Mechanisms downstream of reverse transcription reduce serum levels of HBV DNA but not of HBsAg in chronic hepatitis B virus infection

Abstract

BackgroundHepatitis B virus (HBV) DNA in serum of chronically infected patients declines by 3–4 log10 units at loss of HBe antigen (HBeAg) from serum. The mechanisms behind this decline, and the much smaller decline of surface antigen (HBsAg) levels, are still not well known. The aim of this study was to get a better understanding of this process by analysing both serum and intrahepatic markers of HBV replication.MethodsLevels of HBV DNA and HBsAg in serum, and covalently closed circular DNA (cccDNA), pregenomic RNA (pgRNA) and S-RNA and total intrahepatic HBV DNA (ihDNA) in liver biopsies from 84 chronically infected patients (16 positive and 68 negative for HBeAg) were analysed.ResultsLower HBV DNA levels within HBeAg-positive stage reflected lower levels of cccDNA and pgRNA with strong correlation. In HBeAg-negative patients, ihDNA levels were greater and HBV DNA levels in serum lower than expected from pgRNA levels. A lower HBV DNA/HBsAg ratio corresponded with lower pgRNA/cccDNA (p < 0.01) and higher S-RNA/cccDNA (p < 0.0001) ratios, suggesting that in HBeAg-negative patients transcription of pgRNA, but not of S-RNA, becomes suppressed.ConclusionsThe marked reduction of HBV DNA in serum after loss of HBeAg appears to be due to combined reduction of cccDNA, pgRNA and yet unidentified mechanisms downstream of reverse transcription. Such mechanisms include faster clearance of circulating virus or blocked secretion of virions, the latter supported by the observed relative increase of ihDNA in HBeAg-negative patients. The smaller reduction of S-RNA than of pgRNA partly explains why HBsAg remain high in the HBeAg-negative stage, supporting the possibility of HBsAg synthesis from integrated HBV DNA.

Smaller reduction of hepatitis B virus DNA in liver tissue than in serum in patients losing HBeAg

“…The levels of HBsAg, which mainly represent the levels of SVP, show smaller declines than HBV DNA during antiviral treatment or during the course of infection . Thus, the HBsAg levels are only 1‐2 log 10 units lower in HBeAg negative patients than in HBeAg positive patients, which is much less than the 3‐5 log 10 units difference for HBV DNA …”

“…5,[9][10][11] The levels of HBsAg, which mainly represent the levels of SVP, show smaller declines than HBV DNA during antiviral treatment 12,13 or during the course of infection. 5,14 Thus, the HBsAg levels are only 1-2 log 10 units lower in HBeAg negative patients than in HBeAg positive patients, which is much less than the 3-5 log 10 units difference for HBV DNA. 14 HBV DNA in liver tissue represents several forms of viral DNA, including cccDNA, relaxed circular DNA (rcDNA) in nascent, and mature viral particles, double stranded linear forms 15 (believed to be a nonproductive form that is template for integration) and HBV DNA sequences integrated into the human genome.…”

“…The cccDNA minichromosome is template for the pgRNA that is reverse transcribed to HBV DNA in viral particles, and also for transcripts that are translated into surface antigens embedded in the envelope of both virions and subviral particles (SVP), of which the latter are produced in great excess . The levels of HBsAg, which mainly represent the levels of SVP, show smaller declines than HBV DNA during antiviral treatment or during the course of infection . Thus, the HBsAg levels are only 1‐2 log 10 units lower in HBeAg negative patients than in HBeAg positive patients, which is much less than the 3‐5 log 10 units difference for HBV DNA …”

“…It is currently not known to what extent changes of HBV DNA in serum, which mainly is rcDNA, reflect changes in total intrahepatic HBV DNA (ihDNA), which is heterogeneous and may include significant amounts of integrated DNA. Previous cross‐sectional studies have shown that the levels of total ihDNA are reduced by ≈2 log 10 units at loss of HBeAg, that is, significantly less than HBV DNA in serum . Two previous studies have investigated the reduction of these markers during 1 or several years of nucleoside analog treatment, but similar studies on untreated patients are lacking.…”

“…Previous crosssectional studies have shown that the levels of total ihDNA are reduced by ≈2 log 10 units at loss of HBeAg, that is, significantly less than HBV DNA in serum. 8,14,16,17 Two previous studies have investigated the reduction of these markers during 1 7 or several years of nucleoside analog treatment, 13 but similar studies on untreated patients are lacking. We therefore, investigated HBV markers in stored serum and liver biopsies taken on two occasions during a time period of 3-11 years from patients who did not receive long-term antiviral therapy.…”