DOCK11 and DENND2A play pivotal roles in the maintenance of hepatitis B virus in host cells

Human hepatitis B virus (HBV) infection remains a serious health problem worldwide. However, the mechanism for the maintenance of HBV in a latent state within host cells remains unclear. Here, using single-cell RNA sequencing analysis, we identified four genes linked to the maintenance of HBV in a liver cell line expressing HBV RNA at a low frequency. These genes included DOCK11 and DENND2A, which encode small GTPase regulators. In primary human hepatocytes infected with HBV, knockdown of these two genes decreased the amount of both HBV DNA and covalently closed circular DNA to below the limit of detection. Our findings reveal a role for DOCK11 and DENND2A in the maintenance of HBV.

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Serum Hepatitis B Virus DNA, RNA, and HBsAg: Which Correlated Better with Intrahepatic Covalently Closed Circular DNA before and after Nucleos(t)ide Analogue Treatment?

Journal of Clinical Microbiology ◽

10.1128/jcm.00760-17 ◽

2017 ◽

Vol 55 (10) ◽

pp. 2972-2982 ◽

Cited By ~ 23

Author(s):

Yuhua Gao ◽

Yutang Li ◽

Qinghua Meng ◽

Zhanqing Zhang ◽

Ping Zhao ◽

...

Keyword(s):

Hepatitis B Virus ◽

Hepatitis B ◽

Hepatitis B Surface Antigen ◽

Surrogate Marker ◽

Hbv Dna ◽

Serum Hepatitis ◽

Baseline Serum ◽

Circular Dna ◽

Covalently Closed Circular Dna ◽

B Virus

ABSTRACT The study was designed to investigate whether serum hepatitis B virus (HBV) RNA is a strong surrogate marker for intrahepatic HBV covalently closed circular DNA (cccDNA) compared with serum HBV DNA, hepatitis B surface antigen (HBsAg), and hepatitis B e antigen (HBeAg) in HBeAg-positive chronic hepatitis B (CHB) patients. Serum HBV RNA, HBV DNA, HBsAg, HBeAg, and intrahepatic cccDNA were quantitatively detected at baseline ( n = 82) and 96 weeks ( n = 62) after treatment with nucleos(t)ide analogue (NUC) in HBeAg-positive CHB patients. The correlations among serum HBV RNA, HBV DNA, HBsAg, HBeAg, and intrahepatic cccDNA levels were then statistically analyzed. The results showed that pretreatment intrahepatic cccDNA levels correlated better with serum HBV DNA levels ( r = 0.36, P < 0.01) than with serum HBV RNA levels ( r = 0.25, P = 0.02), whereas no correlations were found between pretreatment intrahepatic cccDNA levels and HBsAg ( r = 0.15, P = 0.17) or HBeAg ( r = 0.07, P = 0.56) levels. At 96 weeks after NUC treatment, intrahepatic cccDNA levels correlated well with HBsAg levels ( r = 0.39, P < 0.01) but not with serum HBV RNA, HBV DNA, and HBeAg levels (all P > 0.05). Besides, the decline in the intrahepatic cccDNA level from baseline to week 96 correlated better with the reduction in the serum HBsAg levels than with the decreases in the levels of the other markers (for the HBsAg decline, r = 0.38, P < 0.01; for the HBV DNA decline, r = 0.35, P = 0.01; for the HBV RNA decline, r = 0.28, P < 0.05; for the HBeAg decline, r = 0.18, P = 0.19). In conclusion, the baseline serum HBV RNA level or its decline after 96 weeks of NUC therapy correlated with the corresponding intrahepatic cccDNA level, while it was less than that seen with serum HBV DNA at baseline and HBsAg (or its decline) at 96 weeks after treatment, respectively.

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Correlation of HBcrAg with Intrahepatic Hepatitis B Virus Total DNA and Covalently Closed Circular DNA in HBeAg-Positive Chronic Hepatitis B Patients

Journal of Clinical Microbiology ◽

10.1128/jcm.01303-18 ◽

2018 ◽

Vol 57 (1) ◽

Cited By ~ 5

Author(s):

Lin Wang ◽

Xi Cao ◽

Zhenzi Wang ◽

Yuhua Gao ◽

Juan Deng ◽

...

Keyword(s):

Chronic Hepatitis ◽

Hepatitis B Virus ◽

Hepatitis B ◽

Hbv Dna ◽

Circular Dna ◽

Hbv Cccdna ◽

B Virus ◽

Positive Chronic Hepatitis ◽

Total Dna ◽

Intrahepatic Hbv

ABSTRACT The purpose of this study was to explore the correlations of serum hepatitis B core-related antigen (HBcrAg) with intrahepatic Hepatitis B virus (HBV) covalently closed circular DNA (cccDNA) and HBV total DNA in hepatitis B e antigen (HBeAg)-positive chronic hepatitis B (CHB) patients. Serum HBcrAg and other parameters, including HBV DNA, HBV RNA, HBeAg, hepatitis B surface antigen (HBsAg), hepatitis B core antibody (anti-HBc), alanine aminotransferase (ALT), and aspartate aminotransferase (AST) were quantitatively measured at baseline and follow-up time points. Intrahepatic HBV cccDNA and total DNA were quantitatively detected at baseline and 96 weeks. Grading of liver necroinflammation and staging of hepatic fibrosis were assessed at baseline and 96 weeks. Correlations between serum HBcrAg and other parameters were analyzed by Pearson’s correlation analysis. The results showed that pretreatment HBcrAg correlated significantly with HBV total DNA levels (r = 0.328, P = 0.003) in 82 CHB patients, and, after removing three outliers, with intrahepatic HBV cccDNA (r = 0.323, P = 0.004; n = 79). Serum HBcrAg correlated better with HBV cccDNA in patients with lower levels of serum HBV DNA (stratified by 7 log IU/ml of HBV DNA; r = 0.656, P = 0.003 versus r = −0.02, P = 0.866). Significant inverse correlations were found between HBcrAg and grade of liver necroinflammation (r = −0.245, P = 0.037), stage of hepatic fibrosis (r = −0.360, P = 0.002) at baseline. Serum HBcrAg presented significant correlation with intrahepatic HBV cccDNA in patients with HBeAg seroconversion at 96 weeks (r = 0.622, P = 0.006). The decrease in HBcrAg showed significant correlation with the decrease in HBV cccDNA after 96-week NA therapy (r = 0.282, P = 0.043). Serum HBcrAg also correlated significantly with other serum markers at baseline and 96 weeks of NA therapy. In conclusion, baseline HBcrAg and its decreased value were significantly correlated with the corresponding intrahepatic HBV cccDNA.

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Diagnosis and Monitoring of Hepatitis B Virus Infection Using the Cobas® HBV Test for Use on the Cobas® 4800 System

Microorganisms ◽

10.3390/microorganisms9030573 ◽

2021 ◽

Vol 9 (3) ◽

pp. 573

Author(s):

Valérie Ortonne ◽

Mélanie Wlassow ◽

Magali Bouvier-Alias ◽

Giovana Melica ◽

Jean-Dominique Poveda ◽

...

Keyword(s):

Clinical Practice ◽

Hepatitis B Virus ◽

Hepatitis B ◽

Limit Of Detection ◽

Clinical Performance ◽

Hbv Dna ◽

Nucleic Acid Amplification ◽

Hbv Genotypes ◽

B Virus ◽

System 2

(1) Background: Sensitive and accurate nucleic acid amplification technologies are now recommended for hepatitis B virus (HBV) DNA detection and quantification in clinical practice to diagnose and monitor hepatitis B infection. The aim of this study was to assess the analytical and clinical performance of the cobas® HBV Test on the cobas® 4800 System. (2) Methods: Standard panel and clinical specimens were tested in parallel with three different real-time commercial PCR assays including the cobas ® HBV Test, the Cobas® AmpliPrep/Cobas® TaqMan HBV Test v2.0 and Alinity™ m HBV assay. (3) Results: The specificity of the cobas® HBV Test was 97.9%. The limit of detection was estimated to be 2.1 IU/mL. Intra-assay and interassay coefficients of variation varied from 0.14% to 1.92% and 2.16% to 12.02%, respectively. HBV DNA levels in patients infected with different HBV genotypes strongly correlated with those measured by the two other commercial comparators assays. (4) Conclusions: The cobas® HBV Test can be confidently used to detect and accurately quantify HBV DNA in clinical practice as well as in clinical trials with the new anti-HBV drugs currently in development.

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Evidence for Dual Effects of DNA-Reactive Bile Acid Derivatives (Bamets) on Hepatitis B Virus Life Cycle in an In Vitro Replicative System

Antiviral Chemistry and Chemotherapy ◽

10.1177/095632020201300605 ◽

2002 ◽

Vol 13 (6) ◽

pp. 371-380 ◽

Cited By ~ 8

Author(s):

Marta R Romero ◽

Maria C Martinez-Diez ◽

Monica G Larena ◽

Rocio IR MacIas ◽

Mercedes Dominguez ◽

...

Keyword(s):

Life Cycle ◽

Bile Acid ◽

Hepatitis B Virus ◽

Hepatitis B ◽

Hbv Dna ◽

Drug Uptake ◽

Host Cells ◽

B Virus ◽

Bile Acid Derivatives ◽

Hepg2 2.2.15

A liver targeting strategy to direct antiviral drugs toward hepatitis B virus (HBV) was investigated. As model drugs we used cisplatin-bile acid derivatives (Bamets) to determine the production of virions by HBV-transfected hepatoblastoma cells (HepG2 2.2.15). Drug uptake was determined using flameless atomic absorption spectrometry to measure platinum cell contents. Cytotoxic effect was determined by formazan formation and neutral red uptake tests. The release of viral surface protein was evaluated by ELISA. The abundance of HBV-DNA in the medium was determined by quantitative real-time PCR and its structure by Southern blot analysis. The uptake of Bamets by HepG2 2.2.15 cells was higher than that of cisplatin. At concentrations lower than 10 μM, distinct Bamets have no toxic effect on host cells, whereas cisplatin dramatically reduced cell viability at concentrations higher than 1 μM. All the drugs tested inhibited the release of viral proteins to the medium, but induced a marked and progressive dose-dependent increase in the amount of viral DNA in the medium. This was mainly due to the release of short fragments of HBV-DNA in the case of cisplatin. On the contrary, Bamets induced an enhanced release of circular forms of HBV-DNA. These findings suggest the existence of a dual effect of Bamets on HBV life-cycle by enhancing the production of DNA replicative intermediates but reducing the secretion of complete virions. Altogether these characteristics recommend consideration of these compounds as a useful experimental tool in the investigation of novel liver targeted therapeutic agents based on bile acid derivatives for the treatment of HBV infections, or to carry out further studies on the HBV life cycle.

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A Highly Prevalent Polymorphism in the Core Region Impairs Quantification of Hepatitis B Virus (HBV) by the cobas TaqMan HBV Assay

Journal of Clinical Microbiology ◽

10.1128/jcm.00647-20 ◽

2020 ◽

Vol 58 (9) ◽

Cited By ~ 1

Author(s):

Laure Boizeau ◽

Annabelle Servant-Delmas ◽

Alexandra Ducancelle ◽

Stéphane Chevaliez ◽

Vincent Thibault ◽

...

Keyword(s):

Hepatitis B Virus ◽

Hepatitis B ◽

Limit Of Detection ◽

Hbv Dna ◽

Cobas Taqman ◽

The Core ◽

B Virus ◽

Reverse Primer ◽

New Generation ◽

Genotype A

ABSTRACT The high genetic variability of hepatitis B virus (HBV) can impair DNA quantification. Here, we investigate a major underquantification of HBV by the cobas TaqMan HBV assay (CTM; Roche). In France, between 2005 and 2017, HBV DNA was detected in 3,102 blood donations by use of the CTM (95% limit of detection [LOD95], 4.8 IU/ml). HBV strains were sequenced in the S region (LOD95, ∼30 IU/ml). Concordant (n = 120) and discordant (n = 45) samples were identified according to the agreement between the plasma viral load (pVL) determined by the CTM and sequencing; all samples were also quantified using the RealTime HBV assay (RTH; Abbott). The viral signature, cloning, and mutagenesis were used to characterize the polymorphism responsible for CTM misquantification. A CTM-RTH discordance (>1 log IU/ml) was found in 14/45 samples that had low pVLs and were successfully genotyped (pVLlow genoS+). PreC/C clones of concordant (C1, C2) and discordant (D1, D2) strains were used to challenge the CTM. Strains D1 and D2 were highly underquantified (42- and 368-fold). In clones, mutating the region corresponding to the CTM reverse primer from a discordant sequence to a concordant sequence restored the levels of quantification to 24% (D1→C1) and 59% (D2→C1) of theoretical levels, while mutating the sequence of a concordant strain to that of a discordant strain led to 78-fold (C1→D1) and 146-fold (C1→D2) decreases in quantification. Moreover, mutating positions 1961 and 1962 was enough to induce a 5-fold underquantification. We conclude that the CTM underestimates pVLs for HBV strains with mutations in the reverse primer target. Specifically, the polymorphism at nucleotides 1961 and 1962 is naturally present in 4.79 and 4.22% of genotype A and D strains, which are highly frequent in Europe, leading to a 5-fold decrease in quantification. Quantification using the new-generation Roche C4800 assay is not affected by this polymorphism.

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The New Aptima HBV Quant Real-Time TMA Assay Accurately Quantifies Hepatitis B Virus DNA from Genotypes A to F

Journal of Clinical Microbiology ◽

10.1128/jcm.02219-16 ◽

2017 ◽

Vol 55 (4) ◽

pp. 1211-1219 ◽

Cited By ~ 9

Author(s):

Stéphane Chevaliez ◽

Claude Dauvillier ◽

Fabienne Dubernet ◽

Jean-Dominique Poveda ◽

Syria Laperche ◽

...

Keyword(s):

Hepatitis B Virus ◽

Hepatitis B ◽

Real Time ◽

Limit Of Detection ◽

Antiviral Treatment ◽

Hbv Dna ◽

Hbv Genotypes ◽

B Virus ◽

Test Version ◽

Related Liver Disease

ABSTRACT Sensitive and accurate hepatitis B virus (HBV) DNA detection and quantification are essential to diagnose HBV infection, establish the prognosis of HBV-related liver disease, and guide the decision to treat and monitor the virological response to antiviral treatment and the emergence of resistance. Currently available HBV DNA platforms and assays are generally designed for batching multiple specimens within an individual run and require at least one full day of work to complete the analyses. The aim of this study was to evaluate the ability of the newly developed, fully automated, one-step Aptima HBV Quant assay to accurately detect and quantify HBV DNA in a large series of patients infected with different HBV genotypes. The limit of detection of the assay was estimated to be 4.5 IU/ml. The specificity of the assay was 100%. Intra-assay and interassay coefficients of variation ranged from 0.29% to 5.07% and 4.90% to 6.85%, respectively. HBV DNA levels from patients infected with HBV genotypes A to F measured with the Aptima HBV Quant assay strongly correlated with those measured by two commercial real-time PCR comparators (Cobas AmpliPrep/Cobas TaqMan HBV test, version 2.0, and Abbott RealTi m e HBV test). In conclusion, the Aptima HBV Quant assay is sensitive, specific, and reproducible and accurately quantifies HBV DNA in plasma samples from patients with chronic HBV infections of all genotypes, including patients on antiviral treatment with nucleoside or nucleotide analogues. The Aptima HBV Quant assay can thus confidently be used to detect and quantify HBV DNA in both clinical trials with new anti-HBV drugs and clinical practice.

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Mechanism of Hepatitis B Virus cccDNA Formation

Viruses ◽

10.3390/v13081463 ◽

2021 ◽

Vol 13 (8) ◽

pp. 1463

Author(s):

Lei Wei ◽

Alexander Ploss

Keyword(s):

Hepatitis B Virus ◽

Hepatitis B ◽

Medical Problem ◽

Circular Dna ◽

Critical Step ◽

Hbv Cccdna ◽

Double Stranded Dna ◽

Cellular Environment ◽

B Virus ◽

Comprehensive Picture

Hepatitis B virus (HBV) remains a major medical problem affecting at least 257 million chronically infected patients who are at risk of developing serious, frequently fatal liver diseases. HBV is a small, partially double-stranded DNA virus that goes through an intricate replication cycle in its native cellular environment: human hepatocytes. A critical step in the viral life-cycle is the conversion of relaxed circular DNA (rcDNA) into covalently closed circular DNA (cccDNA), the latter being the major template for HBV gene transcription. For this conversion, HBV relies on multiple host factors, as enzymes capable of catalyzing the relevant reactions are not encoded in the viral genome. Combinations of genetic and biochemical approaches have produced findings that provide a more holistic picture of the complex mechanism of HBV cccDNA formation. Here, we review some of these studies that have helped to provide a comprehensive picture of rcDNA to cccDNA conversion. Mechanistic insights into this critical step for HBV persistence hold the key for devising new therapies that will lead not only to viral suppression but to a cure.

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Hepatitis B Core-Related Antigen as Surrogate Biomarker of Intrahepatic Hepatitis B Virus Covalently-Closed-Circular DNA in Patients with Chronic Hepatitis B: A Meta-Analysis

Diagnostics ◽

10.3390/diagnostics11020187 ◽

2021 ◽

Vol 11 (2) ◽

pp. 187

Author(s):

Gian Paolo Caviglia ◽

Angelo Armandi ◽

Chiara Rosso ◽

Davide Giuseppe Ribaldone ◽

Rinaldo Pellicano ◽

...

Keyword(s):

Hepatitis B Virus ◽

Hepatitis B ◽

Hepatitis B Surface Antigen ◽

Meta Analysis ◽

Circular Dna ◽

Non Invasive ◽

Hbv Cccdna ◽

B Virus ◽

Chronic Hbv ◽

Intrahepatic Hbv

Hepatitis B virus (HBV) covalently-closed-circular (ccc)DNA is the key molecule responsible for viral persistence within infected hepatocytes. The evaluation of HBV cccDNA is crucial for the management of patients with chronic HBV infection and for the personalization of treatment. However, the need for liver biopsy is the principal obstacle for the assessment of intrahepatic HBV cccDNA. In the last decade, several studies have investigated the performance of hepatitis B core-related antigen (HBcrAg) as a surrogate of HBV cccDNA amount in the liver. In this meta-analysis, we collected 14 studies (1271 patients) investigating the correlation between serum HBcrAg and intrahepatic HBV cccDNA. Serum HBcrAg showed a high correlation with intrahepatic HBV cccDNA (r = 0.641, 95% confidence interval (CI) 0.510–0.743, p < 0.001). In a head-to-head comparison, we observed that the performance of HBcrAg was significantly superior to that of hepatitis B surface antigen (r = 0.665 vs. r = 0.475, respectively, p < 0.001). Subgroup analysis showed that the correlation between HBcrAg and intrahepatic HBV cccDNA was high, both in hepatitis B e antigen-positive and -negative patients (r = 0.678, 95% CI 0.403–0.840, p < 0.001, and r = 0.578, 95% CI 0.344–0.744, p < 0.001, respectively). In conclusion, the measurement of serum HBcrAg qualifies as a reliable non-invasive surrogate for the assessment of an intrahepatic HBV cccDNA reservoir.

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272 Use of LioCyx-M, autologous hepatitis B virus (HBV)-Specific T cell receptor (TCR) T-cells, in advanced HBV-related hepatocellular carcinoma (HCC)

Journal for ImmunoTherapy of Cancer ◽

10.1136/jitc-2020-sitc2020.0272 ◽

2020 ◽

Vol 8 (Suppl 3) ◽

pp. A297-A297

Author(s):

Fu-Sheng Wang ◽

Fanping Meng ◽

Jiehua Jin ◽

Yuanyuan Li ◽

Regina Wanju Wong ◽

...

Keyword(s):

Hepatocellular Carcinoma ◽

T Cells ◽

Hepatitis B Virus ◽

T Cell ◽

Hepatitis B ◽

T Cell Receptor ◽

Dose Level ◽

Cell Receptor ◽

Hbv Dna ◽

B Virus

BackgroundWe have demonstrated the ability of Hepatitis-B-virus (HBV)-specific T cell receptor (TCR) bioengineered T cells to recognize and lyse Hepatocellular carcinoma (HCC) cells expressing HBV antigens derived from HBV-DNA integration in patients with liver transplant.1 LioCyx-M is an immunotherapeutic product composing of autologous T cells transiently modified with in-vitro transcribed mRNA encoding HBV-specific TCR. The transient TCR expression makes LioCyx -M amenable to a dose escalating posology.MethodsThe primary endpoint of this phase 1 trial is to assess the safety and tolerability of LioCyx-M in patients with advanced HBV-HCC without curative treatment options. Eligible patients were diagnosed with Barcelona clinic liver cancer stage B or C HCC (Child-Pugh < 7 points), receiving >1 year antiviral treatment prior to enrollment. These patients had matching HLA class I genotypes which present HBV encoded antigen. Peripheral blood was collected from each patient prior to each dose for LioCyx-M manufacturing. Patients received 4 escalating doses of 1×104 cells/kg, 1×105 cells/kg, 1×106 cells/kg, 5×106 cells/kg bodyweight (BW) in the first treatment cycle, each intravenously administered weekly. Patients underwent 1-month safety assessment post the 4th infusion, according to Common Terminology NCI CTCAE Version 4.0.3. If there were no dose associated toxicities, patients were eligible to continue administration of LioCyx-M at dose of 5 × 106 cells/kg BW weekly. Tumor response per RECIST 1.1 criteria and survival time were assessed.ResultsAt data cutoff (30 April 2020), eight patients were enrolled, with a median age of 53 (range: 49 - 67). These patients received a median number of 6 (range: 4 - 12) infusions of LioCyx-M. 1 patient developed Grade 3 elevations in alanine aminotransferase (ALT), gamma-glutamyl transferase (GGT), aspartate aminotransferase (AST) and bilirubin after receiving LioCyx-M at dose level of 1×105 cells/kg BW. Another patient had Grade 1 transient fever after receiving LioCyx-M at dose level 5×106 cells/kg BW in the 4th, 5th and 6th infusions. No treatment-related adverse events (trAEs) such as cytokine release syndrome or neurotoxicity were observed. No fatal trAEs were observed. The median time to progression was 1.9 months (range: 0.2 - 9.5 months). The median overall survival was 34 months (range: 3 - 38.2 months).ConclusionsThe encouraging clinical outcome and tolerable safety highlight the good benefit-risk profile of LioCyx-M. Therefore, further exploration of efficacy of LioCyx-M treatment for advanced HBV-HCC is warranted in a Phase 2 proof-of-concept clinical study.AcknowledgementsFunding: Lion TCR.Trial RegistrationNCT03899415Ethics ApprovalThe study was approved by Fifth Medical Center of Chinese PLA General Hospital’s Ethics Board, approval number R2016185DI010.ReferenceTan AT, Yang N, Lee Krishnamoorthy T, et al. Use of Expression Profiles of HBV-DNA Integrated Into Genomes of Hepatocellular Carcinoma Cells to Select T Cells for Immunotherapy. Gastroenterology 2019;156(6):1862–1876.e9.

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