Recombinant expression and purification of monoclonal antibodies (mAbs). All heavy and light-chain (HC and LC) sequences were obtained by single-cell repertoire sequencing from individuals on days 7, 21, and 28 post initial BNT162b2 vaccination (second immunization on day 21) 7. Codon-optimized HC and LC variable sequences were cloned into in-house vectors, containing human IgG1 and κ or λ constant regions, respectively. HC and LC plasmids were transfected into Expi293F cells using FectoPro (Polyplus transfection). Cell supernatants were collected after 7 days and purified with AmMag Protein A beads (Genscript). mAb concentrations were measured using a nanodrop spectrophotometer (Thermo Fisher Scientific) and human IgG quantitation ELISAs (Bethyl Laboratories).
ELISA measurements. MaxiSorp 384-well plates (Thermo Fisher Scientific) were incubated for 24h with 1 µg ml− 1 recombinant SARS-CoV-2 RBD (Acro, SPD-C52H3), SARS-CoV-2 S2 (Acro, S2N-C52H5), HCoV-OC43 S (Sino Biological, 40607-V08B), or HCoV-HKU1 S (Sino Biological, 40606-V08B), in carbonate-bicarbonate buffer. Plates were washed with PBST (PBS + 0.1% Tween20) six times between each step. Plates were blocked with PBS + 1% BSA for 1h at RT, then mAbs were added at concentrations of 10, 1, 0.1, and 0.01 µg ml− 1 in PBS + 1% BSA and plates were incubated for 24h at 4°C. Plates were incubated for 1h at RT with secondary HRP-conjugated goat anti-human IgG antibodies (Bethyl Laboratories) and developed with TMB substrate (Thermo Fisher Scientific). Development was stopped with 2 N sulfuric acid. Four dilutions of positive-control plasma and secondary antibody only, as well as BSA only served as positive and negative controls. Plates were read on a GloMax Explorer Microplate Reader (Promega). ELISA assays were performed at least twice, in duplicate or triplicate.
SARS-CoV-2 variants and pseudovirus neutralization Assays. Variant sequences were obtained from www.gsaid.org. Mutations from Wuhan-Hu1 in S and RBD are described in Supplementary Table 1. Full variant S sequences were codon-optimized and replaced the Wuhan-Hu1 S insert in the SARS-CoV-2 spike plasmid (parent plasmids publicly available from J. Bloom laboratory), which was then amplified in E.Coli and checked for correct insertion and sequence integrity by Sanger-sequencing. Pseudotyped lentiviral particles were generated as previously described 7,15. Briefly, LentiX 293T cells in 10-cm tissue culture dishes were transfected 24 h post-seeding using Fugene transfection reagent (Promega) with pHAGE-CMV-Luc2-IRES-ZsGreen-W, lentiviral packaging plasmids (HDM-Hgpm2, HDM-tat1b and pRC-CMV-Rev1b) and wild-type or variant SARS-CoV-2 spike plasmids. 48–60 h post transfection, viral supernatants were collected and spun at 500g for 10 min. Lentiviral supernatants were concentrated with LentiX concentrator (Takara) according to the manufacturer’s instructions. Pellets were resuspended in EMEM at approximately 1/100 of the initial amount of media, and stored at − 80°C until use. Virus was titrated on HeLa-ACE2 cells, provided by Dennis Burton at the Scripps Research Institute.
Neutralization assays were performed as previously described 7,15. Briefly, HeLa-ACE2 cells were seeded at 12,500 cells/well in flat-bottom 96-well plates 20h before viral transduction. mAbs were prepared in EMEM in eight five-fold serial dilutions starting at 50 or 10 µg ml− 1, incubated with SARS-CoV-2 pseudotyped virus for 1h at RT, and then added to HeLa-ACE2 cells in the presence of 5 µg ml− 1 polybrene (Sigma Millipore). After 48h, luciferase activity was measured using the Britelite plus Reporter Gene Assay System (Perkin Elmer), and read on a GloMax Explorer Microplate Reader (Promega). Neutralization assays were performed 1–3 times in at least triplicates for each dilution. mAbs were considered neutralizing only if a considerable decrease in luminescence was measured at concentrations < 10 µg/ml (Supplementary Figs. 1–2).
Human subjects. No human subjects were included in this study. Antibody sequences were derived from our prior study 7, which had been approved by the Institutional Review Board of Stanford University (IRB-3780), and complied with the relevant ethical regulations.
Statistical analysis and software. Statistical analyses were performed with GraphPad Prism v.9.1.0. Significance levels and statistical tests used are indicated in the figure legends. For analysis of ELISA measurements, the areas under the curves (AUC) of serial dilutions were calculated and AUC ± SEM were presented. For neutralization assays, the half-maximum inhibitory concentration (IC50) for each mAb was calculated using least square regression.
All data pertaining to this manuscript are included in the text, figures, and supplementary information.