Total Hepatitis B Core Antigen Antibody, a Quantitative Non-Invasive Marker of Hepatitis B Virus Induced Liver Disease

Non invasive immunologic markers of virus-induced liver disease are unmet needs. We tested the clinical significance of quantitative total and IgM-anti-HBc in well characterized chronic-HBsAg-carriers. Sera (212) were obtained from 111 HBsAg-carriers followed-up for 52 months (28-216) during different phases of chronic-HBV-genotype-D-infection: 10 HBeAg-positive, 25 inactive-carriers (HBV-DNA≤2000IU/ml, ALT<30U/L), 66 HBeAg-negative-CHB-patients and 10 with HDV-super-infection. In 35 patients treated with Peg-IFN±nucleos(t)ide-analogues (NUCs) sera were obtained at baseline, end-of-therapy and week-24-off-therapy and in 22 treated with NUCs (for 60 months, 42-134m) at baseline and end-of-follow-up. HBsAg and IgM-anti-HBc were measured by Architect-assays (Abbott, USA); total-anti-HBc by double-antigen-sandwich-immune-assay (Wantai, China); HBV-DNA by COBAS-TaqMan (Roche, Germany). Total-anti-HBc were detectable in all sera with lower levels in HBsAg-carriers without CHB (immune-tolerant, inactive and HDV-superinfected, median 3.26, range 2.26-4.49 Log10 IU/ml) versus untreated-CHB (median 4.68, range 2.76-5.54 Log10 IU/ml), p<0.0001. IgM-anti-HBc positive using the chronic-hepatitis-cut-off" (0.130-S/CO) were positive in 102 of 212 sera (48.1%). Overall total-anti-HBc and IgM-anti-HBc correlated significantly (p<0.001, r=0.417). Total-anti-HBc declined significantly in CHB patients with response to Peg-IFN (p<0.001) and in NUC-treated patients (p<0.001); the lowest levels (median 2.68, range 2.12-3.08 Log10 IU/ml) were found in long-term responders who cleared HBsAg subsequently. During spontaneous and therapy-induced fluctuations of CHB (remissions and reactivations) total- and IgM-anti-HBc correlated with ALT (p<0.001, r=0.351 and p=0.008, r=0.185 respectively). Total-anti-HBc qualifies as a useful marker of HBV-induced-liver-disease that might help to discriminate major phases of chronic HBV infection and to predict sustained response to antivirals.


Introduction
Hepatitis-B-Virus (HBV) infection elicits a prominent immune response against hepatitis-Bcore-antigen (HBcAg) [1][2][3][4][5]: IgG-antibodies against HBcAg (anti-HBc) persist lifelong in HBV exposed individuals whereas IgM-antibodies decline becoming undetectable after recovery from hepatitis-B [4,[6][7][8]. Commercial assays for IgM-anti-HBc are proposed for diagnosis of acute-hepatitis-B (AHB) that is based on a clinically standardized threshold of 600 Paul-Ehrlich (PEI) [6][7][8][9]. Using the analytical sensitivity cut-off of the assays low levels of IgM-anti-HBc were found to fluctuate in chronic-hepatitis-B (CHB) paralleling the disease remission and reactivation phases [10][11][12][13][14][15]. This prompted the use of different cut-offs values for acute and chronic hepatitis B [10][11][12][13]. However, the quantification range of commercially available IgM-anti-HBc assays, specifically designed for diagnosis of AHB hampers the diagnostic accuracy of detecting low antibody levels in chronic HBsAg-carriers [14][15][16][17]. Thus, a satisfactory non-invasive marker of HBV-induced liver disease in chronic HBV infection remains an unmet need. In clinical practice the combined quantification of serum HBV-DNA and HBsAg is currently used to discriminate HBeAg-negative-CHB from inactive infection and to monitor the switch from CHB to inactive-carrier during antiviral therapy [17][18][19][20]. However, in HBeAgnegative CHB patients undergoing antiviral treatment HBsAg kinetics vary according to HBV genotype and genotype-specific monitoring timeframes and end-of-treatment thresholds have yet to be standardized for a proper response-guided treatment 19]. Recently a new assay based on double antigen sandwich method that detects total (IgG and IgM) anti-HBc was developed and proposed to monitor patients, chronically infected with genotype B and C HBV undergoing antiviral therapy [21][22][23][24]. We tested this assay in a validation study on a panel of sera from well characterized genotype D, HBsAg carriers.

Materials and Methods
Sera (212) were obtained from 111 chronic-HBsAg-carriers infected with HBV-genotype-D (Table 1) followed-up (mean 52-months, 28-216-months). Patients gave an informed consent and the study was approved by the Ethic Committee of the University Hospital of Pisa. All of them were followed with blood tests every 3-6 months, but those with low HBV-DNA levels (<20000 IU/mL) and normal transaminases (ALT) at presentation, underwent monthly testing for at least 1-year for characterization. HBsAg-carriers were classified according to their biochemical and viral profiles [9,17].

Statistics
Data were expressed in median, range and percentiles values. Unpaired t-test and Whitney-U-test were used for total-and IgM-anti-HBc comparisons between groups. The Pearson-correlation  was used for the correlation between total-and IgM-anti-HBc and other continuous variables. ROC analysis was used to analyze the efficacy of total-and IgM-anti-HBc to distinguish different HBsAg-carrier groups. Statistical analysis was performed using SPSS-17.0 software (SPSS, Chicago, IL, USA). All statistical analyses were based on 2-tailed hypothesis tests with a significance level of p<0.05.

Results
All sera tested positive for total-anti-HBc: their levels were significantly lower in HBsAg-carriers without HBV-induced liver disease (median 3.26, range 2.26-4.49Log 10   Overall total-anti-HBc and IgM-anti-HBc correlated significantly (p<0.001, r = 0.417) and both total-anti-HBc and IgM-anti-HBc correlated with ALT p<0.001, r = 0.351 and p = 0.008, r = 0.185 respectively. Using total-anti-HBc and IgM-anti-HBc as binary classifiers we ran two ROC curves separating: A) Inactive carriers (IC) from chronic-hepatitis-B (CHB) patients (
The variations of serum levels of HBsAg, HBV-DNA, total-anti-HBc, IgM anti-HBc from BL to EOT were compared in Peg-IFN±NUCs treated patients according to their treatment response (Fig 3). Overall, serum HBsAg and HBV-DNA the correlated with total anti-HBc significantly (p<0.001 r = 0.303 and p<0.001, r = 0.654 respectively), whereas they did not with IgM-anti-HBc (p = 075, r = 0.123 and p = 0.050, r = 0.135 respectively).
All HBeAg-negative-CHB patients (22)  Finally we studied the dynamic variations of ALT and HBV markers in 18 paired sera from 9 REL after Peg-IFN at the time of their on-treatment disease remission and hepatitis-B-relapse after treatment discontinuation and in 10 paired sera from 5 untreated HBeAg-negative-CHB patients with spontaneous remissions and reactivations. In Peg-IFN-REL all biomarkers showed statistically significant variations, but HBsAg, whereas in untreated patients only ALT variations were statistically significant whereas for total-anti-HBc, IgM anti-HBc and HBV-DNA there was a trend to significance (Fig 4).

Discussion
The dynamic quantification range of the new total-anti-HBc assay allows to distinguish chronic-HBV-infection associated with HBV-induced liver disease, namely chronic-hepatitis-B (CHB), from chronic HBV infection without HBV-induced liver damage, namely noninflammatory HBeAg-positive and inactive HBeAg-negative phases, independently from HBV-genotypes. Our validation study confirms in well characterized HBsAg-carriers infected with genotype-D-HBV the results obtained in Asian patients infected with genotype B and C [21]; the highest levels of total-anti-HBc were detected in CHB, both HBeAg-positive and HBeAg-negative and the lowest levels in inactive-carriers (Fig 1). Very low levels of the antibody were reported in the non-inflammatory, HBeAg-positive immune-tolerant-phase in genotype-B and-C infections [21][22][23]. Consistently in our HBeAg-positive-carriers total-anti-HBc levels comparable to those of inactive-carriers were found only in an immune-tolerant carrier, whereas the remaining CHB-patients showed high antibody levels. Total-anti-HBc declined during antiviral and correlated with response to Peg-IFN±NUC at EOT in both REL and SVR and at EOF in SVR during NUC treatment (Figs 1 and 3). In SVR-patients total-anti-HBc levels declined reaching at EOF values comparable to inactive-carriers. Interestingly in NUC-treated patients, the lower total-anti-HBc serum levels corresponded to the longer follow-up in disease remission. Antibody levels were significantly lower in NUC-treated patients) tested about 60 months after starting therapy as compared with patients with Peg-IFN SVR tested 24-weeks after EOT (p = 0.002). The distribution pattern of IgM-anti-HBc in the same subgroups mimics that of total-anti-HBc, but unfortunately the dynamic quantification range of the IgM-assay hampers the diagnostic accuracy since half of values fall below the analytical specificity cut-off of the assay. Comparing the kinetics of the different HBV-markers according to response to Peg-IFN we found (Fig 3) that the decline of total-anti-HBc during therapy parallels that of HBV-DNA and this may be useful in clinical practice to confirm the effectiveness of Peg-IFN antiviral activity. However, during Peg-IFN-treatment total-anti-HBc is unable to distinguish REL from SVR who are instead identified better by HBsAg-kinetics as reported previously [17][18][19][20]. During NUC therapy the kinetics of total anti-HBc differ from those of the other HBV markers and the total-anti-HBc decline provides an added value to HBV-DNA and HBsAg monitoring beckoning the remission of HBV-induced liver damage under effective antiviral therapy. In patients treated with Entecavir or Tenofovir viral load is consistently suppressed in the very early phase of treatment, whereas HBsAg declines very slowly over time [17][18][19][20]. Interestingly long-term NUC-treated patients with very low levels of total-anti-HBc experienced the HBsAg-clearance with anti-HBs-sero-conversion during follow-up. Thus, quantification of total-anti-HBc might help to predict HBsAg loss and future long-term prospective studies should address the issue in larger cohort of patients. Finally, the findings that total-anti-HBc kinetics correlate with ALT levels as well as IgM-anti-HBc in both naturally-occurring [25][26][27] and therapy-induced remission and reactivation phases [26,27] support the view that total-anti-HBc is a reliable marker of HBV-induced liver disease in chronic HBsAg-carriers. In chronic HBeAg positive hepatitis B baseline total-anti-HBc levels were shown to predict HBeAg to anti-HBe seroconversion in Asiatic HBeAg-positive-CHB patients, treated with Peg-IFN or NUC infected with HBV genotype B and C [24]. Our findings underline the potential of the assay also in the management of chronic HBeAg-negative-CHB of the Mediterranean Area infected with HBV genotype D. In addition our work suggests that Total Anti-HBc and HBV Induced Liver Disease quantification of total-anti-HBc may be helpful to distinguish the inactive HBsAg carrier from HBeAg-negative-CHB which is characterized by intervening phases of disease remission and reactivation [25][26], to identify patients with higher chance of HBsAg clearance and to provide a new tool to answer the unmet needs for treatment tailoring [27][28][29][30].
All available data support the view that q-anti-HBc is complementary to HBsAg quantification. HBsAg is a product of HBV replication, ccc-HBV-DNA transcription and viral mRNAs translation whereas total-anti-HBc is expression of the antiviral immune response against the HBV "core" antigen. Our data suggest that symmetry or asymmetry of the two markers may have important diagnostic implications: high levels of HBsAg associated with low levels of total-anti-HBc are diagnostic for the immune-tolerance or florid non-inflammatory phase of HBeAg-positive HBV-infection, while high levels of both markers identify chronic hepatitis B (either HBeAg-positive and HBeAg-negative). In cirrhotic patients with advanced HBeAg-negative-CHB total-anti-HBc levels are high because of persistent HBV-induced liver disease whereas HBsAg may decline because of HBsAg deletion mutants which hamper HBsAg secretion. In treated patients the decline of total-anti-HBc parallel HBsAg-kinetics in patients who respond to anti-viral treatment (sustained responders to therapy), but is asymmetric with the unchanged HBsAg levels in patients whose hepatitis B recurs after treatment discontinuation (Relaspers). In addition the decline of HBsAg during antiviral therapy is HBV genotype dependent whereas the decline of total-anti-HBc is HBV genotype independent. Thus the combined used of both markers can improve the management of the HBsAg carrier consistently.
In conclusion total-anti-HBc as measured by double-antigen-sandwich-immune-assay is a reliable non invasive marker of HBV-induced liver disease helpful to identify chronic-HBVinfection associated with HBV-induced liver disease. Larger prospective studies are needed address to address its significance particularly in the clinical management of NUC-treated patients.