key: cord-0753128-77uz57tt
authors: Li, Tao; Cui, Zhimin; Jia, Yunfei; Liang, Ziteng; Nie, Jianhui; Zhang, Li; Wang, Meng; Li, Qianqian; Wu, Jiajing; Xu, Nan; Liu, Shuo; Li, Xueli; An, Yimeng; Han, Pu; Zhang, Mengyi; Li, Yuhua; Qu, Xiaowang; Wang, Qihui; Huang, Weijin; Wang, Youchun
title: Aggregation of high‐frequency RBD mutations of SARS‐CoV‐2 with three VOCs did not cause significant antigenic drift
date: 2022-01-28
journal: J Med Virol
DOI: 10.1002/jmv.27596
sha: 1f11502f198481d2c5fa17565bc3d91bcd5ff46b
doc_id: 753128
cord_uid: 77uz57tt

Variants of SARS‐CoV‐2 continue to emerge, posing great challenges in outbreak prevention and control. It is important to understand in advance the impact of possible variants of concern (VOCs) on infectivity and antigenicity. Here, we constructed one or more of the 15 high‐frequency naturally occurring amino acid changes in the receptor‐binding domain (RBD) of Alpha, Beta, and Gamma variants. A single mutant of A520S, V367F, and S494P in the above three VOCs enhanced infectivity in ACE2‐overexpressing 293T cells of different species, LLC‐MK2 and Vero cells. Aggregation of multiple RBD mutations significantly reduces the infectivity of the possible three VOCs. Regarding neutralization, it is noteworthy that E484K, N501Y, K417N, and N439K predispose to monoclonal antibodies (mAbs) protection failure in the 15 high‐frequency mutations. Most importantly, almost all possible VOCs (single RBD mutation or aggregation of multiple mutations) showed no more than a fourfold decrease in neutralizing activity with convalescent sera, vaccine sera, and immune sera of guinea pigs with different immunogens, and no significant antigenic drift was formed. In conclusion, our pseudovirus results could reduce the concern that the aggregation of multiple high‐frequency mutations in the RBD of the spike protein of the three VOCs would lead to severe antigenic drift, and this would provide value for vaccine development strategies.

Concerns about severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the root of the current coronavirus pandemic, have been ongoing. As of November 30, 2021, the global coronavirus disease 2019 (COVID- 19) pandemic had resulted in more than 259 million confirmed cases and more than 5.18 million deaths (https:// covid19.who.int/). SARS-CoV-2 is an RNA virus with a high mutation rate despite its own replication error correction capability. Over the past year, more than 28 000 mutations and 5000 insertion/ deletion changes were detected for this virus (https://bigd.big.ac. cn/ncov/variation/annotation). Spike protein is critical for SARS-CoV-2 to attach to and infect target cells. The receptor-binding domain (RBD) within the spike protein is the most important region and is mainly responsible for binding to the ACE2 receptor in target cells. It is also the primary target of monoclonal antibodies (mAbs) and one of the fastest evolving regions. 1,2 Therefore, mutations in this region can affect the infectivity and antigenicity of the virus, [3] [4] [5] which may lead to reduced vaccine efficacy and the emergence of reinfections. 6 As of March 2021, at the beginning of this study, the most widely spread single mutant variant was N501Y, accounting for more than 820 000 (46%) of the~1.77 million SARS-CoV-2 sequences uploaded in the GISAID database (https://www.epicov. org). The amino acid change N501Y enhances the affinity of the RBD to ACE2 1,7-9 and is more transmissible than other mutant variants. 10, 11 Another important amino acid mutant is E484K, reaching more than 5.7% of total sequences. This mutation causes a significant decrease in the effectiveness of neutralizing antibodies and vaccine protection. 12 

The expression plasmid harboring the SARS-CoV-2 spike gene (GenBank accession no: MN908947.3) was optimized using mammalian codons, and the DNA fragment was constructed on the eukaryotic expression vector pcDNA3.1 using BamHI and XhoI digestion sites to obtain the plasmid pcDNA3.1-SARS-CoV-2 spike. In total, 72 mutation plasmids were constructed on this basis. The site mutation method was the same as that used in our previous studies. 14, 15 The specific mutation sites and corresponding primers (synthesized by China Biotechnology) are shown in Table S2 .

A total of 14 plasmids expressing ACE2 protein were con- 

were constructed in accordance with the method described in our previous study. 15 The day before transfection, 293T cells were digested and their concentration was adjusted to 5-7 × 10 5 cells/ml. 14, 15 Then, 15 ml of the cell culture medium was transferred to T75 cell culture flasks and incubated overnight in an incubator at 37°C and 5% CO 2 . When cells reached 70%-90% confluence, the medium was discarded and 15 ml of G *ΔG-vesicular stomatitis virus (VSV G pseudotyped virus, Kerafast) at a concentration of 7.0 × 10 4 TCID 50 /ml was used for infection. The cells were simultaneously transfected with 30 µg of the SARS-CoV-2 S protein expression plasmid, following the instructions provided with the Lipofectamine 3000 transfection reagent (Invitrogen), and then incubated at 37°C with 5% CO 2 . After 4-6 h, the cell culture medium was discarded and the cells were gently washed twice with phosphate-buffered saline (PBS) + 2% fetal bovine sera (FBS). Next, 15 ml of fresh complete DMEM was added to T75 cell culture flasks, which were incubated at 37°C and 5% CO 2 . After 24 h, culture supernatants containing SARS-CoV-2 pseudovirus were harvested, filtered, divided, and frozen at −80°C for use in subsequent experiments.

Before quantification using RT-PCR, all pseudotyped viruses were purified by 25% sucrose buffer and centrifuged at 100 000g for 3.5 h. 14 

RNA from the SARS-CoV-2 pseudovirus and site mutant pseudovirus was extracted using the QIAamp Viral RNA Mini kit as described above and then reverse-transcribed to obtain complementary DNA (cDNA). After quantification by real-time quantitative fluorescence PCR, the pseudovirus was diluted to the same particle number and added to 96-well cell culture plates at 100 µl per well. After digestion of the 293T cell line and ACE2-overexpressing cells with trypsin, 2 × 10 5 /ml cells were added to each well. Chemiluminescence assays were then performed after incubation for 24 h at 37°C in a 5% CO 2 incubator. Detailed procedures are described in our previous publication. 15 Briefly, 100 μl of luciferase substrate (Perkin-Elmer) was added to the wells, incubated and shaken for 2 min at room temperature, and then transferred to a test white plate for detection using a luminometer (Perkin-Elmer). Each set of experiments was repeated three times.

A total of 12 mAbs that neutralize the SARS-CoV-2 S protein were used.

Among them, mAb CB6 was provided by Jinghua Yan of the Institute of Microbiology, Chinese Academy of Sciences; mAbs DXP-593 and DXP-604 were provided by Sunney Xie of Peking University; mAbs 03-10D12-1C3, 03-9A8, 05-9G11, 09-4E5-1G2, and 09-7B8 were provided by Beijing Biocytogen Co., Ltd. following immunization of mice with spike protein followed by hybridoma cell fusion screening; mAb 9MW3311-

Co., Ltd.; mAbs AM128 and AM180 were provided by ACROBiosystems Co., Ltd.; mAb AbG3 was provided by Fipen Biologics Co., Ltd. 

The test antibody or serum samples are first diluted with PBS solution, then the samples were diluted in a total of six consecutive gradients using fresh DMEM containing serum in threefold gradients, followed by coincubation with VSV pseudotyped virus solution. Virus control wells and cell control wells were included on each 96-well plate. The virus solution (not the test sample) was added to the virus control wells, while only the complete medium (not the virus solution) was added to the cell control wells. After the addition of samples, the 96-well plates were incubated at 37°C for 1 h, and then dispersed Huh7 cells (2 × 10 4 cells/100 μl) were added to each well. Chemiluminescence was detected after incubation at 37°C and 5% CO 2 for 24 h. The effect of mAbs and sera on the inhibition of pseudotyped virus entry was evaluated by detecting a decrease in luciferase expression. 15 The EC 50 or NT 50 values for samples were calculated using the Reed-Muench method.

Packed pseudotyped viruses were collected and viral RNA was first extracted as previously described, reverse-transcribed into cDNA, then viral nucleic acid quantification was performed by real-time fluorescence PCR.

Pseudotyped viruses with the same viral particle number were used for subsequent quantification of the spike protein. Using the sandwich enzyme-linked immunosorbent assay method, standards and samples to be tested were added in accordance with the instructions of the Acro BIOSYSTEMS kit (Cat#TAS-K020), and biotin-labeled anti-SARS-CoV-2 spike protein antibody (Cat#RAS020-C03) was added at the end of the incubation to form antibody-antigen-antibody complexes. After washing the plate, streptavidin-horseradish peroxidase (HRP) was added and the chromogenic solution was added at the end of the incubation. The HRP catalyzed the substrate to produce a blue substance, and the solution turned yellow when the termination solution was added. The absorbance value (OD) was then measured at 450 nm. OD values were positively correlated with the SARS-CoV-2 spike protein content.

Two types of cells were used for fusion experiments, and 293T cells were first transfected with spike protein sequences carrying the D614G variant or a combined single mutant and the GFP1-7 RLN plasmid. Cells stably expressing human ACE2 were transfected with the GFP8-11 RLC plasmid as recipient cells. Cells were cultured at 37°C and 5% CO 2 for 24 h and then isolated with trypsin. Donor and recipient cells were mixed in a 1:1 ratio and seeded in 96-well plates. Fluorescence values reflecting GFP expression were monitored 3 h after mixing. The GFP signal was detected using a BioTek Cytation 5V instrument, as described previously. 17, 18 2.13 | Gene cloning, expression, and protein purification

The pCAGGS plasmid expressing human ACE2-mFc (residues 1-740, GenBank: NP_001358344) protein for surface plasmon resonance LI ET AL. | 2111 (SPR) was constructed in our recent work. 19 The plasmid was transiently transfected into HEK293T cells (ATCC CRL-3216) using PEI and then, 72 h later, the cell supernatants were collected, concentrated, and used in the SPR assays.

The DNA sequence encoding hACE2 (residues 19-615, Gen-Bank:NP_001358344) was inserted into the Baculovirus transfection vector pFastBac1 (Invitrogen) using the EcoRI and XhoI restriction sites. The gp67 signal peptide sequence was added to the N-terminus of the hACE2 gene for protein secretion, and the Hexa-His tag sequence was added to the C-terminus of the hACE2 sequence for protein purification. The hACE2 protein was expressed using the Bacto-Bac Baculovirus expression system and used for crystallization.

The pFastBac1-hACE2 plasmids were transformed into DH10Bac and 150 mM NaCl. 21 

The sitting-drop method was used to obtain high-resolution Gamma-A520S RBD/hACE2 complex crystals. In detail, the purified complex proteins were concentrated to 10 mg/ml. Then, 0.8 ml protein was mixed with 0.8 ml reservoir solution. The resulting solution was sealed and equilibrated against 100 ml of the reservoir solution at 18°C. High-resolution Gamma-A520S RBD/hACE2 complex crystals were grown in 0.1 M MES (pH 6.0), 15% w/v PEG 4000.

Reservoir solution supplemented with 20% (v/v) glycerol was prepared as an antifreezing buffer for freezing crystals. Crystals were picked up from the groove by using a mini loop and soaked in the antifreezing buffer for a few seconds. Then, crystals were picked up and frozen by soaking in liquid nitrogen. Diffraction data were collected at the Shanghai Synchrotron Radiation Facility (SSRF) 02U1.

The data set was processed with HKL2000 software as previously described. 22 The structure of the Gamma-A520S RBD/hACE2 complex was determined by the molecular replacement method using Phaser 23 with a previously reported complex structure of the SARS-CoV-2-RBD complex with human ACE2 (PDB: 6LZG). The atomic models were completed using Coot 24 and refined with Phenix refine in Phenix, 22 and the stereochemical qualities of the final models were assessed using MolProbity. All structural figures were generated using Pymol software (https://pymol.org/2/).

The SPR assays were performed to test the interactions between mFc-fused human ACE2 and SARS-CoV-2 variant RBDs using a The chip was regenerated after each reaction using glycine (pH 1.7).

The equilibrium dissociation constants (binding affinity, K D ) for each pair of interactions were calculated using BIAcore 8K ® evaluation software (GE Healthcare). The K D values were calculated using the model of 1:1 (Langmuir) binding mode. These results were then visualized using Origin 2021.

GraphPad Prism 8.0 (GraphPad Software) was used for plotting and statistical analysis. One-way ANOVA test and Holm-Sidak's multiple comparison test were used to analyze between-group differences. p values less than 0.05 were considered to be significant. The data are presented as the mean ± standard error for the sample mean (SEM) of at least three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.005, ****p < 0.001, ns indicates no significant difference. Figure 1D ). The D614G strain has replaced the SARS-CoV-2 (2019-nCoV) variant as a globally prevalent variant, [25] [26] [27] we used the D614G strain as the control reference in this study. All the pseudovirus variant constructs used in this study are detailed in Table S1 . The RLU values, determined for the infected cells by detecting the variants and their possible mutant strains, were compared with the reference strain D614G. A fourfold difference was considered significant; the dashed lines in the graph represent a 0.25 or fourfold change compared with strain D614G, respectively. As an example of nomenclature, Alpha+2muts (N439K) represents the first two mutations with the highest mutation frequency introduced on the Alpha variant and the last site added is N439K. p values were calculated using the respective variants of concern as the control. Unless otherwise stated, all experiments were performed in triplicate (mean ± SEM). *p < 0.05, **p < 0.01, ***p < 0.005, ****p < 0.001 A fourfold difference was considered significant; the dashed lines in the graph represent a 0.25-or 4-fold change compared with strain D614G, respectively. p values were calculated using the respective variants of concern as the control. Unless otherwise stated, all experiments were performed in triplicate (mean ± SEM). *p < 0.05, **p < 0.01, ***p < 0.005, ****p < 0.001 We first evaluated the effect of A520S on the binding affinity to the human receptor. As previously reported, 21,35 compared with the prototype RBD (WT), all the Alpha, Beta, and Gamma RBDs displayed increased binding strength to the human receptor ( Figure 4C ). When A520S was incorporated into the three VOCs, the affinities to hACE2 were maintained at a similar level to their respective parental ones.

We then solved the complex structure of Gamma-A520S RBD with hACE2 at a resolution of 3.3 Å. The structure of Gamma-A520S RBD-hACE2 was similar to the WT RBD-hACE2 structure with a root-mean-square deviation (RMSD) of 0.323 Å (for 737 Cα atoms, PDB: 6LZG). As indicated in Figure 4D , N501Y in the Gamma RBD induces little conformational change. However, due to the addition of the phenyl, Y501 seems to form a cation-π interaction with hACE2 K353 and a π-π stacking interaction with hACE2 Y41, conferring Gamma RBD higher binding affinity to the receptor than the parental N501, which has been observed by other groups. 35 The residue 520 does not involve the association with hACE2. Consistently, the three VOCs with A520 displayed similar binding affinities with hACE2 to those containing S520 ( Figure 4C) .

Notably, residue 520 at the trimeric S protein locates at the interface with the adjacent protomer NTD. When the complex structure of Gamma-A520S with hACE2 was superimposed with the Cryo-EM structure of S protein (PDB: 6VSB), the standing RBD S520 Cα is 1.7 Å closer to the adjacent NTD loop, compared with the A520 Cα. Accordingly, S520 was 2.1 Å closer to NTD G232, forming stronger interaction with G232 than A520 ( Figure 4D ). Although S520 in the lying RBD also brings RBD a little closer to the adjacent NTD, from 6.8 to 6.1 Å, they still form relatively weak interaction.

Thus, compared with A520, S520 in the standing RBD probably increases the interaction with NTD, thereby stabilizing the RBD at the standing conformation and favoring the binding to the receptor.

Taken together, these may lead to enhanced infectivity of the possible Gamma+A520S virus. Figure 5B ); the mAb 09-4E5-1G2 was escaped by the N439K mutation; and the mAb 09-7B8 was escaped by the S477N, S477R, and T478K mutations. Encouragingly, we also identified a F I G U R E 4 Analysis of the effects of S494P, A520S, and V367F mutations on the spike protein and cell-cell fusion of different mutant strains of pseudotyped viruses. (A) After collecting the pseudotyped viruses, the viral RNA was first extracted and reverse transcribed into cDNA, then quantified by reverse transcription polymerase chain reaction. Finally, the WT, S494P, A520S, and V367F mutant pseudotyped viruses of the different mutant strains were diluted to the same particle number for an enzymw-linked immunosorbent assay assay to quantify the spike protein on the surface of the pseudotyped viruses. (B) The effect of pseudotyped viruses on cell-cell fusion was assessed by the detection of GFP fluorescence values. The 293T cells were transfected with GFP1-7 RLN plasmid and spike mutant plasmids of WT, S494P, A520S, and V367F of different mutant strain variants, and the 293T-ACE2 cells were transfected with GFP8-11 RLC plasmid. The cells were digested 24 h after transfection, and then mixed together and incubated for 3 h for GFP fluorescence detection. Using D614G as the reference strain, the relative GFP expression ratio, which represents the differential change in fusion between cells, was calculated. Data were replicated at least three times (mean ± SEM). *p < 0.05, **p < 0.01, ***p < 0.005, ****p < 0.001. (C) Binding affinity of SARS-CoV-2 RBDs to human ACE2, characterized by surface plasmon resonance. Mouse Fc (mFc)-fused hACE2 in the supernatant was captured in the CM5 chip via its interaction with the preimmobilized anti-mFc antibody. Serially diluted WT RBD, Alpha RBD, Alpha A520S RBD, Beta RBD, Beta A520S RBD, Gamma RBD, and Gamma A520S RBD protein flowed through the chip, and response unit was recorded. K D values were calculated and the representative results from three experiments were shown. The data were presented as the mean ± SEM of three independent replicates (n = 3). (D) The overall structure of Gamma A520S RBD-hACE2. The complex structure of Gamma A520S RBD-hACE2 was superimposed on that of WT RBD-hACE2. Human ACE2, SARS-CoV-2 WT RBD, and Gamma A520S RBD were colored in green, gray, and magenta, respectively. The key contact residues were shown as stick structures and labeled appropriately. The cation-π interaction, π-π stacking interaction, and salt bridge were colored in cyan, yellow, and red, respectively. (E) Superimposition of Gamma A520S RBD in the complex on the Cryo-EM structure of SARS-CoV-2 spike glycoprotein (PDB: 6VSB) suggests that residue 520 in the RBD places in the interface with the adjacent NTD. The three protomers in the S protein were colored in gray, orange, and cyan, respectively. The Gamma A520S RBD was colored magenta. The key residues of RBD-A520, RBD-S520, NTD-D228, and NTD-G232 were marked with yellow dashed lines to show the interaction between them. GFP, green fluorescent protein; RBD, receptor-binding domain F I G U R E 5 Neutralizing monoclonal antibody sets for SARS-CoV-2 used to analyze antigenic changes in variants and possible mutant strains. Before cell processing, monoclonal antibodies were incubated with Alpha (A), Beta (B), and Gamma (C) possible mutant strains of pseudotyped viruses in a cell incubator at 37°C for 1 h before adding Huh7 cells for coculture. Luciferase activity was measured after 24 h to calculate the EC 50 value for each antibody. The EC 50 ratio between the variants or possible mutant strains and the reference strain D614G was calculated and analyzed to generate a heat map using Hem I. 36 Data are the result of at least three replicates. Red and blue boxes indicate enhanced or reduced neutralizing ability, respectively, as indicated by the specific degree of change, as shown on the graph scale mAb-03-9A8-that showed good neutralizing effects ( Figure 5 ) against all possible variants (including Beta and Gamma variants) and did not exhibit neutralizing escape against any pseudotyped virus used in this study. This suggested that mAb 03-9A8 is of clinical interest and deserves further investigation of the key binding sites to explain the mechanism by which the neutralization effect is not affected by these high-frequency RBD mutation sites. All possible multisite VOCs, when superimposed upon the single escape site described for the mAbs above, cause these mAbs to lose neutralization protection. Figure 6A) . Moreover, compared with the D614G variant, the above possible combined Alpha and Gamma variants also showed less than a fourfold decrease in neutralization and did not display immune escape. Relative to the Beta variant, the decrease in the neutralizing activity of the convalescent sera to all Beta multisite combined possible variants was not significant, with a maximum doubling of the average titer ( Figure 6B ). However, it is noteworthy that the proportional decrease in the neutralization titer was more pronounced for the possible Beta multisite variants compared with the D614G strain combined with the second to tenth mutations, although the p values were not statistically significant ( Figure 6B ). The Beta variant showed the greatest decrease in neutralizing activity relative to Alpha and Gamma variants, but aggregation of 13 high-frequency RBD site substitutions on the variant also did not cause a significant decrease in neutralizing activity. The S494P mutation has been reported to enhance the affinity of RBD to ACE2. 2 The V367F mutation was mainly found in the A.23.1

variant, which appears in Uganda and Vietnam, and has been suggested to enhance viral infectivity by increasing human ACE2

receptor-binding affinity. 37, 38 Previous studies in our laboratory have shown that the V367F mutation in the original strain enhances susceptibility to mAbs and to sera from convalescent individuals. 7 In this study, both convalescent patient sera and different antigens immunized guinea pig sera showed significantly higher levels of neutralization with the D614G + V367F variant than the D614G strain ( Figure S2B ), suggesting that V367F does not pose a greater threat of increased protective efficacy in the vaccinated population. However, its elevated infectivity would exacerbate transmission in the unvaccinated population. Furthermore, the elevated infectivity and fusion capability following the combination of the A520S mutation with the Gamma variant was particularly notable. We found that this mutation exerted a slightly reduced binding affinity to the human receptor compared to their corresponding VOCs. Consistently, the complex structure of Gamma-A520S with hACE2 indicates that this residue does not involve receptor binding. This may be similar to the D614G mutation, which reduces the affinity with hACE2, S1 subunit is easier to dissociate and fall off after binding, increasing the chance of S2 subunit exposure and thus enhancing the fusion strength. 39, 40 Meanwhile, in the complex structure, a little conformation change of the A520S-residing loop was observed. Notably, this loop places in the interface with the adjacent protomer NTD. This small conformational shift makes the S520 in the standing RBD closer to the adjacent NTD, thereby probably enhancing the interaction between the standing RBD with the NTD. Thus, the trimeric S with A520S

probably possesses more standing RBD, and more readily interacts with the receptor to initiate the infections. Although this mutation also generally became more responsive to the neutralization of the 

Analysis of antigenic changes in natural variants and possible mutant strains by SARS-CoV-2 vaccine sera and guinea pig sera immunized with different immunogens. The Ad5-Spike vaccine (five cases) based on the adenoviral vector, the mRNA-Spike vaccine (four cases), the 2019-nCoVspike vaccine guinea pig sera (four cases), D614G-spike vaccine guinea pig sera (four cases), (D614G + E484K + N501Y)-spike vaccine guinea pig sera (four cases), and (D614G + K417N + E484K + N501Y)-spike vaccine guinea pig sera (four cases) were inactivated. Then, after threefold series of multiplicative dilutions, the sera were incubated with Alpha (A), Beta (B), and Gamma (C) possible mutant strains of pseudotyped viruses at 37°C for 1 h and then added to Huh7 cells for coculture. Luciferase activity was measured after 24 h to calculate the NT 50 value for each serum. Using D614G as the reference strain, the NT 50 ratio of the variants or possible mutant strains relative to the reference strain D614G was calculated and analyzed, and the neutralization mean values for each group were generated by GraphPad Prism 8.0. The dotted graph indicates the change in neutralization for each inoculator serum, with the dashed lines in the graph representing a fourfold increase or decrease in neutralization capacity compared with the D614G strain, respectively. p values were statistically calculated using the respective VOCs as control groups. Data are the result of at least three replicates (mean ± SEM). *p < 0.05, **p < 0.01, ***p < 0.005, ****p < 0.001 LI ET AL.

| 2123 although a reduced neutralization effect was seen against variants such as Beta and Delta. [44] [45] [46] This may indicate a difference from influenza viruses, for which vaccine strains need to be changed frequently. Certain single amino acid changes on the hemagglutinin (HA) of the influenza virus (e.g., HA T135K) create a significant increase in antigenic distance, resulting in a dramatic decrease in vaccine sera neutralization potency. 47 In conclusion, our study shows no severe decrease in neutralization protection against single mutant or multiple-RBD mutants of the 15 high-frequency in the three VOCs, suggesting that SARS-CoV-2 RBD mutations are not as prone to severe antigenic drift as influenza virus HA region mutations. This may alleviate concern that the vaccine must be updated as mutations in the RBD of spike protein continue to appear. 50 It is undeniable that as Spike proteins continue to accumulate mutations, serious antigenic drift may still occur, not excluding mutations caused by non-RBD regions, so it is suggested that vaccine research can continuously optimize the immunoprotective efficacy of vaccines (e.g., dose and frequency of booster immunization injections) while also focusing on the neutralizing efficacy of heterologous immunizations (different variants or different regions such as NTD-and RBD-specific antigens) to cope with the emerging new viral variants.

We gratefully acknowledge the laboratories that submitted the sequences to GISAID on which this study is based. The original data are available from https://www.gisaid.org. We also appreciate the individuals, laboratories, and companies that provided monoclonal antibodies for this study. 

The authors declare that there are no conflict of interests. 

The data that support the findings of this study are available from the corresponding author upon reasonable request.

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