All COG-UK sequences were downloaded on 24th January 2020, and the translated protein sequences were roughly to the wild-type reference from start and stop codons between nucleotides 21000-25000, and filtered on the mutation 501Y. Sequence alignment was carried out, and identified mutations were plotted as red balls (single point mutations) or black balls (deletions) on the modeled C-alpha positions of the spike structure, size proportional to the logarithm of the incidence. Residues which mutated at an incidence greater than 0.3% compared to the wild-type were labeled explicitly.

The constructs of native RBD and ACE2 are the same as in Zhou et al. (2020). To clone RBD N501Y, a construct of native RBD was used as the template and two primers of RBD (Forward primer 5′-CTACGGCTTTCAGCCCACATACGGTGTGGGCTACCAGCCTT-3′ and reverse primer 5′-AAGGCTGGTAGCCCACACCGTATGTGGGCTGAAAGCCGTAG-3′) and two primers of pNEO vector (Forward primer 5′- CAGCTCCTGGGCAACGTGCT-3′ and reverse primer 5′- CGTAAAAGGAGCAACATAG-3′) were used to do PCR. Amplified DNA fragments were digested with restriction enzymes AgeI and KpnI and then ligated with digested pNEO vector. This construct encodes exactly the same protein as native RBD except the N501Y mutation, as confirmed by sequencing.

Protein expression and purification were performed as described in Zhou et al. (2020) and Dejnirattisai et al., 2021. Briefly RBD and mAb were expressed in 293T cells, His-tagged RBD was purified on Ni-NTA and mAb on protein-A. The Regeneron and AstraZeneca antibodies were supplied by AstraZeneca.

Fab fragments of 269 antibody were digested and purified using Pierce Fab Preparation Kit, following the manufacturer’s protocol.

BLI experiments were run on an Octet Red 96e machine (Fortebio). To measure the binding affinities of monoclonal antibodies with native RBD and RBD N501Y, RBD and RBD N501Y were immobilized onto AR2G biosensors (Fortebio) separately. Monoclonal antibodies were used as analytes. To measure the binding affinities of native RBD and RBD N501Y with ACE2, native RBD and RBD N501Y were immobilized onto AR2G biosensors separately. Serial dilutions of ACE2 were used as analytes. Data were recorded using software Data Acquisition 11.1 (Fortebio) and analyzed using software Data Analysis HT 11.1 (Fortebio) with a 1:1 fitting model.

269 Fab was mixed with RBD or N501Y RBD in a 1:1 molar ratio with a final concentration of 9.9 mg ml−1. After incubation at room temperature for 30 min, the sample was used for initial screening of crystals in Crystalquick 96-well X plates (Greiner Bio-One) with a Cartesian Robot using the nanoliter sitting-drop vapor-diffusion method as previously described (Walter et al., 2003). Crystals of RBD/269 Fab complex were grown in Molecular Dimensions Morpheus screen, condition C6 containing 0.09 M NPS (NaN03; Na2HPO4; (NH4)2SO4), 0.1 M buffer 2 (sodium HEPES, MOPS) and 30% EDO_P8K (ethylene glycol, PEG 8K). Crystals of N501Y RBD/269 Fab complex were obtained from a Molecular Dimensions Proplex screen, condition B10 containing 0.15 M ammonium sulfate, 0.1 M MES pH 6.0 and 15% PEG 4000.

Crystals of N501 RBD/269 Fab were mounted in loops and dipped in solution containing 25% glycerol and 75% mother liquor for a second before being frozen in liquid nitrogen prior to data collection. No cryo-protectant was used for RBD/269 crystals. Diffraction data were collected at 100 K at beamline I03 of Diamond Light Source, UK. Diffraction images of 0.1° rotation were recorded on an Eiger2 XE 16M detector (exposure time of either 0.003 or 0.007 s per image, beam size 80 × 20 μm, 100% beam transmission and wavelength of 0.9763 Å). Data were indexed, integrated and scaled with the automated data processing program Xia2-dials (Winter, 2010; Winter et al., 2018). A dataset of 720° was collected from 2 frozen crystals to 2.19 Å resolution for N501Y RBD/269 Fab complex. 360° of data were collected for the RBD/269 Fab complex from a single crystal to 1.77 Å resolution.

Both structures were determined by molecular replacement with PHASER (McCoy et al., 2007) using search models of SARS-CoV-2 RBD/scFv269 complex (PDB: 7BEM) and the ChCl domains of SARS-CoV-2 RBD/158 complex (PDB: 7BEK) (Dejnirattisai et al., 2021). Cyclic model rebuilding with COOT (Emsley and Cowtan, 2004) and refinement with PHENIX (Liebschner et al., 2019) resulted in the current structures with Rwork/ Rfree = 0.197/ 0.222 and Rwork/ Rfree = 0.185/0.200 for all data to 2.19 Å and 1.77 Å resolution for N501Y RBD/269 Fab and RBD/269 Fab complexes, respectively. Electron density for the side chain of Y501 is weak. However, when the structure was refined with an asparagine at 501, there was strong, dispersed positive density around the side chain, consistent with the presence of a flexible tyrosine residue (Figure S2). Mass spectrometry and biolayer interferometry data confirmed the presence of tyrosine at 501. Data collection and structure refinement statistics are given in Table S1. Structural comparisons used SHP (Stuart et al., 1979), residues forming the RBD/Fab interface were identified with PISA (Krissinel and Henrick, 2007) and figures were prepared with PyMOL (The PyMOL Molecular Graphics System, Version 1.2r3pre, Schrödinger, LLC).

The neutralization potential of an antibody was measured using a Focus Reduction Neutralization Test (FRNT), where the reduction in the number of the infected foci is compared to a no antibody negative control well. Briefly, serially diluted Ab or plasma was mixed with SARS-CoV-2 strain Victoria or B.1.1.7 and incubated for 1 hr at 37°C. The mixtures were then transferred to 96-well, cell culture-treated, flat-bottom microplate containing confluent Vero cell monolayers in duplicate and incubated for further 2 hr, followed by the addition of 1.5% semi-solid carboxymethyl cellulose (CMC) overlay medium to each well to limit virus diffusion. A focus forming assay was then performed by staining Vero cells with human anti-NP mAb (mAb206) followed by peroxidase-conjugated goat anti-human IgG (A0170; Sigma). Finally, the foci (infected cells) approximately 100 per well in the absence of antibodies, were visualized by adding TrueBlue Peroxidase Substrate. Virus-infected cell foci were counted on the classic AID EliSpot reader using AID ELISpot software. The percentage of focus reduction was calculated and IC50 was determined using the probit program from the SPSS package.

Note: The content above has been extracted from a research article, so it may not display correctly.



Q&A
Please log in to submit your questions online.
Your question will be posted on the Bio-101 website. We will send your questions to the authors of this protocol and Bio-protocol community members who are experienced with this method. you will be informed using the email address associated with your Bio-protocol account.



We use cookies on this site to enhance your user experience. By using our website, you are agreeing to allow the storage of cookies on your computer.