Preparation And Functional Analysis Of HBcAg-pulsed Dendritic Cell Vaccine For Therapeutic Application In Patients With Chronic Hepatitis B

Chronic hepatitis B virus (HBV) infection remains a serious challenge to public health worldwide, and patients with chronic hepatitis B (CHB) are at risk of developing serious complications like liver cirrhosis and hepatocellular carcinoma (HCC). However, there is no specific curative therapy for CHB patients currently. Antiviral drugs possess considerable side effects, and have limited efficacy. So, novel therapeutic strategies should be developed to inhibit HBV replication efficiently in CHB patients and to control the disease progression.A dendritic cell (DC) vaccine is defined as DCs loaded with definite antigen which can induce an antigen-specific T cells response in vivo. DC vaccine has been administrated in the clinic, and has been proven to be feasible, nontoxic and effective in some cancer patients. Recent studies have shown that HBV specific DC vaccine pulsed with hepatitis B surface antigen (HBsAg) could efficiently reverse immune tolerance to HBV and induce the HBV-specific cytotoxic T lymphocyte (CTL) responses in transgenic mice, and could prime HBV-specific immune responses in healthy volunteers and patients with CHB. Although these results demonstrated that the HBV-specific DC vaccine has shown great promise for the treatment of CHB patients, however, the standardized preparation processes and quality control of HBV specific DC vaccine have not been described, which are very important for clinical application of DC vaccine. To address aforementioned issues, here, we mainly described the preparation processes and quality control of hepatitis B core antigen (HBcAg)-pulsed monocyte-derived dendritic cells (MoDCs) from HBV infected patients, and further evaluated their function in vitro.First, quality control were implemented in DC vaccine preparation process from patients with HBV-associated HCC in vitro, including the PBMC collection, monocyte adherence, monocyte differentiation into immature MoDC, HBcAg loading, MoDC maturation and harvest, and characteristic of phenotype and function. We found that the purity of MoDCs was more than 70% and viability of MoDCs could reach to 95% during the culture period. In addition, the prepared HBcAg-pulsed DC vaccine (cDCs) had high levels of MHC and costimulatory molecules expression, and had capacities to secrete an amount of IL-12 and to induce autologous T cells proliferation and IFN-γ secretion as compared with imaDCs (immature MoDCs) and conDCs (mature MoDCs without HBcAg pulse). We also found that cryopreservation and thaw did not significantly influence the phenotype and function of cDCs. During the whole process of DC vaccine preparation, the detection of origin of fever, bacterial and fungal contamination were carefully done. These processes provided the basis of preparing qualified DC vaccine in vitro for clinic application.Furthermore, we investigated the capacity of HBcAg-pulsed DC vaccine derived from CHB patients to stimulate CD4 and CD8 T cells to produce IFN-γ and IL-4 in vitro. We found that cDCs derived from CHB patients had more strong capacity to stimulate autologous CD4 and CD8 T cells to produce IFN-γ than to produce IL-4, These results indicated that cDCs derived from CHB patients had capacity to polarize autololgous T cells towards Th1 in vitro. Furthermore, we investigated the ability of cDCs derived from HLA-A2~+ CHB patients to induce HBV specific CTLs from autologous T cells with pentamer staining assay and found that cDCs could induce the expansion of HBV core18-27 specific CTLs in vitro. These induced HBV core18-27 specific CTLs had specific cytotoxicity against T2 cells which had been loaded with HBV core18-27 peptide in vitro. These data strongly suggested that HBcAg-pulsed DC vaccine could induce T cells to differentiate into HBV specific CTLs, secreting antiviral cytokines and exerting specific cytotoxic effect against target cells.Finally, we further investigated that whether CpG-treated plasmacytiod DCs (pDCs) could enhance the function of HBcAg-pulsed DC vaccine in vitro. We first found that CpG-ODN2216 could activate pDCs to secrete an amount of IFN-α. These CpG-treated pDCs could significantly up-regulate the expressions of costimulatory molecules on cDCs and also significantly improve cDCs to produce more IL-12 and to induce more autologous T cells proliferation and secretion of IFN-γ. In addition, we also found that the supernatants from CpG-treated PBMCs had similiar efficency with cDCs. Blockade of IFN-α ould reverse the effects of CpG-treated pDCs or supernatants on cDC function. These data indicated that pDCs activated by CpG-ODN2216 could enhance function of HBcAg-pulsed DC vaccine via secreting a large amount of IFN-α in vitro.Our results indicated that HBcAg-pulsed DC vaccine derived from CHB patients could efficiently induce autologous T cells proliferation, Th1 polarization, and generation of HBV core18-27 specific CTLs. Our data also indicated that CpG-ODN2216 might be used as an efficient adjuvant to enhance the efficacy of HBV-specific DC vaccine in clinical trial. These researches provided an usful clew to prepare DC vaccine with clinic grade.