The Anti-tumoractivity And Immunological Mechanisms Of A Novel Replicative DNA Vaccinesagainst Cancer

With the development of theory and technology in the field of Immunology,Molecular biology and Cell biology,the more efficient and more safer forms ofvaccines are emerging．In recent years,a new type of vector system based on RNAvirus replication components and with the fi．mction of"self-replication"has alreadybeen developed．It combines the advantages of traditional DNA vaccines,RNAvaccines and RNA replicon vaccines:(1)The replicon DNA vaccine has betterstability and ease of production,storage and transportation．(2)The presence of thereplicase genes leads to more mRNA replication resuking in the superior geneexpression．(3)The self-replication and transcription of the replicon DNA vaccineOCCULT in the cytoplasm,which eventually prevents the risk ofintegration into the hostcell genome and greatly improves the safety．(4)With efficient replication andtranslation,most ofthe resources ofthe host cell is consumed,eventually causing theapoptosis ofthe transfected cells,which will be cleared by the bodG and reducing theimmune tolerance．In summary,the replicon DNA vaccine will be expected to bepromising in the development oftherapeutic vaccines．In order to develop a novel anti—tumor therapeutic vaccines,we constructed therecombinant based on the SFV virus and studied the antitumor efficacy and immunemechanism in vitro and in vivo．In part I of this article,we inserted the gene fragments encoding red and greenfluorescent proteins into the PSCK vector and tested its expression in eukaryotic cells．Then,we inserted the mukitarget complex antigen 2PAG fusion gene(containing thecytotoxic T Lymphocyte epitopes of human Survivin and chorionic gonadotropin pchain—CTP37 of human and monky,human GM—CSF and B7．1,human IgG Fc andGPI anchor signal peptide)into the PSCK vector．Finally,we used flow cytometry andimmunohistochemistry to detect the expression offusion antigen．In part II of this article,the full length of human Surviwin and hCGB cDNAfragments with the restriction enzyme position and 6×his tag were amplified by PCRand inserted into eukaryotic expression vector pIRES—neo．After identification of restriction digestion and PC R_The recombinant plasmids pIRES—neo—SUR-(his)6 andpIRES—neo_hCGB一(his)6 were obtained．Then the vectors were tramsfected into B16cells by lipofectamine 2000．After screening culture by G41 8,a stably tramsfected cellline was established,the transcription and expression of the human Survivin andhCGF gene was identified by RT-PC R_Western blot and immunofluorescence assay．Then,we used these two cell lines inoculated with C57BL／6 mouse in differentconcentration to established Survivin+,hCGF'melanoma tumor—bearing mice model．Part III,BALB/C mice were administered with recombinant luciferase expressingplasmid pGL3--CMV or its mock plasmid:pGL3--basic via intramuscular injection,electric pulse and gene gun respectivey 10pg or 100~tg of the plasmids above wereadminstered via f m,or via electric pulse into musculus quadriceps fexoris．Theplasmids were intradermally deliverred by way of gene gun through 3 bullets,2pg ofplasmids and 4．5Mpa for each shoot．The luciferase activities were monitored over aperiod of 24—144 hour after gene delivery into mice by bioluminescence imaging invivoBased on the previously study above,we developed two different strategies ofimmunization(treatment model and preventive model)to studied anti—tumor efficacyofthe replicative anti—tumor DNA vaccine PSCK-2PFcGB．At the same time,we alsostudied the mechanism of vaccine—induced cellular and humoral immunity．First,wedelivered the DNA vaccine into the tumor-bearing C57BL／6 mice musculusquadriceps fexoris via electric pulse,observed and recorded the tumor growth．Second,we used ELISA assay to detect the specific antibody titers in serum of immunizedmice；used LDH assay to discover the spleen lymphocytes specific CTL responses invitro；using immunized mice'S spleen to detect specific secreted IFN7 by Elispot assay．Finally_tumor infiltrating lymphocytes(TIL)in tumor tissues identified by flowcytometry and histopathology stain(H&E)assay in vitro．The results confirmed that:(1)PSCK vector could express red and greenfluorescent protein in vitro,after identification of restriction digestion and PC R_thenovel replicative anti—tumor DNA vaccines PSCK-2PFcGFB was obtained,theexpression of fusion antigen gene and molecular adjuvant was well．(2)Theeukaryotic expression vector plRES--neo--hCGl3--(his)6 and plRES--neo--hCGl3--(his)6was successfully constructed．A stably tramsfected cell line was established and theexpression rate of Survivin,hCGl3 gene was higher than 90％respectively．Theexpression of the target gene provided a solid experimental foundation for further studies on tumor immunotherapy．Meanwhile,we also established a model ofSurviviI,hCGF tumor cells in C57BL／6 mice．(3)The desired high expression ofexogenous gene could be obtained by electric pulse and gene gun,and the expressionlevels via these two routes are much higher than that via f m．These methods arereliable methods for gene delivery~But,the electric pulse is simpler,higher efficiency,lower cost,the storage and transpo~is more convenient．(4)The results ofmorphological and study ofmechanism immunological showed the high antibody titerwas induced,the antigen specific IFN-r release was detected in immunized mice too,and it was detected that a strong correlation of CTL activity with protective efficiencyagainst tumor cells,such as inhibition of tumor growth,long lifespan and SO on inboth two different strategies ofimmunization(treatment model,preventive model)．In this paper,in order to enhance the DNA vaccine efficacy and safety,we did alot ofworks．First,we applied the SFV-based DNA vaccine vector PSCK,this carriercan maximize the expression of foreign genes and relatively safe．About the tumorantigen,we used the fusion antigen fragment 2PFcGB contained the main T cellepitopes of Survivin and human,monkey hCGF—CTP37 genes(2PAG),at the sametime,we also used some immune adhesion molecules,cytokines and costimulatorymolecules to enhance the immune effect such as human IgG Fc,GM—CSF and B7．1．In the choice of the target cell,we have established two stable expression of humanSurvivin,hCGp mouse melanoma(B16)model respectively and Survivin+,hCGp'tumor cells in mice．At the same time,we used bioluminescence technology to chooseone efficient,stable,convenient way to deliver the DNA vaccine．Finally,we havedesigned two different immunization strategies to evaluate the inhibitory effect ofDNA vaccines．In summary,in this study,we had made a lot ofattemption in the application ofvaccine vector,DNA immunization strategies and delivery methods．The recombinantplasmid PSCK-2PFcGB was correctly constructed．The specific cellular and hnmoralimmune responses can be induced in higher levels after immunization with this novelkind ofreplicative anti-tumor DNA vaccine．These resuks provided a basis for furtherstudy in vaccine phaxmacodynamics and pharmacology and lay a solid foundation forclinical application．This kind ofhovel replicative anti—tumor DNA vaccines providesa new method and ideas for the prevention and treatment ofmalignant tumors．...