Identification of a domain containing B-cell epitopes in hepatitis C virus E2 glycoprotein by using mouse monoclonal antibodies

Abstract

Evidence from clinical and experimental studies of human and chimpanzees suggests that hepatitis C virus (HCV) envelope glycoprotein E2 is a key antigen for developing a vaccine against HCV infection. To identify B-cell epitopes in HCV E2, six murine monoclonal antibodies (MAbs), CET-1 to -6, specific for HCV E2 protein were generated by using recombinant proteins containing E2t (a C-terminally truncated domain of HCV E2 [amino acids 386 to 693] fused to human growth hormone and glycoprotein D). We tested whether HCV-infected sera were able to inhibit the binding of CET MAbs to the former fusion protein. Inhibitory activity was observed in most sera tested, which indicated that CET-1 to -6 were similar to anti-E2 antibodies in human sera with respect to the epitope specificity. The spacial relationship of epitopes on E2 recognized by CET MAbs was determined by surface plasmon resonance analysis and competitive enzyme-linked immunosorbent assay. The data indicated that three overlapping epitopes were recognized by CET-1 to -6. For mapping the epitopes recognized by CET MAbs, we analyzed the reactivities of CET MAbs to six truncated forms and two chimeric forms of recombinant E2 proteins. The data suggest that the epitopes recognized by CET-1 to -6 are located in a small domain of E2 spanning amino acid residues 528 to 546.

FIG. 1

SPR analysis for determining the epitope specificities of CET MAbs. (A) Sensorgram showing sequential addition of CET MAbs. Experimental conditions are described in Materials and Methods. SPR response was measured in RU. Buffer flow was maintained at 5μl/min throughout the analysis. The saturation step was performed with two injections of CET-4. CET-4 was bound to gDE2t by the first injection, and the unoccupied binding sites on gDE2t were blocked by the second. CET-5, -3, -2, -6, and -1 were added in turn. Each injection start point and the name of corresponding MAb are indicated. The injection volumes were 25 μl for the first injection of CET-4 and 15 μl in all other cases. (B) Competitive ELISA for binding to hGHE2t. Various amounts of unlabeled CET MAbs were used as competitors. hGHE2t-coated wells were incubated with competitors for 10 min. After incubation, biotin-labeled CET-4 was added to the wells. Percent inhibition = 100 × (A₄₀₅ of CET-4 bound to hGHE2t − A₄₀₅ of CET-4 bound to hGHE2t in presence of competitor)/A₄₀₅ of CET-4 bound to hGHE2t. (C) Reactive pattern matrix showing the binding ability of pairs of CET MAbs to E2t protein. •, pairs of CET MAbs that bind concurrently. ○, pairs of CET MAbs that interfere with binding. (D) Two-dimensional surface-like map of the epitopes on E2t recognized to six CET MAbs. Numbers 1 to 6 correspond to the CET-1 to CET-6; overlapping circles represent MAb groups within which pairs of MAb cannot bind simultaneously.

FIG. 2

Schematic diagrams of expression plasmids. (A) Constructs expressing C-terminally truncated forms of HCV E2 differing in length and form. All constructs were based on Rc/CMVdhfr-TPLgDE2t. The E2t cDNA was modified by replacing the AgeI-XbaI fragment near the full-length E2t gene with PCR products of partially E2t-derived sequences except E2t-1, in which the NdeI-AluI segment encoding HVR-1 of E2t cDNA was replaced by a newly synthesized NdeI-AluI DNA fragment (described in Materials and Methods). Names of recombinant expression plasmids are shown at the left. The striped boxes in E2t-1 and E2D-1 represent the regions replaced with the corresponding sequences of HCV-1. Positions of amino acids at both ends of derivatives of E2t cDNA are shown. P_CMV, human cytomegalovirus promoter; BGH polyA, bovine growth hormone polyadenylation signal; TPL, adenovirus tripartite leader. (B) Alignment of amino acid sequences of the region replaced in E2D-1 obtained from HCV-1 (group 1a) and HCV-N (group 1b). Amino acids are indicated in single-letter code. Positions of amino acids at both ends of the region are shown. Horizontal bars designate common sequences. Amino acid numbering is according to Choo et al. (5).

FIG. 3

Alignment of 19 amino acids (in single-letter code) corresponding to the putative B-cell antigenic domain spanning map positions 528 to 546. The sequence of HCV-N is compared with that from 11 different isolates. Names of strains and subtypes are indicated at the left. Horizontal bars designate common sequences. Amino acid numbering is according to Choo et al.(5). GenBank database accession numbers of the sequences: HCV-1, M62321; HCV-H, M67463; HC-J1, D10749; HCV-J, D90208; HCV-BK, M58335; HC-JK1, X61596; HC-J4, D00832; HC-J5, D10076; HC-J6, D00944; HC-J7, D10076; HC-J8, D01221.