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. 2012 Jun 12;109(24):9499-504.
doi: 10.1073/pnas.1202924109. Epub 2012 May 23.

Structural basis of hepatitis C virus neutralization by broadly neutralizing antibody HCV1

Leopold Kong  1 , Erick Giang, Justin B Robbins, Robyn L Stanfield, Dennis R Burton, Ian A Wilson, Mansun Law
Affiliations

Structural basis of hepatitis C virus neutralization by broadly neutralizing antibody HCV1

Leopold Kong et al. Proc Natl Acad Sci U S A. .

Abstract

Hepatitis C virus (HCV) infects more than 2% of the global population and is a leading cause of liver cirrhosis, hepatocellular carcinoma, and end-stage liver diseases. Circulating HCV is genetically diverse, and therefore a broadly effective vaccine must target conserved T- and B-cell epitopes of the virus. Human mAb HCV1 has broad neutralizing activity against HCV isolates from at least four major genotypes and protects in the chimpanzee model from primary HCV challenge. The antibody targets a conserved antigenic site (residues 412-423) on the virus E2 envelope glycoprotein. Two crystal structures of HCV1 Fab in complex with an epitope peptide at 1.8-Å resolution reveal that the epitope is a β-hairpin displaying a hydrophilic face and a hydrophobic face on opposing sides of the hairpin. The antibody predominantly interacts with E2 residues Leu(413) and Trp(420) on the hydrophobic face of the epitope, thus providing an explanation for how HCV isolates bearing mutations at Asn(415) on the same binding face escape neutralization by this antibody. The results provide structural information for a neutralizing epitope on the HCV E2 glycoprotein and should help guide rational design of HCV immunogens to elicit similar broadly neutralizing antibodies through vaccination.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Biological activities of recombinant mAb HCV1. (A) Binding of mAb HCV1 to E1E2 in ELISA. E1E2 antigens expressed in 293T cells, at their native (closed symbols) or reduced (open symbols) form, were captured onto microwells by lectin. The control mAbs A4 and AR3A recognize a continuous E1 epitope (46) and a discontinuous E2 epitope (19), respectively. (B) HCV1 epitope. The specificity of mAb HCV1 was verified by binding to overlapping peptides (250 ng per well) containing the sequence of the epitope (in bold type). The mAb did not bind peptides with truncated epitope sequences. Note that the amount of captured E1E2 is not equivalent to the directly coated peptides. (C) Neutralization of HCV by mAb HCV1. The recombinant mAb neutralized HCV pseudotype virus particles (HCVpp) displaying the genotype 1a or 1b E1E2, but not the control envelope glycoprotein G from vesicular stomatitis virus (VSVpp). The mAbs AR3A and AR4A are control neutralizing mAbs to E2 and the E1E2 complex, respectively (39). (D) Peptide candidates for crystallography. The binding of mAb HCV1 to three candidate peptides (R12-mer, 15-mer, and 18-mer) was first evaluated by direct ELISA (Left). The mAb bound poorly to the 15-mer and R12-mer when the peptides were coated directly onto microwells. However, when in solution, all three peptides blocked the mAb binding to E1E2 at equivalent levels (Right). The epitope sequence is highlighted in bold in the above peptides.
Fig. 2.
Fig. 2.
Structure of mAb HCV1 in complex with its HCV E2 peptide epitope. Two X-ray structures of a broadly neutralizing antibody in complex with its epitope from two crystals forms, P21 and C2, were solved and refined to1.8-Å resolution. (A) The structure from the P21 crystal is shown in cartoon representation. The peptide epitope (red) is inserted between the heavy chain (blue) CDR2 and CDR3 loops and makes contact with the light chain (green) CDR3 loop. Arg1 in the peptide, which was introduced to enhance the solubility of the peptide and is therefore not part of the HCV antigenic region, is colored gray. (B) B-values of the crystal structure were mapped onto the molecular surface of the paratope (Left) and a stick representation of the peptide (Right) by temperature gradient coloring from 9.8 Å2 (deep blue) to 96.5 Å2 (red). The paratope is shown in gray to highlight the peptide at Right. (C) The adaptive Poisson-Boltzmann solver was used to calculate the surface potential across the solvent-accessible surfaces of both the paratope and the peptide [−3 kT/e (red) to 3 kT/e (blue)]. For the peptide, the surface potential is shown looking from above the antibody (Upper) and from the paratope toward the binding surface, which is a 180° rotation (Lower). The engineered arginine was included for the calculation and is shown here boxed in gray. (D) A comparison of the peptides between the two crystal structures. The peptides are superimposed on each other, with the P21 structure in red and the C2 structure in blue. The two strands of the peptide are separated and reoriented to better visualize the differences in side chains between the structures. The dots indicate where the two strands meet at the turn. Overall, the side chains pointing away from the antibody have greater differences between the structures than those pointing into the binding site. The main chains mostly overlap each other. (E) The peptide forms a β-hairpin, and the backbone hydrogen bonding that stabilizes this structure is indicated.
Fig. 3.
Fig. 3.
Molecular details of antibody binding to the HCV peptide. (A) The Leu413and Trp420 residues on the peptide (red) are shown buried in a hydrophobic depression formed by LC CDR3 residues (green) and by heavy chain FR2, CDR2, and CDR3 residues (blue). (B) Three hydrogen bonds between peptide and antibody also stabilize the interaction, as depicted in wall-eye stereo. Bonds and distances are labeled in black. (C) To further analyze the peptide binding, HCV sequence conservation across 2,161 isolates for this region, crystallographic B-value, rmsd between the two structures, and surface burial by antibody on the peptide are shown. Sequence conservation was taken from the ViPR database (Table S4), whereas the B-values were extracted from the structure and the binding data from ref. . The rmsd was calculated on a residue-by-residue basis in PyMOL. Surface burial corresponds to the accessible surface area of each residue on the peptide in the bound structure (P21 structure) normalized by the surface area calculated after the Fab is removed.
Fig. 4.
Fig. 4.
Viral escape through substitutions at E2 residue 415. (A) Alanine scanning mapping of the HCV1 epitope. (B) Binding of mAb HCV1 to E1E2 variants with substitutions at position 415, including naturally occurring substitutions and Gln. (A and B) Left: Expression of the variants confirmed by mAb AR2A (1 μg/mL) (19). Right: Binding of mAb HCV to the variants. (C) Escape of mAb HCV1 by substitution at E2 Asn415. HCVpp bearing the specific substitutions were generated as described previously (19). The infectivity of the variant panel was compared according to the activity of the reporter gene luciferase (relative light unit, RLU) (Left). Only the K, Q, and E variants produced HCVpp with significantly higher signals (>10-fold) than the control pseudotype virus generated without E1E2. The sensitivity of these variants to mAb HCV1 was determined by incubating them with serially diluted mAb (Right). The results shown are the means ± SD of two independent experiments of triplicate measurement.
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Comment in

  • Unraveling hepatitis C virus structure.
    Fauvelle C, Felmlee DJ, Baumert TF. Fauvelle C, et al. Cell Res. 2014 Apr;24(4):385-6. doi: 10.1038/cr.2014.31. Epub 2014 Mar 14. Cell Res. 2014. PMID: 24626133 Free PMC article.

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