Dendritic Cells Expressing A Combined MUC4/PADRE-Derived Polyepitope DNA Vaccine Induce Multiple Cytotoxic T-Cell Responses

Background:The tumor-associated antigen mucin4 is over-expressed on various epithelial malignancies,making it a potentially broadly applicable candidate for vaccine therapy.We report here the creation of a dendritic cell(DC)-based vaccine,using cells transduced with the universal DR-restricted Th helper epitope(PADRE)combined with HLA-A1-and HLA-A2-restricted epitopes from mucin4(MUC4/PADRE).We examined this vaccine's biological characteristics and immune activity in vitro,finding that infection with the polyepitope adenovirus did not alter the typical morphology of mature DC and the typical markers of these cells(CD86,CD83,CD80 and HLA-DR)were highly expressed on rAd-CMV-MUC4/PADRE transducted DCs.Lymphocytes primed with rAd-CMV-MUC4/PADRE-DCs generated potent cytotoxic responses. By contrast,lymphocytes primed with a GFP expressing adenovirus (rAd-CMV-GFP)or mock-transfected DCs were not cytotoxic. Transduction of DCs with an adenovirus encoding PADRE combined with HLA-A1 and HLA-A2 restricted epitopes may be a potential strategy for immunotherapy of MUC4-associated tumors.Objeetives:Constructing recombinant adenovirus vector containing MUC4/ PADRE polyepitope gene,generating peripheral blood mononuclear cells-derived DC,the capability of DCs transfected with recombined adenovirus were tested in inducing cytotoxic T cell effect against target cells,Therefore the study provides laboratory evidence for the application of tumor-specific immunotherapyMethods:The HLA-A1 and HLA-A2 restricted epitopes of the tumor associated antigen,MUC4,were predicted by BIMAS(http://www-bimas. cit.nih.gov/molbio/hla bind/)and ProPred(http://www.imtech.res.in/ raghava/propredl/);the epitopes were directly linked without flanking sequences in the combined polyepitope sequence.Included a Kozak consensus start sequence,the signal peptide of human mucin4,the universal DR-restricted helper epitope,PADRE,a stop codon,and restriction sites to facilitate future modifications.The 468 bp polyepitope gene was synthesized from Sangon,Shanghai.Recombinant adenovirus was synthesized using the AdEasy system.Gel purified polyepitope DNA was digested with BglⅡand XhoⅠand subcloned into the same sites of pAdTrack-CMV.The ligation product was linearized by PineⅠand transformed into competent E.coli BJ5183 cells;backbone plasmid was then transformed into these cells.The resulting homologously recombined adenoviral plasmid,pAdTrack-MUC4/PADRE,was verified by PacⅠdigest and sequencing.The recombinant vector was amplified in DH5αE coli.Control virus was generated using pAdTrack-GFP vector.293A cells were transfected with PacⅠ-linearized recombinant and lipofectin reagent.After 10 days,the cells were harvested and lysed by four cycles of freeze/thaw to obtain viral supernatants.The virus was amplified by repeated infection of 293A cells.Viral particle concentration was determined by TCID50.The virus transducted of COS7 cells to test the protein expression, Infections were carried out at an MOI of 50.After 48 h of infection, cells were trypsinized,collected for lysis,and analyzed by SDS-Page. rAd-CMV-GFP and mock-transfected COS-7 served as controls.DCs were generated from peripheral blood mononuclear cells (PBMCs);after washing with PBS,cells were transferred to 24-well plates.The non-adherent fractions were harvested and frozen for later use in CTL assays;the adherent cells were cultured in DC medium. Adenovirus infection was carried out by incubation of immature DCs at MOI=100.Then fresh DC medium added with TNF-αto induce maturation.Fluorescence and trypan blue staining was used to determine the infection efficiency and percent viable cells.The DC markers were analyzed by FACS.TNF-αmatured,virus-transduced DCs were irradiated,then mixed with autologous PBLs comprised of the non-adherent PBMCs fraction at DCs:PBLs ratio of 1:20.The cells were distributed into CTL medium. After 3d of culture,and as required thereafter,cultures were fed with CTL medium containing IL-7 and IL-2.PBLs stimulated in week 1 were then re-stimulated weekly with fresh virus-transduced DCs at the same ratio for another 2 weeks.On day 21,PBLs were harvested and evaluated by 51Cr release assay.Effecter cells generated from rAd-CMV-GFP and T lymphocytes stimulated by matured DCs without adenovirus infection were used as a control.Target cells were labeled with[51Cr]sodium chromate.Varying numbers of PBLs were added,at the end of the assay, supernatants were obtained and counted by liquid scintillation counter.A standard Elispot assay was carried to test IFN-γlevel secreted by CTLs; the result was analyzed by Elispot reader.Results:1.Construction and identification of combined polyepitope adenovirus recombinant:PCR,PacⅠdigested and sequence analysis all provided the evidence that the pAdTrack-CMV-MUC4/PADRE vector and the rAd-CMV-MUC4/PADRE recombinant plasmid we obtained contained the right and intact MUC4/PADRE gene sequence.The virus particle titer of rAd-CMV-MUC4/PADRE and rAd-CMV-GFP was 1×10~9pfu/ml determined by Karber's assay with resulting 50%tissue culture infective doses(TCID50).Recombined polyepitopes transfect COS-7 cells at MOI=50,SDS-Page revealed a 16.6kD specific protein corresponding to the predicted mass,this band was absent in the GPF-adenovirus and mock-transfected cells.2.Morphological and phenotypic characteristics of PBMC-derived DCs and virus transduced DCs:After 7 days of culture in medium containing GM-CSF,IL-4,and TNF-α,DCs displayed typical morphological characteristics and predominantly gathered in clusters as non-adherent or loosely adherent cells with a larger cell surface and irregular shape.For virus transduced DCs,GFP was observed in more than 90%of the adenovirus transfected DCs at MOI=200.The morphology of adenovirus-transduced DCs and the frequency of expression of the phenotypic markers were similar to mock-infected DCs.This is consistent with other reports that DC was not adversely affected by adenovirus infection.3.Cytotoxicity assays:Autologous lymphocytes were repeatedly stimulated with adenovirus transduced DC,or mock-transduced DC. After three rounds of 21d stimulation,highly specific anti-tumor CTLs were induced.51Cr release assay demonstrated that cytotoxic activity in the rAd-CMV-MUC4/PADRE group was higher than in the pAd-CMV-GFP and T lymphocytes stimulated by matured DCs without adenovirus infection(p＜0.05).The IFN-γlevel secreted from tumor-specific CTLs was carried by standard Elispot assay,when effecter/target at ratio of 40/1,IFN-γsecretion was significantly increased by effecter cells induced by rAd-CMV-MUC4/PADRE compared with rAd-CMV-GFP or T lymphocytes stimulated with DCs only.Conclusions:Our results above suggested that MUC4/PADRE polyepitope gene carried by adenovirus could be transferred into DC efficiently, MUC4-specific and HLA-A2 restricted CTL could be induced by the polyepitope transfected DCs in vitro,such results could be the foundation for the further immunotherapy of MUC4 expressing tumors,and also provide experimental and theoretical basis for the clinical treatments of MUC4 associated malignancies.