Methods

Cells
Vero E6 and COS-7 cells were purchased from the American Type Culture Collection (Manassas, VA, United States) and cultured in Dulbecco’s Modified Eagle’s Medium (DMEM; Thermo Fisher Scientific, Waltham MA, United States) supplemented with 10% fetal bovine serum (FBS; HyClone, Logan, UT, United States), 100 units/mL penicillin and 100 µg/mL streptomycin (Thermo Fisher Scientific). 293FT cells were purchased from Invitrogen (Carlsbad, CA, United States) and grown in DMEM supplemented with 10% FBS, 100 units/mL penicillin, 100 µg/mL streptomycin and 500 µg/mL geneticin (Thermo Fisher Scientific). Cells were maintained at 37 °C with 5% CO2.

Purification of monoclonal antibody 1A9
The hybridoma for mAb 1A9 was previously generated [20]. All mAbs were purified from cell culture supernatants using HiTrap protein G HP affinity columns (GE Healthcare, Chicago, IL, United States) and stored at −80 °C. The purity of the mAb was confirmed by sodium dodecyl sulphate-polyacrylamide gel electrophoretic (SDS-PAGE) analysis. The concentration of the purified mAb was determined using the Coomassie Plus protein assay reagent (Thermo Fisher Scientific).

Plasmids for expression of full spike protein and fragments
SARS-CoV-2 S-protein-expressing plasmids were codon-optimised and generated by gene synthesis (Bio Basic Asia Pacific, Singapore) according to GenBank accession number: QHD43416.1. One plasmid is for expressing untagged full-length S protein while the other is for expressing a Myc-tagged S-protein fragment consisting of residues 1048–1206 (SARS-CoV-2 numbering). The pXJ40-Myc expression vector was used as an empty vector control and pXJ40-Myc-HBcAg plasmid expressing Myc-tagged hepatitis B virus core antigen (HBcAg) was used as a negative control.

Transient transfection and western blot analysis
293FT cells were seeded onto 6-cm dishes 24 hours before transient transfection using X-tremeGENE HP DNA transfection reagent (Roche, Basel, Switzerland) according to the manufacturer’s protocol. At 24 hours post-transfection, cells were harvested, spun down by centrifugation and washed with cold phosphate buffered saline (PBS) twice. Cells were then resuspended in 2× Laemmli sample buffer, boiled and sonicated. Clarified supernatant containing the protein of interest was obtained by spinning down the cell lysate at 13,000 rpm at 4 °C to remove the cell debris and further analysed by western blot (WB) analysis. Equal amounts of total cell lysates were loaded per lane and resolved using electrophoresis on SDS-PAGE gels and transferred onto nitrocellulose membrane (Bio-Rad, Hercules, CA, United States). The membrane was blocked in 5% skimmed milk in Tris-buffered saline with 0.05% Tween 20 (TBST) for 1 hour at room temperature (RT) and incubated with primary antibodies at 4 °C overnight. After the membrane was washed with TBST, it was incubated with a horseradish peroxidase (HRP)-conjugated secondary antibody (Thermo Fisher Scientific) at RT for 1 hour. The membrane was then washed with TBST again and bound antibodies visualised with enhanced chemiluminescence substrate (Thermo Fisher Scientific) using ChemiDoc MP Imaging System (Bio-Rad).

Transient transfection and immunofluorescence analysis
For immunofluorescence (IF) analysis, COS-7 cells on glass coverslips were transfected as above and fixed at 24 hours post-transfection in 4% paraformaldehyde for 10 min at RT followed by permeabilisation with 0.2% Triton X-100 (Sigma-Aldrich, St. Louis, MO, United States) for 5 min. Fixed cells were then blocked with PBS containing 10% FBS for 30 min at RT. Cells were immunolabelled for 1 hour at RT with the indicated murine mAb and 45 min with Alexa Fluor 488-conjugated goat anti-mouse IgG antibody (Life Technologies, Carlsbad, CA, United States). Immunolabelled coverslips were counterstained with 4′,6-diamidino-2-phenylindole (DAPI; Sigma-Aldrich), and mounted using ProLong Gold Antifade Mountant (Molecular Probes, Eugene, OR, United States). Images were acquired with Olympus CKX53 microscope using Olympus (Tokyo, Japan) LCAch N 20×/0.40 iPC objective lens and Olympus DP27 colour camera with Olympus cellSens software. Each channel was collected separately, with images at 1024 × 1024 pixels.

ELISA
Whole ectodomain of SARS-CoV-2 S protein with His Tag (Sino Biological Inc., Beijing, China; catalogue number: 40589-B08V1) was diluted with coating buffer (0.1 M NaHCO3, 34 mM Na2CO3) and a total of 20 ng of protein was loaded into individual wells of a 96 well plate (Nunc, Roskilde, Denmark) and allowed to coat overnight at 4 °C. Plates were then washed four times with 0.05% Tween 20 in PBS (PBST) and blocked with 5% bovine serum albumin (BSA)/PBST for 30 min before murine antibodies serially diluted with blocking buffer were added to desired wells for 1 hour. Plate were washed four times with PBST before incubation for 1 hour with HRP-conjugated goat anti-mouse IgG (Thermo Fisher Scientific) secondary antibodies diluted in blocking buffer, and washed four times with PBST. Visualisation of bound secondary antibodies was done by the addition of 3,3',5,5'-tetramethylbenzidine (TMB) substrate (Thermo Fisher Scientific) for 5 min in the absence of light and the reaction was stopped with 2 M sulphuric acid. Optical density at 450 nm (OD450nm) was determined by a Tecan (Männedorf, Switzerland) Infinite M1000 reader and normalised OD450nm was obtained by subtracting background absorbances determined in BSA coated wells.

Production of monoclonal antibody CR3022
The human mAb CR3022 was expressed in a similar manner as previously described [22]. The variable heavy (VH; GenBank accession number: DQ168569) and variable light (VL; GenBank accession number: DQ168570) genes of CR3022 were generated by gene synthesis (Bio Basic Asia Pacific) and cloned into pFUSEss-CHIg-hIgG1 and pFUSE2ss-CLIg-hK cloning vectors (InvivoGen, San Diego, CA, United States) respectively. Transfection of suspension FreeStyle 293 cells (Thermo Fisher Scientific) and purification of antibodies by fast protein liquid chromatography is as described in our previous study [23].

Sandwich ELISA
Mab 1A9 was diluted with coating buffer (0.1 M NaHCO3, 34 mM Na2CO3) and 0.1 µg of antibody was coated onto individual wells of a Maxisorp flat-bottom plate (Nunc) overnight at 4 °C. The plate was washed three times with PBST before blocking was done using 5% BSA/PBST at 37 °C for 60 min. Dilutions of His-tagged full length SARS-CoV-2 S protein (Sino Biological Inc., catalogue number: 40589-B08V1) and His-tagged H7N7-HA (Sino Biological Inc., catalogue number: 11082-V08B) were added to desired wells and incubated at 37 °C for 90 min followed by three washes with PBST. 100 µL of CR3022 antibody was added at a concentration of 1 µg/mL and incubated at 37 °C for 60 min followed by three PBST washes before HRP-conjugated goat anti-human IgG (Thermo Fisher Scientific) was added for 60 min at 37 °C. Finally, after three PBST washes, TMB (Sigma-Aldrich) was added for 5 min and the reaction was stopped by 2 M sulphuric acid. The OD450nm was determined by a Tecan Infinite M1000 reader. Statistical analyses were performed using an unpaired, one-tailed Student’s t-test with Welch’s correction for unequal variances. p values < 0.05 were considered statistically significant.

Virus infection and immunofluorescence
All works with live virus were performed in the biosafety level (BSL)3 facility at the Public Health Agency of Sweden. Vero-E6 cells were infected with SARS-CoV-2 (SARS-CoV-2-Iso/01/human/2020/SWE; GenBank accession number: MT093571) at a multiplicity of infection (MOI) of one in DMEM 2% FBS (Thermo Fisher Scientific). At 24 hour post-infection, cells were fixed with chilled methanol/acetone and the cells were kept at −20 °C overnight. Cells were then stained using mAb 1A9 at 5 µg/mL at 37 °C for 30 min in IF buffer (BSA 0.2%, Triton ×100 0.1% in PBS, pH 7.4). The cells were washed three times with PBS and incubated, subsequently with Alexa Fluor 488-conjugated goat anti-mouse IgG antibody (Thermo Fisher Scientific) in IF buffer containing DAPI for an additional 30 min. Cells were washed three times with PBS before visualisation and image acquisition with fluorescent microscopy.

Bioinformatics analysis
S protein reference sequences for SARS-CoV-1, SARS-CoV-2, batRaTG13, Middle East respiratory syndrome (MERS) and human common-cold coronaviruses 229E, NL63, OC43 and HKU1 were downloaded from the National Center for Biotechnology Information (NCBI). A multiple sequence alignment was created with multiple alignment using fast Fourier transform (MAFFT) using the slow but accurate L-INS-I parameter settings [24] and the alignment curated, cut to the target region 1029–1192 (SARS-CoV-1 numbering) and visualised with Jalview [25]. We used Molecular Evolutionary Genetics Analysis (MEGA) X [26] to calculate the number of amino-acid differences for all sequence pairs in the alignment of the mAb target region and the full S protein normalised by the length of the aligned sequence of the respective reference protein to obtain per cent amino acid identities.
To determine SARS-CoV-2 sequence diversity in the S protein within the current pandemic, 230 human and environmental viral sequences were downloaded from GISAID’s EpiCoV database on 1 March 2020. We gratefully acknowledge the authors, originating and submitting laboratories of the sequences on which this part of the research is based. The list is detailed in Supplementary Table 1. The nt sequences were searched with basic local alignment search tool (BLAST)X against the reference S protein. 174 hits covered the full length of the S protein and amino-acid mutations were counted and tabulated using a custom Perl script (Supplementary Table 2).

Ethical statement
Ethical approval was not required for this study.