Endocytosis of hepatitis B immune globulin into hepatocytes inhibits the secretion of hepatitis B virus surface antigen and virions

Abstract

Hepatitis B immunoglobulin is used for prophylaxis against hepatitis B virus (HBV) and is thought to act by neutralization of virions and hepatitis B virus surface antigen (HBsAg)-containing particles in circulation. Using a panel of hepatocyte-derived cell lines, the present study investigated in vitro whether HBs-specific immunoglobulin G (IgG) is internalized in hepatocytes and whether it interacts with HBsAg in the cells. By immunoelectron microscopy and immunoblotting, human IgG and FcRn receptor for IgG were demonstrated on cellular membranes and in cytoplasmic extracts, irrespective of the HBsAg status of the cells. Furthermore, HBsAg and anti-HBs were shown to be colocalized in the same cellular compartment by two-color confocal microscopy. Endocytosis of HBs-specific IgG caused intracellular accumulation of HBsAg in a dose-dependent manner and inhibited the secretion of HBsAg and HBV virions from the cells. These effects were not observed with F(ab)(2) fragments or nonimmune IgG as controls. The specificity of intracellular HBsAg- anti-HBs interaction was further investigated in cells transfected with HBV genomes expressing wild-type HBsAg or immune escape HBsAg (with a G145R mutation). Monoclonal anti-HBs markedly reduced the secretion of wild-type HBsAg, while the secretion of mutant HBsAg was not affected. These results suggest that HBs-specific IgG binds to hepatocytes and interacts with HBsAg within the cells. This may be relevant for the selection of surface antibody escape mutations.

FIG. 1.

Immunoblot of human IgG in cytoplasmic extracts from PLC/PRF/5 cells (lanes 1 to 4) and HuH-7 cells (lanes 5 to 8). The cells were cultured with medium containing human AB serum (lanes 1 and 5), human AB serum plus 1.0 mg of monoclonal anti-HBs IgG/ml (lanes 2 and 6), medium with FCS alone (lanes 3 and 7), or 1.0 mg of monoclonal anti-HBs IgG/ml (lanes 4 and 8). As a standard, monoclonal anti-HBs IgG (mAb) was included in the blot. INT, intensity of the chemiluminescence signal measured by FluorS MultiImager in arbitrary units (au). Cellular IgG, estimated amount of IgG associated with cells; IgG uptake, relative amount of IgG associated with the cells as a percentage of the IgG in culture supernatants. The amount of the latter was calculated on the basis of the known IgG concentration (1.0 mg/ml) and the known volume of medium (2.5 ml) in each well, i.e., 2,500 μg of IgG.

FIG. 2.

Detection of human IgG and FcRn receptor by immunocytochemistry and light microscopy (a to c; magnification, ×65,000) or by IEM (d to f; magnification, ×72,000). (a, b, d, and e) Detection of IgG in HuH-7 cells cultured in the absence of human IgG (a), in the presence of 1.0 mg of polyclonal anti-HBs IgG/ml (b and e), or in medium containing human AB serum (d). (c and f) Detection of human FcRn. Hematoxylin staining of nuclei (light grey) and peroxidase staining (black) are shown. The arrows (the short arrows indicate the cellular membrane; the long arrows indicate membranous invaginations) indicate the positive signals for IgG or FcRn.

FIG.3.

Localization of HBIG and HBV proteins in HBIG-treated HepAD38 cells grown under Tet-off conditions. (A to E) On day 12 of culture, HBIG-treated HepAD38 cells were fixed and processed for IFA. The cells were reacted with monoclonal antibodies to HBV preS-2, followed by reactivity with FITC-conjugated anti-human and Texas Red-X-conjugated anti-mouse immunoglobulins. (A) Uptake of HBIG (arrows) into the cytoplasm of HBIG-treated cells can be detected by fluorescent staining. (B) When identical image collection parameters were applied to view parallel cultures that had not been treated with HBIG, fluorescent staining was not detected. HBIG (arrows) (C) and the HBV pre-S2 protein (arrows) (D) were observed within the cytoplasms of the same cells observed by FITC and Texas Red staining, respectively. (E) Colocalization of HBIG and HBV pre-S2 protein was shown following digital superimposition of the two fluoroprobes (arrows). (F) In addition to the HBV pre-S2 protein, the HBV core protein (arrows) was also detected following reactivity of the cells with rabbit polyclonal antibodies to HBV core. The arrows indicate the positive signals in each panel.

FIG. 4.

Immunoblot analysis of cytoplasmic extracts from PLC/PRF/5 cells cultured in the presence of different concentrations of human monoclonal anti-HBs. The cells were cultured in medium with 0.2 mg of anti-HBs/ml (lanes 1), with 1.0 mg of anti-HBs/ml (lanes 2), with 2.0 mg of anti-HBs/ml (lanes 3), or without human IgG (lanes 4). (a) Amido black staining of proteins. (b) Detection of human IgG. (c) Detection of HBsAg. (d) Detection of FcRn. INT, intensity of the chemiluminescence signal measured by FluorS MultiImager in arbitrary units. The arrows indicate the signals for the γ chain of IgG (50 kDa), for both forms of HBsAg (24 and 27 kDa), and for the heavy chain of FcRn (45 kDa). LM, protein length marker.

FIG. 5.

Immunoblot analysis of cytoplasmic extracts from PLC/PRF/5 cells cultured with different concentrations of polyclonal anti-HBs IgG (HBIG). The cells were cultured in medium without human IgG (lanes 1), with 0.2 mg of HBIG/ml (lanes 2), with 1.0 mg of HBIG/ml (lanes 3), or with 2.0 mg of HBIG/ml (lanes 4). (a) Amido black staining of proteins. (b) Detection of human IgG. (c) Detection of HBsAg. INT, intensity of the chemiluminescence signal measured by FluorS MultiImager in arbitrary units. The arrows indicate the signals for the γ chain of IgG (50 kDa) and for both forms of HBsAg (24 and 27 kDA). LM, protein length marker.

FIG. 6.

RIA quantitation of HBsAg levels in the supernatants of PLC/PRF/5 cells cultured with human monoclonal anti-HBs IgG. During the first period (open bars), the cells were cultured in the presence of medium with FCS and two concentrations of anti-HBs. In parallel, cells were cultured with medium plus FCS only, i.e., without human IgG (FCS) or with nonimmune human IgG (ABS), as controls. After 2 days of culture, the supernatants were collected, and the same cells were maintained in culture for a further two intervals of 2 days each (shaded and solid bars, respectively) in medium with FCS without human IgG. The HBsAg levels in the supernatants were tested at the end of each period, i.e., at three time points. The bars represent the means and standard deviations of two separate experiments, with each experimental condition run in duplicate.

FIG. 7.

Detection of HBV DNA in supernatants and cytoplasmic extracts of HepG2.215 cells. (a) Quantitative real-time PCR for the detection of HBV DNA in cell culture supernatants. HepG2.2.15 cells were cultured for an initial period (open bars) without human IgG (FCS), with nonimmune IgG (ABS), or with 0.1 or 1.0 mg of monoclonal HBs-specific IgG/ml (anti-HBs). In the second period (solid bars), the same cells were maintained in culture for a further two time intervals without human IgG. The bars represent the means and the standard deviations of duplicate samples. (b) Southern blot hybridization for the detection of HBV replicative intermediates in the cytoplasm of HepG2.215 cells cultured without human IgG (FCS), with nonimmune human IgG (ABS), or with monoclonal anti-HBs (mAb). The arrows indicate the signals for single-stranded (ss) and double-stranded (ds) HBV DNAs. INT, intensity of the chemiluminescence signal measured by FluorS MultiImager in arbitrary units.

FIG. 8.

Quantification of HBsAg levels in the culture supernatants of HuH-7 cells transfected with HBV genomes expressing wild-type HBsAg (solid bars) or mutant G145R HBsAg (open bars). (a) HBV-transfected cells were cultured with FCS only as a control (c), with 1.0 mg of human monoclonal anti-HBs IgG/ml, or with 1.0 mg of human anti-HBc IgG/ml. (b) In another experiment, HuH-7 cells were transfected in parallel with the wild-ype HBV or G145R mutant HBV and cultured with 1.0 mg of monoclonal anti-HBs IgG/ml (mAb) or with 1.0 mg of polyclonal anti-HBs IgG/ml (pAb). The bars represent the means and standard deviations of duplicate samples.