Research ArticleSerum hepatitis B core-related antigen (HBcrAg) correlates with covalently closed circular DNA transcriptional activity in chronic hepatitis B patients
Graphical abstract
Introduction
The development of novel antiviral agents and immunomodulatory approaches to cure hepatitis B virus (HBV) infection requires new biomarkers capable of reflecting the intrahepatic activity of the virus and chronic HBV (CHB) stages and defining new meaningful treatment endpoints. Indeed, there is an unmet need for standardized assays able to provide mechanistic insights into the effects of the novel antiviral and immunomodulatory agents and to assess treatment efficacy.1
HBV covalently closed circular (ccc)DNA constitutes the unique template for pregenomic (pg)RNA transcription and viral genome replication. Its persistence in the nucleus of infected cells is responsible for the chronicity of HBV infection.2 So far, antiviral therapies have demonstrated a modest effect on the established cccDNA pool.[3], [4], [5], [6] Measurement of intrahepatic cccDNA levels and transcriptional activity is therefore crucial for the management of patients with CHB and for treatment individualization. The need for liver biopsy strongly limits the evaluation of cccDNA in routine clinical practice. Currently, serum HBV-DNA, hepatitis B surface antigen (HBsAg) and the quantification of HBsAg (qHBsAg) are the most widely used viral markers to diagnose HBV infection and to monitor antiviral therapy.7 Nucleos(t)ides analogues (NAs) block viral polymerase and are highly efficient in achieving viral suppression, despite the continuous presence of cccDNA in infected hepatocytes. Thus, the correlation between intrahepatic cccDNA and serum HBV-DNA is lost and serum HBV-DNA quantification cannot be considered a surrogate marker for cccDNA in NA-treated patients.4 HBsAg is the hallmark of infection and its clearance is considered to be the most important clinical endpoint7 because both spontaneous and therapy-induced HBsAg loss are associated with histological improvement, a reduced risk of hepatocellular carcinoma and prolonged survival.[8], [9], [10], [11] The degree of correlation between qHBsAg and intrahepatic viral markers, in particular cccDNA levels, varies greatly between studies and is still debated,[12], [13], [14], [15], [16], [17], [18] particularly in HBeAg− carriers, where expression from HBV integrants may significantly contribute to HBsAg production, in addition to its expression from the cccDNA template,19 as has been shown in HBV infected chimpanzees.20
Indeed, during NA therapy, the kinetics of qHBsAg decline are much slower in HBeAg− patients than in HBeAg+ patients and slower than those of serum HBV-DNA, reflecting the pool of infected hepatocytes harboring either cccDNA or integrated viral sequences.[21], [22], [23] Quantification of serum HBV RNAs may represent a novel option to predict virological response to both NAs and interferon,[24], [25] but their correlation with intrahepatic viral parameters needs further investigation with well-defined assays. As yet, no surrogate serum marker satisfactorily reflects the pool of transcriptionally active cccDNA in the liver. The so-called hepatitis B core-related antigen (HBcrAg) assay utilizes a mixture of monoclonal antibodies isolated from HBV core antigen-immunized mice26 to detect and quantify HBV core antigen (HBcAg), free HBeAg, HBeAg−antibody complex, and the 22 kDa precore protein (p22cr).[26], [27] Several reports suggest that HBcrAg levels correlate with serum HBV-DNA in untreated patients with CHB and might be useful to differentiate HBeAg− patients with active and inactive disease.[28], [29], [30], [31], [32] A correlation between HBcrAg levels and the size of the intrahepatic cccDNA pool has been suggested in cohorts of Asian patients with genotype B/C CHB, either untreated[33], [34], [35] or undergoing NA therapy.[35], [36], [37] HBcrAg has been also shown to correlate with intrahepatic viral RNA levels in Asian patients treated with NAs.37 No studies are available on HBcrAg and intrahepatic viral parameters in HBV genotypes other than B/C and it remains to be defined whether, and to what extent, HBcrAg serum levels reflect the transcriptional activity of cccDNA.
To better characterize the clinical value of HBcrAg detection, we have investigated the relationship between circulating HBcrAg, HBV-DNA, qHBsAg and intrahepatic cccDNA transcriptional activity, in a cohort of untreated patients with CHB infected with several HBV genotypes.
Section snippets
Patients
Serum samples concomitant to liver biopsy were available for the study in 130 untreated patients with CHB (4 HBeAg+ chronic infection (CI), 32 HBeAg+ chronic hepatitis (CH), 33 HBeAg− CI and 61 HBeAg− CH) (Table S1),7 who underwent needle liver biopsy for routine histology assessment, enrolled at the Hepatology Unit of the “Hospices Civils de Lyon” and at the Gastroenterology and Hepatology Unit of the “Ospedale Maggiore Policlinico” in Milan. Part of the liver tissue was snap frozen and stored
Patients’ characteristics and results of serum and intrahepatic viral marker analysis
Table 1 summarizes patients' demographics and virological parameters in serum and in the liver. Most of the patients were of Caucasian (55/130) and African (41/130) origin with a predominance of HBV genotype D (51/105 tested) (Table 1). HBeAg+ patients (HBeAg+ CI and HBeAg+ CH) had the highest HBcrAg levels, with 78% showing HBcrAg levels >7 LogU/ml, while a wider and heterogeneous distribution was found in HBeAg− patients (HBeAg− CI and HBeAg− CH), with HBcrAg levels <3 LogU/ml in 35% of cases
Discussion
The key determinant of chronicity of HBV infection is the persistence of cccDNA in the infected hepatocytes.2 An accurate monitoring of intrahepatic cccDNA levels and activity during patient management is limited by the need for invasive liver biopsy procedures. So far, no single serum parameter has been shown to accurately reflect the transcriptional activity of the cccDNA pool in the liver, an important target in the perspective of HBV functional cure.1 As a consequence, there is an unmet
Financial support
This work was supported by grants from «Agence Nationale pour la Recherche sur le SIDA et les hepatites virales» (ANRS) to FZ (n°N16003CR) and to FZ and ML (n°ECTZ8323), from the French Research Agency ANR (Agence National de la Recherche) to FZ and ML (RHU cirB-RNA).
Conflict of interest
FZ received research grants and consultancy honoraria from Hoffmann-La-Roche, Gilead Sciences, Janssen, Assembly Biosciences, Arbutus, Contravir, Sanofi and Transgene. ML participates to advisory boards of BMS, Assembly Biosciences, Gilead, Arbutus, Janssen, Galapagos, Medimmune. PL participates to advisor and speaker bureau for Gilead, Roche, BMS, GSK, MSD, Arrowhead and Alnylam.
Authors’ contributions
BT, PL, ML, FZ conceived the experiments. BT, FL, CM, FB performed experiments and analyzed data. FL, SC, SM, FF, AL collected clinical samples and patients’ information. BT, PL, ML, FZ wrote the manuscript.
Acknowledgements
We would like to thank Dr Laura Vernoux (Fujirebio Europe; Gent, Belgium) for providing reagents and technical assistance and Dr. Christophe Combet (INSERM U1052-CRCL) for HBV genotype determination and access to the HBVdb database.
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These authors contributed equally to the work.