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ObjectiveTherapeutic strategies silencing and reducing the hepatitis B virus (HBV) reservoir, the covalently closed circular DNA (cccDNA), have the potential to cure chronic HBV infection. We aimed to investigate the impact of small interferring RNA (siRNA) targeting all HBV transcripts or pegylated interferon-α (peg-IFNα) on the viral regulatory HBx protein and the structural maintenance of chromosome 5/6 complex (SMC5/6), a host factor suppressing cccDNA transcription. In particular, we assessed whether interventions lowering HBV transcripts can achieve and maintain silencing of cccDNA transcription in vivo.DesignHBV-infected human liver chimeric mice were treated with siRNA or peg-IFNα. Virological and host changes were analysed at the end of treatment and during the rebound phase by qualitative PCR, ELISA, immunoblotting and chromatin immunoprecipitation. RNA in situ hybridisation was combined with immunofluorescence to detect SMC6 and HBV RNAs at single cell level. The entry inhibitor myrcludex-B was used during the rebound phase to avoid new infection events.ResultsBoth siRNA and peg-IFNα strongly reduced all HBV markers, including HBx levels, thus enabling the reappearance of SMC5/6 in hepatocytes that achieved HBV-RNA negativisation and SMC5/6 association with the cccDNA. Only IFN reduced cccDNA loads and enhanced IFN-stimulated genes. However, the antiviral effects did not persist off treatment and SMC5/6 was again degraded. Remarkably, the blockade of viral entry that started at the end of treatment hindered renewed degradation of SMC5/6.ConclusionThese results reveal that therapeutics abrogating all HBV transcripts including HBx promote epigenetic suppression of the HBV minichromosome, whereas strategies protecting the human hepatocytes from reinfection are needed to maintain cccDNA silencing.

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.

ObjectiveA growing body of evidence points to a role for herpesviruses in the development of Alzheimer’s disease (AD) and a reduced risk of AD among patients receiving antiherpetic medications. We investigated the association between herpes simplex virus type 1 (HSV-1) and AD using real-world data (RWD) from USA.DesignIn a matched case–control study, patients with AD aged ≥50 years diagnosed between 2006 and 2021 were identified from the IQVIA PharMetrics Plus claims database. Controls were matched in a 1:1 ratio with subjects with AD on age, sex, region, database entry year and healthcare visit numbers.ResultsThe study included 344 628 AD case–control pairs. History of HSV-1 diagnosis was present in 1507 (0.44%) patients with AD compared with 823 (0.24%) controls. HSV-1 diagnosis was found to be associated with AD (adjusted OR 1.80; 95% CI 1.65 to 1.96). Patients with HSV-1 who used antiherpetics were less likely to develop AD compared with those who did not use antiherpetics (adjusted HR 0.83, 95% CI 0.74 to 0.92).ConclusionsFindings from this large RWD study implicate HSV-1 in the development of AD and highlight antiherpetic therapies as potentially protective for AD and related dementia.

Chronic hepatitis B (CHB) affects approximately 300 million people worldwide and current therapies rarely cure it [...].Chronic hepatitis B (CHB) affects approximately 300 million people worldwide and current therapies rarely cure it [...].

Chronic hepatitis B virus (HBV) infection is characterized by the presence of high circulating levels of non-infectious lipoprotein-like HBV surface antigen (HBsAg) particles thought to contribute to chronic immune dysfunction in patients. Lipid and metabolomic analysis of humanized livers from immunodeficient chimeric mice (uPA/SCID) revealed that HBV infection dysregulates several lipid metabolic pathways. Small molecule inhibitors of lipid biosynthetic pathway enzymes acetyl-CoA carboxylase (ACC), fatty acid synthase, and subtilisin kexin isozyme-1/site-1 protease in HBV-infected HepG2-NTCP cells demonstrated potent and selective reduction of extracellular HBsAg. However, a liver-targeted ACC inhibitor did not show antiviral activity in HBV-infected liver chimeric mice, despite evidence of on-target engagement. Our study suggests that while HBsAg production may be dependent on hepatic de novo lipogenesis in vitro , this may be overcome by extrahepatic sources (such as lipolysis or diet) in vivo . Thus, a combination of agents targeting more than one lipid metabolic pathway may be necessary to reduce HBsAg levels in patients with chronic HBV infection.

Nucleos(t)ide analogs are standard-of-care for the treatment of chronic hepatitis B and can effectively reduce hepatitis B virus (HBV) replication but rarely leads to cure. Nucleos(t)ide analogs do not directly eliminate the viral episome, therefore treatment cessation typically leads to rapid viral rebound. While treatment is effective, HBV DNA is still detectable (although not quantifiable) in the periphery of the majority of nucleos(t)ide analog treated HBV patients, even after prolonged treatment. Addressing whether the detectable HBV DNA represents infectious virus is a key unknown and has important implications for the development of a curative treatment for HBV. The minimum HBV genome equivalents required to establish infection in human liver chimeric mice was determined by titration of HBV patient sera and the infectivity in chimeric mice of serum from patients (n = 7) suppressed to the limit of detection on nucleos(t)ide analog therapy was evaluated. A minimum of 5 HBV genome equivalents were required to establish infection in the chimeric mice, confirming this model has sufficient sensitivity to determine whether serum from virally suppressed patients contains infectious virus. Strikingly, serum from 75% (n = 3 out of 4) of nucleos(t)ide-treated HBV patients with DNA that was detectable, but below the lower limit of quantitation, also established infection in the chimeric mice. These results demonstrate that infectious virus is still present in some HBV patients on suppressive nucleos(t)ide therapy. This residual virus may support viral persistence via continuous infection and explain the ongoing risk for HBV-related complications despite long-term suppression on therapy. Thus, additional treatment intensification may facilitate HBV cure.

Current therapies for chronic hepatitis B virus (HBV) infections are effective at decreasing the viral load in serum, but do not lead to viral eradication. Recent studies highlighted the therapeutic or “adjuvant” potential of immune-modulators. Our aim was to explore the direct anti-HBV effect of Toll-Like-Receptors (TLR) agonists in hepatocytes. HBV-infected primary human hepatocytes (PHH) or differentiated HepaRG cells (dHepaRG) were treated with various TLR agonists. Amongst all TLR ligands tested, Pam3CSK4 (TLR1/2-ligand) and poly(I:C)-(HMW) (TLR3/MDA5-ligand) were the best at reducing all HBV parameters. No or little viral rebound was observed after treatment arrest, implying a long-lasting effect on cccDNA. We also tested Riboxxol that features improved TLR3 specificity compared to poly(I:C)-(HMW). This agonist demonstrated a potent antiviral effect in HBV-infected PHH. Whereas, poly(I:C)-(HMW) and Pam3CSK4 mainly induced the expression of classical genes from the interferon or NF-κB pathway respectively, Riboxxol had a mixed phenotype. Moreover, TLR2 and TLR3 ligands can activate hepatocytes and immune cells, as demonstrated by antiviral cytokines produced by stimulated hepatocytes and peripheral blood mononuclear cells. In conclusion, our data highlight the potential of innate immunity activation in the direct control of HBV replication in hepatocytes, and support the development of TLR-based antiviral strategies.

Identifying signaling pathways that induce B cell response can aid functional cure strategies for chronic hepatitis B infection (CHB). TLR8 activation with ssRNA was shown to enhance follicular helper T cell (T FH ) function leading to improved B cell responses in vitro . We investigated whether this mechanism can rescue an exhausted immune response in CHB infection. Effect of TLR8 agonism on supporting cytokines and T FH and B cells were evaluated using ex vivo and in vitro assays. The ability of an oral TLR8 agonist to promote T FH and B cell response was tested in samples from phase 1b clinical trial. TLR8 agonism induced T FH polarizing cytokine IL-12 in monocytes. Treatment of peripheral blood mononuclear cells (PBMCs) from CHB patients with TLR8 agonists induced cytokine IL-21 by T FH cells with enhanced IL-21 + BCL-6 + and ICOS + BCL-6 + co-expression. Mechanistically, incubation of isolated naïve CD4 + T cells with TLR8 triggered monocytes resulted in their differentiation into IL-21 + ICOS + BCL-6 + T FH in an IL-12 dependent manner. Furthermore, co-culture of these IL-21 producing T FH with autologous naïve B cells led to enhanced memory (CD19 + CD27 + ) and plasma B cell generation (CD19 + CD27 ++ CD38 + ) and IgG production. Importantly, in T FH from CHB patients treated with an oral TLR8 agonist, HBsAg-specific BCL-6, ICOS, IL-21 and CD40L expression and rescue of defective activation induced marker (AIM) response along with partial restoration of HBsAg-specific B cell ELISPOT response was evident. TLR8 agonism can thus enhance HBV-specific B cell responses in CHB patients by improving monocyte-mediated T FH function and may play a role in achieving HBV functional cure.

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).

It is well established that humoral immunity targeting hepatitis B virus surface antigen (HBsAg) plays a critical role in viral clearance and clinical cure. However, the functional changes in HBsAg-specific B cells before and after achieving functional cure remain poorly understood. In this study, we characterized circulating HBsAg-specific B cells and identified functional shifts and B-cell epitopes directly associated with HBsAg loss. The phenotypes and functions of HBV-specific B cells in patients with chronic HBV infection were investigated using a dual staining method and the ELISpot assay. Epitope mapping was performed to identify B cell epitopes associated with functional cure. Hyperactivated HBsAg-specific B cells in patients who achieved HBsAg loss were composed of enriched resting memory and contracted atypical memory fractions, accompanied by sustained co-expression of multiple inhibitory receptors and increased IL-6 secretion. The frequency of HBsAb-secreting B cells was significantly increased after achieving a functional cure. The rHBsAg displayed a weaker immunomodulatory effect on B cells than rHBeAg and rHBcAg . Notably, sera from patients with HBsAg loss reacted mainly with peptides S60, S61, and S76, suggesting that these are dominant linear B-cell epitopes relevant for functional cure. Intriguingly, patients reactive with S76 showed a higher frequency of the HLA class II DQB1*05:01 allele. Taken together, HBsAg-specific B cells were partially restored in patients after achieving a functional cure. Functional cure-related epitopes may be promising targets for developing therapeutic vaccines to treat HBV infection and promote functional cure.