Structural Basis of Zika Virus-Specific Antibody Protection - PubMed (original) (raw)

Structural Basis of Zika Virus-Specific Antibody Protection

Haiyan Zhao et al. Cell. 2016.

Abstract

Zika virus (ZIKV) infection during pregnancy has emerged as a global public health problem because of its ability to cause severe congenital disease. Here, we developed six mouse monoclonal antibodies (mAbs) against ZIKV including four (ZV-48, ZV-54, ZV-64, and ZV-67) that were ZIKV specific and neutralized infection of African, Asian, and American strains to varying degrees. X-ray crystallographic and competition binding analyses of Fab fragments and scFvs defined three spatially distinct epitopes in DIII of the envelope protein corresponding to the lateral ridge (ZV-54 and ZV-67), C-C' loop (ZV-48 and ZV-64), and ABDE sheet (ZV-2) regions. In vivo passive transfer studies revealed protective activity of DIII-lateral ridge specific neutralizing mAbs in a mouse model of ZIKV infection. Our results suggest that DIII is targeted by multiple type-specific antibodies with distinct neutralizing activity, which provides a path for developing prophylactic antibodies for use in pregnancy or designing epitope-specific vaccines against ZIKV.

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Figures

Figure 1. Profile of neutralizing mAbs against ZIKV

A. Cells were infected with DENV-1, DENV-2, DENV-3, DENV-4, or ZIKV (H/PF/2013), and stained with indicated anti-ZIKV mAbs or isotype controls and processed by flow cytometry. The data is representative of several independent experiments. Blue arrows indicate binding of cross-reactive mAbs (ZV-13 or WNV E53) to DENV and ZIKV-infected cells; Red arrows indicate binding of ZIKV mAbs to ZIKV-infected cells. B. The indicated flavivirus proteins (ZIKV E, ZIKV E-FL (fusion loop mutant), ZIKV DIII, WNV E, and DENV-4 E) were incubated with the indicated anti-ZIKV mAbs or controls (WNV E60 (flavivirus cross-reactive) and WNV E24 (WNV type-specific). Binding was determined by ELISA and the results are representative of two independent experiments performed in triplicate. C. Focus reduction neutralization tests (FRNT). Different ZIKV strains (H/PF/2013, Paraiba 2015, Dakar 41519, and MR-766 were incubated with increasing concentrations of mAbs for 1 h at 37°C prior to infection of Vero cells. Subsequently, an FRNT assay was performed (see Experimental Procedures). The results reflect pooled data from two or more independent experiments performed in triplicate.

Figure 2. Differential binding and ADE activity of different anti-ZIKV mAbs

A. Quantitative analysis of DIII binding to anti-ZIKV mAbs by BLI. Shown in the top panel are binding curves obtained by passing different concentrations of DIII over biotinylated anti-ZIKV antibody immobilized on a biosensor surface. The kinetic values were obtained by simultaneously fitting the association and dissociation responses to a 1:1 Langmuir binding model (KD, kinetic). The lower panels show the steady-state analysis results (KD, equilibrium). Plotted in the lower panels (open circles) is the binding response (nm) versus concentration of DIII offered. In each case the binding was saturable. Lower panel insets, Scatchard plots, suggest a single binding affinity for each interaction. The data is representative of two independent experiments per mAb. B. (Left) ZIKV SVPs were adsorbed to 96-well plates and detected with the indicated biotinylated anti-ZIKV or control (WNV E60 (flavivirus cross-reactive) and WNV E16 (WNV type-specific) mAbs by ELISA. (Right) The relative avidity of binding was calculated. Data are representative of five independent experiments, and the avidity values reflect the mean of the five experiments. Error bars indicate standard deviations. C. ADE studies. Serial dilutions of anti-ZIKV or control (WNV E60 (flavivirus cross-reactive) and WNV E16 (WNV type-specific) mAbs were mixed with (left) ZIKV H/PF/2013 or (right) DENV-2 RVPs (which encode for GFP) prior to infection of FcγRIIa+ human K562 cells and processing by flow cytometry. One representative experiment of two is shown. Error bars indicate the range of duplicate technical replicates. See also Fig S1.

Figure 3. Structures of anti-ZIKV Fabs and scFv complexed with DIII

A. Ribbon diagrams of four ZIKV DIII (H/PF/2013) complexes with antibody fragments. The crystal structure of (outer left) ZV-2 Fab (green), (inner left) ZV-48 scFv (cyan), (inner right) ZV-64 Fab (cyan), and (outer right) ZV-67 Fab (magenta) are shown with light chains rendered in paler colors. DIII is colored dark blue with contact segments labeled. B. Docking of the ZV-2, ZV-48, and ZV-64 complexes onto ZV-67-DIII. DIII is rendered as a molecular surface with each mAb contact surface color-coded. Simultaneous docking of ZV-2 and ZV-67 with either ZV-48 or ZV-64 buries nearly half of the solvent surface of DIII and creates no van der Waal contacts between adjacent mAbs. C. Five mAbs were probed for competitive and non-competitive binding against the DIII antigen by BLI. In one experiment, biotin-labeled ZV-67 was captured on the streptavidin sensor, the antibody was then loaded with ZIKV DIII followed by either ZV-54 or ZV-64, and finally ZV-2 was added. In another experiment, ZV-48 was immobilized and ZV-64 or ZV-67 was added after DIII followed by ZV-2. Additional BLI signal indicates an unoccupied epitope (non-competitor), whereas no binding indicates epitope blocking (competition). In this experiment, ZV-48 competed with ZIKV-64 as expected given that they both bind nearly identical epitopes, while ZV-67 competed with its presumed sibling clone ZV-54. A dash (−) represents that no 2nd or 3rd antibody was offered. See also Fig S2 and Tables S1, S2, S3, and S4.

Figure 4. Structural definition of ZIKV-specific DIII epitopes

A. Sequence alignment of DIII from our ZIKV immunizing stains (H/PF/2013 and MR-766), WNV, DENV-1, DENV-2, DENV-3, and DENV-4 and highlighting of structurally defined DIII epitopes. The ABDE sheet epitope of ZV-2 is shown in green, the C–C′ loop epitope of ZV-48 and ZV-64 is shown in cyan, and the LR epitope of ZV-67 is shown in magenta. DIII residues are colored if they make van der Waals contact of 3.90-Å distance or less, and the total number of contacts for each epitope residue are shown below the ZIKV sequences. For comparison, the same structurally defined DIII epitopes of WNV E16 (magenta, LR), DV1-E106 (magenta, LR), DV1-E111 (cyan, C–C′ loop), DV2 1A1D-2 (pink, A-strand), DV3 2H12 (light-green, AB-loop), and DV4 4E11 (pink, A-strand) are displayed. The ZIKV β-strands are labeled and shown in dark blue above the sequences. B. Delineation of the epitope contact regions on the ZIKV DIII structures of ZV-2 (ABDE sheet), ZV-48 (C–C′ loop), ZV-64 (C–C′ loop) and ZV-67 (LR). DIII epitope residues are colored as in A, with side chains drawn as sticks and labeled if they make eight or more van der Waals contacts. See also Fig S2.

Figure 5. Accessibility of ZIKV DIII epitopes

A. Mapping of the three distinct ZIKV DIII epitopes onto the mature virion (5IRE) (Sirohi et al., 2016). The surface distribution of the ABDE sheet (green), C–C′ loop (cyan), and LR (magenta) epitopes are rendered on the three symmetrically unique E proteins colored olive, wheat, and grey. While the ABDE sheet and C–C′ loop epitopes are dominantly buried in all three symmetry environments, the LR epitope is solvent accessible on the mature virion. B. Docking of the ZV-2-DIII complex onto the crystal structure of dimeric ZIKV (5JHM) (Dai et al., 2016). Shown above is the ZV-2 Fab docked to a soluble E monomer, which indicates that the ABDE sheet epitope is occluded by DI with clashes by the VH domain. Below the ZIKV dimer is depicted, showing how it would sterically clash with the ZV-2 VL domain. ZV-2 CDR loops contact several of the same DIII residues that are contacted by the DII fusion loop in the dimer. C. Docking of the ZV-64-DIII and ZV-67-DIII complexes onto the cryo-electron microscopy model of the M-E dimer that forms the mature virion. ZV-67 binding to the LR epitope allows for the projection of the Fab away from the viral membrane whereas ZV-64 binding to the C–C′ loop epitope positions the Fab in the plane of the viral envelope and membrane. D. Comparative docking of the DV1-E111 Fab-DIII complex (Austin et al., 2012) onto the cryptic C–C′ loop epitope suggests similar steric clashes as predicted for ZV-64. E. Comparative docking of the WNV-E16 Fab-DIII complex (Nybakken et al., 2005) onto the exposed LR epitope. F. Comparative docking of the DV2-1A1D-2 Fab-DIII complex (Lok et al., 2008) and DV4-4E11 scFv-DIII complex (Cockburn et al., 2012) onto the exposed A-strand epitope.

Figure 6. In vivo protection of anti-ZIKV mAbs

Four to five week-old C57BL/6 mice were passively transferred 2 mg of anti-Ifnar1 mAb and 250 μg of the indicated mAbs (CHK-166, ZV-54, or ZV-57) via an intraperitoneal injection one day before subcutaneous inoculation with 105 FFU of ZIKV Dakar 41519. A. On day 3 after infection, serum was collected for analysis of viral RNA by qRT-PCR. B. Daily weights were measured. For A and B, statistical significance was analyzed by a one-way ANOVA with a Dunnett’s multiple comparisons test (**, P < 0.01; ***, P < 0.001). C. ZV-54 and ZV-67 protected against ZIKV infection compared to the control CHK-166 mAb (***, P < 0.001, log rank test). The results are pooled from two independent experiments with n = 8 to 9 mice for each treatment condition.

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Structural Basis of Zika Virus-Specific Antibody Protection - PubMed (original) (raw)