Chronic hepatitis B virus infection (CHB) remains a global health concern, with currently available antiviral therapies demonstrating limited effectiveness in preventing hepatocellular carcinoma (HCC) development. Two primary challenges in CHB treatment include the persistence of the minichromosome, covalently closed circular DNA (cccDNA) of the hepatitis B virus (HBV), and the failure of the host immune response to eliminate cccDNA. Recent findings indicate several host and HBV proteins involved in the epigenetic regulation of cccDNA, including HBV core protein (HBc) and HBV x protein (HBx). Both proteins might contribute to the stability of the cccDNA minichromosome and interact with viral and host proteins to support transcription. One potential avenue for CHB treatment involves the utilization of therapeutic vaccines. This paper explores HBV antigens suitable for epigenetic manipulation of cccDNA, elucidates their mechanisms of action, and evaluates their potential as key components of epigenetically-driven vaccines for CHB therapy. Molecular targeted agents with therapeutic vaccines offer a promising strategy for addressing CHB by targeting the virus and enhancing the host\'s immunological response. Despite challenges, the development of these vaccines provides new hope for CHB patients by emphasizing the need for HBV antigens that induce effective immune responses without causing T cell exhaustion.

Covalently closed circular DNA (cccDNA) exists as a stable episomal minichromosome in the nucleus of hepatocytes and is responsible for hepatitis B virus (HBV) persistence. We recently reported a technique involving recombinant cccDNA (rcccDNA) of HBV by site-specific DNA recombination. A floxed monomeric HBV genome was engineered into a precursor plasmid (prcccDNA) which was excised via Cre/loxP-mediated DNA recombination to form a 3.3-kb rcccDNA bearing a loxP-chimeric intron. The foreign sequence was efficiently removed during RNA splicing, rendering a functionally seamless insertion. We characterized rcccDNA formation, effective viral transcription, and replication induced by rcccDNA both in vitro and in vivo. Furthermore, we closely simulated chronic hepatitis by using a replication-defective recombinant adenoviral vector to deliver rcccDNA to the transgenic mice expressing Cre recombinase, which led to prominent HBV persistence. Here, we describe a detailed protocol about how to construct and evaluate Cre/loxP-based recombinant HBV cccDNA system both in vitro and in vivo.

Hepatitis B virus (HBV) infection remains a global public health issue, and approximately 294 million individuals worldwide are chronically infected with HBV. Approved antivirals rarely cure chronic HBV infection due to their inability to eliminate the HBV covalently closed circular DNA (cccDNA), the viral episome, in the nucleus of infected hepatocytes. The persistence of cccDNA underlies the chronic nature of HBV infection and the frequent relapse after the cessation of antiviral treatment. However, drug development targeting cccDNA formation and maintenance is hindered by the lack of sufficient biological knowledge on cccDNA, and of its reliable detection due to its low abundance and the presence of high levels of HBV DNA species similar to cccDNA. Here, we describe a Southern blot method for reliably detecting the HBV cccDNA even in the presence of high levels of plasmid DNA and other HBV DNA species, based on the efficient removal of plasmid DNA and all DNA species with free 3\' ends. This approach also allows the detection of certain potential intermediates during cccDNA formation.

To achieve a virological cure for hepatitis B virus (HBV), innovative strategies are required to target the covalently closed circular DNA (cccDNA) genome. Guanine-quadruplexes (G4s) are a secondary structure that can be adopted by DNA and play a significant role in regulating viral replication, transcription, and translation. Antibody-based probes and small molecules have been developed to study the role of G4s in the context of the human genome, but none have been specifically made to target G4s in viral infection. Herein, we describe the development of a humanized single-domain antibody (S10) that can target a G4 located in the PreCore (PreC) promoter of the HBV cccDNA genome. MicroScale Thermophoresis demonstrated that S10 has a strong nanomolar affinity to the PreC G4 in its quadruplex form and a structural electron density envelope of the complex was determined using Small-Angle X-ray Scattering. Lentiviral transduction of S10 into HepG2-NTCP cells shows nuclear localization, and chromatin immunoprecipitation coupled with next-generation sequencing demonstrated that S10 can bind to the HBV PreC G4 present on the cccDNA. This research validates the existence of a G4 in HBV cccDNA and demonstrates that this DNA secondary structure can be targeted with high structural and sequence specificity using S10.

UNASSIGNED: Cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) attenuates cytotoxic T lymphocyte (CTL) activation. This study was performed to examine the relationships between CTLA-4 genotypes/haplotypes, hepatitis B surface antigen (HBsAg), and hepatitis B core-related antigen (HBcrAg) levels, and their potential impact on the clinical course of chronic HBV infection.
UNASSIGNED: We recruited 145 treatment-naïve patients with genotype B or C chronic HBV infection who were initially hepatitis B e-antigen (HBeAg)-positive and had been followed from a mean age of 7.08 years for a total of 4,787 person-years in the study cohort. We also recruited another 69 treatment-naïve adults with genotype B or C chronic HBV infection as a validation cohort. We assessed the CTLA-4 gene single nucleotide polymorphisms rs4553808 (-A1661G)/rs5742909 (-C318T) in both cohorts, and the serum HBsAg and HBcrAg levels in the study cohort.
UNASSIGNED: CTLA-4 promoter haplotypes were associated with HBsAg and HBcrAg levels at 10 and 15 years of age in the study cohort. Patients with the CTLA-4 AA/CC haplotype showed earlier spontaneous HBeAg seroconversion (hazard ratio = 1.58; p = 0.02), and a more rapid annual decline in the serum HBsAg level than other patients (0.09 vs. 0.03 log10 IU/ml/year, p = 0.02). The CTLA-4 AA/CC haplotype was also predictive of HBeAg seroconversion in the validation cohort (p = 0.01).
UNASSIGNED: Chronic HBV-infected patients with a CTLA-4 AA/CC haplotype had lower serum HBsAg and HBcrAg levels in childhood and earlier spontaneous HBeAg seroconversion.
UNASSIGNED: The role of cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) in chronic HBV-infected children has not been studied previously. In a very long-term cohort followed from childhood to adulthood, we showed that CTLA-4 haplotypes are associated with HBV biomarker levels in childhood and are correlated with the clinical course of chronic HBV infection. CTLA-4 pathway may serve as a future target for the development of therapeutic agents against HBV infection.

Chronic hepatitis B (CHB) is a global health challenge that can result in significant liver-related morbidity and mortality. Despite a prophylactic vaccine being available, patients already living with CHB often must engage in lifelong therapy with nucleoside analogues. However, the potential of RNA interference (RNAi) therapeutics as a promising avenue for CHB treatment is being explored. RNAi, particularly using small interfering RNA (siRNA), targets viral RNA that can be used to inhibit hepatitis B virus (HBV) replication. Several candidates are currently being studied and have exhibited varying success in reducing hepatitis B surface antigen (HBsAg) levels, with some showing sustained HBsAg loss after cessation of therapy. The dynamic evolution of RNAi therapy presents a promising trajectory for the development of effective and sustained treatments for CHB. This review highlights recent findings on RNAi therapeutics, including modifications for stability, various delivery vectors, and specific candidates currently in development.

Hepatitis B virus (HBV) chronically infects 296 million people worldwide and causes more than 820,000 deaths annually due to cirrhosis and hepatocellular carcinoma. Current standard-of-care medications for chronic hepatitis B (CHB) include nucleos(t)ide analogue (NA) viral DNA polymerase inhibitors and pegylated interferon alpha (PEG-IFN-α). NAs can efficiently suppress viral replication and improve liver pathology, but not eliminate or inactivate HBV covalently closed circular DNA (cccDNA). CCC DNA is the most stable HBV replication intermediate that exists as a minichromosome in the nucleus of infected hepatocyte to transcribe viral RNA and support viral protein translation and genome replication. Consequentially, a finite duration of NA therapy rarely achieves a sustained off-treatment suppression of viral replication and life-long NA treatment is most likely required. On the contrary, PEG-IFN-α has the benefit of finite treatment duration and achieves HBsAg seroclearance, the indication of durable immune control of HBV replication and functional cure of CHB, in approximately 5% of treated patients. However, the low antiviral efficacy and poor tolerability limit its use. Understanding how IFN-α suppresses HBV replication and regulates antiviral immune responses will help rational optimization of IFN therapy and development of novel immune modulators to improve the rate of functional cure. This review article highlights mechanistic insight on IFN control of HBV infection and recent progress in development of novel IFN regimens, small molecule IFN mimetics and combination therapy of PEG-IFN-α with new direct-acting antivirals and therapeutic vaccines to facilitate the functional cure of CHB.

Though the world-wide hepatitis B virus (HBV) vaccination program has been well completed for almost thirty years in many nations, almost HBV-related hepatocellular carcinoma (HCC) occurs in unvaccinated middle-aged and elderly adults. Apparently, treating 80% of qualified subjects could decrease HBV-related mortality by 65% in a short period. Nevertheless, globally, only 2.2% of CHB patients undergo antiviral therapy. The HBV markers related to HCC occurrence and prevention are as follows: the HCC risk is the highest at a baseline of HBV DNA of 6-7 log copies/mL, and it is the lowest at a baseline of an HBV DNA level of >8 log copies/mL and ≤4 log copies/mL (parabolic, and not linear pattern). The titer of an HBV core-related antigen (HBcrAg) reflecting the amount of HBV covalently closed circular DNA (ccc DNA) in the liver is related to HCC occurrence. The seroclearance of HBs antigen (HBsAg) is more crucial than HBV DNA negativity for the prevention of HCC. In terms of the secondary prevention of hepatitis B-related HCC involving antiviral therapies with nucleos(t)ide analogues (NAs), unsolved issues include the definition of the immune-tolerant phase; the optimal time for starting antiviral therapies with NAs; the limits of increased aminotransferase (ALT) levels as criteria for therapy in CHB patients; the normalization of ALT levels with NAs and the relation to the risk of HCC; and the relation between serum HBV levels and the risk of HCC. Moreover, the first-line therapy with NAs including entecavir (ETV), tenofovir disoproxil fumarate (TDF), and tenofovir alafenamide (TAF) remains to be clarified. Discussed here, therefore, are the recent findings of HBV markers related to HCC occurrence and prevention, unsolved issues, and the current secondary antiviral therapy for the prevention of HBV-related HCC.

Hepatitis B virus cccDNA is the key target for the necessary development of antiviral therapies aimed at curing chronic hepatitis B. The CRISPR-based system to produce covalently closed circular (cccDNA)-like extrachromosomal DNAs described in this report enables large-scale screens of chemical libraries to identify drug candidates with the potential to permanently inactivate cccDNA. Moreover, this approach permits investigations on unresolved problems as described in this report concerning cccDNA biology including mechanisms of SMC5/6-dependent transcriptional silencing and the contributions of the SMC5/6 complex to cccDNA stability in resting and dividing hepatocytes.

UNASSIGNED: Chronic hepatitis B virus (HBV) infection is one of the common infectious diseases in the world. HBV covalently closed circular DNA (cccDNA) is the initial template of HBV replication, which can exist in human hepatocytes for a long time and is difficult to be completely removed. It has been shown that HBV RNA can directly respond to the levels and transcriptional activity of cccDNA in hepatocytes and can be used as a surrogate marker of cccDNA transcriptional activity. At present, the detection techniques used for quantitative HBV RNA mainly include reverse transcription quantitative polymerase chain reaction (RT-qPCR) and simultaneous amplification and testing (SAT).
UNASSIGNED: In this study, we verified the performance of the SAT method for detecting HBV RNA and the clinical effectiveness of SAT and RT-qPCR, and compared the correlation and consistency of the two detection methods for HBV RNA detection.
UNASSIGNED: The results showed that the limit of detection for HBV RNA by SAT method was 50 copies/mL, with a linear range of 1 × 102-1 × 108 copies/mL. There was no difference in HBV RNA levels detected by the two methods. The correlation and consistency of the results were good, with the coefficient of determination of 0.7787 in HBeAg positive group and 0.8235 in HBeAg negative group.
UNASSIGNED: Therefore, this study confirmed that the SAT method and RT-qPCR for detecting HBV RNA have good agreement, which are both reliable methods to detect HBV RNA and can replace each other.