Hepatitis B Viral DNA Decline at Loss of HBeAg Is Mainly Explained by Reduced cccDNA Load – Down-Regulated Transcription of PgRNA Has Limited Impact

BACKGROUND Quantification of hepatitis B virus (HBV) DNA and surface antigen (HBsAg) serum levels have become increasingly important for the assessment of clinical stage and response to treatment for chronic hepatitis B. Effective immune clearance results in reduction of viremia by 4-5 log units and HBsAg levels by 2 log, but these processes are not well understood. Thus, it is uncertain to what extent mechanisms that inhibit transcription of the pregenomic RNA (pgRNA), an RNA intermediate, contribute to suppression of viremia. Likewise, it is unclear if transcriptional regulation is important for the excessive production of surface antigen (HBsAg) that is a hallmark of HBV infection. METHODS HBV RNA and cccDNA were quantified in 19 liver biopsies from patients with chronic HBV infection, as well as in transfected Huh7.5 cells and in PLC/PRF/5 cells carrying integrated HBV genome. RESULTS Patients negative for HBeAg had 2.15 log lower levels of cccDNA in liver tissue, 4.84 log lower serum levels of HBV DNA and 1.45 log lower serum levels of HBsAg, than HBeAg-positive patients. The pgRNA in liver tissue correlated strongly with cccDNA (R(2) = 0.87, p<0.0001) and HBV DNA levels in serum (R(2) = 0.81, p<0.0001), whereas S-RNA correlated strongly with cccDNA (R(2) = 0.65, p<0.0001) and HBsAg levels (R(2) = 0.57, p = 0.0003). The S-RNA/pgRNA ratio was higher in HBeAg-negative patients (ratio 40 vs. 3, p = 0.01) and in PLC/PRF/5 cells, and was in transfected Huh7.5 cells not influenced by mutations in the HBV core promoter. CONCLUSION The reduction of viremia that is observed after loss of HBeAg was mainly explained by reduced cccDNA load in the liver, whereas the contribution of down-regulation of pgRNA transcription was relatively small. Enhanced transcription of S-RNA does not explain excessive production of HBsAg.