Neutralization test of HMAb C10 to Zika virus

The neutralization activity of the HMAb C10 on Zika virus strain H/PF/2013 was determined by PRNT. Two-fold serially diluted HMAb C10 starting at 0.5 μg ml−1 were incubated with equal volumes of virus at 37 °C for 0.5 h. One hundred microlitres of each mixtures were then layered on BHK-21 cells in a 24-well plate and incubated at 37 °C for 1.5 h. The infected cells were washed with phosphate-buffered saline, overlaid with carboxyl-methyl cellulose and incubated at 37 °C for 5 days. Cells were fixed and stained, and the plaques were counted. Percentage neutralization was determined from the comparison of the number of plaques in specific antibody dilutions to the control (without antibody). PRNT 50 is the concentration of the antibody that causes 50% reduction in plaque numbers.

Virus sample preparation

Aedes albopictus C6/36 cells (ATCC) were grown in RPMI 1640 media supplemented with 2% fetal bovine serum at 29 °C. At about 80% confluency, the cells were inoculated with ZIKV strain H/PF/2013 at a multiplicity of infection of 0.5 and incubated at 29 °C for 4 days. The virus-containing media was clarified by centrifugation at 12,000g for 1 h. Virus was precipitated overnight from the supernatant using 8% (w/v) polyethylene glycol 8000 in NTE buffer (10 mM Tris-HCl pH8.0, 120 mM NaCl and 1 mM EDTA) and the suspension was centrifuged at 14,334g for 1 h. The resulting pellet containing the virus was resuspended in NTE buffer and then purified through a 24% (w/v) sucrose cushion followed by a linear 10-30% w/v potassium tartrate gradient. The virus band, visualized by its light scattering ability, was extracted, buffer exchanged into NTE buffer and concentrated using a concentrator with 100-kDa molecular weight cut-off filter. All steps of the purification procedure were done at 4 °C. The concentrations and purity of the E protein were estimated with Coomassie blue-stained SDS-PAGE using different known concentrations of bovine serum albumin solution as standards.

Monoclonal antibody C10 production

Monoclonal antibody (mAb) C10 was synthesized in transfected human cells from cloned plasmids (Lake Pharma, Belmont, CA, USA). Briefly, previously published heavy and light chain variable region sequences14 were cloned into plasmids containing the human IgG1 heavy and the human lambda 2 light chain constant regions. HEK293T cells (ATCC) were transiently co-transfected with both the heavy chain and light chain plasmids, and soluble antibody was collected and protein A purified. The antibody was resuspended in a buffer containing 200 mM HEPES, 100 mM NaCl, 50 mM NaOAc, pH7.0.

Preparation of the C10 Fab fragments

The Fab regions of C10 IgG were produced by papain digestion. Briefly, the whole IgG (8 mg ml−1) was incubated overnight with immobilized papain (Thermo scientific) at 37 °C. After digestion, the Fab fragment was purified with anion exchange chromatography (resource Q, GE Healthcare) and gel filtration (Superdex 200 increase 10/300 GL, GE Healthcare) on an AKTA purifier system.

CryoEM sample preparation

The Fab C10 was mixed with ZIKV at a molar ratio of 1.5 Fab to every E protein. The mixture was incubated for 30 min at 37 °C followed by ∼1 h on ice, and then applied to a cryoEM grid (pre-cooled to 4 °C) for 10 s prior to adjusting the pH. The final pH of the virus was reached by addition of a volume ratio of 1.5 μl of 50 mM MES buffer at respective pH (pH5.0 or pH6.5) to every 1 μl of the virus-Fab mixture. The pH-adjusted samples were left on the grid for another 15 s. The grid was then blotted with filter paper and flash frozen in liquid ethane by using the Vitrobot Mark IV plunger (FEI, the Netherlands). The corresponding controls (ZIKV without Fab) for each pH were prepared similarly.

Cryoelectron microscopy and image processing

The images of the frozen ZIKV complexes were taken with the FEI Titan Krios electron microscope, equipped with 300 kV field emission gun, at nominal magnification of 47,000 for pH5.0 Fab C10 ZIKV complex, and 59,000 for pH6.5 and pH8.0 complex samples. A 4096 × 4096 FEI Falcon II direct electron detector was used to record the images.

Leginon23 was used to carry out the data collection. Images for pH8.0 and pH6.5 complexes were collected in movie mode, with total exposure of 1.6 s and total dose 38 e− Å−2 for pH8.0, total exposure of 1.05 s and total dose of 43 e− Å−2 for pH6.5 complex. The pH5.0 complex was collected at single image mode, with the dose of 20 e− Å−2. The frames from each ‘movie’ were aligned using MotionCorr24 to produce full dose images used for particle selection and orientation search, and images from the first several frames amounting to the dose of about 18 e− Å−2 to use in 3D reconstruction. The images were taken at underfocus in 0.5∼2.5 μm range. The astigmatic defocus parameters were estimated with Gctf25 and accounted for in orientation search and 3D reconstruction procedures in MPSA26 and Relion27. In total, 3,257, 2,540 and 2,865 micrographs were collected for Zika-C10 complex at pH8.0, pH6.5 and pH5.0, respectively. The virus-Fab particles were picked with automatic selection tool Gautomatch (from Dr K. Zhang, author of Gctf), run through 2D classification in Relion27 to produce 2D class averages, broken and classes containing nonviral particles and broken particles were removed, 49,100, 45,867 and 23,810 individual particles in the Fab complex samples which were incubated at pH8.0, pH6.5 and pH5.0, respectively, were selected for further processing. The 3D reconstruction of the pH8.0 and pH6.5 complex structure was done with MPSA26, whereas Relion was used for the pH5.0 complex structure. Uncomplexed ZIKV (EMDB ID EMD-8139) was used as the starting model. The gold standard protocol27 for structure refinement was used for all complexes. The 3D reconstruction procedure produced the complex structures with resolutions of 4.0, 4.4 and 12 Å, for pH8.0, pH6.5 and pH5.0, respectively—using the Fourier shell correlation cutoff of 0.143 for the pH8.0 and pH6.5 cryoEM maps and 0.5 for the pH5.0 cryoEM map (Extended Data Fig. 2).

Protein structure building

The pH8.0 and pH6.5 ZIKV-C10 structures were interpreted by fitting in the uncomplexed ZIKV (PDB ID 5IZ7) and Fab C10 (PDB ID 4UT9) first as rigid bodies in Chimera28 and then finer adjustment were made by using the program Coot29. The 12 Å resolution cryoEM map of the ZIKV-C10 complex at pH5.0 was interpreted by first fitting in the entire E-protein raft complex with Fab molecules at pH8 structure by using the ‘fit-in-map’ function in Chimera. The individual Fabs complexed with A-C′and also Fab complexed with B-B′ dimers within a raft were kept as separate rigid bodies groups and their fit into the density were independently optimized by using the ‘fit-in-map’ function in Chimera.

Electrostatic potential calculations

Electrostatic potentials of protein surfaces were calculated using Adaptive Poisson-Boltzmann Solver(APBS)30 and PDB2PQR31 packages. The structures of uncomplexed ZIKV (PDB ID 5IZ7) and the ZIKV complexed with Fab C10 were processed with the PDB2PQR web server (nbcr-222.ucsd.edu/pdb2pqr_2.0.0/) to prepare the PDB files for APBS. A PARSE force field was applied and PROPKA (v3.0) was used to assign pKa values. APBS was then used to calculate the electrostatic properties of the protein surface.

Data availability

The cryoEM maps and the atomic models of the ZIKV-C10 complex at pH8.0, 6.5 and 5.0 have been deposited in the Electron Microscopy Data Bank (EMD) and the Protein Data Bank (PDB) under the accession codes EMD-9575, EMD-9573, EMD-9574 and 5H37, 5H30, 5H32, respectively. The data that support the findings of this study are available from the corresponding authors on request.