Hepatitis B virus (HBV) is a major global health concern, and the development of curative therapeutics is urgently needed. Such efforts are impeded by the lack of a physiologically relevant, pre-clinical animal model of HBV infection. Here, we report that expression of the HBV entry receptor, human sodium-taurocholate cotransporting polypeptide (hNTCP), on macaque primary hepatocytes facilitates HBV infection in vitro, where all replicative intermediates including covalently closed circular DNA (cccDNA) are present. Furthermore, viral vector-mediated expression of hNTCP on hepatocytes in vivo renders rhesus macaques permissive to HBV infection. These in vivo macaque HBV infections are characterized by longitudinal HBV DNA in serum, and detection of HBV DNA, RNA, and HBV core antigen (HBcAg) in hepatocytes. Together, these results show that expressing hNTCP on macaque hepatocytes renders them susceptible to HBV infection, thereby establishing a physiologically relevant model of HBV infection to study immune clearance and test therapeutic and curative approaches.

T cell therapy is a promising means to treat chronic hepatitis B virus (HBV) infection and HBV-associated hepatocellular carcinoma. T cells engineered to express an HBV-specific T cell receptor (TCR) may cure an HBV infection upon adoptive transfer. We investigated the therapeutic potential and safety of T cells stably expressing high-affinity HBV envelope– or core–specific TCRs recognizing European and Asian HLA-A2 subtypes. Both CD8⁺ and CD4⁺ T cells from healthy donors and patients with chronic hepatitis B became polyfunctional effector cells when grafted with HBV-specific TCRs and eliminated HBV from infected HepG2-NTCP cell cultures. A single transfer of TCR-grafted T cells into HBV-infected, humanized mice controlled HBV infection, and virological markers declined by 4 to 5 log or below the detection limit. Engineered T cells specifically cleared infected hepatocytes without damaging noninfected cells when, as in a typical clinical setting, only a minority of hepatocytes were infected. Cell death was compensated by hepatocyte proliferation, and alanine amino transferase levels peaking between days 5 and 7 normalized again thereafter. Cotreatment with the entry inhibitor myrcludex B ensured long-term control of HBV infection. Thus, T cells stably transduced with highly functional TCRs have the potential to mediate clearance of HBV-infected cells, causing limited liver injury.

Available treatments for hepatitis B can control the virus but are rarely curative. This led to a global initiative to design new curative therapies for the 257 million patients affected. Discovery and development of these new therapies is contingent upon functional in vitro and in vivo hepatitis B virus (HBV) infection models. However, low titer and impurity of conventional HBV stocks reduce significance of in vitro infections and moreover limit challenge doses in current in vivo models. Therefore, there is a critical need for a robust, simple and reproducible protocol to generate high-purity and high-titer infectious HBV stocks. Here, we outline a three-step protocol for continuous production of high-quality HBV stocks from supernatants of HBV-replicating cell lines. This purification process takes less than 6 h, yields to high-titer stocks (up to 1 × 10¹¹ enveloped, DNA-containing HBV particles/mL each week), and is with minimal equipment easily adaptable to most laboratory settings.

Background: Type III interferons (IFNs) (λ1–3) activate similar signaling cascades as type I IFNs (α and β) via different receptors. Since IFN-α and lymphotoxin-β activate cytosine deamination and subsequent purging of nuclear hepatitis B virus (HBV) DNA, we investigated whether IFN-β and -λ may also induce these antiviral effects in differentiated HBV-infected hepatocytes. Methods: After determining the biological activity of IFN-α2, -β1, -λ1, and -λ2 in differentiated hepatocytes, their antiviral effects were analyzed in HBV-infected primary human hepatocytes and HepaRG cells. Results: Type I and III IFNs reduced nuclear open-circle DNA and covalently closed circular DNA (cccDNA) levels in HBV-infected cells. IFN-β and -λ were at least as efficient as IFN-α. Differential DNA-denaturing polymerase chain reaction and sequencing analysis revealed G-to-A sequence alterations of HBV cccDNA in IFN-α, -β, and -λ–treated liver cells indicating deamination. All IFNs induced apolipoprotein B messenger RNA–editing enzyme–catalytic polypeptide-like (APOBEC) deaminases 3A and 3G within 24 hours of treatment, but IFN-β and -λ induced longer-lasting expression of APOBEC deaminases in comparison to IFN-α. Conclusions: IFN-β, IFN-λ1, and IFN-λ2 induce cccDNA deamination and degradation at least as efficiently as IFN-α, indicating that these antiviral cytokines are interesting candidates for the design of new therapeutic strategies aiming at cccDNA reduction and HBV cure.