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. 2004 Sep;78(17):9030-40.
doi: 10.1128/JVI.78.17.9030-9040.2004.

Inhibition of hepatitis C virus-like particle binding to target cells by antiviral antibodies in acute and chronic hepatitis C

Daniel Steinmann  1 , Heidi Barth, Bettina Gissler, Peter Schürmann, Mohammed I Adah, J Tilman Gerlach, Gerd R Pape, Erik Depla, Dirk Jacobs, Geert Maertens, Arvind H Patel, Geneviève Inchauspé, T Jake Liang, Hubert E Blum, Thomas F Baumert
Affiliations

Inhibition of hepatitis C virus-like particle binding to target cells by antiviral antibodies in acute and chronic hepatitis C

Daniel Steinmann et al. J Virol. 2004 Sep.

Abstract

Hepatitis C virus (HCV) is a leading cause of chronic viral hepatitis worldwide. The study of antibody-mediated virus neutralization has been hampered by the lack of an efficient and high-throughput cell culture system for the study of virus neutralization. The HCV structural proteins have been shown to assemble into noninfectious HCV-like particles (HCV-LPs). Similar to serum-derived virions, HCV-LPs bind and enter human hepatocytes and hepatoma cell lines. In this study, we developed an HCV-LP-based model system for a systematic functional analysis of antiviral antibodies from patients with acute or chronic hepatitis C. We demonstrate that cellular HCV-LP binding was specifically inhibited by antiviral antibodies from patients with acute or chronic hepatitis C in a dose-dependent manner. Using a library of homologous overlapping envelope peptides covering the entire HCV envelope, we identified an epitope in the N-terminal E2 region (SQKIQLVNTNGSWHI; amino acid positions 408 to 422) as one target of human antiviral antibodies inhibiting cellular particle binding. Using a large panel of serum samples from patients with acute and chronic hepatitis C, we demonstrated that the presence of antibodies with inhibition of binding activity was not associated with viral clearance. In conclusion, antibody-mediated inhibition of cellular HCV-LP binding represents a convenient system for the functional characterization of human anti-HCV antibodies, allowing the mapping of envelope neutralization epitopes targeted by naturally occurring antiviral antibodies.

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Figures

FIG. 1.
FIG. 1.
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.
FIG. 2.
FIG. 2.
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.
FIG. 3.
FIG. 3.
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.
FIG. 4.
FIG. 4.
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%).
FIG. 5.
FIG. 5.
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.
FIG. 6.
FIG. 6.
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.
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