Search Results (121)

Hepatitis B virus (HBV) persists in infected hepatocytes through covalently closed circular DNA (cccDNA), a stable episomal form that serves as the transcriptional template for viral replication. Accurate and sensitive quantification of intrahepatic cccDNA is crucial for evaluating antiviral therapies, particularly those targeting a functional cure. Here, we report the development of a novel, cccDNA-specific detection system combining recombinase polymerase amplification (RPA) with CRISPR/Cas12a-based fluorescence detection. We designed and validated CRISPR RNAs (crRNAs) targeting HBV cccDNA-specific regions conserved across genotypes A–D. Reaction conditions for both RPA and Cas12a detection were optimized to enhance sensitivity, specificity, and accuracy. The system reliably detected as few as 10 copies of cccDNA-containing plasmid per reaction and showed no cross-reactivity with non-cccDNA forms in serum or plasma, indicating assay specificity. When applied to liver tissue samples from 10 HBV-infected and 6 non-HBV patients, the RPA-CRISPR/Cas12a assay exhibited a high sensitivity (90%) and a strong correlation with qPCR results (R² = 0.9155), confirming its accuracy. In the conclusion, the RPA-CRISPR/Cas12a system provides a robust, cost-effective, and scalable platform for sensitive and specific quantification of intrahepatic HBV cccDNA. This method holds promises for research and high-throughput therapeutic screening applications targeting cccDNA clearance. Full article

Chronic hepatitis B virus (HBV) infection remains a global public health challenge, and the currently approved medications can not achieve a cure. Synthetic triterpenoids have shown promising therapeutic potential for liver pathologies. In our search for novel antiviral agents against HBV, we found that two triterpenoids, 2-cyano-3,12-dioxooleana-1,9-dien-28-oic acid (CDDO) and CDDO-ethyl amide (CDDO-EA), significantly inhibited HBV DNA replication. Further mechanistic investigation indicated that these two compounds did not significantly alter the levels of total HBV pgRNA, but dramatically reduced extracellular pgRNA and intracellular encapsidated pgRNA in a dose-dependent manner. Western blot analysis indicated minimal effects on core protein expression. Interestingly, using a particle gel assay, we observed that CDDO and CDDO-EA promoted the formation of empty capsids with no alteration in electrophoretic mobility. Moreover, we demonstrated that both compounds modulated the phosphorylation status of the core protein. Further cellular thermal shift assay (CETSA), surface plasmon resonance (SPR) assay, and molecular docking analyses collectively suggested that CDDO and CDDO-EA could bind directly to the dimer–dimer interfaces of HBV core protein. Finally, a synergistic effect was observed between CDDO-EA and lamivudine in reducing intracellular and extracellular HBV DNA levels. Our findings indicate that triterpenoids CDDO and CDDO-EA are new mechanistically type of HBV capsid assembly modulators and warranted for further development as lead compounds against HBV. Full article

Hepatitis B virus (HBV) represents a significant global health challenge, affecting over 254 million individuals and contributing to 1.1 million deaths from liver-related complications in 2022. The World Health Organization has set ambitious targets to reduce HBV infections and mortality by 2030. However, only a small proportion (13%) of infected individuals receives timely diagnosis and treatment. HBV elimination efforts necessitate substantial improvements in HBV diagnosis, particularly in identifying early-stage infections, occult HBV infections (OBI), and breakthrough cases. The hepatitis B surface antigen (HBsAg) is a key biomarker in HBV diagnosis, serving as a reliable indicator of infection status and treatment response. Conventional HBsAg assays, with a lower limit of detection (LoD) between 0.03 and 250 IU/mL, often fail to detect OBI and HBV reactivation. In contrast, ultrasensitive HBsAg assays, with an LoD as low as 0.005 IU/mL, can improve the identification of low concentration levels of HBsAg, facilitating earlier diagnosis, monitoring of therapeutic response, and assessment for functional cure. Research confirms the superiority of ultrasensitive assays in detecting HBV in cases missed by conventional assays, detecting NAT-yield samples, and enabling earlier detection of HBV reactivation. This review examines the challenges in HBV diagnostics and the clinical utility of ultrasensitive HBsAg assays in improving progress toward global HBV elimination. Full article

Chronic hepatitis B poses a serious threat to human health and life, and early diagnosis is essential to improving patient cure rates. Hepatitis B virus (HBV) and Alpha-fetoprotein (AFP) are two key biomarkers for diagnosing chronic hepatitis B. In this study, we propose a silicon nanowire array field effect transistor (SiNW-array FET) biosensor that enables highly sensitive, real-time, and low-cost joint detection of both HBV and AFP. The SiNW-array FET is fabricated using traditional micro-nano fabrication techniques such as self-limiting oxidation and anisotropic etching, and its morphology and electrical properties were tested. The results show that the diameters of the fabricated silicon nanowires (SiNWs) are uniform and the SiNW-array FET exhibits a strong output signal and high signal-to-noise ratio. Through specific chemical modification on the surface of SiNWs, the SiNW-array FET is highly sensitive and specific to HBV-DNA fragments and AFP, with ultralow detection limits of 0.1 fM (HBV-DNA) and 0.1 fg/mL (AFP). The detection curve of the SiNW-array FET exhibits good linearity within the HBV-DNA concentration range of 0.1 fM to 100 pM and AFP concentration range of 0.1 fg/mL to 1000 pg/mL. More importantly, the device could also detect HBV-DNA successfully in serum samples, laying a solid foundation for the highly sensitive clinical detection of chronic hepatitis B. Full article

Chronic hepatitis B (CHB) remains a major global health challenge, largely due to the persistence of covalently closed circular DNA (cccDNA) and impaired host immunity. Interferon-α (IFN-α), a key antiviral cytokine, not only directly restricts HBV replication but also orchestrates innate and adaptive immune responses. This review summarizes current advances in IFN-α-mediated immune regulation, highlighting its effects across diverse immune cell populations. Evidence indicates that IFN-α can reprogram immune responses to promote viral clearance, although clinical efficacy is limited by modest response rates and adverse effects. Recent progress in cytokine engineering, subtype research, and rational combination strategies—including nucleo(s/t)ide analogs, RNA interference therapeutics, antisense oligonucleotides, therapeutic vaccines, and beyond—has expanded opportunities to improve treatment outcomes. While challenges remain, these advances lay the foundation for optimizing IFN-α–based interventions and highlight IFN-α as a key driver for innovative therapies aimed at achieving a functional cure of chronic hepatitis B. Full article

Chronic hepatitis B virus (HBV) infection remains a major global health challenge, substantially contributing to liver-related morbidity and mortality. Background/Objectives: Developing therapeutic strategies that overcome immune tolerance and achieve functional cures is an urgent priority. Methods: In this study, we report a therapeutic vaccine comprising hepatitis B surface antigen (HBsAg) formulated with the dual adjuvant system CpG 1018S and QS-21. The immunogenicity and therapeutic efficacy of this vaccine were systematically evaluated in an rAAV8-HBV1.3-established chronic HBV mouse model. Results: The vaccine elicited a robust Th1-skewed immune response, characterized by elevated anti-HBs IgG2b titers and an increased IgG2b/IgG1 ratio. Notably, immunized mice showed markedly reduced circulating HBsAg levels. Mechanistically, the CpG 1018S and QS-21 adjuvant system enhanced dendritic cell activation, maturation, and antigen presentation, expanded HBV-specific CD4⁺ and CD8⁺ T cell populations, and attenuated the expression of the exhaustion markers TIM-3 and TIGIT. Additionally, immunized mice exhibited restored T cell polyfunctionality, with an increased secretion of effector cytokines, including TNF-α and IL-21. These responses collectively contributed to the reversal of T cell exhaustion and breakdown of immune tolerance, facilitating sustained viral suppression. Conclusions: Our findings demonstrate that the CpG 1018S/QS-21-adjuvanted vaccine induces potent humoral and cellular immunity against chronic HBV infection and represents a promising candidate for clinical chronic HBV (CHB) treatment. Full article

Chronic hepatitis B virus (HBV) infection remains a major global health burden, affecting millions and contributing significantly to liver-related morbidity and mortality. While substantial progress has been made in elucidating the virology and natural history of HBV, the management of chronic hepatitis B (CHB) continues to present clinical challenges. The development of potent nucleos(t)ide analogs and pegylated interferon has improved viral suppression and delayed disease progression, yet a definitive cure remains elusive due to the persistence of covalently closed circular DNA (cccDNA). Recent research has focused on novel antiviral agents, immunomodulatory therapies, and combination strategies aimed at achieving a functional cure. This review summarizes current therapeutic approaches, recent advancements, and emerging directions in CHB management. Full article

The burden of hepatitis delta virus (HDV) infection is currently unknown and may affect 12 to 72 million people distributed across various hot spots in different regions of the globe. Screening for antibodies to HDV infection in patients positive for the hepatitis B surface antigen (HBsAg) is generally available in most parts of the world, but systematic testing for HDV is needed. Chronic HDV infection is associated with a higher risk of progression to cirrhosis, liver failure, and hepatocellular carcinoma compared to hepatitis B virus (HBV) mono-infection. Bulevirtide is the recently available treatment against hepatitis delta. The results of efficacy studies and new drugs (lonafarnib) are under discussion. New therapeutic strategies are in development, revealing a critical need for valid next-generation treatments to cure HDV. Full article

Background/Objectives: Hepatitis B virus (HBV) infection poses a major global health challenge, with current therapies like nucleos(t)ide analogs and pegylated interferon alpha offering limited functional cure rates due to persistent HBsAg-driven immune tolerance. This study aimed to develop a targeted immunoadsorption system using a high-affinity humanized anti-HBsAg monoclonal antibody for efficient HBsAg and viral particle clearance, providing a novel approach to overcome therapeutic bottlenecks in chronic hepatitis B (CHB). Methods: A murine anti-HBsAg monoclonal antibody was humanized via complementarity-determining region grafting, resulting in HmAb-12 (equilibrium dissociation constant, K_D = 0.36 nM). A stable Chinese Hamster Ovary K1 (CHO-K1) cell line was established for high-yield expression (fed-batch yield: 8.31 g/L). The antibody was covalently coupled to agarose microspheres (coupling efficiency > 95%) to prepare the immunoadsorbent. Efficacy was evaluated through in vitro dynamic circulation assays with artificial sera and preclinical trials using an integrated blood purification system in two CHB participants. Clearance rates for HBsAg and HBV DNA were quantified, with safety assessed via blood component monitoring. Results: In vitro, a single treatment cycle achieved HBsAg clearance rates of 70.14% (high antigen load, >10⁵ IU/mL) and 92.10% (low antigen load, ~3000 IU/mL). Preclinically, one treatment session resulted in acute HBsAg reductions of 78.30% and 74.31% in participants with high and moderate antigen loads, respectively, alongside HBV DNA decreases of 65.66% and 73.55%. Minimal fluctuations in total protein and albumin levels (<15%) confirmed favorable safety profiles, with no serious adverse events observed. Conclusions: Preliminary findings from this study indicate that the HBsAg-specific immunoadsorption system can achieve efficient HBV antigen clearance with an initial favorable safety profile in a small cohort. These results support its further investigation as a potential therapeutic strategy for functional cure in CHB. Future work will focus on validating these findings in larger studies and exploring the system’s combinatory potential with existing blood purification platforms. Full article

Hepatitis B virus (HBV) remains a major global health challenge, with over 296 million people chronically infected worldwide. Despite the availability of antiviral therapies, a functional cure is rarely achieved, highlighting the need for novel therapeutic strategies. RNA 5-methylcytosine (m⁵C) is a pivotal epitranscriptomic mark implicated in RNA stability, transport, and translation. Emerging evidence shows that m⁵C is conserved within HBV RNA and plays critical roles in the viral life cycle. This review provides a comprehensive overview of the molecular mechanisms governing m⁵C deposition and recognition, summarizes recent advances in m⁵C biology, and highlights the emerging role of epitranscriptomic m⁵C regulation in HBV infection. We discuss the identification of HBV-specific m⁵C sites, the functions of key regulatory enzymes, and their interplay in viral RNA stabilization and evasion of innate immune responses. Interplay between m⁵C and other RNA modifications—particularly N6-methyladenosine (m⁶A)—is examined alongside virus-specific m⁵C regulation in EV71, HIV, HCV, EBV, and SARS-CoV-2. Potential links between m⁵C dysregulation and HBV-induced hepatocarcinogenesis are outlined, and emerging therapeutic strategies targeting the m⁵C machinery are highlighted. Together, these insights position the epitranscriptomic landscape as a promising avenue for innovative antiviral strategies. Full article

Background: Hepatitis presents a major health and economic challenge in Saudi Arabia, necessitating insight into its epidemiology, risk factors, and control measures. This review aims to synthesize current evidence on the epidemiology, risk factors, and prevention strategies for viral hepatitis in Saudi Arabia. It evaluates the effectiveness of existing interventions and proposes data-driven approaches to advance national hepatitis elimination goals. Methods: This study reviewed data from 2016 to 2024, sourced from PubMed, Google Scholar, ResearchGate, and ScienceDirect, focusing on hepatitis epidemiology and prevention in Saudi Arabia. Studies relevant to Saudi-specific trends and prevention strategies were included. Results: Saudi Arabia has achieved significant reductions in viral hepatitis prevalence, notably HBV (1.3%) due to universal infant vaccination (98% coverage), and HCV (0.124%) through the Saudi National Hepatitis Program (SNHP), which provides free DAAs (95% cure rate) and has screened 5 million people. However, challenges persist: HAV susceptibility is rising in adults (seroprevalence 33.1%), HDV affects 7.7% of HBV patients, and key risk factors include socioeconomic disparities (higher HAV/HEV in rural/low-income areas), intravenous drug use (30–50% of HCV cases), unsafe medical/cultural practices (e.g., Hijama), and limited healthcare access for migrants/rural populations. While interventions like water sanitation initiatives (58% HAV decline) and prenatal screening are effective, advancing elimination goals requires addressing gaps in HDV/HEV surveillance, outdated seroprevalence data, equitable treatment access (35% lower in rural areas), stigma reduction, and targeted strategies for high-risk groups to meet WHO 2030 targets. Conclusions: Saudi Arabia has made significant progress in hepatitis control through vaccination and public health efforts, but challenges persist. Strengthening healthcare systems, improving community engagement, and ensuring equitable access are key to sustaining elimination efforts. Full article

The Hepatitis B Virus (HBV) presents a formidable global health challenge, impacting hundreds of millions worldwide and imposing a considerable burden on healthcare systems. The elusive nature of the virus, with its ability to establish chronic infection and evade immune detection, and the absence of curative agents have prompted efforts to develop novel therapeutic approaches beyond current antiviral treatments. This review addresses the challenging concept of a functional cure for HBV, a state characterized by the suppression of HBV and HBsAg, mitigating disease progression and transmission without a complete cure. We provide an overview of HBV epidemiology and its clinical impact, followed by an exploration of the current treatment landscape and its limitations. The immunological basis of a functional cure is then discussed, exploring the intricate interplay between the virus and the host immune response. Emerging therapeutic approaches, such as RNA interference-based interventions, entry inhibitors, nucleic acid polymers, and therapeutic vaccines, are discussed with regard to their success in achieving a functional cure. Lastly, the review underscores the urgent need for innovative strategies to achieve a functional cure for HBV. Full article

Human viral infections remain a significant global health challenge, contributing to a substantial number of cancer cases worldwide. Among them, infections with oncoviruses such as hepatitis B virus (HBV) and hepatitis C virus (HCV) are key drivers of hepatocellular carcinoma (HCC). Despite the availability of an effective HBV vaccine since the 1980s, millions remain chronically infected due to the persistence of covalently closed circular DNA (cccDNA) as a reservoir in hepatocytes. Current antiviral therapies, including nucleos(t)ide analogs and interferon, effectively suppress viral replication but fail to eliminate cccDNA, underscoring the urgent need for innovative therapeutic strategies. Direct-acting antiviral agents (DAAs), which have revolutionized HCV treatment with high cure rates, offer a promising model for HBV therapy. A particularly attractive target is the intrinsically disordered region (IDR) of the HBx protein, which regulates cccDNA transcription, viral replication, and oncogenesis by interacting with key host proteins. DAAs targeting these interactions could inhibit viral persistence, suppress oncogenic signaling, and overcome treatment resistance. This review highlights the potential of HBx-directed DAAs to complement existing therapies, offering renewed hope for a functional HBV cure and reduced cancer risk. Full article

Available interventions for the management of chronic hepatitis B (hepB) exhibit limited efficacy and barriers to vaccination against the hepatitis B virus (HBV) have hampered prophylaxis programmes. Development of potent therapeutics capable of functional cure of chronic hepB thus remains a relevant medical objective. RNA interference (RNAi) can be exploited to effect potent and specific silencing of target genes through the introduction of RNA sequences that mimic the natural activators of the pathway. To achieve a therapeutic effect, artificial primary microRNAs (pri-miRNAs) have been used extensively to target various viruses, including HBV. To date artificial pri-miRNAs have exclusively been produced from DNA expression cassettes. Although this achieves impressive silencing, eventual translation of this platform to the clinic is complicated by the requirement for viral vectors to deliver DNA. Consequently, clinical translation has been slow. Recently, the use of in vitro transcribed RNA, specifically to produce mRNA vaccines at industrial scale, has gained significant interest. We therefore sought to evaluate the feasibility of using in vitro transcribed artificial pri-miRNAs for the inhibition of HBV gene expression. Artificial HBV-targeting pri-miR-31 sequences, which are highly effective when expressed in cells from a DNA template, demonstrated modest silencing of viral replication when incorporated into mRNA that was transcribed in vitro. Off-target effects were also observed. Characterisation revealed that intracellular processing of the artificial pri-miRNAs was inefficient and non-specific effects were caused by stimulation of the interferon response. Nevertheless, optimised nuclear delivery of the artificial pri-miRNAs should improve their processing and achieve better anti-hepB efficacy. Full article

Background/Objectives: In chronic hepatitis B infection (CHB), the hepatitis B surface antigen (HBsAg) continuously exhausts the hepatitis B surface antibody (HBsAb), which leads to the formation of immune tolerance. Accordingly, the hepatitis B virus (HBV) infection can be blocked by inhibiting the binding of the hepatitis B surface pre-S1/pre-S2 antigen to the hepatocyte receptor NTCP, but the clinical cure rate of pre-S-based vaccines for CHB is limited. Methods: In this study, we designed and prepared multivalent hepatitis B therapeutic mRNA vaccines encoding three hepatitis B surface antigen proteins (L, M, and S) at the cell membrane, verified via in vitro transfection and expression experiments. An in vivo immunization experiment in HBV transgenic (Tg) mice was first completed. Subsequently, an adeno-associated virus plasmid vector carrying the HBV1.2-fold genome (pAAV HBV1.2) model and the adeno-associated virus vector carrying HBV1.3-fold genome (rAAV HBV1.3) model were constructed and immunized with mRNA vaccines. The HBV antigen, antibodies, and HBV DNA in serum were detected. Indirect (enzyme-linked immunosorbent assay) ELISA were made to analyze the activated antigen-specific IgG in HBV Tg mice. Antigen-dependent T-cell activation experiments were carried out, as well as the acute toxicity tests in mice. Results: The L protein/pre-S antigens could be stably presented at the cell membrane with the support of the S protein (and M protein). After vaccinations, the vaccines effectively reactivated the production of high levels of HBsAb, disrupted immune tolerance, and activated the production of high-affinity antibodies against structural pre-S antigen in HBV Tg mice. The HBsAg seroconversion and serum HBV DNA clearance were achieved in two HBV mice models. Furthermore, pre-S antigen-dependent T-cell response against HBV infection was confirmed. The therapeutic vaccine also showed safety in mice. Conclusions: A novel therapeutic mRNA vaccine was developed to break through HBsAg-mediated immune tolerance and treat CHB by stably presenting the pre-S antigen at the membrane, and the vaccine has great potential for the functional cure of CHB. Full article