Research ArticleVIROLOGY

Genetic and structural insights into broad neutralization of hepatitis C virus by human VH1-69 antibodies

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Science Advances  02 Jan 2019:
Vol. 5, no. 1, eaav1882
DOI: 10.1126/sciadv.aav1882
  • Fig. 1 Crystal structures of HK6a E2c3 and AR3 Fab complexes.

    (A) Schematic representations of the E2 core domain (amino acids 412 to 645) colored by structural components, with VRs in gray, front layer in cyan, β sandwich in red, CD81 loop in blue, and back layer in green. The E2 HVR1, stalk, and transmembrane region (amino acids 384 to 410 and 646 to 746) are not shown. The E2 N-linked glycans are indicated (29). (B) Modeling of the HVR1, VR2, and the Man6GlcNac2 N-linked glycans in the complex structure of H77 E2c, indicating the accessibility of AR3. The Man6GlcNac2 N-linked glycans were modeled on the basis of the N430 glycan in the H77 E2c structure [Protein Data Bank (PDB) entry 4MWF]. The AR3 is marked by a dashed line. The glycans that surround the AR3 are colored in dark gray. (C) KD values of various AR3 immunoglobulin G (IgG) binding to E2ΔTM, E2c, and E2c3 from genotypes 1a and 6a. (D) Neutralization breadth of AR3A–D, U1, and AR2A mAbs tested by HCVcc assays. Dendrograms shown for each mAb are colored on the basis of their half maximal inhibitory concentration (IC50) values. (E) Superposition of the crystal structures of HK6a E2c3-AR3A (blue), -AR3B (green), or -AR3D (pink) complexes with H77 E2c-AR3C (orange) illustrating the differences in the angle of approach of the AR3A–D mAbs to E2c. The structures of the HK6a E2c3-AR3A, -AR3B, or -AR3D complexes were superimposed with H77 E2c-AR3C on the basis of E2, and for each complex, the angle between the Cα atoms of HC V111, HC F54, and light chain (LC) I106 was calculated. The angle of approach was determined as the relative change in the angle between AR3A, AR3B, and AR3D mAbs to AR3C. For clarity, only the Fab VRs and the HK6a E2c3 structure are shown. (F) AR3 epitopes. The E2c structures are shown in surface representation, and the interacting residues in the epitopes were colored and labeled. (G) Schematic overview of the interactions between E2 and the AR3 HCs. The CDR sequences are aligned, and E2 interacting residues are highlighted in blue (hydrogen bonds) and green (hydrophobic interactions).

  • Fig. 2 Features that enable broad neutralization by AR3 mAbs.

    (A) Alignment of the CDRH2 sequences of AR3 bNAbs and mAbs U1 and AR1A. The hydrophobic motif in CDRH2 is highlighted in red. (B) Surface area buried by the CDRs of AR3 mAbs on E2s. CDRH3 length is indicated in brackets. (C) Correlation between CDRH3 length and LC surface area buried. CDRH3 is colored in red. (D) Interactions between E2 and CDRH1-3 of AR3A–D.

  • Fig. 3 The inferred GL of AR3 mAbs.

    (A) Alignment of the HC CDRs of AR3, AR3GL (inferred GL), and AR31–69 (reversion of only the VH gene to the VH1-69 sequence). The interacting residues in the mature mAbs are colored in blue (hydrogen bonds) and green (hydrophobic interactions). (B) Biolayer interferometry analysis of AR31–69 and AR3GL IgGs binding to H77 and HK6a E2c. (C) Single-dose (50 μg/ml) neutralization assay of AR31–69 and AR3GL IgGs against the eight HCVcc JFH1-based Core-NS2 recombinants of genotypes 1 to 6. Error bars represent SEM.

  • Fig. 4 Analysis of B cell repertoires of HCV-infected donors.

    (A) Quantitative B cell repertoire distribution of HC GL V gene usage, degree of SHM, and CDRH3 length and (B) identity/divergence analysis (to AR mAb/VH GL) of the prepanned and panned phage display Ab libraries. #a.a., number of amino acids. The panning experiments were performed using E1E2. (C) Schematic representation of the epitopes and the HC variable genes encoding the AR1–5 mAbs. The AR1–3 epitopes are shown on the E2 structure (16, 17, 31). bNAbs that target the AR3 epitopes are listed. (D) The distribution of CDRH3 length in the total HC GL V genes and (E) the three largest HC GL gene families of five patients with chronic HCV with high anti-E1E2 Ab titers and cross-neutralizing activity (top) and five healthy donors (bottom) as controls.

Supplementary Materials

  • Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/5/1/eaav1882/DC1

    Fig. S1. AR3 epitopes.

    Fig. S2. Binding of AR3A–D and AR1A IgGs to E2.

    Fig. S3. Binding and neutralization of AR3 mAbs to HCV isolates from genotypes 1 to 6.

    Fig. S4. Crystal structures of HK6a E2c3 with AR3 Fabs.

    Fig. S5. Features that enable broad neutralization by AR3.

    Fig. S6. The inferred GL genes of the AR3 mAbs.

    Fig. S7. Identity/divergence analysis of the phage display library from an HCV-infected donor.

    Fig. S8. B cell repertoire of healthy and HCV-infected donors.

    Fig. S9. Primers that were used for amplification of the HC regions of the Fab phage libraries in the different panning steps.

    Fig. S10. Summary of the statistic analysis of the NGS of HCV chronically infected and healthy donors.

    Table S1. Amino acid sequence variation of the E2 protein of HCV genotypes 1 to 7.

    Table S2. Data collection and refinement statistics for HK6a E2c3 AR3 Fab complex structures.

    Table S3. Hydrogen bond and hydrophobic interactions between E2 and the HC of AR3 Fabs.

    Table S4. NGS of HCV-immune Ab repertoire of an HCV-infected patient.

    Table S5. NGS of HCV-immune Ab repertoires.

    References (6568)

  • Supplementary Materials

    This PDF file includes:

    • Fig. S1. AR3 epitopes.
    • Fig. S2. Binding of AR3A–D and AR1A IgGs to E2.
    • Fig. S3. Binding and neutralization of AR3 mAbs to HCV isolates from genotypes 1 to 6.
    • Fig. S4. Crystal structures of HK6a E2c3 with AR3 Fabs.
    • Fig. S5. Features that enable broad neutralization by AR3.
    • Fig. S6. The inferred GL genes of the AR3 mAbs.
    • Fig. S7. Identity/divergence analysis of the phage display library from an HCV-infected donor.
    • Fig. S8. B cell repertoire of healthy and HCV-infected donors.
    • Fig. S9. Primers that were used for amplification of the HC regions of the Fab phage libraries in the different panning steps.
    • Fig. S10. Summary of the statistic analysis of the NGS of HCV chronically infected and healthy donors.
    • Table S1. Amino acid sequence variation of the E2 protein of HCV genotypes 1 to 7.
    • Table S2. Data collection and refinement statistics for HK6a E2c3 AR3 Fab complex structures.
    • Table S3. Hydrogen bond and hydrophobic interactions between E2 and the HC of AR3 Fabs.
    • Table S4. NGS of HCV-immune Ab repertoire of an HCV-infected patient.
    • Table S5. NGS of HCV-immune Ab repertoires.
    • References (6568)

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