Development Of Dendritic Cell Vaccine Presenting Multi-cytotoxic T Lymphocyte Epitopes Against Hepatitis C Virus

Infection with hepatitis C virus (HCV) has become a global healthproblem with a prevalence of170million people infected worldwide.HCV-infected patients can be treated with pegylated interferon-α (PEG-IFN) incombination with ribavirin (RBV). However, this therapy is lengthy, expensive,and only effective in around50%of patients expressing the most commongenotypes (1a/1b). There are many side effects which lead to early withdrawalsor dose reductions that compromise the sustained virological response (SVR).No vaccine is currently available to prevent hepatitis C, therefore, thedevelopment of effective preventive and therapeutic vaccines for HCV is crucial.Multi-epitope DNA vaccine based on epitope prediction and syntheticepitope gene is a hotspot for new vaccines. Epitope, also known as antigenicdeterminant, is a specific site in the antigen which can be recognized by immune cells, and can bind with the virus receptor. Recombinant multi-epitope vaccinecarry antigen epitopes and help epitopes, it can effectively cope with thevariability of pathogenic microorganisms to overcome the genetic restriction ofthe major histocompatibility complex (MHC) molecules, laid the foundation forspecific immunotherapy and gene therapy. In order to stimulate the body’s CD8T cell-mediated immune response potently and widely, you must have multipleepitopes of the different segments of HCV.We have previously reported multi-CTL HCV epitopes for designingHCV vaccines predicted by bioinformatics. We also observed the proliferationof PBMCs and detected the level of IFN-γ in the supernatant of cells stimulatedwith each predicted peptide. These studies showed that the HCV epitopes, NS4B(1793-1801) and P7(774-782) could induce high frequencies of IFN-γproducing T cells, and the specific CTL for other epitopes were not detected inperipheral blood lymphocytes from patients with HCV.DC are professional antigen-presenting cells with potent immunostimulatorycapabilities. Because of their pivotal role in antigen processing and antiviralimmunity, use of ex vivo genetically manipulated DC to augment the immuneresponse is an attractive approach for immunotherapy. Use of viral vectors are amore promising strategy for antigen loading, as viral vectors efficiently expressfull-length endogenous antigen in DC. Expression of full-length antigen allowsDC to present a wide variety of both MHC class I and II epitopes. Researchershave shown that DC infected with adenovirus efficiently elicit a strong andspecific CTL response, and the safety and efficiency of Ad-based DC vaccineshave been documented in many clinical trials.Based on these studies, we constructed recombinant Ads expressingmulti-CTL HCV epitopes and infected DC, which stimulated antiviral T-cell responses.The development of Ad-based DC vaccines in vitro provides a basisfor further immunization studies in vivo.1. Culture of DCPeripheral blood mononuclear cells (PBMC) obtained from the peripheralblood of healthy adults donors were isolated by Ficoll-Hypaque (Sigma) densitygradient separation. CD14+monocytes were isolated using CD14isolation beads(Miltenyi Biotec). The purified cells were adjusted to1×106cells/mL andcultured in serum-free X-vivo15medium (Lonza,04-744Q) in the presence ofrecombinant granulocyte macrophage colony stimulating factor (GM-CSF,PeproTech,1000U/ml) and interleukin-4(IL-4, PeproTech,1000U/ml) insix-well plates at37℃in a5%CO2atmosphere for5days. Add a maturationcocktail to the imDC for the additional final2days. The maturation cocktailconsisted of10ng/mL IL-1β, IL-6, and TNF-α, as well as1μg/mL PGE2(allfrom PeproTech). The cells were evaluated by fluorescence microscopy andflow cytometry.2. Stimulate DC with adenovirus loaded with HCV multi-epitopeRecombinant Ads (Ad-HCV, Ad-positive and Ad-negative) were added tothe immature DC at varying multiplicity of infection (MOI) ranging from50to1,000. After24hours, the Ad-infected cells were evaluated by fluorescencemicroscopy and flow cytometry. The target sequence was detected by specificPCR analysis, and the protein expression was detected by western blot.3. DC stimulate T cells in vitroCollect DC infected with recombinant adenovirus as stimulating cells, takeCD14-PBMC as effector cells in mixed lymphocyte culture. Detect IL-12p70inDC supernatant and IFN-γ in mixed lymphocyte culture supernatant by ELISA.CTL activity was assessed by LDH release assay. Results:1. DC grew half suspended with irregular cell body, the membranesprotruded like tree. Upon examination by fluorescent microscopy, cells showedidentical morphologic changes. DC surface markers CD83, CD86and CD80andHLA-DR were measured by flow cytometry, the results show that surfacemarker expressions of mature DC and adenoviral infected DC were significantlyincreased than immature DC.2. Recombinant adenovirus effectively infected DC, fluorescencemicroscope showed GFP expression. Using specific primers, specificamplification bands at150bp were consistent with size of purpose gene. Usemouse anti-FLAG antibody as first antibody and anti-HRP labeled goatanti-mouse IgG as secondary antibody, Western blot analysis total protein of DCinfected with recombinant adenovirus after48h. Results showed10KD proteins,which are consistent with the size of sequence1-FLAG and sequence2-FLAGfusion protein, indicating that DC successfully expressed sequence1andsequence2protein.3. DC can stimulate T cell proliferation, secretion of IL-12p70byadenovirus infected DC increased, secretion of IFN-γ by stimulated T cellsincreased. DC infected with adenovirus expressing multi-CTL epitopes caninduce specific cellular immune responses.In summary, adenovirus expressing multi-CTL epitopes can effectivelyinfect DC in vitro, promoting T cell responses, to lay the foundation for thedevelopment of anti-HCV DC vaccine.