’s immuneresponse to target tumor antigens, thearaputic vaccines could induce specific immuneresponse in the affected individuals, clear pathogens or abnormal cells and furthermorecure the disease. Therapeutic vaccines have become an important technical means toprevent and treat postoperative recurrence and metastasis of cancers. Due to thesignificant advangtages of the specificity for target antigens, the higher applicationsafety and convenience of manufacturing, several kinds of DNA vaccines as a specialhigh-tech biological therapy have been evaluated in multiple preclinical models andclinical trials. Aiming at―immune tolerance‖, the the core problem in cancer treatmentand based on independently developed replicable DNA vaccine vector system, ourgroup have carried out the immune efficiency strategy research of therapeutic vaccinesand have successfully constructed a novel replicable double-plasmid DNA vaccineagainst tumors. Using the heterologous antigens design technology, we have chosenhCG and Survivin, two tumor antigen widely expressed in a variety of solid tumors,and VEGFR2closely related to tumor angiogenesis, and have constructedmulti-targeted anti-tumor xenogeneic homologous gene complex antigen. Throug theabove strategy, tumor immune tolerance to self antigens could be effectively broken,and cellular and humoral immune responses could be efficiently activated. Furthermore,the clinical cure rate of tumors could be improved.Objective: On the basis of preliminary solid work, we aimed to construct areplicable double-plasmid DNA vaccine consist of CAVA and CAVE. The acquireddouble-plasmid DNA vaccie was delivered into the mice through the combinedapplication of intramuscular injection and electroporation pulse. And then we preliminarily studied the anti-tumor activity and immune mechanism for the DNAvaccines in vitro and in vivo.Methods:1. Human Umbilical Vein Endothelial Cells (HUVEC) were isolated and acquiredby filling Trypsin solution into the lumen of umbilical veins and then were co-culturedwith supernatants of HepG2cells to differentiate them into Tumor derived-EndothelialCells (Td-EC). HUVEC was examined with observation, and its cell biologicalcharacteristic was identified through fluorescence staining Ⅷ-related antigen and flowcytometry assay in vitro. Tumor Endothelial Marker (TEM) expression of Td-EC wasexamined through RT-PCR assay..2. The gene of1-4IgG like domains of mouse VEGFR2and β-hcg were amplifiedand cloned into pET-42a vector respectively. The acquired recombinant prokaryoticexpression plasmids were transformed into E.coli.BL21(DE3) respectively. Theexpression of the two fusion proteins was induced by IPTG. The fusion proteins wereidentified by SDS-PAGE and Western blotting and purified by affinitychromatography respectively. The antigenicity of the purified proteins werecharacterized by ELISA respectively.3. On the solid basis of the plasmid pVAX1-mVEGFR2-GFc-hIL12and the novelreplicable plasmid vector PSVK，the replicable DNA vaccine targeting the xenogeneichomologous gene mVEGFR2D1-4, named CAVE, was recombined.4. The acquired DNA vaccie CAVE and CAVA previously constructed weredelivered into the mice through the combined application of intramuscular injectionand electroporation pulse. The anti-tumor efficacy of the DNA vaccine was observedand the immune mechanism was preliminarily studied: Specific antibody titers andcytokine levels in the serum of mice immunized were detected ELISA assay. Theimmuniaze mice spleen lymphocyte-specific IFN-gamma secretion was detected byELISPOT assay. The anti-angiogenic effect was evaluated by alginate wrapped tumorcell graft and CD31immunohistochemical staining of tumor tissue displaying tumorcapillary density. Result：1. A large number of high purified HVECs have been acquired. And they could beinduced into Td-ECs with satisfactory biological activity.2. The fusion proteins of mVEGFR2D1-4/GST and β-hcg/GST with satisfactorybiological antigenicity were successfully induced and purified respectively.3. The replicable DNA vaccine CAVE was successfully constructed and itseukaryotic expression was demonstrated by Western Blot assay.4. After C57BL/6mice were immunized by combination-or single-use of theCAVA and CAVE plasmids, the morphological results were record. Compared with thePSVKand PBS controls, the inhibitory effect of tumor growth in vaccine groups,especially in the double-plasmid group, was significantly enhanced. The special anti-Survivin, β-hcg and-VEGFR2antibodies were detected in the vaccinated mice serumrespectively. The antigen specific IFN-γ which released by T cells was also detected invaccine immunized mice. Meanwhile, alginate-encapsulated tumor cells assay andCD31immunohistochemical staining of tumor tissue demonstrated that CAVE couldeffectively inhibit tumor angiogenesis.Conclusion：Compared with separate application groups and control groups, therecombinant novel replicable double-plasmid DNA vaccine can induce higher cellularand humoral immune responses and furthermore inhibit tumor growth afterimmunization. This kind of novel DNA vaccines provides new choice to activeimmunotherapy of tumors.