key: cord-0838866-cqqygvzt
authors: Vogel, Monique; Augusto, Gilles; Chang, Xinyue; Liu, Xuelan; Speiser, Daniel; Mohsen, Mona O.; Bachmann, Martin F.
title: Molecular definition of severe acute respiratory syndrome coronavirus 2 receptor‐binding domain mutations: Receptor affinity versus neutralization of receptor interaction
date: 2021-07-27
journal: Allergy
DOI: 10.1111/all.15002
sha: 5ad90c1a1e8d4c9df01d6964f02025b07d55a615
doc_id: 838866
cord_uid: cqqygvzt

BACKGROUND: Several new variants of SARS‐CoV‐2 have emerged since fall 2020 which have multiple mutations in the receptor‐binding domain (RBD) of the spike protein. It is unclear which mutations affect receptor affinity versus immune recognition. METHODS: We produced wild type RBD, RBD with single mutations (E484K, K417N, or N501Y) or with all three mutations combined and tested their binding to ACE2 by biolayer interferometry (BLI). The ability of convalescent sera to recognize RBDs and block their interaction with ACE2 was tested as well. RESULTS: We demonstrated that single mutation N501Y increased binding affinity to ACE2 but did not strongly affect its recognition by convalescent sera. In contrast, single mutation E484K had almost no impact on the binding kinetics, but essentially abolished recognition of RBD by convalescent sera. Interestingly, combining mutations E484K, K417N, and N501Y resulted in a RBD with both features: enhanced receptor binding and abolished immune recognition. CONCLUSIONS: Our data demonstrate that single mutations either affect receptor affinity or immune recognition while triple mutant RBDs combine both features.

B.1.6.1.7, 5,6 which show mutations in the receptor-binding domain (RBD) and receptor-binding motif (RBM) of the spike (S) glycoprotein (Table 1, Figure 1 ). RBD and in particular RBM are responsible for interaction with the cellular receptor ACE2 and are the primary target of neutralizing antibodies 7 (Figure 1 ). Mutations altering the RBD conformation have been shown to permit SARS-CoV-2 to escape antibody neutralization and for the rapid infectivity and transmission of SARS-CoV-2. 8 Mutant viruses may spread more efficiently because they show increased affinity for the receptor or because they escape neutralizing antibody responses. 9 The importance of receptor affinity has been illustrated by SARS-CoV-1, which showed a fourfold lower affinity for ACE2 compared to SARS-CoV-2 and also was much less contagious and showed strongly reduced transmission than SARS-CoV-2. 10 Viruses that escape neutralization are typically called serotypes and usually may only occur when a large proportion of individuals show antibody-based immunity against the original strain and further spread may only be possible by escape of neutralizing antibody responses. 11 For SARS-CoV-2, it remains to be shown de- Here we assessed the molecular basis for antibody escape and how the RBD mutations present in two variants of concern (B.1.1.7 and P.1) influence the affinity to the receptor.

The SARS-CoV-2 receptor-binding domain of the wild-type RBD (RBD WT ), the single RBD mutants (RBD K417N , RBD E484K , and RBD N501Y ,), and the triple RBD mutant (RBD TRIP ) were expressed using Expi293F cells (Invitrogen, ThermoFisher Scientific). The genes that encode SARS-CoV-2 RBD WT (residues Arg319-Phe541) or RBD mutants with a C-terminal 6-His-tag were inserted into pTwist CMV BetaGlobin WPRE Neo vector (Twist Bioscience). The construct plasmids were transfected into Expi293F cells using ExpiFectatmine 293 transfection kit (Gibco, ThermoFisher Scientific). The supernatant of cell culture containing the secreted RBDs was purified using His-Trap HP column (GE Healthcare). Collected RBD WT or RBD mutated proteins were equilibrated in PBS and kept at −20°C.

ACE2-mFc was purchased from Sino Biological. Biotinylated and non-biotinylated soluble human ACE2 fused to mouse IgG2a Fc proteins were kindly provided by PD Dr. Alexander Eggel (University

• In this study, we tested RBD of the spike protein with single mutations or with all three mutations combinations for binding to sera from naïve donors and convalescent COVID-19 patients. • Convalescent sera recognize RBD of the early SARS-CoV-2 emerged from Wuhan, China. Single mutations (E484K or N501Y) either affect receptor affinity to ACE2 or immune recognition of RBD by convalescent sera. Clinic of Rheumatology and Immunology, Inselspital) who received the plasmid from Prof. Peter Kim (Stanford University).

Human sera were obtained from 11 COVID-19 convalescent patients which were recruited at the University Hospital of Bern, Bern, Switzerland as described. 12 Participants were recruited via three dif- 

Corning half area 96-well plates were coated with 1 μg/ml RBD WT or mutated RBDs in PBS overnight at 4°C and then blocked with PBS/0.15% casein. Convalescent human sera were added, serially 

The analysis of binding kinetics of RBD WT and RBD TRIP to ACE2-mFc was performed by BioLayer Interferometry (BLI) using an Octet RED96e (Fortebio) instrument. High precision Streptavidin (SAX, ForteBio) biosensors were saturated with 7.5 μg/ml biotinylated ACE2-mFc in BLI assay buffer (PBS, 0.1% BSA, 0.02% Tween 20) for 10 min. RBD WT and RBD TRIP were prepared as twofold serial dilution (typically 50, 25, 12.5, 6.25, and 3.125 nM) in BLI assay buffer plus buffer blanks. Kinetic values were calculated by ForteBio data analysis software using a 1:1 binding model.

The ability of the sera of the COVID convalescent patient to compete with ACE2 for binding to RBD WT and RBD TRIP was tested in a sandwich format assay on the Octet RED96e (Fortebio). Anti-penta-His (HIS1K) biosensors were loaded for 10 min with RBD WT and RBD TRIP at a concentration of 7.5 μg/ml in BLI assay buffer followed by addition of samples (diluted 1:20 in BLI assay buffer) from convalescent human sera.

To assess whether the sera can inhibit the binding of ACE2 to RBD WT and RBD TRIP , ACE2-mFc (50 nM) was added to biosensor. For control two additional sensors with BLI buffer were used, one for baseline and one without serum sample to determine binding of ACE2-mFc alone.

The results are expressed of single individual. The response data were normalized using ForteBio data analysis software version1.2.0.1.55. Figure 1A ). 13 In order to assess the role of each of these mutations on the binding of the RBD to ACE2, we generated single RBD mutants each containing one of the above mentioned mutations (RBD K417N , RBD E484K , and RBD N501Y ). All RBDs were purified to homogeneity, and the affinity to recombinant ACE2 was determined by Biolayer Interferometry using Octet technology. 14 The BLI assays showed that the affinity of ACE2 for RBD TRIP (shown in Figure 2B ,F, Table 2 , K D ≈10 nM) was about twice as high as for RBD WT (shown in Figure 2A ,F, Table 2 , K D = 20.5 nM). The affinity of the SARS-CoV-2 for ACE2 has been reported to be only fourfold higher compared to SARS-CoV-1; thus, a difference of two reported here between RBD WT and RBD Trip is expected to be biologically significant and most likely reflects enhanced infectivity. In contrast, the introduction of a single E484K mutation in the RBD (RBD E484K ) did not affect receptor affinity (shown in Figure 2D ,F). For comparison, the affinity observed for RBD N501Y was threefold lower (K D ≈6 nM, shown in Figure 2C ,F, Table 2 ). Interestingly, K417N mutation in the single RBD mutant (RBD K417N ) resulted in completely altered binding properties (shown in Figure 2E ). RBD K417N showed much lower association rates and plateau levels and a non-monovalent pattern of dissociation rates (shown in Figure 2E , 

To determine whether RBD WT -specific immune sera might have a reduced ability to bind to mutated RBDs we performed ELISA and Biolayer Interferometry using sera from convalescent patients (shown in Figure 3 ). As expected RBD WT was well recognized by convalescent sera in ELISA experiments. In contrast, RBD K417N and RBD N501Y were recognized in a slightly impaired fashion (shown in Figure 3A ). In marked contrast, mutation at position 484 essentially abolished recognition of both RBD E484K and RBD TRIP . Corresponding results were obtained using Biolayer Interferometry (shown in Figure 3B ). RBD-specific neutralizing antibodies typically block interaction of RBD with the viral receptor ACE2. We therefore assessed whether reduced binding of convalescent sera to RBD TRIP was paralleled by reduced ability of these antibodies to block binding of ACE2 to the triple mutant (shown in Figure 3C ). These experiments demonstrate that human convalescent sera essentially failed to block binding of ACE2 to RBD TRIP, explaining why SARS-CoV-2-induced antibodies largely fail to neutralize the triple mutant variants.

The newly emerging mutant RBDs may affect the affinity for the viral receptor. In addition, such mutations at the virus-receptor interaction face may alter the ability of RBD-specific antibodies-induced by previous infection-to neutralize the mutant viruses. When we investigated whether distinct mutations may affect receptor affinity, we found that N501Y mutation enhanced affinity for the viral receptor ACE2 both as a single mutation and as a triple mutation, while E484K mutation alone did not affect the interaction with ACE2. In addition, such mutations at the virus-receptor interaction interface may alter the ability of RBD-specific antibodies to recognize and neutralize the mutant variants. 15 A previous study has shown that serum neutralization is not compromised by N501Y (also found in the strain B.1.1.7). 16 In contrast, E484K (found B.1.1.7 and in P.1 strains) was associated with reduced neutralization by monoclonal antibodies and reduced recognition as shown here. [17] [18] [19] Interestingly, studies applying in vitro pressure produced similar mutations as those that occurred naturally. 20 In this study, we showed that convalescent sera have reduced ability to recognize RDB TRIP variants explaining why the mutant SARS-CoV-2 strain P.1 is more infectious 21 and less susceptible to neutralization by antibodies induced with RBD WT . 22 In summary, our data demonstrate that distinct mutations may affect receptor affinity which likely affects viral infectiv- 

We thank Marianne Zwicker for production of wild-type and mutant

RBDs. We thank PD Dr. Alexander Eggel and Dr. Daniel Brigger for providing biotinylated and non-biotinylated ACE2-mFc. 

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Molecular definition of severe acute respiratory syndrome coronavirus 2 receptor-binding domain mutations: Receptor affinity versus neutralization of receptor interaction