Volume 133, Issue 3, Pages 843-852 (September 2007) Impaired Intrahepatic Hepatitis B Virus Productivity Contributes to Low Viremia in Most HBeAg-Negative Patients  Tassilo Volz, Marc Lutgehetmann, Paul Wachtler, Anna Jacob, Alexander Quaas, John M. Murray, Maura Dandri, Joerg Petersen  Gastroenterology  Volume 133, Issue 3, Pages 843-852 (September 2007) DOI: 10.1053/j.gastro.2007.06.057 Copyright © 2007 AGA Institute Terms and Conditions

Figure 1 Relationship between viral titers and intrahepatic viral loads in HBeAg-positive and HBeAg-negative patients. Correlation between HBV DNA in serum and amounts of (A) intrahepatic rcDNA per cell or (B) intrahepatic cccDNA per cell. Dots represent single patient measurements. Open dots represent HBeAg-negative (n = 77) and closed dots represent HBeAg-positive patients (n = 42). Linear regression lines were determined by Pearson correlations. In B, correlations are displayed separately (solid line for HBeAg positive and dashed line for HBeAg-negative patients). r (Spearman rank) and P values are indicated. Gastroenterology 2007 133, 843-852DOI: (10.1053/j.gastro.2007.06.057) Copyright © 2007 AGA Institute Terms and Conditions

Figure 2 HBeAg-negative patients have significantly lower viral productivity. (A) Intrahepatic levels of rcDNA per cccDNA in the liver of HBeAg-positive (n = 42) and HBeAg-negative patients (n = 77). (B) Ratios of pgRNA to cccDNA measured in random liver biopsy specimens from 17 of 42 HBeAg-positive and 43 of 77 HBeAg-negative patients. Median levels are indicated by bars and were 49 versus 9 rcDNA/cccDNA and 214 versus 38 pgRNA/cccDNA in HBeAg-positive and HBeAg-negative patients, respectively. Dots represent single patient measurements. (C) Relationship between intrahepatic amounts of pgRNA and rcDNA measured in each liver biopsy sample. Linear regression lines were determined by Pearson correlations. r and P values are indicated. Gastroenterology 2007 133, 843-852DOI: (10.1053/j.gastro.2007.06.057) Copyright © 2007 AGA Institute Terms and Conditions

Figure 3 Production and secretion of subviral particles is not impaired in HBeAg-negative patients. (A) Serum HBsAg concentrations normalized for cccDNA contents measured in the liver of corresponding patients. Median values (bars) were 25 and 70 μg/mL serum in HBeAg-positive and HBeAg-negative individuals, respectively. Each dot represents a single patient measurement. (B) Intrahepatic levels of subgenomic preS/S RNA per cccDNA molecules determined in the same biopsy samples. Median values are 357 (HBeAg positive) versus 768 (HBeAg negative) preS/S RNA copies/cccDNA. Gastroenterology 2007 133, 843-852DOI: (10.1053/j.gastro.2007.06.057) Copyright © 2007 AGA Institute Terms and Conditions

Figure 4 Relationship between HBsAg concentrations in serum and intrahepatic levels of cccDNA. Box plots represent 3 groups of patients with different HBsAg concentrations as indicated. Shown are the 25th, 50th, and 75th percentiles of patients with log HBV cccDNA copies/cell. Median cccDNA levels were 0.07, 0.3, and 1.8 cccDNA copies/cell in groups 1, 2, and 3, respectively. By applying the Mann–Whitney test, we found that differences between groups were highly significant (group 1 vs 2, P = .004; group 2 vs 3, P < .0001). Gastroenterology 2007 133, 843-852DOI: (10.1053/j.gastro.2007.06.057) Copyright © 2007 AGA Institute Terms and Conditions

Figure 5 Accumulation of BCP mutations restores high viral productivity and increases viremia in HBeAg-negative patients. (A) Comparison of virion productivity (rcDNA/cccDNA) between HBeAg-positive and HBeAg-negative patients harboring the same pattern of PC/BCP mutations. Box plots show comparison of HBeAg-positive (white boxes) and HBeAg-negative (gray boxes) patients without emergence of HBeAg variants (group 1; P < .0001), harboring cccDNA pools with both wild-type and PC mutations (group 2) or with BCP variants (group 3) or with PC + BCP mutants (group 4). Median virion productivity in HBeAg-negative patients in groups 2, 3, and 4 was significantly lower (P = .004) compared with HBeAg-positive patients within the same groups. Among HBeAg-negative patients, significantly higher ratios (P = .002) of rcDNA to cccDNA levels were found in 6 patients without detectable wild-type cccDNA sequences in the PC and BCP regions (group 5). Shown are the 25th, 50th, and 75th percentiles of patients with log rcDNA/cccDNA. The number of patients in each group is indicated. (B) Comparison of log rcDNA levels produced per cccDNA among patients displaying similar viremia. Three groups of viremia ranges are indicated. Median rcDNA/cccDNA levels were 64 and 5.7 in group 1 (P = NS), 59 and 10.6 in group 2 (P = .014), and 36 and 70.6 in group 3 (P = NS) by comparing HBeAg-positive (white boxes) and HBeAg-negative patients (light gray boxes), respectively. HBeAg-negative patients without detectable wild-type cccDNA sequences in the PC and BCP regions are shown in groups 1 (n = 1) and 2 (n = 5, dark gray box) and had a median of 176 rcDNA/cccDNA. Gastroenterology 2007 133, 843-852DOI: (10.1053/j.gastro.2007.06.057) Copyright © 2007 AGA Institute Terms and Conditions