Computational identification of HCV neutralizing antibodies with a common HCDR3 disulfide bond motif in the antibody repertoires of infected individuals

Abstract

Despite recent success in hepatitis C virus (HCV) treatment using antivirals, an HCV vaccine is still needed to prevent reinfections in treated patients, to avert the emergence of drug-resistant strains, and to provide protection for people with no access to the antiviral therapeutics. The early production of broadly neutralizing antibodies (bNAbs) associates with HCV clearance. Several potent bNAbs bind a conserved HCV glycoprotein E2 epitope using an unusual heavy chain complementarity determining region 3 (HCDR3) containing an intra-loop disulfide bond. Isolation of additional structurally-homologous bNAbs would facilitate the recognition of key determinants of such bNAbs and guide rational vaccine design. Here we report the identification of new antibodies containing an HCDR3 disulfide bond motif using computational screening with the Rosetta software. Using the newly-discovered and already-known members of this antibody family, we review the required HCDR3 amino acid composition and propose determinants for the bent versus straight HCDR3 loop conformation observed in these antibodies.

Fig. 1. Analysis of HCDR3 loop diversity in the obtained heavy chains sequences library and sequences of the Abs selected for testing.

a, d, f Alignments of the amino acid sequences of the heavy chain variable regions of a HEPC3 with its sibling Abs, d HEPC3 with selected by the P3SM approach HEPC3-like Abs, and f AR3C with selected by the P3SM approach AR3C-like Abs. Dots indicate the same amino acid as in the reference Ab sequence. Positions of the HCDR loops (IMGT definition) are marked above the alignment. b, c, e Amino acid frequency distribution in b all found HEPC3-like, c 8 selected by the P3SM approach HEPC3-like, and e all found AR3C-like HCDR3 loops. Amino acid frequency distributions were analyzed and visualized using WebLogo (https://weblogo.berkeley.edu/logo.cgi) web server.

Fig. 2. HEPC3.1, HEPC3.4, and sibling HEPC3 Abs display broad binding activity and neutralize multiple HCV strains.

a Heat map showing the binding of Abs to a panel of HCV genotype 1 E2 glycoproteins. The EC₅₀ value for each E2-Ab combination is shown, with dark red, orange, yellow, or white shading indicating high, intermediate, low, or no detectable binding, respectively. The “>” symbol indicates EC₅₀ values >10 µg/mL or combinations were OD₄₅₀ values at the highest antibody concentration tested were lower than 0.5. One experiment representative of two independent experiments is shown. b Heat map showing neutralization activities of Abs measured using a panel of genotype 1 HCVpp. The IC₅₀ value for each E2-Ab combination is shown, with dark red, orange, yellow, or white shading indicating high, intermediate, low, or no detectable neutralization activity, respectively. The “>” symbol indicates IC₅₀ values >100 µg/mL or combinations in which percent neutralization at the highest antibody concentration tested was lower than 50%. c Binding of HEPC3.1, HEPC3.4, and reference bNAbs to a 1a157 E2 glycoprotein ectodomain (1a157 E2ecto) and 1a157 E2 variant that contains alanine substitutions that disable binding of HEPC3-like bNAbs that target AR3 antigenic site (1a157 E2ecto ΔFRLY). Introduced alanine substitutions in 1a157 E2ecto ΔFRLY do not disrupt the binding of HC33.1-like bNAbs targeting the antigenic site 412 (AS412) that lies in close proximity to the E2 front layer. One experiment representative of two independent experiments is shown. Source data are provided in the Source Data file. d Binding data from (c) is shown as the area under the curve (AUC).

Fig. 3. The HCDR3 loop in HEPC3.1 and HEPC3.4 Abs adopts a bent orientation.

Side (top) and top (bottom) views of Fab apo structures of HEPC3 (PDB ID: 6MED), HEPC3.1 (this study), HEPC3.4 (this study), and AR3C (PDB ID: 6MEF). The crystal structures were superimposed on their V_H domains. Protein backbones are shown as ribbons, and CDR loops are blue (HCDR1), orange (HCDR2), and red (HCDR3). Disulfide bonds are shown as yellow sticks.

Fig. 4. Interactions of the HCV neutralizing V_H1-69 gene-encoded Abs encompassing a common HCDR3 disulfide bond motif with the front layer of HCV glycoprotein E2.

a All six Abs of the described family demonstrate the same pattern of antigen recognition mediated by their HCDR3 loop. Shown is the overlay of the HCV glycoproteins E2 from the co-crystal structures with bNAbs HEPC3, HEPC74, AR3A, AR3C, AR3X, and HC11. The glycoproteins are shown as a gray cartoon. The backbones of the HCDR3 loops of the Abs are shown as sticks. b, c The HCDR3 loop tips of the unliganded HEPC3.1 (b) and HEPC3.4 (c) Fabs were aligned with the observed common antigen recognition pattern of the V_H1-69 gene-encoded Abs with an HCDR3 disulfide bond motif. The absence of major clashes suggests that HEPC3.1 and HEPC3.4 Abs might interact with the antigen in the same way as other front layer-specific bNAbs. d–i Polar interactions between HCV glycoprotein E2 and HCDR3 loops of d AR3A, e AR3C, f HC11, g AR3X, h HEPC74, and i HEPC3 Abs, as seen in the crystal structures.

Fig. 5. Analysis of the Ab structures with an HCDR3 loop CXXXXC motif.

a The four points on an Ab that were used for the description of the overall shape of the HCDR3 loops in the dataset on the example of HEPC74 (PDB ID 6MEE). The HCDR3 loop is colored. The Cα atoms used for calculations are shown as spheres. The four calculated points are shown as black spheres and are connected by black lines. b, c Calculated angles and dihedrals for HCDR3 loops of all 27 found Abs. Source data are provided in the Source Data file. b The values for the HCV-specific Abs are shown as blue dots. The unrelated Abs that are following the same trend as the HCV-specific Abs are shown as black dots. Also shown are the estimated linear correlation curve (dashed line) for the proposed trend and +/−3σ uncertainty around the estimated curve (shadowed area). c The Abs that seem to follow the trend of the rigid body HCDR3 loop rotating away from the heavy chain and the Ab Fv region, in general, are color-coded based on the distance (blue—closer, green— further away). A side view (d) and a top view (e) on the aligned Fv regions of the Ab heavy chains. The HCDR3 loops are colored according to the legend on the panel (c). The ultra-long HCDR2 loop of AR3X is not shown. f Sequences of the HCDR3 loops (IMGT definition) of the Abs shown on the panels (d) and (e). The two cysteines in each HCDR3 loop are underscored. Prolines in the first half of the loop and glycines in the second half of the loop are colored blue.

Fig. 6. HCDR3 loop conformational dynamics.

a 2D-histograms of individual Ab simulations projected onto the first two time-lagged independent components of the combined Ab TICA-transformed conformational space. Histograms corresponding to simulations initialized with the crystallographic conformations are outlined in blue. b Cartoon representation of the structures of the Abs HEPC3, HEPC3.1, and HEPC3.4 as observed in the crystal structures of the apo Fabs. HCDR1, HCDR2, and HCDR3 loops are colored light blue, blue, and dark blue, respectively. c Ensembles of 10 structures from representative conformational clusters. Structures are shown as cartoons. HCDR1, HCDR2, and HCDR3 loops are colored light blue, blue, and dark blue, respectively. Numbers in (a) and (c) correspond to minima sampled to display ensembles.