Identification and application of a pair of non-competing monoclonal antibodies that bind extensively to the nucleocapsid proteins of SARS-CoV-2 variants, including Omicron | Journal of Virology

Blood samples

Donor P301 was infected with WT SARS-CoV-2. She had a fever for 6 days, then was admitted to Shenzhen Third People’s Hospital on January 31, 2020. She was discharged from the hospital on February 14, 2020. Blood samples were taken on February 12, 2020 during the period of convalescence. Plasma samples were stored at -80°C and PBMCs were maintained in freezing medium and stored in liquid nitrogen at Shenzhen Third People’s Hospital Biobank.

Flow cytometry

PBMCs from the COVID-19 patient were collected and incubated with LIVE/DEAD™ Fixable Dead Cell Stain Reagent (Thermo Scientific) to exclude dead cells. Cells were then stained with His-tagged WT SARS-CoV-2 NP (Sino Biological) and fluorescently labeled antibodies including CD19-PE/Cy7, CD3-Pacific Blue, CD8-Pacific Blue, CD14-Pacific Blue , CD27-APC/Cy7, IgG-FITC (BD Biosciences), followed by incubation with His-specific antibodies labeled APC and PE (Abcam). Single NP-specific B cells were checked as CD19+CD3-CD8-CD14-CD27+IgG+NP+ and sorted into 96-well PCR plates containing lysis buffer (Invitrogen) using a FACSAriaII flow cytometer (BD Biosciences). Plates were then flash frozen on dry ice and stored at −80°C until reaction at room temperature.

For intracellular staining, 293 T cells transfected with NP expression vectors were fixed and permeabilized using the Fixing/Permeabilization Solution Kit (BD Biosciences), stained with NP-specific mAbs (P301-F7 or P301-H5) or polyclonal antibody (pAb) (Sino Biological), followed by staining with an APC-conjugated secondary antibody (Life Technologies). After washing, the cells were resuspended and subjected to acquisition with a FACSCalibur flow cytometer (BD Biosciences). Data were analyzed with FlowJo V10.6 software (BD Biosciences).

Single B cell PCR, cloning and expression of mAbs

To amplify the IgG variable genes, we first performed the RT reaction in the 96-well PCR plate. Then, the heavy chain and light chain genes were amplified, sequenced, synthesized and cloned separately into the backbone of antibody expression vectors containing human IgG1 constant regions (Sangon Biotech). mAbs were produced by transient transfection of 293 F cells (Life Technologies) with equal amounts of paired heavy and light chain plasmids, and purified by affinity chromatography using protein A bead columns (Senhui Microsphere Tech) according to the manufacturer’s protocol.

Enzyme immunoassay (ELISA)

Native recombinant core proteins (NPs) of SARS-CoV, SARS-CoV-2 or variants, MERS-CoV, HKU1, OC43, NL63, and 229E (Sino Biological) (2 μg/mL) or denatured by treatment with denaturing buffer containing 0.5% SDS and 40 mM DTT [18] (New England Biolabs) at 95°C for 10 min were coated in 96-well half-zone plates overnight at 4°C. The plates were then blocked with blocking buffer (PBS containing 4% skimmed milk) at 37°C for 1 h. Five or three times serially diluted NP-specific mAbs or rabbit pAbs against SARS-CoV-2 NP (Sino Biological) were added to the plates in duplicate and incubated for 1 h at 37°C. Then, a HRP-conjugated goat anti-Human IgG (ZSGB-BIO) or goat anti-Rabbit IgG (TransGen Biotech) secondary antibody was added to the plates and incubated at 37°C for 1 h. For competitive ELISA, after blocking, serially diluted P301-F7, P301-H5, and human IgG1 were mixed with HRP-conjugated P301-F7, then added to the plates and incubated at 37°C for 1 h. The enzymatic reaction was developed with the substrate TMB (BD Biosciences) and stopped by 2 MH2SO4. Optical density was measured at 450 nm (OD. 450 nm) with a Varioskan™ LUX multi-mode microplate reader (Thermo Scientific).

Surface Plasmon Resonance (SPR) Binding Analysis

Binding assays of mAbs to SARS-CoV-2 and SARS-CoV NPs were performed using the Biacore 8 K system (GE Healthcare). Specifically, a flow cell of CM5 sensor chips was covalently coated with NPs (Sino Biological) in 10 mM sodium acetate buffer (pH 5.0) for a final RU (response units) of about 250, while the other flow cell was left uncoated and blocked as a control. All assays were performed at a flow rate of 30 µL/min in HBS-EP buffer (10 mM HEPES pH 7.4, 150 mM NaCl, 3 mM EDTA and 0.05% Tween-20). Serially diluted antibodies were injected for 60 s respectively and the resulting data was fitted in a 1:1 binding model with Biacore evaluation software (GE Healthcare). Each measurement was made three times and the individual values ​​were used to produce the mean affinity constant and standard deviation.

Structure model prediction

The complete SARS-CoV-2 NP contains 419 amino acids, but a complete structure has not been resolved because it contains many flexible domains. To predict the model of the full-length SARS-CoV-2 NP, Coot was used to connect the existing crystal structures of the N-terminal domain (NTD, aa44-174, PDB code: 7CDZ) and C-terminal domain (CTD, aa255-364, PDB code: 7CE0), and the remaining parts have been labeled with loops. Then, the model served as a model to predict the complete structure of SARS-CoV-2 NP using AlphaFold2 (https://alphafold.ebi.ac.uk). The image was represented using the PyMOL visualization software (http://www.pymol.org).

Western blot analysis

For recombinant protein, 2 μg of His-tagged NPs of SARS-2-CoV, SARS-CoV and MERS-CoV (Sino Biological) were loaded on 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE ), transferred to polyvinylidene difluoride (PVDF) using the Mini-PROTEAN® Tetra system (Bio-Rad). After blocking with 5% skim milk for 1 h at room temperature, membranes were incubated with HRP-conjugated anti-His mAb (Sangon Biotech) or P301-F7, P301-H5 overnight at 4°C, followed by incubation with HRP-IgG goat anti-human conjugate (ZSGB-BIO) for 1 hour at room temperature. Proteins were visualized with a chemiluminescent substrate (Thermo) and a ChemiDoc™ MP imaging system (Bio-Rad).

Immunofluorescence

Vero E6 cells were infected with SARS-CoV-2 at an MOI of 0.01 when treated with different doses of Remdesivir. 48 h after infection, cells were fixed with 4% paraformaldehyde for 30 min at room temperature, permeabilized with Perm/Wash (BD Biosciences) containing 0.1% Triton X-100 for 10 min at room temperature . After washing, cells were stained with 2 μg/mL of NP-specific mAb (P301-F7) or pAb (Sino Biological) for 1 h at room temperature, followed by staining with Alexa Fluor® conjugated secondary antibody. 488 (Invitrogen) for 1 h at RT. After washing, the cells were incubated with Hoechst 33258 (Life Technologies). The microscopic images were obtained under an EVOS digital inverted microscope (Life technologies).

Focus reduction override test

The SARS-CoV-2 Concentration Reduction Neutralization (FRNT) test was performed in a biosafety level 3 certified laboratory. Antibodies were serially diluted three times, mixed with an equal volume of SARS-CoV-2 or variants in 96-well U-bottom plates, and incubated for 60 min at 37°C. The mixture (containing 200 focus-forming units of live virus) was then transferred to a 96-well plate seeded with Vero E6 cells and absorbed for 1 h at 37°C before removal. After washing, the overlay medium (MEM containing 1.6% carboxymethylcellulose, 2% fetal bovine serum) was added, then the cells were incubated at 37°C for 24 h. After removing the overlay medium, cells were fixed with 4% paraformaldehyde solution, permeabilized with Perm/Wash buffer (BD Biosciences) containing 0.1% Triton X-100, incubated with P301-F7 conjugated to HRP. The reactions were developed with KPL TrueBlue Peroxidase substrates (Seracare Life Sciences). The number of SARS-CoV-2 foci or variants was calculated using an EliSpot reader (Cellular Technology Ltd).

SARS-CoV-2 WT and variant strains

The original strain (WT) of SARS-CoV-2 and the variant strains Alpha, Delta, Omicron were separated in our biosafety level 3 laboratory, and the Beta strain was kindly donated by the Provincial Center for Control and Prevention of Guangdong Diseases, Guangdong Center for Human Pathogens Cultural Collection (GDPCC). The complete genome sequences of the WT and Alpha, Delta variant strains have been deposited in the Genome Warehouse of the National Genomics Data Center, Beijing Institute of Genomics, Chinese Academy of Sciences/China National Center for Bioinformation, whose accession numbers are indicated below and have been publicly available on https://ngdc.cncb.ac.cn/gwh. The clade of the isolated Omicron variant was determined using the Nextclade web application (https://clades.nextstrain.org/) and sequence information has not yet been downloaded.

WT strain:

Beta/Shenzhen/SZTH-003/2020, EPI_ISL_406594 at GISAID;

Alpha strain:

SZTH008, Accession No. GWHBFWX01000000;

Beta strain:

GDPCC-nCoV84, Accession No. GWHBDSE01000000;

Delta strain:

SZTH012, access number GWHBFWZ01000000.

Antibody amino acid sequences

Heavy chain P301-F7:

QLQLQESGPGLVKPSETLSLTCTVSGGSISSTSYYWGWIRQPPGKGLEWIGSIYYSGSTYYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARFSLYCSSTSCYENWFDPWGQGTLVTVSS

P301-F7 light chain:

QSVLTQPPSVSAAPGQKVTISCSGSSSNIGNNYVSWYQQLPGTAPKLLIYDNNKRPSGIPDRFSGSKSGTSATLGITGLQTGDEADYYCGTWDSSLSAGQVVFGGGTKLTVL

Heavy chain P301-H5:

EVQLVESGGGLVQPGGSLKLSCAASGFTFSGSAMHWVRQASGKGLEWVGRIRSKANSYATAYAASVKGRFTISRDDSKNTAYLQMNSLKTEDTAVYYCNFRGAFDYWGQGTLVTVSS

P301-H5 light chain:

SYELTQPPSVSVSPGQTARITCSSGDALPKQYAYWYQQKPGQAPVLVIYKDSERPSGIPERFSGSSSGTTVTLTISGVQAEDEADYYCQSADSSGTYVVFGGGTKLTVL


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