The structural maintenance of chromosome 5/6 complex (Smc5/6) is a restriction factor that represses hepatitis B virus (HBV) transcription. HBV counters this restriction by expressing HBV X protein (HBx), which targets Smc5/6 for degradation. However, the mechanism by which Smc5/6 suppresses HBV transcription and how HBx is initially expressed is not known. In this study we characterized viral kinetics and the host response during HBV infection of primary human hepatocytes (PHH) to address these unresolved questions. We determined that Smc5/6 localizes with Nuclear Domain 10 (ND10) in PHH. Co-localization has functional implications since depletion of ND10 structural components alters the nuclear distribution of Smc6 and induces HBV gene expression in the absence of HBx. We also found that HBV infection and replication does not induce a prominent global host transcriptional response in PHH, either shortly after infection when Smc5/6 is present, or at later times post-infection when Smc5/6 has been degraded. Notably, HBV and an HBx-negative virus establish high level infection in PHH without inducing expression of interferon-stimulated genes or production of interferons or other cytokines. Our study also revealed that Smc5/6 is degraded in the majority of infected PHH by the time cccDNA transcription could be detected and that HBx RNA is present in cell culture-derived virus preparations as well as HBV patient plasma. Collectively, these data indicate that Smc5/6 is an intrinsic antiviral restriction factor that suppresses HBV transcription when localized to ND10 without inducing a detectable innate immune response. Our data also suggest that HBx protein may be initially expressed by delivery of extracellular HBx RNA into HBV-infected cells.

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GS-9688 (selgantolimod) is an oral selective small molecule agonist of toll-like receptor 8 (TLR8) in clinical development for the treatment of chronic hepatitis B (CHB). In this study, we evaluated the antiviral efficacy of GS-9688 in woodchucks chronically infected with woodchuck hepatitis virus (WHV), a hepadnavirus closely related to hepatitis B virus (HBV). WHV-infected woodchucks received eight weekly oral doses of vehicle, 1 mg/kg GS-9688 or 3 mg/kg GS-9688. Vehicle and 1 mg/kg GS-9688 had no antiviral effect, whereas 3 mg/kg GS-9688 induced a >5 log 10 reduction in serum viral load and reduced WHV surface antigen (WHsAg) levels to below the limit of detection in half of the treated woodchucks. In these animals, the antiviral response was maintained until the end of the study (>5 months after the end of treatment). GS-9688 treatment reduced intrahepatic WHV RNA and DNA levels by >95% in animals in which the antiviral response was sustained after treatment cessation, and these woodchucks also developed detectable anti-WHsAg antibodies. The antiviral efficacy of weekly oral dosing with 3 mg/kg GS-9688 was confirmed in a second woodchuck study. The antiviral response to GS-9688 did not correlate with systemic GS-9688 or cytokine levels but was associated with transient elevation of liver injury biomarkers and enhanced proliferative response of peripheral blood mononuclear cells (PBMC) to WHV peptides. Transcriptomic analysis of liver biopsies taken prior to treatment suggested that T follicular helper cells (T FH) and various other immune cell subsets may play a role in the antiviral response to GS-9688. Conclusion: Finite, short-duration treatment with a clinically relevant dose of GS-9688 is well tolerated and can induce a sustained antiviral response in WHV-infected woodchucks. The identification of a baseline intrahepatic transcriptional signature associated with response to GS-9688 treatment provides insights into the immune mechanisms that mediate this antiviral effect. Approximately 260 million individuals are chronically infected with hepatitis B virus (HBV), and over half a million people are estimated to die each year due to liver diseases associated with chronic hepatitis B (CHB), such as cirrhosis and hepatocellular carcinoma (HCC). Immunological control of CHB ("functional cure") is defined as sustained loss of HBV surface antigen (HBsAg) off treatment, with or without seroconversion to anti-HBs antibody. Several nucleos(t)ide analogs, as well as interferon-alpha (IFN-α), are approved for the treatment of CHB. These therapies reduce viremia and

BaCKgRoUND aND aIMS: GS-9688 (selgantolimod) is a toll-like receptor 8 agonist in clinical development for the treatment of chronic hepatitis B (CHB). Antiviral activity of GS-9688 has previously been evaluated in vitro in HBVinfected hepatocytes and in vivo in the woodchuck model of CHB. Here we evaluated the potential of GS-9688 to boost responses contributing to viral control and to modulate regulatory mediators.appRoaCH aND ReSUltS: We characterized the effect of GS-9688 on immune cell subsets in vitro in peripheral blood mononuclear cells of healthy controls and patients with CHB. GS-9688 activated dendritic cells and mononuclear phagocytes to produce IL-12 and other immunomodulatory mediators, inducing a comparable cytokine profile in healthy controls and patients with CHB. GS-9688 increased the frequency of activated natural killer (NK) cells, mucosalassociated invariant T cells, CD4 + follicular helper T cells, and, in about 50% of patients, HBV-specific CD8 + T cells expressing interferonγ. Moreover, in vitro stimulation with GS-9688 induced NK-cell expression of interferonγ and TNFα, and promoted hepatocyte lysis. We also assessed whether GS-9688 inhibited immunosuppressive cell subsets that might enhance antiviral efficacy. Stimulation with GS-9688 reduced the frequency of CD4 + regulatory T cells and monocytic myeloid-derived suppressor cells (MDSCs). Residual MDSCs expressed higher levels of negative immune regulators, galectin-9 and programmed death-ligand 1. Conversely, GS-9688 induced an expansion of immunoregulatory TNFrelated apoptosis-inducing ligand + NK cells and degranulation of arginase-I + polymorphonuclear MDSCs.CoNClUSIoNS: GS-9688 induces cytokines in human peripheral blood mononuclear cells that are able to activate antiviral effector function by multiple immune mediators (HBVspecific CD8 + T cells, CD4 + follicular helper T cells, NK cells, and mucosal-associated invariant T cells). Although reducing the frequency of some immunoregulatory subsets, it enhances the immunosuppressive potential of others, highlighting potential biomarkers and immunotherapeutic targets to optimize the antiviral efficacy of GS-9688. (Hepatology 2021;0:1-17). C hronic hepatitis B (CHB) remains a global health concern with an estimated 260 million people infected worldwide. CHB

Hepatitis B X protein (HBx) plays an essential role in the hepatitis B virus (HBV) replication cycle, but the function of HBx has been elusive until recently. It was recently shown that transcription from the HBV genome (covalently-closed circular DNA, cccDNA) is inhibited by the structural maintenance of chromosome 5/6 complex (Smc5/6), and that a key function of HBx is to redirect the DNA-damage binding protein 1 (DDB1) E3 ubiquitin ligase to target this complex for degradation. By doing so, HBx alleviates transcriptional repression by Smc5/6 and stimulates HBV gene expression. In this review, we discuss in detail how the interplay between HBx and Smc5/6 was identified and characterized. We also discuss what is known regarding the repression of cccDNA transcription by Smc5/6, the timing of HBx expression, and the potential role of HBx in promoting hepatocellular carcinoma (HCC).