Chronic hepatitis B virus (HBV) infection continues to be a major health burden worldwide; it can cause various degrees of liver damage and is strongly associated with the development of liver cirrhosis and hepatocellular carcinoma. The molecular mechanisms determining HBV persistence are not fully understood, but these appear to be multifactorial and the unique replication strategy employed by HBV enables its maintenance in infected hepatocytes. Both the stability of the HBV genome, which forms a stable minichromosome, the covalently closed circular DNA (cccDNA) in the hepatocyte nucleus, and the inability of the immune system to resolve chronic HBV infection are believed to be key mechanisms of HBV chronicity. Since a true cure of HBV requires clearance of intranuclear cccDNA from infected hepatocytes, understanding the mechanisms involved in cccDNA biogenesis, regulation and stability is mandatory to achieve HBV eradication. This review will summarize the state of knowledge on these mechanisms including the impact of current treatments on the cccDNA stability and activity. We will focus on events challenging cccDNA persistence in dividing hepatocytes.

Bulevirtide (BLV) is a first-in-class entry inhibitor and the only approved treatment for patients chronically infected with HDV in Europe. We aimed to investigate the efficacy of BLV treatment in paired liver biopsies obtained at baseline and after 24 or 48 weeks of treatment. We performed a combined analysis of 126 paired liver biopsies derived from three clinical trials. In the phase II clinical trial MYR202, patients with chronic hepatitis D were randomised to receive 24 weeks of BLV at 2 mg, 5 mg or 10 mg/day. Patients in MYR203 (phase II) and MYR301 (phase III) received 48 weeks of BLV at 2 mg or 10 mg/day. Tenofovir disoproxil fumarate monotherapy or delayed treatment served as comparators. Virological parameters and infection-related host genes were assessed by qPCR and immunohistochemistry. At week 24, median intrahepatic HDV RNA decline from baseline was 0.9Log₁₀ with 2 mg (n = 7), 1.1Log₁₀ with 5 mg (n = 5) and 1.4 Log₁₀ with 10 mg (n = 7) of BLV. At week 48, median reductions were 2.2Log₁₀ with 2 mg (n = 27) and 2.7Log₁₀ with 10 mg (n = 37) of BLV, while HDV RNA levels did not change in the comparator arms. Notably, a drastic decline in the number of hepatitis delta antigen-positive hepatocytes and a concomitant decrease in transcriptional levels of inflammatory chemokines and interferon-stimulated genes was determined in all BLV-treatment arms. Despite the abundance of HBsAg-positive hepatocytes, replication and covalently closed circular DNA levels of the helper virus HBV were low and remained unaffected by BLV treatment. Blocking viral entry diminishes signs of liver inflammation and promotes a strong reduction of HDV infection within the liver, thus suggesting that some patients may achieve HDV cure with long-term treatment. Chronic infection with HDV causes the most severe form of viral hepatitis, affecting approximately 12 million people worldwide. The entry inhibitor bulevirtide (BLV) is the only recently approved anti-HDV drug, which has proven efficacious and safe in clinical trials and real-word data. Here, we investigated paired liver biopsies at baseline and after 24 or 48 weeks of treatment from three clinical trials to understand the effect of the drug on viral and host parameters in the liver, the site of viral replication. We found that BLV treatment strongly reduces the number of HDV-infected cells and signs of liver inflammation. This data implies that blocking viral entry ameliorates liver inflammation and that prolonged treatment regimens might lead to HDV cure in some patients. This concept will guide the further development of therapeutic strategies and combination treatments for patients with CHD. NCT03546621, NCT02888106, NCT03852719.

Chronic infection with hepatitis B virus (HBV) is caused by the persistence of closed circular DNA (cccDNA) in the nucleus of infected hepatocytes. Despite available therapeutic anti-HBV agents, eliminating the cccDNA remains challenging. Thus, quantifying and understanding the dynamics of cccDNA are essential for developing effective treatment strategies and new drugs. However, such study requires repeated liver biopsy to measure the intrahepatic cccDNA, which is basically not accepted because liver biopsy is potentially morbid and not common during hepatitis B treatment. We here aimed to develop a noninvasive method for quantifying cccDNA in the liver using surrogate markers in peripheral blood. We constructed a multiscale mathematical model that explicitly incorporates both intracellular and intercellular HBV infection processes. The model, based on age-structured partial differential equations, integrates experimental data from in vitro and in vivo investigations. By applying this model, we roughly predicted the amount and dynamics of intrahepatic cccDNA within a certain range using specific viral markers in serum samples, including HBV DNA, HBsAg, HBeAg, and HBcrAg. Our study represents a significant step towards advancing the understanding of chronic HBV infection. The noninvasive quantification of cccDNA using our proposed method holds promise for improving clinical analyses and treatment strategies. By comprehensively describing the interactions of all components involved in HBV infection, our multiscale mathematical model provides a valuable framework for further research and the development of targeted interventions.

Chronic hepatitis B virus (HBV) infection affects millions worldwide despite the availability of effective vaccines. The stability of HBV’s genomic minichromosome (cccDNA) within hepatocytes, the persistence of integrated viral sequences capable of producing viral antigens, and the ability of the virus to evade immune control all contribute to the difficulty in achieving a functional cure. Existing antiviral treatments have minimal impact on HBV transcription, allowing persistent viral antigen production and immune dysfunction. Emerging RNA interference (RNAi) therapies targeting HBV RNAs reduce viral replication, antigen expression, and, in turn, cccDNA activity, providing a potential path to a functional cure.

Cells have developed various mechanisms to counteract viral infections. In an evolutionary arms race, cells mobilize cellular restriction factors to fight off viruses, targeted by viral factors to facilitate their own replication. The hepatitis B virus (HBV) is a small dsDNA virus that causes acute and chronic infections of the liver. Its genome persists in the nuclei of infected hepatocytes as a covalently closed circular DNA (cccDNA) minichromosome, thus building up an episomal persistence reservoir. The chromosomal maintenance complex SMC5/6 acts as a restriction factor hindering cccDNA transcription, whereas the viral regulatory protein HBx targets SMC5/6 for proteasomal degradation, thus relieving transcriptional suppression of the HBV minichromosome. To date, no curative therapies are available for chronic HBV carriers. Knowledge of the factors regulating the cccDNA and the development of therapies involving silencing the minichromosome or specifically interfering with the HBx-SMC5/6 axis holds promise in achieving sustained viral control. Here, we summarize the current knowledge of the mechanism of SMC5/6-mediated HBV restriction. We also give an overview of SMC5/6 cellular functions and how this compares to the restriction of other DNA viruses. We further discuss the therapeutic potential of available and investigational drugs interfering with the HBx-SMC5/6 axis.

The role of hepatocytes in producing chemokines and triggering liver inflammation and damage in chronic hepatitis D (CHD) is not fully understood. Herein, we investigated the contribution of primary human hepatocytes (PHHs) infected with HDV in triggering inflammation by producing the chemokines CXCL9–11. We performed quantitative PCR, RNA in situ hybridisation, activation-induced marker (AIM) assays, and FACS analysis to investigate the CXCR3/CXCL9–11 receptor/ligand axis of T cells in peripheral blood and livers from patients with chronic hepatitis B (n = 27 and 18, respectively) and CHD (n = 20 and 18, respectively). Chemokine expression was investigated in cultured HDV-infected PHHs and in livers of HBV- or HBV/HDV-infected humanised mice in the presence or absence of adoptively transferred human immune cells (n = 35 in total). In patient and chimeric mouse livers, higher expression levels of CXCL9–11 were found in an HBV/HDV-coinfected vs. HBV-mono-infected setting. Similarly, high levels of CXCL9–11 were observed in HDV-infected PHHs in vitro. Analysis by RNA in situ hybridisation on patient livers revealed that HDV-infected hepatocytes were a significant contributor to the chemokine expression. The corresponding chemokine receptor CXCR3 was found upregulated specifically on peripheral bulk CD4 T cells of patients with CHD. CXCR3 upregulation was unspecific and was not detected on HDAg- or HBsAg-specific CD4 T cells by activation-induced marker assay. Lastly, adoptive transfer of human T cells in humanised mice led to recruitment of non-HBV/HDV-specific CD4+ T cells only in the setting of HBV/HDV coinfection, but not in HBV-mono-infected mice. HDV infection upregulated the intrahepatic expression of the CXCL9–11/CXCR3 receptor/ligand axis. Higher amounts of HBV/HDV-unspecific CD4 T cells expressing CXCR3 may contribute to the aggravated liver inflammation frequently observed in patients with CHD. Chronic hepatitis D (CHD) causes the most severe form of viral hepatitis, and treatment options are still limited; therefore, a more precise understanding of CHD immunopathology is needed. In this study, we demonstrated that HDV infection triggers CXCL9–11 expression in hepatocytes and liver infiltration of CXCR3-expressing CD4 T cells in preclinical models as well as patient biopsies. Because recruitment of Th1-polarised CD4 T cells to the liver has been also described for other severe liver diseases, such as autoimmune hepatitis, it may represent an important mechanism of aggravating liver diseases. The data of this study set hereby the basis for future studies analysing phenotype and function of intrahepatic T cells in CHD.