This is an author version of the contribution published on:
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[Minerva Biotecnologica, 30(1), 2018, DOI : 10.23736/S1120-4826.17.02306-0]
ovvero [Gian Paolo Caviglia, Daniele Noviello, Rinaldo Pellicano, Antonella Olivero, 30,
ed. Minreva Medica, 2018, pagg.29-35]
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La versione definitiva è disponibile alla URL:
Role of serum hepatitis B core-related antigen (HBcrAg) in chronic
hepatitis B infection
Gian Paolo CAVIGLIA1,*, Daniele NOVIELLO1, Rinaldo PELLICANO2, Antonella OLIVERO1
1Department of Medical Sciences, University of Turin, Turin, Italy
2Unit of Gastroenterology, Molinette-SGAS Hospital, Turin, Italy
*Corresponding author: Gian Paolo Caviglia, Department of Medical Sciences, University of Turin, Turin 10100, Italy. E-mail: [email protected]
Abstract
Hepatitis B core-related antigen (HBcrAg) is a novel serum marker consisting of hepatitis B core antigen, hepatitis e antigen (HBeAg) and a 22 kDa precore protein without C-terminal arginine rich domain. All these core-related proteins can be simultaneously detected in serum and quantified by chemiluminescence enzyme immunoassay. Current available data indicate that HBcrAg serum levels vary according to the different phases of chronic hepatitis B (CHB) infection showing a strong correlation with both serum HBV DNA and intrahepatic HBV covalently closed circular DNA (cccDNA). In addition, HBcrAg levels seem able to identify CHB patients likely to respond to
interferon-based therapy and those treated with nucleos(t)ide analogues that have higher possibility of successful treatment cessation. Finally, recent data pointed out an association between HBcrAg levels and hepatocellular carcinoma (HCC) development and recurrence.
In this review we report and discuss novel findings and clinical applications of HBcrAg determination in patients with chronic hepatitis B virus infection.
Key words: Biomarkers; Hepatitis B core-related antigen; Hepatitis B virus; Antiviral therapy;
Hepatitis B virus (HBV) is the first greatest cause of chronic viral hepatitis worldwide.1 Despite
decades of vaccination, the estimated number of HBV infected patients in the world is approximately 257,000,000.2 Chronic hepatitis B (CHB) is the result of an acute, unresolved infection, that overtime
may lead to cirrhosis and its complications such as liver failure and hepatocellular carcinoma (HCC).3
The introduction of novel potent nucleos(t)ide analogues (NAs) with high genetic barrier to resistance and the implementation of several reliable serological and molecular biomarkers allowed remarkable advances in the management of CHB infected patients.4
Principal biomarkers for treatment monitoring include hepatitis B surface antigen (HBsAg), hepatitis B e antigen (HBeAg), alanine-aminotransferase (ALT) and HBV DNA. HBsAg represents the serological hallmark of HBV infection. HBsAg-positivity for more than six months determines a chronic infection. In the last years, quantitative HBsAg became clinically important to predictHBsAg loss and seroconversion to anti-HBs.5 HBeAg is a secretory viral protein considered a marker of HBV
replication and infectivity. Seroconversion from HBeAg-positivity to anti-HBe is usually associated with liver disease remission.6 ALT level reflects liver damage; patients in immune tolerance phase of
infection show normal ALT values, whereas those in immune clearance phase have persistently elevated ALT levels.7 A biochemical response to treatment is defined as a sustained ALT
normalization.8 HBV DNA is the principal criteria for virologic response to treatment (<2000 IU/mL).
Sustained low HBV DNA levels in association to HBeAg-negativity, anti-HBe-positivity and normal serum ALT characterize the inactive chronic HBsAg carriers that usually do not have indication for antiviral treatment.9
Beside conventional serological markers for hepatitis B, novel biomarkers such as microRNA and other epigenetic factors have been investigated in order to provide clinicians novel tools for optimal CHB patients management.10 However, results are conflicting and far from a potential use in
long amino-acid sequence with HBeAg and hepatitis B core antigen (HBcAg), has been recently described as the main capsid protein in viral DNA-negative Dane particles (Figure 1).11,12 A fully
automated chemiluminescence enzyme immunoassay (CLEIA) has been developed for the simultaneous quantitation of HBeAg, HBcAg and p22cr, namely hepatitis B core-related antigen (HBcrAg) assay.13
The aim of this review is to report and discuss novel findings and clinical applications of HBcrAg determination in patients with chronic HBV infection.
HBcrAg assay
HBV core-related proteins present in the serum of HBV infected patients can be detected and
quantified by CLEIA technology through a two-step immunoassay method on the Lumipulse® G
System (Lumipulse® G HBcrAg, Fujirebio Inc, Tokyo, Japan) using monoclonal antibodies that
recognize denatured HBeAg, HBcAg and p22cr common epitopes.14
Before testing, 150 µL of serum specimen is incubated with 150 µL of pre-treatment solution containing detergent at 60°C for 30 minutes. After heat treatment, core-related antigens in specimen specifically bind to anti-HBcr antibodies present in assay cartridge micro-particles suspension.
Following the formation of antigen-antibody immune-complexes, alkaline phosphatase (ALP)-labeled anti-HBcr antibodies specifically bind to HBcrAg and additional immune-complexes are formed. After washing, substrate solution [AMPPD: 3-(2’spiroadamantane)-4-methoxy-4-(3”-phosphoryloxy)phenyl-1,2-dioxetane disodium salt] is added and mixed with the particles. AMPPD contained in substrate solution is dephosphorylated by catalysis of ALP indirectly conjugated to particles. Luminescence (at a maximum wavelength of 477 nm) is generated by cleavage reaction of dephosphorylated AMPPD. The luminescence signal reflects the amount of HBcrAg and concentration is calculated by comparison with a standard curve generated using recombinant pro-HBeAg. The immunoreactivity of pro-HBeAg at 10
fg/mL is defined as 1 U/mL. HBcrAg values are expressed as Log U/mL with an analytic measurement range between 2.0-7.0 Log U/mL.
HBcrAg in the natural history of chronic hepatitis B infection
Several reports showed that the different phases of CHB infection can be distinguished according to HBcrAg serum levels.15,16 Seto et al investigated HBcrAg levels in a cohort of 404 Asian
treatment-naïve CHB patients with no evidence of concomitant liver disease grouped according to different disease phases of CHB infection: immune tolerance phase (HBeAg-positive and normal ALT), immune clearance phase (HBeAg-positive and elevated ALT), HBeAg-negative hepatitis phase (elevated ALT), HBeAg-negative quiescent phase (normal ALT) and known CHB with HBsAg
seroclearance. Among HBeAg-positive disease group, median HBcrAg levels were significantly higher in immune tolerance than in immune clearance phase (8.54 vs. 7.82 Log U/mL, p<0.001). Within HBeAg-negative disease group, median HBcrAg levels were significantly lower in HBeAg-negative quiescent than in HBeAg-negative hepatitis phase (2.60 vs. 4.92 Log U/mL, p<0.001), whereas among patients that experienced HBsAg seroclearance, only 12 (21.8%) had detectable HBcrAg.15 Similarly,
in a large European cohort predominantly infected with HBV genotypes A and D, higher HBcrAg levels were found in immune tolerance and immune clearance phases (8.41 [7.42-9.61] vs. 8.11 [4.57-10.34] Log U/mL; p=0.002), whereas significantly lower levels were observed in HBeAg-negative CHB (4.82 [2.00-7.73] Log U/mL) and in inactive HBV carriers (2.00 [2.00-5.35] Log U/mL).16
Interestingly, several studies pointed out a moderate correlation between HBcrAg and HBsAg (from r=0.199 to r=0.765) and a good correlation between HBcrAg and HBV DNA (from r=0.498 to r=0.854), both in HBeAg-positive (r=0.765) and -negative patients (r=0.635).13,15-17 Finally, some body
of evidence suggest that HBcrAg may be useful for predicting the pathological status of liver tissues in CHB patients. Zhang et al enrolled 205 Chinese CHB patients (121 positive and 84
HBeAg-negative) to evaluate HBcrAg effectiveness in predicting the degree of liver inflammation and fibrosis.18 In HBeAg-positive patients, serum HBcrAg accuracies (AUCs) for severe
necro-inflammation and advanced fibrosis were 0.711 (cut off ≤ 4.68 Log U/mL, sensitivity [Se]=0.697 and specificity [Sp]=0.716) and 0.785 (cut off ≤ 4.39 Log U/mL, Se=0.818 and Sp=0.778), respectively. In HBeAg-negative patients, serum HBcrAg AUCs for significant necro-inflammation and significant fibrosis were 0.952 (cut off ≥2.23 Log U/mL, Se=0.929 and Sp=0.964) and 1.000 (cut off ≥1.60 Log
U/mL, Se=1.000 and Sp=1.000).18
Finally, two recent independent studies investigated HBcrAg usefulness for the identification of inactive carriers among HBeAg-negative HBV infected patients.19,20 The combination of a single
measurement of HBcrAg ≤3 Log U/mL and HBV DNA ≤2000 IU/mL yielded both a positive
predictive value and diagnostic accuracy greater than 85% for identifying inactive carriers regardless of HBV genotype, in comparison to HBsAg levels <3 Log IU/mL that were only useful for identifying genotype-D inactive carriers.19 Similarly, Oliveri et al found a significant higher proportion of patients
with HBcrAg levels <3 Log UI/mL in inactive carriers in comparison to HBeAg-negative patients with active infection (96.5% vs. 77.3%, p=0.001),and the combination of a single measurement of HBcrAg <3 Log U/mL and HBV DNA <2000 IU/mL showed a diagnostic accuracy of 86.8%.20
HBcrAg as surrogate biomarker for HBV cccDNA
Measurement of HBV covalently closed circular DNA (cccDNA) holds important clinical implications but it is mainly limited by liver biopsy availability and the lack of sensitive standardized PCR methods. Recently, a significant correlation between HBcrAg and intrahepatic HBV cccDNA has been
reported.21-23
Suzuki et al investigated the correlation between HBcrAg and intrahepatic HBV cccDNA in 57 CHB patients, including 13 HBsAg-negative/HBs-seroclearance patients, and they found a positive
correlation between the two parameters (r=0.692, p<0.001). In addition, a significant correlation was observed also in HBV DNA-negative patients (r=0.482, p=0.006), with different HBcrAg levels between HBsAg-positive and HBsAg-negative patients (5.05±0.21 Log U/mL vs. 3.11±0.21 Log U/mL, p<0.001) suggesting a potential role of HBcrAg in subjects with occult HBV infection.24,25 Also
Chen et al reported an high correlation between HBcrAg and HBV cccDNA in 139 untreated CHB patients (r=0.929, p<0.001).26 In addition, evaluating a subgroup of 32 entecavir (ETV)-treated patients,
authors found that on-treatment HBcrAg dynamic changes were significantly associated to HBV cccDNA decline (r=0.665, p<0.001).26
More recently, Matsuzaki et al found a correlation between HBcrAg and HBV cccDNA levels (r=0.616, p<0.001) in patient who underwent liver transplant (LT) for HBV-related liver disease.22
After excluding from analysis preoperative state samples, HBcrAg remained significantly correlated to HBV cccDNA (r=0.402, p=0.046). In addition, HBcrAg and HBV cccDNA levels showed similar dynamics during pre- and post-transplantation, denoting HBcrAg reliability as a surrogate biomarker for HBV cccDNA monitoring in the setting of LT.
A large scale study including 305 liver biopsies and corresponding serum samples collected from 138 NAs-treated patients showed a median HBcrAg reduction at ≥6 years of therapy comparable to the magnitude of HBV cccDNA decay.23 HBcrAg was strongly correlated with intrahepatic HBV
cccDNA (r=0.70, p<0.0001). According to HBeAg status, HBcrAg was significantly correlated to HBV cccDNA levels both in the 133 HBeAg-positive patients (r=0.66, p<0.0001) and in the 172 -negative patients (r=0.45, p<0.0001). Moreover, analyzing 130 samples of HBV DNA-negative patients receiving antiviral therapy, authors observed that HBcrAg was still detectable in 101 of them and HBcrAg was significantly correlated with HBV cccDNA levels (r=0.42, p<0.0001).23
Finally, serum HBcrAg showed a positive correlation not only with intrahepatic HBV cccDNA (0.77, p<0.0001) but also with HBV cccDNA productivity, defined as the total intrahepatic HBV
DNA/cccDNA ratio (r=0.69, p<0.0001).27 These findings suggest that HBcrAg may represent not only
a useful surrogate marker for intrahepatic HBV cccDNA quantitation, but also an important tool for HBV cccDNA transcriptional and replicative activity investigation.
HBcrAg role in chronic hepatitis B treatment
Currently available drugs for CHB treatment include the immune-modulatory interferon-α (IFN-α) and NAs which are HBV reverse transcriptase inhibitors causing termination of HBV DNA chain
prolongation.28 In order to guide treatment decisions, the role of HBV biomarkers in therapy outcome
prediction has been investigated.29 However, results are not entirely satisfactory. It has been shown that
HBV DNA is not a reliable predictor of sustained response to IFN-based therapy because of similar kinetics between non-responder patients and those who relapse, whereas in NA-treated patients, HBV DNA becomes rapidly undetectable without excluding the possibility of viral replication reactivation in case of therapy cessation.30 HBsAg quantification has been proposed as surrogate marker of outcome
prediction in patients treated with pegylated (PEG)-IFN,31-33 while evidence from studies involving
Asian population mainly infected with HBV genotypes B and C, suggests that HBcrAg could be a potential marker for NAs therapy cessation.34,35 Low HBcrAg levels were associated with favorable
ETV treatment outcome, response to NAs/interferon-α sequential therapy and with lower risk of hepatitis reactivation following discontinuation of lamivudine (LAM) in CHB patients or
immunosuppressive therapy in occult HBV carriers.36,37 Guidelines for avoiding risks resulting from
discontinuation of NAs have been proposed and incorporated in the official Japan Society of Hepatology (JSH) guidelines for the management of HBV infection.38,39 Such recommendations are
based on a score that includes HBsAg and HBcrAg levels for NAs therapy cessation in CHB patients with at least two years of NAs administration, undetectable serum HBV DNA and negative serum HBeAg.39 Accordingly, patients with HBsAg load <1.9 Log IU/mL and HBcrAg <3.0 Log U/mL have
a low risk of relapse (predicted success rate 80-90%) whereas patients with HBsAg >2.9 Log IU/mL and HBcrAg >4.0 Log U/mL must continue treatment (predicted success rate 10-20%) (Table I).39
Previously, studying a longitudinal cohort of NAs treated CHB genotype D patients, we identified a subgroup of patients who experienced an early HBcrAg decline reaching undetectable values between months 6 and 12 (<2 Log U/mL) whereas another group maintained sustained HBcrAg values at last
follow up (4.4±0.6 Log U/mL at month 36) irrespectively from HBV DNA and HBsAg kinetics.13
According to JSH guidelines for NAs therapy cessation, all HBcrAg rapid decliner patients were classified into the moderate risk group (relapse risk ≈50%), while patients of month-36 HBcrAg-positive group were classified into the high risk of relapse group (p=0.0005),13 suggesting a potential
clinical role for HBcrAg kinetic also in CHB genotype D patients undergoing NAs treatment.
HBcrAg has been investigated also in HBeAg-positive and -negative CHB patients receiving PEG-IFN. Chuaypen et al retrospectively analyzed data of HBeAg-positive CHB patients of Thai nationality (n=46) receiving PEG-IFN for 48 weeks and found similar HBcrAg baseline levels between responders and non-responders, but responders showed more rapid HBcrAg decline during and after treatment.40 Moreover, negative predictive values (NPVs) of achieving virological response (HBeAg
clearance and HBV DNA <2000 IU/mL at week 72) were 89.5% and 100% at week 12 and 24,
respectively.40 Similarly, van Campenhout et al studied 133 HBeAg-negative CHB patients treated with
PEG-IFN ± ribavirin for 48 weeks and found that HBcrAg decline at week 12 was stronger in patients who achieved a sustained response (ALT normalization and HBV DNA <2000 UI/mL at week 72) compared to non-responders (-1.5 Log U/mL vs. -1.0 Log U/mL, p=0.04).41 In addition, other studies
showed that a baseline HBcrAg 3.45 Log U/ml threshold allowed the prediction of PEG-IFN-based therapy with a positive predictive value of 80.3% and a NPV of 76.5%.42 The identification of CHB
patients likely to respond to PEG-IFN-based therapy could allow a more individualized treatment increasing response rates in selected patients.
HBcrAg for hepatocellular carcinoma prediction
To date, the use of serum biomarkers in the setting of early HCC detection and prediction is still a matter of debate.43-45 Beside well knows risk factors for HCC development such as age, gender, alcohol
consumption and genetic susceptibility, several HBV biomarkers including basal core promoter mutations, HBeAg positivity and HBV DNA levels were reported as factors associated with tumor development.46-52
Interestingly, recent data pointed out a relationship between HBcrAg levels and HCC. In a cohort of 55 patients who developed HCC during NAs therapy and underwent either curative resection or percutaneous ablation, Hosaka et al found that HBcrAg ≥4.8 Log U/mL at the time of tumor
diagnosis (hazard ratio [HR]=8.96, 95%confidence interval [CI] 1.94-41.4) and portal vein invasion were independent risk factors for HCC recurrence.53 Higher HBcrAg levels have been reported in
HBV-related HCC in comparison to non-HCC matched patients (p=0.005),54 and high on-treatment
HBcrAg levels were significantly associated with HCC development in CHB patients undergoing NAs therapy.55,56 Recently, Tada et al further clarified the predictive power of HBcrAg for HCC
development.57Authors retrospectively enrolled a total of 1031 untreated CHB patients with no other
causes of liver disease and a follow-up duration greater than 1 year. They found that only HBcrAg >2.9 Log U/mL and basal core promoter mutation were significantly and independently associated with
HCC incidence (HR=5.05, 95%CI 2.40-10.63 and HR=28.85, 95%CI 4.00-208.20, respectively).57
Conclusions
The wide spectrum of potential clinical applications in the setting of CHB makes HBcrAg extremely compelling. Noteworthy is the strong correlation between serum HBcrAg and intrahepatic cccDNA. Currently, the major treatment endpoint is the “functional cure” of CHB defined as long-term
suppression of HBV replication, ALT normalization and HBsAg seroclearance. Considering that novel antiviral drugs for definitive cccDNA elimination are under development in order to obtain the so-called “complete cure”, HBcrAg may represent a reliable non-invasive approach to monitor intrahepatic virological status.
Conflicts of interest. The authors certify that there is no conflict of interest with any financial
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Table I. HBcrAg- and HBsAg-based score for the evaluation of relapse risk following NAs therapy
cessation
HBsAg (IU/mL) Score HBcrAg (U/mL) Score Total Score Success rate
<1.9 Log 0 <3 Log 0 0
(Low risk)
80-90%
≥1.9 Log, <2.9 Log 1 ≥3 Log, <4 Log 1 1-2
(Moderate risk)
≈50%
≥2.9 Log 2 ≥4 Log 2 3-4
(High risk)
10-20%
Figure 1. HBV related biomarkers
Modified from Caviglia et al.12
HBcAg (21.5 kDa) is the main viral capsid structural protein that is translated from pregenomic HBV RNA. Similarly, HBeAg (17 kDa) is a secretory non-structural protein with immune-modulatory proprieties that is a product of precore/core gene. Also p22cr (22 kDa) is translated from the same mRNA, but includes an uncleaved signal and lacks the C-terminal arginine-rich domain that is
into capsid particles. Such empty particles are enclosed within HBsAg envelope and released into the circulation likewise Dane particles.
Abbreviations: HBcAg, hepatitis B core antigen; HBcrAg, hepatitis B core-related antigen; HBeAg, hepatitis B e antigen; HBsAg, hepatitis B surface antigen.
