Amino acid residue-specific neutralization and nonneutralization of hepatitis C virus by monoclonal antibodies to the E2 protein

Abstract

Antibodies to epitopes in the E2 protein of hepatitis C virus (HCV) reduce the viral infectivity in vivo and in vitro. However, the virus can persist in patients in the presence of neutralizing antibodies. In this study, we generated a panel of monoclonal antibodies that bound specifically to the region between residues 427 and 446 of the E2 protein of HCV genotype 1a, and we examined their capacity to neutralize HCV in a cell culture system. Of the four monoclonal antibodies described here, two were able to neutralize the virus in a genotype 1a-specific manner. The other two failed to neutralize the virus. Moreover, one of the nonneutralizing antibodies could interfere with the neutralizing activity of a chimpanzee polyclonal antibody at E2 residues 412 to 426, as it did with an HCV-specific immune globulin preparation, which was derived from the pooled plasma of chronic hepatitis C patients. Mapping the epitope-paratope contact interfaces revealed that these functionally distinct antibodies shared binding specificity for key amino acid residues, including W(437), L(438), L(441), and F(442), within the same epitope of the E2 protein. These data suggest that the effectiveness of antibody-mediated neutralization of HCV could be deduced from the interplay between an antibody and a specific set of amino acid residues. Further understanding of the molecular mechanisms of antibody-mediated neutralization and nonneutralization should provide insights for designing a vaccine to control HCV infection in vivo.

Fig 1

Peptide specificity and binding affinity of the MAbs. (A) Peptides used for the present study. Peptide A corresponding to amino acid residues 412 to 447 of the E2 protein of HCV H strain (H77) was used to immunize mice for generating the MAbs tested in the present study. The truncated forms of peptide A, i.e., peptide B, B short, and peptide D, are indicated. The locations of epitope I and epitope II within peptide A, as mapped in our previous studies (30, 31), are shown. (B) Peptide A specificity of the MAbs in an ELISA. Biotin-conjugated peptide A was added to streptavidin-coated 96-well plates (200 ng/well). Each MAb (ascites fluid) was diluted 1:1,000 and used as the primary antibody. The y axis indicates the absorbance at 405 nm obtained in the ELISA, representing specific binding of a given antibody to peptide A. The data shown represent three independent experiments. (C) Peptide B specificity of the MAbs in an ELISA. Instead of peptide A, biotin-conjugated peptide B was used for the ELISA as described in panel B. (D) The affinities of B-specific antibodies to peptide A and B were determined by an ELISA using sodium thiocyanate (NaSCN) as a chaotropic agent as described in reference . An ELISA was performed with 1:20,000-diluted ascites fluid in the presence or absence of various concentrations of NaSCN as indicated. The specific binding affinity was calculated based on the values obtained with or without NaSCN. The data shown represent three independent experiments. Error bars represent the standard deviation.

Fig 2

Neutralization of chimeric viruses by MAbs in Huh 7.5 cells. (A) Neutralization of genotype 1a/2a virus. Each antibody at the concentrations of 0.1, 1, 10, and 100 μg/ml was incubated with the appropriately diluted genotype 1a/2a virus before the mixture was added to Huh 7.5 cells. Cell culture medium (DMEM) was used as a negative control for the antibody. The x axis indicates the particular antibody tested. The y axis indicates the relative infectivity of the virus (%), i.e., the percentage of the negative control. Error bars represent the standard error of the mean. (B) Peptide B-specific neutralization of genotype 1a/2a virus by antibody 41. Antibody 41 was adsorbed with (+) or without (−) peptide B prior to performing an ELISA to test its binding to peptide B (left panel) and a neutralization assay to assess its neutralizing activity in Huh 7.5 cells (right panel). Each of these samples shown on the x axis was tested at the dilution of 1:10⁵ in an ELISA. The y axis indicates the absorbance at 405 nm, representing the specific binding of the antibody to peptide B. The data shown represent at least three independent experiments. Error bars represent the standard deviation. For the neutralization assay (right panel), the supernatant was diluted at 1:400, followed by incubation with the genotype 1a/2a virus before the mixture was added to Huh 7.5 cells. The cell culture medium (Med) was used as the negative control in place of the tested antibodies. The x axis indicates the samples tested. The y axis indicates the relative infectivity of the virus (%), i.e., the percentage of the negative control. The data shown represent the results from three independent experiments. The error bars represent the standard error of the mean. The statistical significance of the difference in infectivity is indicated. (C) Inability of the antibodies to cross-neutralize the J6/JFH1 virus. The antibodies were tested for their abilities to neutralize J6/JFH1, a genotype 2a virus, in Huh 7.5 cells with the procedure described in panel A. The data shown represent three independent experiments. The error bars represent the standard error of the mean.

Fig 3

Reduction of virus neutralizing activity of Ch1587 plasma and D-eluate by nonneutralizing antibody 12. Ch1587 plasma was prepared at the ID₅₀ (1:400 dilution) and used as a positive control antibody. The plasma was mixed with an equal (1:1) or 4-fold volume (1:4) of antibody 12 (A) or antibody 50 (B) and incubated with genotype 1a/2a virus in a final assay volume of 100 μl at 37°C for 1 h, after which, the mixture was added to the Huh 7.5 cells. The cell culture medium (Med) was used as the negative control. (C) Ch1587 plasma was affinity purified by using biotinylated peptide D. The resulting D-eluate was confirmed in the ELISA for its specific binding to peptide D. (D) The IC₅₀ of the D-eluate was predetermined to be 0.348 μg/ml. At the IC₅₀, the D-eluate was mixed with various concentrations of nonneutralizing MAb 12 (D) or with MAb 50 (E). The x axis indicates the samples used in this assay. The relative infectivity, i.e., the percentage of the negative control indicated by the y axis, was calculated as described in panel A. The P values were determined for the differences in the infectivity (*, P < 0.05; **, P < 0.01). The data shown represent at least three independent experiments. The error bars represent the standard errors of the mean.

Fig 4

Effects of nonneutralizing antibodies on virus neutralization by HCIGIV and antibody 41. HCIGIV at the ID₅₀ (1:3,000 dilution) was used to neutralize genotype 1a/2a virus in the presence or absence of different concentrations of antibody 12 (A) or antibody 50 (B). The cell culture medium (Med) was used as the negative control. (C) The ability of antibody 41 to neutralize genotype 1a/2a virus was measured in the presence or absence of nonneutralizing antibody 12 or antibody 50 according to the procedure described in Fig. 3. Antibody 41 was diluted at 1:400 in DMEM, and then mixed with antibody 12 or antibody 50 with the ratios of 1:1 and 1:4 (vol/vol). The antibody mixture was subsequently incubated with genotype 1a/2a chimeric virus at 37°C for 1 h before being added to Huh 7.5 cells. The data shown represent three independent experiments, and the standard errors of the mean are indicated by error bars. The x axis indicates the samples used in this assay. The y axis indicates the relative infectivity of the virus (%).

Fig 5

Epitope mapping by screening random peptide phage display libraries. The amino acid sequences of phage clusters identified after at least three rounds of screening phage-display libraries (12-mer and 7-mer) with neutralizing antibodies 8 and 41 and nonneutralizing antibodies 12 and 50 are listed. The numbers of peptides identified over the total numbers of peptides sequenced are shown in parenthesis. The candidate core residues at the epitope-paratope contact interfaces are indicated in bold font. The letter “X” denotes any amino acid residue other than L at that position.

Fig 6

Use of mutational analysis to identify the residues that are critical for antibody recognition. (A) Biotin-conjugated peptides were chemically synthesized to represent the truncated peptide B, i.e., B short, from the E2 protein of HCV genotype 1a (H77 strain) and its mutations. The B short mutant peptides contained a single alanine substitution at positions 437, 438, 440, 441, and 442, respectively. A hyphen indicates an amino acid residue identical to that of the H77 sequence. (B) Biotin-conjugated B short peptide and its mutants were added to streptavidin-coated 96-well plates at 200 ng/well in an ELISA. Each MAb (ascites fluid) was diluted 1:10⁵ dilution, and applied as the primary antibody. The data shown represent at least three independent experiments. The x axis indicates the antibodies used in the assay. The y axis indicates the absorbance at 405 nm, representing specific binding of a given antibody to each individual peptide.

Fig 7

Summary of the amino acid residues critical for antibody functions. (A) Key residues identified in peptide B that are critical for the binding property of each antibody. (B) Alignment of amino acid sequences of the E2 region from aa 412 to 446 of various HCV genotypes. Key residues identified in the present study are indicated in boldface underlined letters. A hyphen indicates an amino acid residue identical to that of the H77 sequence.

Fig 8

Effect of the W437F switch on antibody binding. (A) Schematic representation of the peptide mutations used in the ELISA. Biotin-conjugated peptides were chemically synthesized to represent peptide B, the truncated peptide B (B short), and B short with specific single mutations at position 437. A hyphen indicates an amino acid residue identical to that of the H77 sequence. (B) Detection of the effect of the W437F switch on antibody binding in an ELISA. Biotin-conjugated peptide B, B short peptide, and its mutants were added to streptavidin-coated 96-well plates at 200 ng/well. Each antibody (in ascites fluid) was used at a 1:10⁵ dilution as the primary antibody in the ELISA. Cell culture medium was used as the negative control of the antibody. The x axis indicates the antibodies used in this assay. The y axis indicates the absorbance obtained at 450 nm, which represents the measurement of specific binding of a given antibody to each individual peptide. The ELISA results of the W437A mutant here were comparable to the ELISA results obtained separately in Fig. 6B.