Depletion of interfering antibodies in chronic hepatitis C patients and vaccinated chimpanzees reveals broad cross-genotype neutralizing activity

Abstract

Using human immune globulins made from antihepatitis C virus (HCV)-positive plasma, we recently identified two antibody epitopes in the E2 protein at residues 412-426 (epitope I) and 434-446 (epitope II). Whereas epitope I is highly conserved among genotypes, epitope II varies. We discovered that epitope I was implicated in HCV neutralization whereas the binding of non-neutralizing antibody to epitope II disrupted virus neutralization mediated by antibody binding at epitope I. These findings suggested that, if this interfering mechanism operates in vivo during HCV infection, a neutralizing antibody against epitope I can be restrained by an interfering antibody, which may account for the persistence of HCV even in the presence of an abundance of neutralizing antibodies. We tested this hypothesis by affinity depletion and peptide-blocking of epitope-II-specific antibodies in plasma of a chronically HCV-infected patient and recombinant E1E2 vaccinated chimpanzees. We demonstrate that, by removing the restraints imposed by the interfering antibodies to epitope-II, neutralizing activity can be revealed in plasma that previously failed to neutralize viral stock in cell culture. Further, cross-genotype neutralization could be generated from monospecific plasma. Our studies contribute to understanding the mechanisms of antibody-mediated neutralization and interference and provide a practical approach to the development of more potent and broadly reactive hepatitis C immune globulins.

Fig. 1.

Key residues for the binding of epitope-I-specific neutralizing antibody. (A) Eluate I, an antibody solution, was obtained by affinity purification of HCIGIV with the chemically synthesized peptide containing epitope I (4). With eluate I, peptidyl mimics of epitope-I were selected by screening a random peptide phage display library using a panning method (4). The key residues within both epitope I and peptidyl mimics are indicated (bold and underline). The numbers indicate the position of these peptides in HCV polyprotein. (B) Chemically synthesized peptides containing epitope I and its mutants at key residues are indicated. (C) ELISA analysis revealed critical residues in epitope I. Biotin-conjugated peptides listed in B were added to streptavidin-coated 96-well plates (200 ng/well), individually. Eluate I at 1:50 dilution or HCIGIV at 1:2,000 dilution was used as the primary antibody in the ELISA. The x axis indicates individual peptides tested, and the y axis shows corresponding absorbance at 450 nm (A450 nm) of each mutant relative to that of epitope I (in percent of control).

Fig. 2.

Presence of HCV epitope-specific antibodies in plasma of patients with chronic HCV infection. (A) Biotin-conjugated peptides encompassing epitope II, epitope I, or the mutant of epitope I, SW>AA (Fig. 1 B and C) were added to streptavidin-coated 96-well plates (200 ng/well), individually. SW>AA was used as a NC for the assay. Human patient plasma samples, H77 and numbers 1 to 8, were diluted 1:800 and 1:100, respectively, and used as the primary antibody. Diluted HCIGIV lot A (1:2,000), eluate I, and eluate II, an HCIGIV fraction affinity-purified by using epitope II peptide (4), were used at 1:50 dilution as controls. The y axis indicates A450 nm obtained in the ELISA, representing specific binding of a given plasma to each individual peptide. (B) HCIGIV lots (A–F) were diluted 1:2,000 and used as the primary antibodies in the ELISA, in which biotin-conjugated peptides encompassing epitope II, epitope I, or SW>AA were added to streptavidin-coated 96-well plates (200 ng/well). Peptide SW>AA was used as a NC. The ratios of antibody against epitope II versus that against epitope I are presented. (C) Plasma samples collected from patient H before and after HCV infection were diluted 1:100 except for the sample of d 5266, which was diluted at 1:400, and used as the primary antibodies. Plasma levels of antibodies directed against epitope II, epitope I, or SW>AA, as indicated by values of A450 nm, were determined in the ELISA. SW>AA was used as a negative peptide control. Diluted HCIGIV lot A at 1:2,000 and eluates I and II at 1:50 were used as controls for primary antibodies.

Fig. 3.

Recovery of cross-genotype neutralizing activity from patient H plasma. (A) epitope-II-specific antibodies in plasma 5266 were specifically blocked by epitope II peptide or left untreated as a control. These samples were then tested for their abilities to neutralize genotype 1a/2a or 2a virus in HCV cell culture. A normal IGIV at 1:400 dilution was used as NC. The x axis indicates the plasma sample used in this assay at 1:400 dilution. The y axis indicates the infectivity expressed as percentage of NC. Statistical significance of difference in infectivity is indicated. (B) Biotin-conjugated epitope II and epitope I peptides were added to streptavidin-coated 96-well plates (200 ng/well). Plasma 5266, before and after 3 rounds of absorption with epitope II peptide, was diluted at 1:800 and used as primary antibody in ELISA. HCIGIV lot A, at 1:2,000 dilution, was used as the positive control. The y axis indicates absorbance at 450 nm, representing specific binding of a given plasma sample to each individual peptide. (C) Plasma 5266 was absorbed with epitope II peptide to deplete epitope-II-specific antibodies. These absorbed samples, along with those left untreated, were diluted at 1:400 and assayed for their abilities to neutralize the 2a virus in HCV cell culture. A normal IGIV at 1:400 dilution was used as NC. The neutralizing activity was expressed as the relative infectivity of the virus, i.e., percent of NC. Statistical significance of difference in infectivity is indicated.

Fig. 4.

Recovery of cross-genotype neutralizing activity from plasma of rE1/E2 vaccinated chimpanzees. Epitope II peptide (500 ng/mL) was incubated with the plasma of 2 vaccinated chimpanzees, Ch1587 and Ch1601, to block epitope-II-specific antibodies wherein. These samples, at 1:400 dilution, were then tested for their abilities to neutralize the 1a/2a, 1b/2a, or 2a virus in HCV cell culture. (A) Neutralization with plasma from Ch1587 and (B) neutralization with plasma from Ch1601. For comparisons, control plasma samples and virus stocks were mock treated before the neutralization assay. A normal IGIV at 1:400 dilution was used as NC. The neutralizing activity was expressed as the relative infectivity of the virus, i.e., percent of NC. Statistical significance of difference in infectivity is indicated.