Potent Neutralizing Antibodies against SARS-CoV-2 Identified by High-Throughput Single-Cell Sequencing of Convalescent Patients’ B Cells

Materials Availability

There are restrictions on the availability of antibodies due to limited stock and continued consumption. We are glad to share remaining antibodies with reasonable compensation for processing and shipping upon completion of a Material/Data Transfer Agreement for non- commercial usage.

PBMCs from blood

All human blood samples were collected for laboratory assessments according to the doctor’s instruction from Beijing Youan Hospital, China. Peripheral blood mononuclear cells (PBMCs) were isolated immediately from fresh blood. Primary human B lymphocytes were obtained by purification of human blood CD19⁺ B cells or memory CD19⁺ CD27⁺ B cells, by immunomagnetic bead isolation (Catalog #19054 and #17864, STEMCELL), after Ficoll-Hypaque sedimentation according to standard density-gradient centrifugation methods (GE Healthcare). For each sample, the cell viability exceeded 85%. Several patients’ PBMCs were stored frozen and thawed before use. Cryopreserved PBMCs were thawed in RPMI 1640 medium supplemented with 20% fetal bovine serum.

Recombinant RBD and full-length Spike protein from SARS-CoV-2

Spike/RBD recombinant proteins were purchased from Sino Biological Inc. with > 95% purity. Full-length Spike/RBD protein was expressed in the Baculovirus-Insect cell system using the DNA sequence encoding the SARS-CoV-2 spike protein (S1+S2 ECD) or RBD protein. The protein is expressed with a polyhistidine tag at the C terminus and purified in sterile 20 mM Tris, 300 mM NaCl, 10% glycerol, pH 8.0.

SARS-CoV-2 pseudovirus

The SARS-CoV-2 pseudovirus is a gift from the Division of HIV/AIDS and Sex-transmitted Virus Vaccines, National Institutes for Food and Drug Control (NIFDC). The pseudovirus was constructed as previously described using the spike genes from strain Wuhan-Hu-1 (GenBank: {"type":"entrez-nucleotide","attrs":{"text":"MN908947","term_id":"1798172431","term_text":"MN908947"}}MN908947)(Nie et al., 2020). Briefly, the SARS-CoV-2 spike gene was codon-optimized and cloned into a eukaryotic expression plasmid. 293T cells were transfected by the plasmid and later infected with a VSV pseudotyped virus (G^∗ΔG-VSV), which substituted the VSV-G gene with luciferase expression cassettes. The culture supernatants were harvested and filtered 24 h post-infection. The SARS-CoV-2 pseudovirus could not be neutralized by VSV-G antibodies, and no G^∗ΔG-VSV was mixed with the SARS-CoV-2 pseudovirus stock (Nie et al., 2020).

Authentic SARS-CoV-2 virus

The authentic SARS-CoV-2 virus (2019-nCoV BetaCoV/Wuhan/AMMS01/2020) was isolated from a COVID-19 patient from Beijing. The RNA sequence of the isolated virus was verified by Sanger sequencing. All experiments associated with the authentic virus were conducted in Biosafety Level 3 laboratory with standard operating procedures.

CD27⁺ memory B cell enrichment

CD27⁺ memory B cells were isolated from purified B cells by immunomagnetic positive selection according to the manufacturer’s protocol (EasySep Human Memory B Cell Isolation Kit, STEMCELL). Briefly, CD27⁺ B cells are labeled with magnetic beads combined with CD27 antibodies and separated using an EasySep magnet. Purified CD27⁺ B cells were eluted and washed in PBS containing 2% (v/v) fetal bovine serum (FBS) and 1 mM EDTA. CD27⁺ B cells were counted by using 0.4% (w/v) trypan blue stain and Countess Automated Cell Counter according to the manufacturer’s protocol.

Antigen-binding B cells enrichment

Biotinylated Spike/RBD recombinant proteins were purchased from Sino Biological Inc. Fresh antigen/streptavidin M-280 Dynabeads (Thermofisher) complex was made every time before B cell enrichment. Briefly, 100 μL of M-280 beads containing 6.5 × 10⁷ beads were vortexed for 30 s and brought to room temperature before use. Beads were washed with 1 mL 1x PBS twice on a magnetic rack, and eluted in 100 μL 1x PBS. Beads (100 μL) were mixed with 20 μg of biotinylated spike/RBD protein and incubated at room temperature for 30 mins. After incubation, the complex was washed three times using 500 μL 1x PBS on a magnetic rack. The washed complex was eluted in 100 μL 1x PBS and stored on ice until use.

Before antigen-enrichment, the complex was equilibrated at room temperature. The amount of beads complex used was calculated based on a 1:1 ratio with the number of purified B cells. The spike/RBD magnetic beads complex was added directly into the B cell mixture, mixed and incubated at 4°C on a thermomixer for 30 mins. After the incubation, the mixture was put on a magnetic rack, and the supernatant was removed. The beads were washed times by mixing off the magnet four times in total. The final antigen-enrichment B cells were eluted in 1x PBS containing 2% (v/v) fetal bovine serum (FBS) and 1 mM EDTA. Enriched B cells were counted by using 0.4% (w/v) trypan blue stain and Countess Automated Cell Counter according to the manufacturer’s protocol.

Single-cell 5′ mRNA and VDJ sequencing

After CD27⁺ memory B cell enrichment or antigen-specific enrichment, all B cells were loaded on a 10X Chromium A Chip. If the total cell number exceeded 20,000, two individual runs would be performed. Since SARS-CoV-2 may still appear in convalescent patients’ blood (An et al., 2020, Zhang et al., 2020), we performed all the experimental processes before library preparation in a P3-level laboratory. Single-cell lysis and RNA first-strand synthesis were performed using 10X Chromium Single Cell 5′ Library & Gel Bead Kit according to the manufacturer’s protocol. After RNA first-strand synthesis, all viruses should be denatured since all samples were heated to 85°C for 5 mins. RNA samples were wiped with 80% EtOH for three times and sealed in a plastic box for sample transportation.

The following RNA and VDJ library preparation is performed according to the manufacturer’s protocol (Chromium Single Cell V(D)J Reagent Kits, 10X Genomics) in a P2 laboratory. All libraries were quantified by using Qubit 3.0 (ThermoFisher), Fragment Analyzer, and qPCR. Sequencing was performed on a Hiseq 2500 platform running Rapid SBS Kit V2 2x100bp kit (Illumina), with a 26x91 pair-end reading mode. Sequencing results were obtained within two days. Average sequencing depth aimed for the mRNA library is 10,000 read pairs per cell and 5,000 read pairs per cell for the VDJ libraries (Table S3).

In Vitro expression of mAbs

The selected paired heavy- and light-chain’s cDNA was codon-optimized and cloned into expression vectors containing the IgG1 constant regions of human. IgG mAbs were expressed by transfecting HEK293 cells with equal amounts of heavy- and light-chain plasmids. The mAbs were resuspended into PBS and analyzed by SDS-PAGE.

The numbering of mAbs is based on the order of DNA synthesis submission. BD 1-175 were mAbs produced from the experiments without using antigen enrichment (Table S2). BD 176 – 425 were mAbs produced from the Batch 1-6 with antigen enrichment, and were all selected from clonotypes that have an enrichment frequency larger than 1 (Table S2). BD 492 – 515 were mAbs selected based on CDR3_H structural similarity to SARS-CoV neutralizing antibodies (Table S2). BD 426 – 491, which were not shown, were attempts trying to substitute rare DNA mutations in the FR region of the verified neutralizing mAbs, aiming for higher neutralization potency. None of the mAbs showed improvements in terms of neutralization potency, and the results were not shown. Also, the antibodies in each group may have discontinuous numbering. The excluded mAbs were the antibodies selected from clonotypes that consist of only IgM- or IgG3-presenting B cells. None of mAbs showed neutralizing ability and thus were excluded from the results.

ELISA quantification

ELISA plates were coated with SARS-CoV-2 RBD or S protein at 0.01 μg/mL and 1 μg/mL in PBS at 4°C overnight. After standard washing and blocking, 100 μL 1 μg/mL antibodies was added to each well. After a 2 h incubation at room temperature, plates were washed and incubated with 0.08 μg/mL goat anti-human IgG (H+L)/HRP (JACKSON) for 1 h incubation at room temperature. Chromogen solution was used as the substrate, and absorbance at 450nm was measured by a microplate reader. A mAb is defined as ELISA-positive when the OD₄₅₀ is saturated using 1 μg/mL RBD/S protein.

mAbs binding affinity measurement

The dissociation coefficient is measured by using surface plasmon resonance (SPR). Biotinylated RBD/S (Sino Biological Inc.) was immobilized to a SA sensor chip (GE Healthcare) at a level of ∼100 response units (RUs) using a Biacore T200 (GE Healthcare). Running buffer is composed of PBS pH 7.4 and 0.005% (v/v) P20. Serial dilutions of purified antibody were injected, ranging in concentration from 50 to 0.78 nM. The resulting data were fit to a 1:1 binding model using Biacore Evaluation Software.

ACE2 competition assay

Biotinylated RBD (0.3 μg/mL, Sino Biological Inc.) was immobilized to an ELISA plate. After standard washing, 0.05 μg/mL His-tagged hACE2 protein was added to the wells, and then the diluted mAbs were added and mixed immediately. After 2 h of incubation at room temperature, plates were washed, and 0.08 μg/mL anti-His/HRP was added. After 1 h of incubation at room temperature, the chromogen solution was used as the substrate, and absorbance at 450 nm was measured by a microplate reader. The ACE2/RBD-binding inhibition rate was calculated by comparing to the mAbs negative control well.

Protein expression and purification for cryo-EM

The SARS-CoV-2 S gene was synthesized by Sino Biological Inc. The DNA fragment encoding the ectodomain of S (residues 1-1208) was cloned into a pcDNA vector. Two stabilizing proline substitutions at residues 986-987 and a “GSAS” substitution at the furin cleavage site (residues 682–685) were introduced as previously described to improve protein expression (Wrapp et al., 2020). A C-terminal T4 fibritin trimerization motif and an 8 × His tag were also engineered to stabilize trimer formation and facilitate protein purification.

HEK293F cells were cultured in SMM 293T-I medium (Sino Biological Inc.) at 37°C, with 5% CO2 and 55% humidity. The plasmid was transfected into the cells using polyethylenimine (Polysciences) when the cell density reached 1 × 10⁶ cells per mL. Four days after transfection, the conditioned media were collected by centrifugation at 500 × g, concentrated using a Hydrosart Ultrafilter (Sartorius), and exchanged into the binding buffer (25 mM Tris-HCl, pH 8.0, 200 mM NaCl). The S protein was subsequently isolated using the Ni-NTA resin, and purified using a Superose 6 10/300 gel filtration column (GE Life Sciences). The final buffer used for the gel filtration step is 20 mM HEPES, pH 7.2, 100 mM NaCl.

To obtain BD23-Fab, BD-23 (2 mg/mL) was digested with papain (0.1 mg/mL) at 37°C for 2 h in 50 mM phosphate buffer saline, pH 7.0, 2 mM EDTA, and 5.5 mM cysteine. The reaction was terminated using 30 mM iodoacetamide at room temperature for 20 minutes. The Fc region was removed by protein A chromatography, and BD23-Fab was further purified using a Superdex 200 Increase 10/300 gel filtration column (GE Life Sciences) and eluted using the final buffer. Ab23-Fab was incubated with the purified S protein in a 1.5:1 molar ratio overnight on ice. After that, the complex was purified using the Superose 6 10/300 column and eluted with the final buffer.

Cryo-EM data collection and processing

Holy-carbon gold grids (Quantifoil, R1.2/1.3) were glow-discharged with a Solarus 950 plasma cleaner (Gatan) for 30 s. Four-microliter aliquots of the S/BD23-Fab complex (0.6 mg/mL) were applied to the grids, blotted with filter paper at 4°C and 100% humidity, and plunged into the liquid ethane using a Vitrobot Mark IV (FEI). The grids were first screened on a 200 kV Talos Arctica microscope equipped with Ceta camera (FEI). Data were collected using a Titan Krios electron microscope (FEI) operated at 300 kV. Movies were recorded with a K2 Summit direct electron detector (Gatan) in the super-resolution mode using the SerialEM software (Mastronarde, 2005). Statistics for data collection are summarized in (Table S4).

The workflow of data processing was illustrated in Figure S7. Raw movie frames were aligned and averaged into motion-corrected summed images using MotionCor2 (Zheng et al., 2017). The Gctf program (v1.06) was used to estimate the contrast transfer function (CTF) parameters of the motion-corrected images (Zhang, 2016). Relion (v3.07) was used for all the following data processing (Zivanov et al., 2018). The S trimer (PDB ID: 6VSB) was used as a reference for the 3D classifications. The local resolution map was analyzed using ResMap (Kucukelbir et al., 2014) and displayed using UCSF Chimera (Pettersen et al., 2004).

A structural model of BD23-Fab was generated using the Phyre2 server (Kelley et al., 2015). This BD23-Fab model and the S trimer structure (PDB ID: 6VSB) were docked into the cryo-EM density using UCSF Chimera. One round of rigid-body refinement was performed using the real-space refinement in Phenix (Liebschner et al., 2019). Further structure refinement of BD23-Fab was not performed due to limited resolution in this region. Nevertheless, it is evident that BD23-Fab engages an epitope on RBD that predominantly overlaps with the ACE2-binding site.

Pseudovirus neutralization assay

The pseudovirus neutralization assays were performed using Huh-7 cell lines. Huh-7 are human hepatocellular carcinoma cells that express both ACE2 and TMPRSS2 (Matsuyama et al., 2018). Various concentrations of mAbs (3-fold serial dilution using DMEM, 50 μL aliquots) were mixed with the same volume of SARS-CoV-2 pseudovirus with a TCID₅₀ of 1.3 × 10⁴ in a 96 well-plate. The mixture was incubated for 1 h at 37°C, supplied with 5% CO₂. Negative control wells were supplied with 100 μL DMEM (1% (v/v) antibiotics, 25 nM HEPES, 10% (v/v) FBS). Positive control wells were supplied with 100 μL DMEM. Pre-mixed Huh-7 cells (100 μL, 2 × 10⁵ in DMEM) were added to all wells, and the 96-well plates were incubated for 24 h at 37°C supplied with 5% CO₂. After the incubation, 150 μL of supernatants were removed, and 100 μL D-luciferin reagent (Invitrogen) was added to each well and incubated for 2 mins. After the incubation, every well is mixed 10 times by pipetting, and 150 μL of the mixture was used to measure luciferase activity using a microplate spectrophotometer (Perkinelmer EnSight). The inhibition rate is calculated by comparing the OD value to the negative and positive control wells. IC₅₀ and IC₈₀ were determined by a four-parameter logistic regression using GraphPad Prism 8.0 (GraphPad Software Inc.). The raw data of a representative neutralization assay on BD-368-2 is described in Table S5.

Authentic SARS-CoV-2 PRNT

Plaque reduction neutralization test (PRNT) was performed using a clinical isolate of SARS-CoV-2 (BetaCoV/Wuhan/AMMS01/2020) (Roehrig et al., 2008). Briefly, 5-fold serial dilutions of mAbs were added to the same volume of approximately 100 PFU of SARS-CoV-2 and incubated for 1 h at 37°C. The mixture was added to a monolayer of Vero cells in a 24-well plate and incubated for 1 h at 37°C. The mixture was removed, 0.5 mL of 1.0% (w/v) LMP agarose (Promega) in 2 × DMEM supplied with 4% (v/v) FBS was added onto the infected cells. After further incubation at 37°C supplied with 5% CO₂ for 2 days, the wells were stained with 1% (w/v) crystal violet dissolved in 4% (v/v) formaldehyde to visualize the plaques. IC₅₀ values were determined using four-parameter logistic regression analysis by using GraphPad Prism 8.0 (GraphPad Software Inc.). All experiments were performed in a Biosafety Level 3 facility.

Authentic SARS-CoV-2 neutralization CPE assay

A neutralization assay of authentic SARS-CoV-2 was performed using a cytopathic effect (CPE) assay. Briefly, various concentrations (3-fold serial dilution using DMEM) of mAbs were mixed with the same volume of 100 PFU of authentic SARS-CoV-2 and incubated at 37°C for 1 h. The mixture was added to a monolayer of Vero-E6 cells (5x10³ cell per well) in a 96-well plate and incubated for 1 h at 37°C. The supernatant was removed, and 200 μL of DMEM supplied with 2% (v/v) FBS was added onto the infected cells. After incubation at 37°C supplied with 5% CO2 for 5 days, all wells examine for the CPE effect. All experiments were performed in a Biosafety Level 3 facility.

hACE2 in vivo SARS-CoV-2 neutralization assay

A total of 9 hACE2 transgenic mice were used in this study. The mice were equally split into three groups for prophylactic and therapeutic study, as well as the negative control. For the prophylactic group, BD-368-2 (20 mg/kg) was intraperitoneally injected one day before infection. For the therapeutic group, BD-368-2 (20 mg/kg) was intraperitoneally injected 2 h after infection. For the negative control group, HG1K (20 mg/kg, Sino Biology), which is an anti-influenza antibody, was intraperitoneally injected 2 h after infection. The hACE2 mice were inoculated intranasally with the SARS-CoV-2 stock virus with a dosage of 10⁵ TCID₅₀. Body weights were recorded daily for all infected mice for 5 continuous days. The mice were sacrificed at 5 dpi, and the lungs were collected for viral load analysis.

Viral load detection was performed by qRT-PCR on RNA extracted from lung homogenate supernatants, as described previously (Bao et al., 2020). Briefly, lung homogenates were prepared by homogenizing perfused whole lung using an electric homogenizer. The supernatant was collected, and total RNA was extracted. A quantitative real-time reverse transcription-PCR (qRT-PCR) reactions were performed using the previously described primers and protocol. qRT-PCR was performed on each mouse with two replicates. All experiments were performed in a Biosafety Level 3 facility.

Author Contributions

Y.C. and X.S.X. conceptualized the project and designed and coordinated the experiments. X.S.X. obtained the funding for the project. R.J. and Y.F. organized the collection of convalescent patients’ blood samples. B.S. and X.G. prepared B cells and performed 10X experiments. Y.W. and L.Q. arranged sample transportation. Y.C. and C.G. performed scRNA and scVDJ library preparation. X.Z. and Y.C. optimized the antigen pull-down experiment. W.S. and X.C. analyzed the sequencing data. Y.C. selected the antibody sequences for synthesis. Y.Z. performed the pseudovirus neutralization assays. R.S.H. performed SPR assays. Z.D. and C.Q. coordinated the experiments on the authentic virus, which was developed by C.Q. Y.D., X.L., and Y.T. performed the PRNT on the authentic virus in the P3 laboratory. C.Q. designed and J.L. coordinated the animal experiment. L.B., H.Z., W.D., and Y.X. performed the animal experiment in the P3 laboratory. Q.L. performed the CPE assay in the P3 laboratory. D.S. and J.X. led the cryo-EM structure experiment. Q.Z., G.W., X.D., and N.G. collected and analyzed the structure data. Y.C. and X.S.X. wrote the manuscript with the help of all authors. L.S. arranged the figures.