PBMC and serum sample preparation
Blood samples were collected 3-4 days after donors were discharged from the hospital and separated into plasma and peripheral blood mononuclear cells (PBMCs) by Ficoll-Hypaque gradient centrifugation. Plasmas and PBMCs were maintained in freezing media and stored at -80oC. The plasma was heat-inactivated at 56oC for 1h before use.
Titer measurements by ELISA
The ELISA protocol was adapted from previously established protocols. 384 well plates were coated overnight at 4°C with PBS containing 1 μg/mL of the respective protein (S1 RBD-mFc tag, Full length S-his and S1-mFc from Sinobiological, S1-his from Kactus). The next day the plate was washed 4 times with washing buffer (PBS and 0.05% Tween) and then incubated 1 hour at 37°C in blocking buffer (PBS with 2% BSA). After two washes the plate was incubated for 1 hour at 37 °C with the serum or the positive control ACE2 protein (The human serum samples were diluted to 1:100 in PBS + 2% BSA followed by 5-fold serial dilutions. The plates were then washed 4 times and incubated for 1 hour in detection reagent (Mouse anti-Human IgG Fc HRP labeled (Thermo Fisher 05-4220) at 0.2 μg/mL in PBS with 0.05% Tween and 1% BSA) for 1 hour at room temperature. Following this the plate was washed again 4 times and developed in TMB substrate for 5 min before stopping the reaction with the stop solution.
Flow cytometry for B cell immune profiling
PBMC was thawed at 37°C and then centrifuged at 450 g for 8 min. The supernatant was discarded, and the cells resuspended in 200 μL of DMEM. Following the addition of 1μL of Dnase I cells were incubated for 3 min and spun down again. The pellet was resuspended in 20μL of FcR Blocking Reagent, incubated for 10 mins and centrifuged. The cells were suspended in 200μL PBS. 3μL of anti-CD19 (FITC labeled, eBioscience 11-0199-42), anti-CD27 (APC labeled, eBiosciences 17-0279-42), anti-CD38 (PE labeled mouse IgG1 isotype control, eBioscience 12-0388-42) or its isotype (PE Mouse isotype control, BioLegend 400114 and FITC labeled mouse IgG1 isotype control, ebioscience 11-4714-41 and APC labeled mouse IgG1 isotype control, BD 550854) was then added, and incubated for 30 min at room temperature. Following centrifugation, cells were resuspended in a 100μL of 4% PFA. After 10 min the cells were washed twice by centrifugation and finally resuspended in PBS and ready for flow cytometry analysis using a Cytoflex, Beckman Coulter. The median fluorescence intensity (MFI) was calculated with FlowJo.
Patient sample information for FACS
|
labeling
|
Cell Number
|
Viability (%)
|
comments
|
PBMC
|
Patient 4
|
2.54×105/mL, 200μL
|
84
|
Patient
|
PBMC
|
Patient 20
|
7.1×105/mL, 200μL
|
90
|
Patient
|
PBMC
|
Healthy donor
|
7.0×105/mL, 200μL
|
92
|
Healthy Volunteer
|
Isolation of S protein specific B cells
Avitag and His tag SARS-CoV-2 S protein was expressed in HEK293F cells and biotinylated using Biotin-Protein Ligase (GeneCopoeia, BI001). The B cells were stained with biotinylated S protein and incubated at 4℃ for 1 hour. After incubation, the cells were washed three times with PBS. Subsequently, the cells were labeled with Streptavidin microbeads (Miltenyi Biotec) at 4℃ for 1 hour. After the incubation, the cell suspension was loaded onto a MACS column which is placed on a magnetic field of a MACS separator. The column were washed three times so the magnetically labeled B cells was retained in the column and unlabeled cells passed through. After removing from the MACS separator, the magnetically labeled B cells were eluted. The isolated B cells were counted by using 0.4% (w/v) Trypan blue stain.
Single-cell BCR sequencing
Enriched S protein specific B cells were individually co-compartmentalized in droplets with single barcoded hydrogel beads and lysis and reverse transcription reagents using a microfluidic device as described15. Droplets of ~1 nL volume were formed at 250 s-1. The droplets were collected in a 1.5mL tube containing HFE-7500 and 0.1% surfactant, UV photo-cleaved for 90 seconds (OmniCure ac475-365) and incubated at 50°C for cell lysis and cDNA synthesis. Reverse transcription of VH and VL mRNAs from single B cells took place in droplet using barcoded primers carrying the T7-SBS12 sequences followed by barcode and gene-specific primer sequences complementary to heavy chain J genes and light chain constant region sequences.
The emulsion containing the barcoded cDNA was broken by adding one volume of 1H,1H,2H,2H-Perfluoro-1-octanol after the droplet RT reaction finished. The pooled, barcoded cDNAs were purified with Agencourt RNA CleanUp beads (Beckman, A63987) at a 1:1 ratio (vol/vol) twice and eluted in 60 μL DNase- and RNase-free H2O. The sequencing library was generated by two-step nested PCR using GoTaq Polymerase (Promega). In the first PCR, forward primers were priming on the T7 and with reverse primers priming on the VH, Vλ and Vκ leader and framework 1 sequenced of the V genes. In the second PCR, the forward primer appends the Illumina P7 and Illumina index sequences by priming on the SBS12 sequence and the reverse primer appends the Illumina P5 and SBS3 sequence. The approximately 550 bp final PCR products were extracted by agarose gel electrophoresis (Qiagen, 28606). The constructed NGS libraries were sent out for sequencing using Illumina MiSeq PE300 which allows sequencing of the entire VH and VL domain as well as the barcode sequence (GeneScript sequencing service supplier) with data varying from 6-12 million reads per samples. The resulting FASTQ data was analyzed by a bioinformatics pipeline enable trimming, merging, barcode extraction and clustering as described16. Briefly, paired-end reads were first trimmed at the 3’ end to remove low quality score bases then merged using the program FLASH requiring at least 10bp overlap. The barcodes were extracted form merged reads followed by clustering requiring the DNA sharing at least 93% identify. The consensus sequence was created from clusters by aligning up to 200 sequencing using ClustalO, and each antibody sequence was characterized for immunoglobulin content using VDJFasta. We also applied a minimum number of reads 10 for VH and VL. VH-VL pairing was carried out by identifying the most abundant VH and VL consensus sequence (by number of reads that contributed to that in each barcode cluster).
Production of recombinant antibody
The DNA of variable regions of the heavy and light chains were synthesized by Genewiz and cloned into expression plasmids containing the human IgG1 heavy chain and kappa light chain constant regions. The antibodies were expressed in 293F cell for 5 days after the co-transfection of both heavy and light chain expression plasmids. Antibodies were purified from cell culture supernatants using Protein-A affinity chromatography.
Antibody binding and competition with receptor ACE2
The binding affinity of antibodies to S protein was analyzed by ELISA. 384 well plate (Corning#3700), was coated overnight at 4°C with PBS containing 30μL 20nM of the SARS-CoV-2 Spike S1+S2 ECD, his Tag protein. The next day the plate was washed 5 times with washing buffer (PBS and 0.05% Tween) and then incubated 1 hour at room temperature in blocking buffer (PBS with 2% BSA). After 5 washes the plate was incubated with serial dilution of purified antibodies for 1 hour at room temperature. The plates were then washed 5 times and incubated for 1 hour in detection reagent (Mouse anti-Human IgG Fc HRP labeled (Thermo Fisher 05-4220) at 0.2 μg/mL in PBS with 0.05% Tween and 1% BSA) for 1 hour at room temperature. Following this the plate was washed again 5 times and developed in TMB substrate for 5 min before stopping the reaction with the stop solution. The OD values were determined using Thermo MultiSkan or MD SpectraMax i3X at 450nm wavelength.
The blocking with receptor ACE2 was performed using cell surface expressed ACE2. 10nM SARS-CoV-2 spike S1, mFc tag protein was incubated with serial dilution of purified antibodies at room temperature for 1 hour and then added to Vero E6 cells (approximately 105 per well) in duplicate. Then detection reagent rabbit anti mouse IgG Fc-AF647 was used. Half-maximal inhibitory concentration (IC50) of the evaluated antibodies was determined with Beckman Cytoflex and FlowJo software analysis.
Antibody neutralization activity against pseudovirus
Murine leukemia virus-based SARS-CoV-2 S pseudotyped virus were prepared by GenScript as previous described. Neutralization assay were performed by incubating pseudovirus with serial dilution of purified antibodies at room temperature for 1hour. ACE2 overexpression Hela cells (approximately 8×104 per well) were cultured in DMEM containing 10% FBS, 1μg/mL puromycin were added in triplicate into virus-antibody mixture. Following infection at 37oC for 48 hours, luciferase activity was determined using Promega Bio-Glo luciferase assay system and half-maximal inhibitory concentration (IC50) was calculated with GraphPad Prism.
Live virus assay
Vero E6 cells were incubated with SARS-CoV-2 at 100×TCID50 in absence or presence of diluted antibodies for 1 hour, fixed with 4% paraformaldehyde diluted in PBS (pH=7.2) for 15 minutes at room temperature followed by penetrating solution containing 0.25% triton-X 100 for 10-15 minutes. After three washes, cells were blocked at 37 ℃ for 1 hour using PBS containing 5% BSA, then incubated with in-house prepared anti-SARS-CoV-2 NP rabbit serum as primary antibody and FITC-conjugated goat anti-mouse IgG antibody as the secondary antibody. Cell nuclei were strained using Hoechst 33258 at room temperature for 10 minutes. Images were taken under an inverted fluorescence microscope (Nikon).
Surface plasmon resonance analysis.
SPR experiments were performed using Biacore T200 system (GE Healthcare). In brief, experiments were performed at 25°C in HBS-EP+ buffer. Antibody was immobilized onto a protein A sensor chip. Serially diluted SARS-CoV-2 RBD (WT), RBD (N354D/D364Y), RBD (R408I), RBD (W436R), RBD (V367F) or SARS-CoV-2 spike S1 domain (D614G) were injected through flow cells for 60s of association followed by a 150s dissociation phase at a flow rate of 30 μL min−1. Prior to next cycle, the sensor surface was regenerated with Glycine-HCl (pH 1.5) for 30s at a flow rate of 30 μL min−1.KD values were calculated using the 1:1 binding kinetics model.