Hepatitis B virus (HBV) persistence is facilitated by exhaustion of CD8 T cells that express the inhibitory receptor programmed cell death-1 (PD-1). Improvement of the HBV-specific T cell function has been obtained in vitro by inhibiting the PD-1/PD-ligand 1 (PD-L1) interaction. In this study, we examined whether in vivo blockade of the PD-1 pathway enhances virus-specific T cell immunity and leads to the resolution of chronic hepadnaviral infection in the woodchuck model. The woodchuck PD-1 was first cloned, characterized, and its expression patterns on T cells from woodchucks with acute or chronic woodchuck hepatitis virus (WHV) infection were investigated. Woodchucks chronically infected with WHV received a combination therapy with nucleoside analogue entecavir (ETV), therapeutic DNA vaccination and woodchuck PD-L1 antibody treatment. The gain of T cell function and the suppression of WHV replication by this therapy were evaluated. We could show that PD-1 expression on CD8 T cells was correlated with WHV viral loads during WHV infection. ETV treatment significantly decreased PD-1 expression on CD8 T cells in chronic carriers. In vivo blockade of PD-1/PD-L1 pathway on CD8 T cells, in combination with ETV treatment and DNA vaccination, potently enhanced the function of virus-specific T cells. Moreover, the combination therapy potently suppressed WHV replication, leading to sustained immunological control of viral infection, anti-WHs antibody development and complete viral clearance in some woodchucks. Our results provide a new approach to improve T cell function in chronic hepatitis B infection, which may be used to design new immunotherapeutic strategies in patients. Chronic hepatitis B virus (HBV) infection is still one of the major public health problems. Two billion people worldwide have been infected with HBV, of whom more than 360 million developed chronic infection. Every year, approximately one million of these individuals will die from HBV-associated liver diseases such as cirrhosis and hepatocellular carcinoma (HCC). Treatment of chronic hepatitis B remains a clinical challenge, and alternative strategies to treat chronic HBV infection are urgently needed. Here, we designed a new combination strategy to enhance the patient's own antiviral immune response and to achieve long-term viral suppression. The therapeutic effect of our combination therapy strategy for chronic hepadnaviral infection was tested in the woodchuck model. We demonstrated that our novel combination therapy could elicit potent antiviral immune response and achieved a strong antiviral effect, leading to sustained immunological control of chronic hepadnaviral infection and complete viral clearance in treated woodchucks. The results of this study may have an impact on clinical trials of the immunotherapy in chronically HBV-infected patients.

A potent therapeutic T-cell vaccine may be an alternative treatment of chronic hepatitis B virus (HBV) infection. Previously, we developed a DNA prime-adenovirus (AdV) boost vaccination protocol that could elicit strong and specific CD8⁺ T-cell responses to woodchuck hepatitis virus (WHV) core antigen (WHcAg) in mice. In the present study, we first examined whether this new prime-boost immunization could induce WHcAg-specific T-cell responses and effectively control WHV replication in the WHV-transgenic mouse model. Secondly, we evaluated the therapeutic effect of this new vaccination strategy in chronically WHV-infected woodchucks in combination with a potent antiviral treatment. Immunization of WHV-transgenic mice by DNA prime-AdV boost regimen elicited potent and functional WHcAg-specific CD8⁺ T-cell response that consequently resulted in the reduction of the WHV load below the detection limit in more than 70% of animals. The combination therapy of entecavir (ETV) treatment and DNA prime-AdV boost immunization in chronic WHV carriers resulted in WHsAg- and WHcAg-specific CD4⁺ and CD8⁺ T-cell responses, which were not detectable in ETV-only treated controls. Woodchucks receiving the combination therapy showed a prolonged suppression of WHV replication and lower WHsAg levels compared to controls. Moreover, two of four immunized carriers remained WHV negative after the end of ETV treatment and developed anti-WHs antibodies. These results demonstrate that the combined antiviral and vaccination approach efficiently elicited sustained immunological control of chronic hepadnaviral infection in woodchucks and may be a new promising therapeutic strategy in patients. Chronic hepatitis B virus (HBV) infection is one of the major causes of liver cirrhosis and liver cancer worldwide. Recommended treatment regimens of chronic hepatitis B based on interferon alpha and nucleot(s)ide analogues do not lead to the satisfactory results. Over the last 20 years, continuous efforts have been undertaken to develop new immunotherapeutic approaches for the treatment of chronic hepatitis B, however, without satisfactory results. We proposed here that the combination of potent antivirals with a prime-boost vaccination protocol that is inducing appropriate virus-specific T-cell responses may restore immune control over HBV. To test this hypothesis we performed a proof-of-principle experiment using woodchucks, a widely accepted animal model of chronic HBV infection. We pretreated animals with entecavir to suppress viral replication and immunized them by a prime-boost regimen with DNA vaccines expressing woodchuck hepatitis virus (WHV) surface and core antigens and adenoviral vectors expressing WHV core antigen. Consistent with our hypothesis, the combination therapy achieved a stronger antiviral effect than the monotherapy alone, leading to sustained immunological control of chronic WHV infection and viral clearance in some animals. These data are encouraging and implicate the feasibility and usefulness of the immunotherapeutic strategies for the treatment of chronically HBV-infected patients.

Nowadays, dried blood spots (DBS) are primarily used to obtain diagnostic access to risk collectives such as intravenous drug users, who are prone to infections with hepatitis B virus (HBV), hepatitis C virus (HCV), and human immunodeficiency virus (HIV). Before DBS analyses can be used in this diagnostic context, however, a comprehensive evaluation of its performance characteristics must be conducted. To the best of our knowledge, the current study presents for the first time such essential data for the Abbott ARCHITECT system, which is currently the worldwide leading platform in this field of infection diagnostics.