Inhibition of HCV-LP binding by purified human anti-HCV IgG. (A) Inhibition of cellular HCV-LP binding by anti-HCV-positive or control serum (dilution, 1:25). (B) Inhibition of cellular HCV-LP binding by purified anti-HCV or control IgG (1 mg/ml) from the same sera shown in panel A. (C) Concentration-dependent inhibition of HCV-LP binding by purified anti-HCV IgG (squares) and serum containing anti-HCV antibodies (circles). Analysis of inhibition of HCV-LP binding was performed as described in the legend to Fig. 2. Serum IgG concentrations were determined as described in Materials and Methods.

Inhibition of cellular HCV-LP binding and virus clearance. (A) HCV-LP inhibition of binding activity and outcome of infection in acute hepatitis C. The graph shows the percentages of patients with sera inhibiting cellular HCV-LP binding at a titer of ≥1:50 in relation to the outcome of infection (self-limited infection versus chronic infection). (B) Inhibition of HCV-LP binding and phase of infection. The graph shows the percentages of patients with sera inhibiting cellular HCV-LP binding at a titer of ≥1:50 in relation to the phase of infection (acute versus chronic). (C and D) Serial analyses of antibodies with inhibition of HCV-LP binding activity in two patients with acute self-limited hepatitis C and virus clearance. Alanine aminotransferase (ALT) levels (triangles) are indicated on the left y axis. Titers of antibody-mediated inhibition of HCV-LP binding were determined as described in the legend to Fig. 2 and are indicated on the right y axis (circles). The x axis represents time points after the diagnosis of acute hepatitis C. The presence (+) or absence (−) of HCV RNA and anti-HCV antibodies is shown at the top; +/−, weakly positive. (C) Virus clearance in the absence of antibodies with inhibition of HCV-LP binding activity. (D) Virus clearance in the presence of antibodies with inhibition of HCV-LP binding activity.

Antibody-mediated inhibition of cellular HCV-LP binding. HCV-LPs were incubated with anti-HCV-positive serum or a pool of anti-HCV-negative control sera (dilution, 1:50). HCV-LP-antibody complexes were added to HuH-7 cells for 1 h at 4°C. After removal of nonbound HCV-LP-antibody complexes by washing the cells in PBS-2% BSA, the binding of HCV-LPs was detected by flow cytometry using the mouse monoclonal anti-E2 antibody AP33 and PE-conjugated anti-mouse IgG (A and B) or human polyclonal anti-HCV and FITC-conjugated anti-human IgG antibody (C). The fluorescence intensity and relative cell numbers (Counts) are shown on the x and y axes, respectively. NC, negative control, corresponding to HuH-7 cells incubated with control insect cell preparations (GUS) and control serum. (D) For the assessment of concentration-dependent antibody-mediated inhibition of binding, HCV-LPs (genotype 1b) were incubated in subsaturating concentrations with an anti-HCV-positive serum or a pool of anti-HCV-negative control sera (at various dilutions as indicated on the x axis) for 1 h at 37°C. HCV-LP-antibody complexes were added to HuH-7 cells for 1 h at 4°C. HCV-LP binding to the HuH-7 cells was detected by flow cytometry as described above. Inhibition of cellular HCV-LP binding (y axis) was calculated as described in Materials and Methods.

Strain-dependent inhibition of cellular HCV-LP binding. For the assessment of antibody-mediated neutralization of binding, HCV-LPs derived from the HCV-J strain (genotype 1b) (A and C) or clone H77c (genotype 1a) (B and D) were incubated in subsaturating concentrations with anti-HCV-positive sera from two patients infected with HCV genotype 1a or 1b or a pool of anti-HCV-negative control sera (NC; dilution, 1:50). HCV-LP-antibody complexes were added to HuH-7 cells. After the removal of nonbound HCV-LP-antibody complexes by washing the cells in PBS-2% BSA, binding of HCV-LPs was detected by flow cytometry using mouse monoclonal anti-E2 or human polyclonal anti-HCV antibodies (insets) as described in the legends to Fig. 1 and 2. (A and B) Genotype-dependent inhibition of HCV-LP binding by serum from a patient infected with HCV genotype 1b. (C and D) Genotype-independent inhibition of cellular HCV-LP binding by serum from a patient infected with genotype 1a. Fluorescence intensity (FL2-Height) and relative cell numbers (Counts) are shown on the x and y axes, respectively. The insets show the corresponding results of serum-induced inhibition of HCV-LP binding detected by human polyclonal anti-HCV antibody (HCV-LP binding in the presence of control serum [Control] = 100%).

Epitope mapping of antibodies inhibiting cellular binding of HCV-LPs. (A) To identify HCV-LP envelope epitopes targeted by human antibodies inhibiting cellular HCV-LP binding, an anti-HCV-positive serum (HCV+) with marked inhibition of binding activity (Fig. 3) was incubated with overlapping 15-mer peptides of the HCV envelope glycoproteins (comprising amino acid positions 201 to 758) derived from the HCV-J strain (26) or PBS (as a control) for 1 h at RT (peptide concentration, 100 μg/ml; serum dilution, 1:50). HCV-LPs (derived from the homologous isolate as the peptides) were then added to the peptide-antibody complexes and incubated for 1 h at 37°C. The HCV-LPs, antibodies, and peptides were added to HuH-7 cells and incubated for 1 h at 4°C. Following the removal of nonbound HCV-LPs, antibodies, and peptides by washing the cells in PBS, binding of HCV-LPs was detected by flow cytometry as described above. Inhibition of HCV-LP binding (as indicated on the y axis) was calculated as described in the legend to Fig. 2. The error bars indicate standard deviations of results of a representative experiment performed in triplicate. (B) Concentration-dependent reversion of HCV-LP inhibition of binding activity by E2 peptide 408. Anti-HCV-positive serum (dilution, 1:50) was preincubated with E2 peptide 408 (SQKIQLVNTNGSWHI; corresponding to amino acid positions 408 to 422) at the peptide concentrations indicated on the x axis. Cellular HCV-LP binding (corresponding to ΔMFI, indicated on the y axis) in the presence of the antiviral antibody-peptide complex was determined as described in Materials and Methods.

Concentration-dependent and saturable binding of HCV-LPs to and entry into human hepatoma cells. (A and B) Binding of HCV-LPs to target cells is concentration-dependent and saturable. HuH-7 (A) and HepG2 (B) cells were incubated with increasing concentrations of HCV-LPs of genotype 1a (triangles), as well as genotype 1b (squares), and particle binding was analyzed by flow cytometry as described in Materials and Methods. On the y axis, net MFI values for each HCV-LP E2 concentration were calculated by subtracting the MFI of the negative control with anti-E2 and PE-conjugated anti-mouse IgG antibodies from that obtained with the respective HCV-LP concentration (x axis). (C and D) Analysis of HCV-LP entry into target cells using anti-E2-specific immunofluorescence and cross section laser scanning confocal microscopy. (C) Cellular binding of HCV-LP was assessed by incubation of HuH-7 cells with HCV-LPs (genotype 1a) at 4°C for 40 min. (D) For temperature-dependent HCV-LP entry, cells were incubated for an additional 60 min at 37°C. After removal of nonbound HCV-LPs by washing the cells with ice-cold PBS, the cells were added to poly-l-lysine-coated cover slides, fixed, permeabilized, and stained for HCV-LPs using anti-E2 and Cy3-conjugated anti-mouse IgG antibodies (arrows). For the costaining of the cytoskeleton, cells were coincubated with a rabbit anti-actin and FITC-conjugated anti-rabbit IgG antibody. For costaining of the nucleus, the cells were incubated with DRAQ-5, a highly permeable DNA-interactive agent.