Characterization of a novel chemiluminescent enzyme immunoassay for the quantitation of antibodies to hepatitis B core antigen class IgG and correlation with intrahepatic HBV covalently-closed-circular DNA

Background and aims: Non-invasive biomarkers for the cure and the management of chronic hepatitis B (CHB) infection are an unmet need. Our aim was to characterize a novel HBV assay for the quantitation of anti-HBc IgG and to assess the correlation between serum anti-HBc IgG and intrahepatic HBV cccDNA, in comparison to quantitative hepatitis B surface antigen (qHBsAg) and hepatitis B core-related antigen (HBcrAg) in patients with CHB infection. Method: Serum samples and liver specimens were collected from 35 CHB patients (26M/9F; median age 52 [20-70] years; 25 chronic hepatitis and 10 cirrhosis). Intrahepatic HBV cccDNA was measured by digital-droplet PCR (Bio-Rad, USA) following total DNA digestion with plasmid safe ATP-dependent DNase. Serum qHBsAg and HBcrAg were measured by CLEIA on Lumipulse® G600 II analyzer (Fujirebio, Japan). qHBsAg limit of sensitivitywas 0.005 IU/ml. The lower limit of detection (LLoD) and the measurement range of HBcrAg were 2 Log U/ml and 3-7 Log U/ml, respectively. The WHO 1st International Standard for anti-HBc (NIBSC code 95/522) was used for the calibration of anti-HBc IgG assay (Lumipulse® G HBcAb-N, Fujirebio). Results: The newassay for anti-HBc IgG quantitation showed a linear dynamic range (R2 = 0.997, p < 0.001); lower limit of detection (LLoD) and quantitation (LLoQ) were estimated at 0.5 IU/ml and 0.8 IU/ml, respectively. The coefficient of variation (CV) for repeatability was 3.1% whereas the CV for reproducibility was 4.0%. In liver specimens, mean HBV cccDNA levels were 3.11 ± 1.14 Log copies/105 cells; in serum samples, mean qHBsAg, HBcrAg and anti-HBc IgG values were 3.13 ± 1.31 Log IU/ml, 3.8 ± 1.9 Log U/ml and 3.68 ± 0.83 Log IU/ml, respectively. In 18/35 (51%) patients, HBcrAg was below the measurement limit of the assay (<3 Log U/ml). HBV cccDNA correlated significantly with qHBsAg (r = 0.624, p < 0.001), HBcrAg (r = 0.734, p < 0.001) and anti-HBc IgG (r = 0.553, p < 0.001). In patients with HBcrAg values <3.0 Log U/ml, intrahepatic HBV cccDNA correlated significantly with anti-HBc IgG (r = 0.752, p < 0.001) but not with qHBsAg (r = 0.384, p = 0.116). Conclusion: Anti-HBc IgG quantitation by CLEIA was a sensitive and accurate assay. Among the investigated biomarkers, HBcrAg was confirmed as reliable surrogate marker of intrahepatic HBV cccDNA. In patients with low HBcrAg levels (<3.0 log), anti-HBc IgG quantitation by CLEIA may be proposed as alternative marker for intrahepatic HBV cccDNA measurement.