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Antibody isolation study design

This study was designed to isolate SARS-CoV-2 antigen-specific human mAbs from peripheral plasmablasts in humans with natural SARS-CoV-2 infection, to characterize the antigenic specificity and phenotypic activity of SARS-CoV-2 spike-reactive mAb, and to determine the structure of antibody in complex with viral antigen.

The infection of patients/individuals by SARS-CoV-2 was confirmed by positive real-time RT-PCR analyses of respiratory samples, according to the guidelines of the Taiwan Centers for Disease Control (https://www.cdc.gov.tw/En). The study protocol and informed consent were approved by the ethics committee at the Chang Gung Medical Foundation and the Taoyuan General Hospital, Ministry of Health and Welfare, Taiwan. Each patient provided signed informed consent. The study and all associated methods were carried out in accordance with the approved protocol and the Declaration of Helsinki and Good Clinical Practice guidelines.

Sorting of plasmablasts and production of human IgG mAbs

Fresh peripheral blood mononuclear cells (PBMCs) were separated from whole blood by density gradient centrifugation and cryo-preserved PBMCs were thawed. PBMCs were stained with a mix of fluorescent-labeled antibodies to cellular surface markers (all from BD Biosciences) including anti-CD3, anti-CD19, anti-CD27, anti-CD20, anti-CD38, anti-IgG and anti-IgM. Plasmablasts were selected by gating on CD3CD20CD19+CD27hiCD38hiIgG+IgM events and were isolated in chamber as single cells, as previously described31. Sorted single cells were used to produce human IgG mAbs as previously described31. Expression vectors that carry variable domains of heavy and light chains were transfected into the 293T cell line for expression of recombinant full-length human IgG monoclonal antibodies in serum-free transfection medium.

To determine the individual gene segments employed by VDJ and VJ rearrangements and the number of nucleotide mutations and amino acid replacements, the variable domain sequences were aligned with germline gene segments using the international ImMunoGeneTics (IMGT) alignment tool (http://www.imgt.org/IMGT_vquest/input).

Protein cloning, expression and purification

All plasmids were sequenced to confirm clones were correct.

EY6A IgG used for neutralization and making Fab

Antibody was expressed using the ExpiCHO expression system (Life Technologies) according to the manufacturer’s protocol and purified using a Protein A MabSelect SuRE column (GE Healthcare). The wash buffer contained 20 mM Tris, 150 mM NaCl buffered to pH 8.6 and the elution was done using 0.1 M citric acid pH 2.5. The eluate was neutralized immediately using 1.5 M Tris pH 8.6 and then buffer-exchanged to PBS using 15 ml 30-kDa MWCO centrifugal filter (Merck Millipore).

Preparation of Fab-EY6A from IgG

EY6A Fab was digested from IgG with papain using a Pierce Fab Preparation Kit, following the manufacturer’s standard protocol.

Expression and purification of EY6A-6His Fab

Plasmids encoding the heavy and light chains of EY6A-6His Fab were amplified in Escherichia coli DH5α, then extracted and purified using a Qiagen HiSpeed Plasmid Giga Kit. HEK293T cells were transfected with the two plasmids. The medium was harvested and dialyzed into 1.7 mM NaH2PO4, 23 mM Na2HPO4, 250 mM NaCl, pH 8.0 at 4 °C overnight. The sample was then applied to a 5-ml HisTrap nickel column (GE Healthcare). Initially purified EY6A-6His Fab was then loaded onto a Superdex 75 HiLoad 16/60 gel filtration column (GE Healthcare) for further purification using 10 mM HEPES pH 7.4, 150 mM NaCl. Fractions containing EY6A-6His Fab were collected and concentrated.

RBD, ACE2, spike ectodomain and CR3022 cloning

Constructs are as described in ref. 14.

Nanobody

This was derived from a naive library followed by affinity maturation as described in ref. 19.

Production of RBD and ACE2

Plasmids encoding these constructs were transiently expressed in Expi293 (Thermo Fisher Scientific) and proteins were purified from culture supernatants by immobilized metal affinity chromatography using an automated protocol implemented on an ÄKTAxpress system (GE Healthcare)32, followed by size-exclusion chromatography using a Hiload 16/60 Superdex 75 or a Superdex 200 10/300GL column equilibrated in PBS pH 7.4 buffer. Recombinant spike ectodomain was expressed by transient transfection in HEK293S GnTI− cells (ATCC CRL-3022) for nine days at 30 °C. Conditioned medium was dialyzed against 2× PBS pH 7.4 buffer. The spike ectodomain was purified by immobilized metal affinity chromatography using Talon resin (Takara Bio) charged with cobalt followed by size-exclusion chromatography using a HiLoad 16/60 Superdex 200 column in 150 mM NaCl, 10 mM HEPES pH 8.0, 0.02% NaN3 at 4 °C, before buffer exchange into 2 mM Tris pH 8.0, 200 mM NaCl4.

De-glycosylation of RBD or ACE2

A 10 μl volume of endoglycosidase F1 (~1 mg ml−1) was added to protein (~2 mg ml−1, 3 ml) and incubated at room temperature for 2 h. The sample was then loaded to a Superdex 75 HiLoad 16/600 gel filtration column (GE Healthcare) for further purification using 10 mM HEPES pH 7.4, 150 mM NaCl. Purified RBD or ACE2 was concentrated using 10-kDa ultra centrifugal filters (Amicon) to 12 mg ml−1.

Enzyme-linked immunosorbent assay

The ELISA plates (Corning 96-well Clear Polystyrene High Bind Stripwell Microplates) were coated with SARS-CoV-2 antigen (Sino Biological) or SARS antigen (Sino Biological) or Middle East respiratory syndrome coronavirus (MERS) antigen (Sino Biological, 40069-V08B) or human coronavirus OC43 antigen (Sino Biological, 40607-V08B) at optimal concentrations in carbonate buffer and incubated at 4 °C overnight. The next day, unbound antigens were removed by pipetting to avoid the risk of forming aerosols. Non-specific binding was blocked with a solution of PBS with 3% BSA at room temperature for 1 h on a shaker. After removing blocking buffer, mAb-containing cell culture supernatant or purified mAb preparation was added and incubated at 37 °C for 1 h. The non-transfected cell culture supernatant, anti-influenza human monoclonal antibody BS 1A (in house), anti-SARS spike monoclonal antibody CR3022 and convalescent serum were used as antibody controls for each experiment. After incubation, the plate was washed and incubated with horseradish peroxidase (HRP)-conjugated rabbit anti-human IgG (Rockland Immunochemicals) as secondary antibody. After incubation, the plate was washed and developed with 3,3′,5,5′-tetramethylbenzidine substrate reagent (BD Biosciences). Reaction was stopped by 0.5 M hydrochloric acid and the optical density (OD) was measured at 450 nm on a microplate reader. The well that yielded an OD value four times the mean absorbance of negative controls (BS 1A) was considered positive.

Immunofluorescence assay

SARS-CoV-2 (strain CDC-4)-infected Vero E6 cells were prepared and fixed with acetone in a Biosafety Level 3 (BSL-3) laboratory following biosafety rules and guidelines28. The fixed cells on the cover slips were incubated with anti-SARS-CoV-2 spike EY6A mAb-containing cell culture supernatant or anti-influenza human monoclonal antibody BS 1A control (produced in house). Following incubation and washing, the cells were stained with FITC-conjugated goat anti-human IgG secondary antibody (Invitrogen) and Evans Blue dye as counterstain. Binding antibodies were detected by fluorescence microscopy.

Quantitative polymerase chain reaction-based neutralization assay

Neutralization activity of mAB-containing supernatant was measured using a SARS-CoV-2 (strain CDC-4) infection of Vero E6 cells28. Briefly, Vero E6 cells were pre-seeded in a 96-well plate at a concentration of 2 × 104 cells per well. On the following day, mAb-containing supernatant was mixed with an equal volume of 100-TCID50 virus preparation and incubated at 37 °C for 1 h. The mixture was added into seeded Vero E6 cells and incubated at 37 °C for five days. The cell control, virus control and virus back-titration were set up for each experiment. At day 5, the culture supernatant was collected from each well and the viral RNA was extracted by the automatic LabTurbo system (Taigen) following the manufacturer’s instructions for the most part, except that the specimen was pretreated with proteinase K before RNA extraction. Real-time RT-PCR was performed in a 25-μl reaction containing 5 μl of RNA33. The primers and probe used to amplify the E gene were as follows: E_Sarbeco_F, 5′- ACAGGTACGTTAATAGTTAATAGCGT-3′; E_Sarbeco_R, 5′- ATATTGCAGCAGTACGCACACA-3′; E_Sarbeco_P1, FAM-ACACTAGCCATCCTTACTGCGCTTCG-BBQ.

Cell-based ACE2 or RBD blocking assays

MDCK-SIAT1 cells were stably transfected using a second-generation lentiviral vector, with human ACE2 cDNA or with a construct corresponding to RBD (amino acids 340–538 NITN.GPKK) fused to the transmembrane and cytoplasmic domain of hemagglutinin H7 (A/HongKong/125/2017) (EPI977395) via a short linker for surface expression (TGSGGSGKLSSGYKDVILWFSFGASCFILLAIVMGLVFICVKNGNMRCTICI*) using the method described above. ACE2-expressing cells were sorted by fluorescent activated cell sorting (FACS), post staining with RBD-6xH, followed by a secondary anti-His AlexaFluor 647 labeled antibody. RBD-expressing cells were FACS-sorted using the CR3022 antibody. Cells (3 × 104 per well) were seeded the day before the assay in flat-bottomed 96-well plates.

Serial half-log dilutions (ranging from 1 μM to 0.1 nM) of antibodies and controls were performed in 30 μl volumes. PBS supplemented with 0.1% BSA (37525, Thermo Fisher Scientific) was used for dilution of all antibodies. RBD or ACE2-Fc was biotinylated using EZ-link sulfo-NHS-biotin (A39256, Life Technologies). A 30 μl volume of biotinylated RBD at 25 nM or Ace2-Fc at 5 nM was added to titrated antibodies. Cells were washed with PBS and 50 μl of each mixture of biotinylated protein and antibodies was transferred to the MDCK-ACE2 and incubated for 1 h at room temperature. Cells were then washed with PBS and incubated for 1 h with a second layer of streptavidin-HRP antibody (434323, Life Technologies) diluted to 1:1,600 and developed with BM POD substrate (11484281001, Roche) for 5 min before stopping with 1 M H2SO4. Plates were then read on a ClarioStar Plate Reader.

Graphs were plotted as percent binding of biotinylated protein (ACE2 or RBD) to its respective ligand on the cell surface. Binding % = (X − min)/(max − min) × 100 where X = measurement of the competing component, min = buffer without binder biotinylated protein and max = biotinylated protein alone. Inhibitory concentration at 50% (IC50) of the antibodies was determined using nonlinear regression [inhibitor] versus a normalized response curve fit using GraphPad Prism 8. Non-biotinylated ACE2-Fc-6H and VHH72-Fc were used as positive controls.

Surface plasmon resonance

SPR experiments were performed using a Biacore T200 system (GE Healthcare). All assays were performed using Sensor Chip protein A (GE Healthcare), with a running buffer of PBS pH 7.4 supplemented with 0.005% vol/vol surfactant P20 (GE Healthcare) at 25 °C. To determine the binding kinetics between the RBD of SARS-CoV-2 and EY6A mAb, two different experimental settings were attempted. The first set-up had RBD-Fc immobilized on the sample flow cell and the reference flow cell was left blank. The EY6A Fab was injected over the two flow cells at a range of five concentrations prepared by serial twofold dilution from 50 nM, at a flow rate of 30 μl min−1, using a single-cycle kinetics program with an association time of 75 s and dissociation time of 900 s. Running buffer was also injected using the same program for background subtraction. The second set-up had EY6A IgG immobilized on the sample flow cell and the reference flow cell was left blank. The RBD was injected over the two flow cells at a range of five concentrations prepared by serial twofold dilution from 100 nM, at a flow rate of 30 μl min−1, using a single-cycle kinetics program with an association time of 90 s and dissociation time of 60 s. Running buffer was also injected using the same program for background subtraction. All data were fitted to a 1:1 binding model using Biacore T200 Evaluation Software 3.1.

In the competition assay, where either CR3022 IgG or ACE2-hIgG1Fc was used as the ligand, the following samples were injected: (1) a mixture of 1 µM EY6A Fab and 0.1 µM RBD, (2) a mixture of 1 µM (anti-Caspr2) E08R Fab and 0.1 µM RBD, (3) 0.1 µM RBD, (4) 1 µM EY6A Fab and (5) 1 µM E08R Fab. In the competition assay where EY6A IgG was used as the ligand, the following samples were injected: (1) a mixture of 1 µM CR3022 Fab and 0.1 µM RBD, (2) a mixture of 1 µM ACE2 and 0.1 µM RBD, (3) a mixture of 1 µM de-glycosylated ACE2 and 0.1 µM RBD, (4) a mixture of 1 µM E08R Fab and 0.1 µM RBD, (5) 0.1 µM RBD, (6) 1 µM CR3022 Fab, (7) 1 µM ACE2 and (8) 1 µM E08R Fab. All injections were performed with an association time of 60 s and a dissociation time of 600 s. All curves were plotted using GraphPad Prism 8.

Plaque reduction neutralization test (PHE, Porton Down)

SARS-CoV-2 (Australia/VIC01/2020)34 at passage 3 was diluted to a concentration of 933 p.f.u. ml−1 (70 p.f.u./75 μl) and mixed 50:50 in minimal essential medium (MEM) (Life Technologies) containing 1% FBS (Life Technologies) and 25 mM HEPES buffer (Sigma) with doubling antibody dilutions in a 96-well V-bottomed plate, repeated in triplicate. The plate was incubated at 37 °C in a humidified box for 1 h to allow neutralization to take place before the virus–antibody mixture was transferred into the wells of a twice Dulbecco’s PBS-washed 24-well plate containing confluent monolayers of Vero E6 cells (ECACC 85020206; PHE) that had been cultured in MEM containing 10% (vol/vol) FBS. Virus was allowed to adsorb onto cells at 37 °C for a further hour in a humidified box, and overlaid with MEM containing 1.5% carboxymethyl cellulose (Sigma), 4% (vol/vol) FBS and 25 mM HEPES buffer. After five days of incubation at 37 °C in a humidified box, the plates were fixed overnight with 20% formalin/PBS (vol/vol), washed with tap water and then stained with 0.2% crystal violet solution (Sigma) and plaques were counted. Median neutralizing titers (ND50) were determined using the Spearman–Karber formula35 relative to virus-only control wells.

Plaque reduction neutralization test (Oxford)

Plaque reduction neutralization tests were performed using passage 4 of SARS-CoV-2 Victoria/01/202034. Virus suspension at appropriate concentrations in DMEM containing 1% FBS (D1; 100 μl) was mixed with antibody (100 μl) diluted in D1 at a final concentration of 50 μg ml−1, 25 μg ml−1, 12.5 μg ml−1 or 6.125 μg ml−1, in triplicate, in wells of a 24-well tissue culture plate, and incubated at room temperature for 30 min. Thereafter, 0.5 ml of a single-cell suspension of Vero E6 cells in D1 at 5 × 105 ml−1 was added and incubated for 2 h at 37 °C before being overlain with 0.5 ml of D1 supplemented with carboxymethyl cellulose (1.5%). Cultures were incubated for a further four days at 37 °C before plaques were revealed by staining the cell monolayers with amido black in acetic acid/methanol.

Crystallization, data collection and X-ray structure determination

Purified and de-glycosylated RBD and EY6A Fab were combined in an approximate molar ratio of 1:1 at a concentration of 6.5 mg ml−1. Nb was also combined with EY6A-6His Fab and RBD in a 1:1:1 molar ratio with a final concentration of 5.7 mg ml−1. These two complexes were separately incubated at room temperature for 1 h. Initial screening of crystals was performed in Crystalquick 96-well X plates (Greiner Bio-One) with a Cartesian Robot using the nanoliter sitting-drop vapor-diffusion method as previously described36,37. Crystals for the binary complex were initially obtained from a Hampton Research Index screen, condition B7 containing 0.04 M NaH2PO4 and 0.96 M K2HPO4 and further optimized to produce better crystals in 0.02 M NaH2PO4 and 0.98 M K2HPO4. Good crystals for the ternary complex were also obtained from the Index screen condition G1 containing 25% (wt/vol) PEG 3350, 0.2 M NaCl and 0.1 M Tris pH 8.5.

Crystals were soaked in a solution containing 25% glycerol and a 75% reservoir solution for a few seconds and then mounted in loops and frozen in liquid nitrogen before data collection. Diffraction data were collected at 100 K at Beamline I03 (wavelength 0.97625) at the Diamond Light Source. Diffraction images of 0.1° rotation were recorded on an Eiger2 XE 16M detector with an exposure time of 0.01 s per frame, beam size of 80 × 20 μm and 100% beam transmission. Data were indexed, integrated and scaled with the automated data-processing program Xia2-dials38,39. The binary complex structure (Table 1) was determined by molecular replacement with PHASER40 using search models of antibody CR3022 Fab and the RBD of the RBD–CR3022 Fab complex (PDB 6YLA14). There are three RBD–EY6A complexes in the crystal asymmetric unit, resulting in a crystal solvent content of ~75%. For the ternary complex, data were collected on Beamline I03 with an exposure time of 0.008 s per 0.1° frame, beam size of 80 × 20 μm and 100% beam transmission. There is one RBD–EY6A–Nb complex in the asymmetric unit and a solvent content of ~61%.

X-ray crystallographic refinement and electron density map generation

One cycle of REFMAC541 was used to refine atomic coordinates after manual correction in COOT42 to the protein sequence from the search model. For both the binary and ternary complexes, the final refinement used PHENIX43. There is well-ordered density for a single glycan at glycosylation site N343 in the RBD. Data collection and structure refinement statistics are provided in Table 1.

EY6A Fab–spike complex preparation and cryo-electron microscopy data collection

Following size-exclusion chromatography purification, spike protein was buffer-exchanged into 2 mM Tris pH 8.0, 200 mM NaCl, 0.02% NaN3 buffer using a desalting column (Zeba, Thermo Fisher). A final concentration of 0.18 mg ml−1 was incubated with EY6A Fab (in the same buffer) in a 6:1 molar ratio (Fab to trimeric spike) at room temperature for 5 h. Control grids of spike alone after incubation at room temperature for 5 h were also prepared.

Each grid was prepared using a 3-μl sample applied to a freshly glow-discharged (on high for 20 s; Plasma Cleaner PDC-002-CE, Harrick Plasma) holey carbon-coated 200-mesh copper grid (C-Flat, CF-2/1, Protochips) and excess liquid was removed by blotting for 5–5.5 s with a blotting force of −1 using Vitrobot filter paper (grade 595, Ted Pella) at 4.5 °C and 100% relative humidity, then it was immediately plunge-frozen in ethane slush using a Vitrobot Mark IV (Thermo Fisher).

Grids were screened on a Titan Krios microscope using the SerialEM program, operating at 300 kV (Thermo Fisher). Videos were collected on a K3 detector on the Titan Krios microscope operating at 300 kV in super-resolution mode, with a calibrated super-resolution pixel size of 0.415 Å per pixel at both 0° and 30° tilt. To compensate for the poorer contrast with tilted data, it was necessary to use a higher dose rate for the latter dataset.

Cryo-electron microscopy data processing

Alignment and motion correction were performed using Relion3.1’s implementation of motion correction44, with a five-by-five patch-based alignment. All frames were binned by two, resulting in a final calibrated pixel size of 0.83 Å per pixel. The contrast transfer function (CTF) of full-dose and non-weighted micrographs was estimated within a CryoSPARC wrapper for Gctf-v1.0645. Images were then manually inspected and those with poor CTF fits were discarded. Particles were then picked by unbiased blob-picking in CryoSPARC v.2.14.146 and subjected to rounds of 2D classification.

For the spike–EY6A dataset (structure A), 2,096,246 spike-like particles were used to make a template to pick particles from the untilted dataset, which were then filtered by 2D classification to 110,096 particles and then further refined by 3D classification with an ab initio model set. For the 30° dataset, 124,194 particles were used as a template and filtered by 2D classification to a set of 84,230 particles and then, as before, further refined by unbiased 3D classification. The two particle sets were then refined together, with a final set of 144,680 particles.

For B and C (triangular ring and ‘dimeric’ form), particles from both the zero and 30° datasets were combined in a similar manner to the spike–EY6A dataset using the ‘Exposure Group Utilities’ module in CryoSPARC. Both particle sets (B, 41,372 particles and C, 119,343 particles) were then reclassified and the best class refined with non-uniform refinement. For B, C3 symmetry was imposed at this final refinement stage, resulting in an appreciable improvement in resolution, as indicated by inspection and the gold-standard FSC = 0.143 (4.7 versus 5.9 Å, see Table 2).

Cryo-electron microscopy model building and refinement

The electron microscopy density of spike–EY6A was fitted with the structure of a closed form of spike (PDB 6VXX6), apart from the RBDs and EY6A Fab, which were fitted with RBD–EY6A of the ternary crystal structure using COOT42. Because of the lower resolution, RBD and EY6A are only fitted to the ‘dimeric’ and ‘trimeric’ electron microscopy density. The spike–EY6A structure was refined with PHENIX43 real-space refinement, first as a rigid body and then by positional and B-factor refinements. Only rigid body refinement was applied to the ‘dimeric’ and ‘trimeric’ complexes. The statistics of EM data collection and structure refinement are shown in Table 2.

Illustrations and figures

Structural comparisons used SHP47, residues forming the RBD/Fab interface were identified with PISA48, figures were prepared with PyMOL (The PyMOL Molecular Graphics System, Version 1.2r3pre, Schrödinger). Sequence alignments were generated using Clustal Omega49 and colored with ESPript50.

Reporting Summary

Further information on research design is available in the Nature Research Reporting Summary linked to this article.



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