(A) Fc-mediated effector functions are not required for 2H5-A14 to protect mice from HDV infection. The experiment was performed similarly to that described in Figure 3A. Both 2H5-A14 and Fc mutant 2H5-A14-DANA were tested at 5 mg/kg. The copy numbers shown in the Y-axes are from 20 ng of total liver RNA for each sample. (B) Schematic diagram illustrating HBV challenge, bleeding and antibody treatment schedules in the mouse study. Three animals were used in each group. Recipient hFRG mice were challenged with HBV viruses (genotype D) at 2 × 109 GE. Twice weekly treatment with PBS, 2H5-A14 (5 mg/kg), or 2H5-A14-DANA (5 mg/kg) began at 33 dpi and lasted for 4 weeks. (C) HBV DNA titers and HBsAg levels in serum. Blood samples were collected at the indicated time points for measuring HBV DNA titers and/or HBsAg levels. (D) Antibody concentrations. The antibody concentrations in serum samples were measured by ELISA using antibody standards with known concentrations. Note, for dpi 40 and dpi 54, the blood samples were collected at three days after Ab administration; for dpi 60, the blood samples were collected 1 hr after Ab administration. The horizontal dotted lines indicate the lowest detection limits; the vertical dotted lines and the grey-shaded areas indicate the treatment window (C–D). (E) IHC staining of human cytokeratin-18 (hCK18) and HBsAg in serial sections of liver tissues from sacrificed hFRG mice at the end of the experiment (dpi 89). Intrahepatic HBsAg was detected by a specific anti-HBsAg mouse mAb, followed by HRP-anti-mouse secondary Ab and stained brown using DAB substrate, nuclei were stained blue by Hematoxylin. Human hepatocytes in consecutive tissue sections were visualized by staining with a human-specific hCK18 mAb and DAB substrate in blue violet, nuclei were stained with Nuclear Fast Red. Each image shown represents the staining result for one mouse of each group. (F) Southern blot analysis of intrahepatic viral DNA. Total HBV DNA was extracted from mouse liver tissues at dpi 89. The extracted DNA samples were analyzed by Southern blotting with an [α-32P]dCTP-labeled full-length HBV DNA probe. DNA samples prepared from normal mouse livers were used as a negative control. 100 pg each of 3.2 kb, 2.1 kb, and 1.7 kb HBV DNA fragments were used as DNA markers. rcDNA (relaxed circular DNA), cccDNA, and ssDNA (single-strand DNA) intermediates are labeled. (G) qPCR quantification of intrahepatic viral cccDNA level. 500 ng of total DNA prepared from the liver tissues collected at dpi 89 was digested by PSAD and 1/10 of the digested samples were used to quantify HBV cccDNA by qPCR using specific primers (see Materials and methods). The hNTCP gene copy numbers, which represent the amount of human hepatocytes in the liver tissues of chimeric mice, were used to normalize the cccDNA level in the human hepatocytes for each sample. The cccDNA copy number shown in the Y-axis is the relative value normalized to 1000 copies of hNTCP gene in ~50 ng total DNA samples. Each square represents one mouse. The horizontal dotted line indicates the reliable detection limit.