HBV-CpG Induces Immunity Responses Against Hepatitis B Virus

Persistent infection with the HBV (Hepatitis B Virus) has become a severe public health problem, and more than360million people worldwide affect HBV. The infected individuals tend to progression to liver cirrhosis and hepatocellular carcinoma. The current rHBsAg (recombinant hepatitis B surface antigen) vaccine provides protection against HBV infection, but～10%of people cannot produce neutralizing antibody after vaccination, moreover, the vaccine cannot help HBV-infected patients. Now, antiviral drugs, including the nucleoside analogous and IFN-a, used for the treatment of HBV can suppress viral replication and reduce hepatic symptoms. However, the persistence of HBV cccDNA (covalently closed circular DNA) and defective immune responses lead to treatment failure and progression to severe liver disease. Consequently, it is necessary to develop more efficient therapeutic strategies to eradicate HBV infection.HBV is unlike other enveloped viruses, which seems to avoid inducing strong innate immune responses including the type I IFN (interferon). Therefore, it may play a critical role in finding ways to induce vigorous immune responses against HBV.The unmethylated CpG (cytosine-phosphate-guanosine) motifs presented in microbial DNA can stimulate the immune system by interacting with the TLR9(pattern-recognition receptor Toll-like receptor9). Unmethylated CpG DNA can trigger immune cascades that improves antigen presentation and the secretion of cytokines, including high levels of the type I IFN. A lot of investigations indicate that CpG ODNs can provide not only a basis for improved vaccines, but also immunotherapy for infectious diseases. However, the responses to CpG ODN-based treatments were generally not sustained and was always accompanied by side effects such as toxic shock or deleterious autoimmune reactions. So far, the clinical application of CpG ODNs to HBV has not been achieved.The genome of HBV is partially double-stranded DNA, and also contains CpG motifs. Whether these HBV genome-derived CpG ODNs can induce type I IFN has not been described. In this study, we successfully identified some CpG ODNs from the HBV genome (named HBV-CpG) that are capable of inducing IFN-a production. In addition to identifying HBV-CpG, We also identified inhibitory guanosine-rich ODNs from HBV DNA (named HBV-ODNs) which have capable of inhibiting HBV-CpG-mediated IFN-α production. Nanoparticle-encapsulated HBV-CpG (termed NP(HBV-CpG)) reversed the HBV-ODN-mediated suppression of IFN-α production and activated the innate immune system. In the following study, we focused on the function of NP(HBV-CpG) in prevention and treatment of HBV infection.In our study, we designed and screened HBV-CpG and HBV-ODN by ELISA and flow cytometry. The mechanism of inhibition of HBV-ODN was analyzed by the immunofluorescence.NP(HBV-CpG) reversing the HBV-ODN-mediated-block of IFN-a production was analyzed by ELISA. ELISA and intracellular cytokine staining were used to examine the secretion of IFN-a from HBV patient-derived PBMCs. RIA and ELISA were used to detect the antibody response to HBsAg. HBV carrier mouse models were established by hydrodynamically tail intravenously injection with the pAAV/HBV1.2plasmid. ELISA and flow cytometry were used to detect the production of serum IFN-a and the activation of lymphocytes. A double-emulsion method encapsulates HBV-CpG into nanoparticles. HBsAg and anti-HBsAg antibodies were measured by RIA. The level of HBV DNA and HBcAg was tested by RT-PCR and RIA. Tissue injury was assessed by H&E staining and the measurement of ALT and bilirubin. The intracellular cytokine staining was used to examine the HBsAg-specific IFN-y-producing CD8+T cells. The major findings and conclusions of our study are shown as follows:1. HBV-CpG potently induces the production of IFN-α by human pDCs.We hypothesized that endogenous CpG ODNs from the HBV genome could interact with TLR9to induce immune responses, so an extensive screen was performed to identify the HBV-CpG in the HBV genome. Therefore, we found two candidates to potently induce IFN-α release by PBMCs. We next measured which cell type within the PBMC populations produced IFN-α in response to HBV-CpG. IFN-α was exclusively produced by Lineagel-, CD123+, HLA-DR+cell populations (pDCs) determined by flow cytometry after stimulation with HBV-CpG. So pDCs are the only cells within the PBMC populations that can response to HBV-CpG to produce IFN-α.To determine whether TLR9acted as the HBV-CpG ligand, TLR9expression was determined by flow cytometry after PBMCs were stimulated with HBV-CpG for8h. We found TLR9was significantly upregulated in PBMCs treated with HBV-CpG compared to PBMCs treated with IL-3. What is more, when we made use of chloroquine to block TLR7/9signaling, HBV-CpG-induced IFN-α production completely inhibited. We also found that there was little change in expression of TLR7on pDCs determined by flow cytometry when PBMCs were incubated with HBV-CpG.These data indicate that HBV-CpG induced a potent IFN-a response by human pDCs in a TLR9-dependent but TLR7-independent manner.2. HBV-ODN specifically blocked the HBV-CpG-mediated induction of IFN-a.Guanosine-rich ODNs specifically inhibiting TLR9signaling has been reported. Based on the considerations, we investigated HBV genome and found a high frequency of guanosine repetitive elements. We screened eight inhibitory ODNs derived from HBV genomic sequences (named HBV-ODNs) with guanosine-rich motifs. When we incubated PBMCs with HBV-CpG and different inhibitory HBV-ODNs, we found that the HBV-CpG-induced IFN-a production was inhibited. However, HBV-ODNs could not inhibit CpG-2216-induced IFN-a production. When Gen2.2cells incubated with HBV-CpG, we found that HBV-CpG colocalized with TLR9by the immunofluorescence and confocal microscopy. However, when Gen2.2incubated with HBV-CpG and HBV-ODN, HBV-ODN suppressed the uptake of HBV-CpG by Gen2.2and blocked the colocalization of HBV-CpG with TLR9.Taken together, these data suggest that inhibitory HBV-ODN specifically blocked the endogenous HBV-CpG-mediated induction of IFN-a.3. NP(HBV-CpG) reversed the HBV-ODN-mediated-block of IFN-a production.We used a double-emulsion method to encapsulate HBV-CpG into nanoparticles (termed NP(HBV-CpG)). The coadministration of PBMCs with NP(HBV-CpG) or HBV-CpG and inhibitory HBV-ODNs at a1:1ratio resulted in IFN-a production at barely detectable levels. However, the incubation of NP(HBV-CpG) and HBV-ODNs at a2:1ratio increased IFN-a production. Additional, NP(HBV-CpG) could activate NK, pDCs and T cells to a greater extent than nanoparticles and HBV-CpG. Furthermore, we also found that NP(HBV-CpG) could induce IFN-a production in HBV patient-derived PBMCs.Together, these results suggested that NP(HBV-CpG) could suppress the activity of inhibitory HBV-ODNs and stimulate strong immune responses from PBMCs. 4. NP(HBV-CpG) exerted strong immunostimulatory effects on the lymphocytes of mice.In our previous study we found that NP(HBV-CpG) could activate human PBMCs in vitro. Subsequently, we challenged wild-type mice with NP(HBV-CpG) in vivo. Mice treated intravenously with NP(HBV-CpG) displayed higher levels of CD40and CD80on pDCs and cDCs than mice treated with PBS. Meanwhile, the expression of ICOS and CD69was strongly upregulated on the NK and T cells treated with NP(HBV-CpG) compared to the PBS treated. Furthermore, the coincubation of splenic lymphocytes with NP(HBV-CpG) increased the expression of CD80and CD40on pDC and cDC but also the expression of CD69on NK and T cells in agreement with the in vivo experiment. NP(HBV-CpG) treated also increased the viability of the pDCs and cDCs.These experiments indicate that NP(HBV-CpG) can induce a strong immunostimulatory effect on murine lymphocytes in vitro and in vivo.5. NP (HBV-CpG) enhanced the antibody response to HBsAg.BALB/c and C57BL/6mice were divided into four groups, including the rHBsAg vaccine plus NP(HBV-CpG), the rHBsAg vaccine plus NPs, rHBsAg vaccine alone and untreated control. The level of anti-HBsAg antibodies in the sera of the mice was examined2and4weeks after the final immunization. Immunization with the rHBsAg vaccine plus NP(HBV-CpG) greatly increased the anti-HBsAg antibody levels in both mice. Significantly, NP(HBV-CpG) administration was a large increase in the production of Thl-dependent IgG2a-antibodies.Together, these results suggested that NP(HBV-CpG) as an adjuvant can assist the rHBsAg vaccine in inducing vigorous Thl-biased anti-HBsAg antibody responses.6. NP (HBV-CpG) stimulated robust innate immune responses in HBV carrier mice.Given that NP(HBV-CpG) exerted a strong immunostimulatory effect on wild-type mice, we evaluated the effect of NP(HBV-CpG) on HBV carrier mice. The mouse model established by hydrodynamically tail intravenously injection with the pAAV/HBV1.2plasmid. The HBV carrier mice were administered NP(HBV-CpG), NPs, HBV-CpG or PBS respectively. The NP(HBV-CpG) treatment increased the expression of CD40and CD80on cDCs, ICOS and CD69on NK cells and CD69on CD4+and CD8+T cells. Significantly, high levels of serum IFN-α, but not IFN-β, were observed in the NP(HBV-CpG) treatment.These results indicated that NP(HBV-CpG) can trigger a robust immune response in HBV carrier mice.7. NP(HBV-CpG)-based theatment effectively cleared HBV in HBV carrier mice.Previous study had reported that the HBV carrier mice exhibit tolerance toward HBsAg because HBsAg failed to induce an immune response in these mice. However, our study indicated that NP(HBV-CpG) could stimulate the activation of lymphocyte, especially IFN-α expression in HBV carrier mice. Therefore, we speculated NP(HBV-CpG) may have the effect on the clearance of HBV. In fact, after we treated HBV carrier mice with NP(HBV-CpG) combined with rHBsAg or NPs combined with rHBsAg, we found that the HBsAg level in the serum of carrier mice treated with NP(HBV-CpG)-baesd treatment declined significantly. Actually HBV surface antigenemia disappeared in about90%of carrier mice within the NP(HBV-CpG)-based trerapy. However, the HBsAg level remained high in the mice treated with rHBsAg combined with NPs. Moreover, the HBcAg level in the liver tissues and the HBV DNA level in the serum declined significantly and were undetectable in some of the samples receiving the NP(HBV-CpG)-based treatment. Furthermore, anti-HBsAg antibodies began to appear after HBsAg clearance in the groups treated with NP(HBV-CpG) combined with rHBsAg. Moreover, the percentage of HBsAg-specific IFN-y-producing CD8+T cells upregulated by the NP(HBV-CpG)-based therapy that suggest that the combination treatment augmented CTL function. Meanwhile, the livers of the HBV carrier mice receiving the NP(HBV-CpG) combined administration showed normal architecture with mild inflammatory infiltrate. Normalization of serum bilirubin and ALT further suggested that HBV carrier mice had good tolerance to the NP(HBV-CpG)-based treatment.Taken together, these data suggest that NP(HBV-CpG)-based theatment can effectively eradicates HBV infection, thereby exerting strong anti-HBV activity.