Figures & data
Figure 1 Bio-panning of anti-ZIKV DIII protein Fabs from the full-human Fab library. (A) The individual E protein (black oval), which consists of DI, DII and DIII, is indicated. DI, DII and DIII are shown in red, yellow, and blue, respectively. (B) Polyclonal phage ELISA showing the phage enrichment of four rounds (Rounds 1–4) by panning. (C) Binding of Fabs m301–m312 to ZIKV DIII protein according to ELISA. (D) Western blot analysis of binding activity for Fabs m301–m304. Domain, D; enzyme-linked immunosorbent assay, ELISA; Zika virus, ZIKV.
Figure 2 Binding kinetics of IgGs m301–m304 to ZIKV-DIII (A) or control antigen (B), as measured by BLI using OctetRED96. Purified ZIKV-DIII-hFc or control antigen (influenza H3N2 HA) was immobilized on activated AR2G biosensors. The analytes consisted of serial dilutions of IgGs between 100 and 1.2 nM. Binding kinetics were evaluated using a 1:1 Langmuir binding model by Fortebio Data Analysis 7.0 software. Biolayer interferometry, BLI; domain, D; immunoglobulin G, IgG; Zika virus, ZIKV.
Figure 3 Immunogenetic analysis of the heavy- and light-chain variable regions of m301 and m302 using the IMGT tool.
Table 1 Genetic analysis of the heavy and light chain variable regions of ZIKV DIII-specific antibodies
Figure 4 Neutralization activity of anti-ZIKV antibodies (m301–m304) to different ZIKV strains by plaque reduction assays. (A) The ZIKV Asian lineage SZ01 was incubated with 300 μg/mL m301–m304 antibodies. (B) Four kinds of ZIKV strains, including PAN2015, FLR, R103451, and PRVABC59, were incubated with 175 μg/mL m301. ZIKV E serum (1:50) served as a positive control. (C) PAN2015 was mixed with four-fold serial dilutions of m301 for 1.5 h at 37 °C prior to infection of Vero E6 cells. Subsequently, neutralization activity was evaluated by plaque reduction assays in duplicate. (D) Neutralization activity of mAbs alone or in a cocktail of m301 and m302 to ZIKV SZ01 strain. Data are represented as the mean±s.e.m. The figure represents three independent experiments performed in duplicate. Monoclonal antibodies, mAbs; Zika virus, ZIKV.
Figure 5 A competition assay was performed among different IgGs to DIII. Immobilized ZIKV-DIII-hFc was first saturated with 100 nM of m301 (A), m302 (B), m303 (C) or m304 (D). The capacity of the second IgG binding to the antigen was monitored by measuring further shifts after injecting the second IgG (100 nM) in the presence of the first IgG (100 nM). The red dotted vertical line represents the second IgG loading. domain, D; immunoglobulin G, IgG; Zika virus, ZIKV.
Figure 6 The structural basis and epitopes of anti-ZIKV Fabs m301 and m302 on ZIKV DIII. (A) Docking diagrams of m301 Fab (left) and m302 Fab (right) onto ZIKV DIII (PDB identifier 5 kvg) complexes with antibody fragments are shown. The heavy chain of Fab is in red, and the light chain is in green. DIII is colored dark blue with contact segments labeled. (B) Sequence definition of binding epitopes on ZIKV-specific DIII. DIII residues are colored if they make van der Waals contact within 3 Å distance, and the total number of contacts for each epitope residue are shown below the DIII sequences. (C) The binding capacity of m301 with wild-type and a panel of mutant DIII determined by ELISA. Data are represented as the mean±s.e.m. The figure represents three independent experiments performed in duplicate. (D) The structure of the mature ZIKV E protein (PDB 4ire) is shown. The C–C′ loop epitope residues are colored in cyan in each symmetry group. Domain, D; enzyme-linked immunosorbent assay, ELISA; antigen-binding fragment, Fab; Zika virus, ZIKV.
Figure 7 In vivo protection and therapeutic activity of anti-ZIKV mAbs against ZIKV infection. Four- to eight-week-old AG6 mice were inoculated with 105 PFU of ZIKV by i.p. route. At 12 h post ZIKV infection or 4 h before 103 PFU ZIKV infection, mice were administered m301 or a cocktail of m301 and m302 via i.p. injection. (A, C) Mortality of AG6 mice was monitored after infection. (B, D) Mice were weighed daily, and weights are expressed as the percentage of body weight prior to infection. Abbreviations: intraperitoneal, i.p.; monoclonal antibodies, mAbs; plaque-forming units, PFU; Zika virus, ZIKV.
