Today News 24. All Right Reserved.

Structural and functional ramifications of antigenic drift in recent SARS-CoV-2 variants

By admin on August 13, 2021

Defenses against SARS-CoV-2 variants

Our key defense against the COVID-19 pandemic is neutralizing antibodies against the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus elicited by natural infection or vaccination. Recent emerging viral variants have raised concern because of their potential to escape antibody neutralization. Wang et al. identified four antibodies from early-outbreak convalescent donors that are potent against 23 variants, including variants of concern, and characterized their binding to the spike protein of SARS-CoV-2. Yuan et al. examined the impact of emerging mutations in the receptor-binding domain of the spike protein on binding to the host receptor ACE2 and to a range of antibodies. These studies may be helpful for developing more broadly effective vaccines and therapeutic antibodies.

Science, abh1766, this issue p. eabh1766, abh1139, this issue p. 818

Abstract

Neutralizing antibodies (nAbs) elicited against the receptor binding site (RBS) of the spike protein of wild-type severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are generally less effective against recent variants of concern. RBS residues Glu484, Lys417, and Asn501 are mutated in variants first described in South Africa (B.1.351) and Brazil (P.1). We analyzed their effects on angiotensin-converting enzyme 2 binding, as well as the effects of two of these mutations (K417N and E484K) on nAbs isolated from COVID-19 patients. Binding and neutralization of the two most frequently elicited antibody families (IGHV3-53/3-66 and IGHV1-2), which can both bind the RBS in alternative binding modes, are abrogated by K417N, E484K, or both. These effects can be structurally explained by their extensive interactions with RBS nAbs. However, nAbs to the more conserved, cross-neutralizing CR3022 and S309 sites were largely unaffected. The results have implications for next-generation vaccines and antibody therapies.

The COVID-19 pandemic has already lasted for more than a year, but new infections are still escalating throughout the world. Although several different COVID-19 vaccines have been deployed globally, a major concern is the emergence of antigenically distinct severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants of concern (VOCs). In particular, the B.1.1.7 lineage that arose in the UK (1) and quickly became dominant, the B.1.351 (also known as 501Y.V2) lineage in South Africa (2), the B.1.1.28 lineage (and its descendant B.1.1.28.1, also known as P.1/501Y.V3) in Brazil (3), and B.1.232/B.1.427/B.1.429 (also called CAL.20C and CAL.20A) in the United States (4) have raised serious questions about the nature, extent, and consequences of antigenic drift in SARS-CoV-2. In the receptor binding site (RBS) of the spike (S) protein receptor-binding domain (RBD), the B.1.1.7 lineage has acquired an Asn501 → Tyr (N501Y) mutation, the B.1.351 and P.1 lineages share this mutation along with Lys417 → Asn/Thr (K417N/T) and Glu484 → Lys (E484K), whereas the California variants have a Leu452 → Arg mutation that is also present in the B.1.617 variant, which was first isolated in India, with Glu484 → Gln (5). E484K has also been detected in a few B.1.1.7 genomes (1) (Fig. 1A). We therefore investigated the structural and functional consequences of such mutations on neutralizing antibodies (nAbs) isolated from COVID-19 convalescent patients, as well as their effect on angiotensin-converting enzyme 2 (ACE2) receptor binding.

Fig. 1 Emergent SARS-CoV-2 variants escape two major classes of neutralizing antibodies.

(A) Emergent mutations (spheres) in the RBS of B.1.351 and P.1 lineages are mapped onto a structure of SARS-CoV-2 RBD (white) in complex with ACE2 (green) (PDB 6M0J) (90). Binding affinities of Fc-tagged human ACE2 against SARS-CoV-2 RBD wild type and mutants were assayed by biolayer interferometry (BLI) experiments. Detailed sensorgrams are shown in fig. S1. (B) Distribution of IGHV gene usage. Numbers of RBD-targeting antibodies encoded by each IGHV gene are shown. The frequently used IGHV3-53 and IGHV3-66 genes are highlighted in blue, and IGHV1-2 in orange. The IGHV gene usage in 1593 SARS-CoV-2 RBD-targeting antibodies (11, 14–44) relative to healthy individuals (baseline) (76) (fold enrichment) is shown as black line segments. Hashtags (#) denote IGHV gene frequencies in healthy individuals that were not reported in (76). Asterisks denote IGHV genes that are significantly enriched over the baseline repertoire (76) (P < 0.05, one-sample proportion test with Bonferroni correction). A fold enrichment of 1 (red dashed line) represents no difference over baseline. (C) Effects of single mutations on the neutralization activity and binding affinity of each neutralizing antibody. “–” denotes an increase in half-maximal inhibitory concentration (IC50) or KD by a factor of 100. Results in red with “×” indicate that no neutralization activity or binding was detected at the highest amount of immunoglobulin G used. N.C., not categorized in the original studies; N.S., no structure available. (D) Neutralization of pseudotyped SARS-CoV-2 virus and variants carrying K417N or E484K mutations. We tested a panel of 17 neutralizing antibodies, including four mode-1 IGHV3-53 antibodies (blue), two mode-2 IGHV3-53 antibodies (purple), and two IGHV1-2 antibodies (orange). The discrepancy between CV05-163 neutralizing SARS-CoV-2 pseudotyped virus (IC50 = 0.47 μg/ml) and authentic virus (IC50 = 0.02 μg/ml) reported in our previous study (17) is possibly due to different systems (pseudovirus versus authentic virus) and host cells (HeLa cells versus Vero E6 cells) used in these experiments.

” data-icon-position=”” data-hide-link-title=”0″>

Fig. 1 Emergent SARS-CoV-2 variants escape two major classes of neutralizing antibodies.

(A) Emergent mutations (spheres) in the RBS of B.1.351 and P.1 lineages are mapped onto a structure of SARS-CoV-2 RBD (white) in complex with ACE2 (green) (PDB 6M0J) (90). Binding affinities of Fc-tagged human ACE2 against SARS-CoV-2 RBD wild type and mutants were assayed by biolayer interferometry (BLI) experiments. Detailed sensorgrams are shown in fig. S1. (B) Distribution of IGHV gene usage. Numbers of RBD-targeting antibodies encoded by each IGHV gene are shown. The frequently used IGHV3-53 and IGHV3-66 genes are highlighted in blue, and IGHV1-2 in orange. The IGHV gene usage in 1593 SARS-CoV-2 RBD-targeting antibodies (11, 14–44) relative to healthy individuals (baseline) (76) (fold enrichment) is shown as black line segments. Hashtags (#) denote IGHV gene frequencies in healthy individuals that were not reported in (76). Asterisks denote IGHV genes that are significantly enriched over the baseline repertoire (76) (P < 0.05, one-sample proportion test with Bonferroni correction). A fold enrichment of 1 (red dashed line) represents no difference over baseline. (C) Effects of single mutations on the neutralization activity and binding affinity of each neutralizing antibody. “–” denotes an increase in half-maximal inhibitory concentration (IC50) or KD by a factor of 100. Results in red with “×” indicate that no neutralization activity or binding was detected at the highest amount of immunoglobulin G used. N.C., not categorized in the original studies; N.S., no structure available. (D) Neutralization of pseudotyped SARS-CoV-2 virus and variants carrying K417N or E484K mutations. We tested a panel of 17 neutralizing antibodies, including four mode-1 IGHV3-53 antibodies (blue), two mode-2 IGHV3-53 antibodies (purple), and two IGHV1-2 antibodies (orange). The discrepancy between CV05-163 neutralizing SARS-CoV-2 pseudotyped virus (IC50 = 0.47 μg/ml) and authentic virus (IC50 = 0.02 μg/ml) reported in our previous study (17) is possibly due to different systems (pseudovirus versus authentic virus) and host cells (HeLa cells versus Vero E6 cells) used in these experiments.

N501Y was previously reported to enhance binding to the human ACE2 receptor (6, 7). Here, we quantified binding of K417N, E484K, N501Y, and double and triple combinations in the RBD to ACE2 by biolayer interferometry (Fig. 1A and fig. S1). N501Y indeed increased RBD binding to ACE2 relative to wild-type RBD (binding affinity KD = 3.3 nM versus 7.0 nM), whereas K417N substantially reduced ACE2 binding (41.6 nM). E484K slightly reduced binding (11.3 nM). N501Y could rescue binding of K417N (9.0 nM), and the triple mutant K417N/E484K/N501Y (as in B.1.351) had similar binding (6.5 nM) to the wild type (Fig. 1A and fig. S1). Consistently, K417N/T mutations are associated with N501Y in naturally circulating SARS-CoV-2. Among 585,054 SARS-CoV-2 genome sequences in the GISAID database (5 March 2021) (8), about 95% of K417N/T mutations occur with N501Y, despite N501Y being present in only 21% of all analyzed sequences. In contrast, only 36% of E484K mutations occur with N501Y.

We and others have shown that most SARS-CoV-2 nAbs that target the RBD and their epitopes can be classified into different sites and subsites (9–12). Certain IGHV genes are highly enriched in the antibody response to SARS-CoV-2 infection, with IGHV3-53 (11, 13–16) and IGHV3-66, which differ by only one conservative substitution (V12I), and IGHV1-2 (11, 17, 18) being the most enriched IGHV genes used among 1593 RBD-targeting antibodies from 32 studies (11, 14–44) (Fig. 1B). We investigated the effects of the prevalent SARS-CoV-2 mutations on neutralization by these major classes of antibodies found in multiple donors, and the consequences for current vaccines and therapeutics.

K417N and E484K in VOCs B.1.351 and P.1 have been reported to decrease the neutralizing activity of sera as well as neutralizing monoclonal antibodies (mAbs) isolated from COVID-19 convalescent plasma and vaccinated individuals (45–62). Relative to wild-type SARS-CoV-2, B.1.351 is more resistant to neutralization by convalescent plasma and mRNA vaccine sera by a factor of ~8 to 14, and P.1 is more resistant by a factor of ~2.6 to 5 (63–68). Some variants are able to escape neutralization by some nAbs (e.g., LY-CoV555, 910-30, COVOX-384, S2H58, C671, etc.), whereas others retain activity (e.g., 1-57, 2-7, mAb-222, S309, S2E12, COV2-2196, C669, etc.) (50, 57, 63, 66, 69, 70). Here, we selected a representative panel of 17 human nAbs isolated from COVID-19 patients or humanized mice to study the escape mechanism. These nAbs cover all known neutralizing sites on the RBD and include those encoded by V genes that are the most frequently used and also significantly enriched (Fig. 1B). We tested the activity of a panel of nAbs against wild-type (Wuhan strain) SARS-CoV-2 pseudovirus and single mutants K417N and E484K (Fig. 1C). Binding and neutralization of four and five antibodies out of the 17 tested were abolished by K417N and E484K, respectively. Strikingly, binding and neutralization by all six highly potent IGHV3-53 antibodies (71) that we tested were abrogated by either K417N (RBS-A/class 1) or E484K (RBS-B/class 2) (Fig. 1, C and D, and fig. S2). In addition, binding and neutralization of IGHV1-2 antibodies was severely reduced for the E484K mutation (Fig. 1, C and D, and fig. S2).

We next examined 54 SARS-CoV-2 RBD-targeting human antibodies with available structures. The antibody epitopes on the RBD can be classified into six sites—four RBS subsites, RBS-A, B, C, and D; CR3022 site; and S309 site (fig. S3) (72)—that are generally related to the four classes assigned in (10) (Fig. 1C). Of 23 IGHV3-53/3-66 antibodies, 21 target RBS-A (Fig. 2A). All IGHV1-2 antibodies with known structures bind to the RBS-B epitope. A large fraction of antibodies in these two main families make contact with Lys417, Glu484, or Asn501 in their epitopes (Fig. 2A) (73). Almost all RBS-A antibodies interact extensively with Lys417 and Asn501, whereas most RBS-B and RBS-C antibodies contact Glu484, and most RBS-C antibodies interact with Leu452. We also examined the buried surface area (BSA) of Lys417, Glu484, and Asn501 upon interaction with these RBD-targeting antibodies (fig. S3C). The extensive BSA confirmed why mutations at positions 417 and 484 affect binding and neutralization. Antibodies targeting RBS-D, or the cross-neutralizing S309 and CR3022 sites, are minimally or not involved in interactions with these four RBD mutations (Fig. 2A and fig. S3C).

Fig. 2 Antibody binding to the SARS-CoV-2 RBS.

(A) Antibodies making contact with RBD residues Lys417, Glu484, and Asn501 are represented by blue, red, and yellow boxes, respectively (cutoff distance = 4 Å). Antibodies encoded by the most frequently elicited IGHV3-53/3-66 and IGHV1-2 in convalescent patients are shown in green and orange boxes, respectively. Antibodies are ordered by epitopes originally classified in (9) with an additional epitope, RBS-D, that maps to a region in the RBS above or slightly overlapping with the S309 site. Details of the epitope classifications are shown in fig. S3A. Structures of RBD-targeting antibodies that were isolated from patients are analyzed (91). (B and C) Residues that are mutated in recently circulating variants are integral to the binding sites of IGHV3-53 antibodies. Representative structures are shown for IGHV3-53 binding mode 1 [CC12.1 (PDB 6XC3), CC12.3 (PDB 6XC4) (13), and COVA2-04 (PDB 7JMO) (75)] (B) and binding mode 2 [COVA2-39 (PDB 7JMP) (75)] (C). The SARS-CoV-2 RBD is in white and Fabs in different colors. Residues Lys417 and Glu484 are represented by blue and red spheres, respectively. Hydrogen bonds and salt bridges are represented by black dashed lines.

” data-icon-position=”” data-hide-link-title=”0″>

Fig. 2 Antibody binding to the SARS-CoV-2 RBS.

(A) Antibodies making contact with RBD residues Lys417, Glu484, and Asn501 are represented by blue, red, and yellow boxes, respectively (cutoff distance = 4 Å). Antibodies encoded by the most frequently elicited IGHV3-53/3-66 and IGHV1-2 in convalescent patients are shown in green and orange boxes, respectively. Antibodies are ordered by epitopes originally classified in (9) with an additional epitope, RBS-D, that maps to a region in the RBS above or slightly overlapping with the S309 site. Details of the epitope classifications are shown in fig. S3A. Structures of RBD-targeting antibodies that were isolated from patients are analyzed (91). (B and C) Residues that are mutated in recently circulating variants are integral to the binding sites of IGHV3-53 antibodies. Representative structures are shown for IGHV3-53 binding mode 1 [CC12.1 (PDB 6XC3), CC12.3 (PDB 6XC4) (13), and COVA2-04 (PDB 7JMO) (75)] (B) and binding mode 2 [COVA2-39 (PDB 7JMP) (75)] (C). The SARS-CoV-2 RBD is in white and Fabs in different colors. Residues Lys417 and Glu484 are represented by blue and red spheres, respectively. Hydrogen bonds and salt bridges are represented by black dashed lines.

IGHV3-53/3-66 RBD antibodies can adopt two different binding modes (9, 10), which we refer here to as binding modes 1 and 2 (74), with distinct epitopes and approach angles (Fig. 2B and fig. S4). All IGHV3-53/3-66 RBD antibodies to date with binding mode 1 have a short, heavy-chain complementarity-determining region 3 (CDR H3) of <15 amino acids (Kabat numbering) and bind RBS-A (13, 16, 32), whereas those with binding mode 2 have a longer CDR H3 (≥15 amino acids) and target RBS-B (9, 10, 75). These dual binding modes enhance recognition of this antibody family for the SARS-CoV-2 RBD, although most IGHV3-53/3-66 RBD antibodies adopt binding mode 1 (Fig. 2 and fig. S4). Lys417 is a key epitope residue for antibodies with IGHV3-53/3-66 binding mode 1 (Fig. 2B and fig. S4). IGHV3-53 germline residues VH Tyr33 and Tyr52 make hydrophobic interactions with the aliphatic moiety of Lys417, and its ε-amino group interacts with CDR H3 through a salt bridge (Asp97 or Glu97), hydrogen bond (H-bond), or cation-π interaction (Phe99) (Fig. 2B). K417N/T would diminish such interactions and therefore affect antibody binding and neutralization, providing a structural explanation for K417N escape in IGHV3-53/3-66 antibodies with binding mode 1 (Fig. 1, C and D, Fig. 2B, and fig. S2). In contrast, IGHV3-53 antibodies with binding mode 2 do not interact with RBD-Lys417 (fig. S4) but with Glu484 through H-bonds with CDR H2 (Fig. 2C). Consistently, binding and neutralization of IGHV3-53 antibodies with binding mode 2 (fig. S4) are abolished by E484K but not K417N (Fig. 1, C and D, and fig. S2). Interestingly, unlike most IGHV3-53 antibodies that are sensitive to K417N/T or E484K, a recently discovered IGHV3-53-encoded mAb-222, which binds RBS, retains activity against P.1 and B.1.351. The mAb-222 light chain could largely restore the neutralization potency of other IGHV3-53 antibodies, which suggests that light-chain interactions can compensate for loss of binding of K417N/T by the heavy chain (63). However, this antibody may represent only a small portion of IGHV3-55/3-66 antibodies that can neutralize VOCs.

Among the IGHV genes used in RBD antibodies, IGHV1-2 is also highly enriched over the baseline frequency in the antibody repertoire of healthy individuals (76) and is second only to IGHV3-53/3-66 (Fig. 1B). We compared three structures of IGHV1-2 antibodies, namely 2-4 (27), S2M11 (30), and C121 (10), that target RBS-B. Despite being encoded by different IGK(L)V genes, 2-4 (IGLV2-8), S2M11 (IGKV3-20), and C121 (IGLV2-23) share a nearly identical binding mode and epitope (Fig. 3A). Structural analysis reveals that the VH Gly26-Tyr-Thr-Phe-Thr-Gly-(Tyr)-Tyr33, Trp50-(Ile)-Asn/Ser-(Pro)-X-Ser-X-Gly-Thr57, and Thr73-Ser-(Ile)-Ser/Thr76 motifs are important for RBD binding (fig. S5, A to D). Although only a small part of the epitope interacts with the light chains of 2-4, S2M11, and C121, VL residues 32 and 91 (and also residue 30 in some antibodies) play an important role in forming a hydrophobic pocket together with VH residues for binding RBD-Phe486, which is another key binding residue in such classes of antibodies (9) (fig. S5, E to I). Three other IGHV1-2 antibodies, 2-43, 2-15, and H4, also bind in a similar mode (77), further highlighting the structural convergence of IGHV1-2 antibodies in targeting the same RBD epitope. All IGHV1-2 antibodies to date form extensive interactions with Glu484 (Fig. 3A and fig. S3C). In particular, germline-encoded VH Tyr33, Asn52 (somatically mutated to Ser52 in C121), and Ser54 are involved in polar interactions with the RBD-Glu484 side chain that would be altered by substitution with Lys (Fig. 3A) and thereby diminish binding and neutralization of IGHV1-2 antibodies against E484K (Fig. 1, C and D, and fig. S2).

Fig. 3 Glu484 is critical for RBD recognition of IGHV1-2 antibodies.

(A) Binding mode 1; (B) binding mode 2. Heavy and light chains of antibody 2-4 (PDB 6XEY) (27) are shown in pink and light pink, respectively; S2M11 (PDB 7K43) (30) in orange and yellow; C121 (PDB 7K8X) (10) in dark and light green; and CV05-163 in cyan and light cyan. The RBD is shown in white. Glu484 and Lys417 are highlighted as red and blue spheres, respectively. Hydrogen bonds are represented by dashed lines; hydrogen bonds are not shown for C121 because of limited resolution (3.9 Å).

” data-icon-position=”” data-hide-link-title=”0″>

Fig. 3 Glu484 is critical for RBD recognition of IGHV1-2 antibodies.

(A) Binding mode 1; (B) binding mode 2. Heavy and light chains of antibody 2-4 (PDB 6XEY) (27) are shown in pink and light pink, respectively; S2M11 (PDB 7K43) (30) in orange and yellow; C121 (PDB 7K8X) (10) in dark and light green; and CV05-163 in cyan and light cyan. The RBD is shown in white. Glu484 and Lys417 are highlighted as red and blue spheres, respectively. Hydrogen bonds are represented by dashed lines; hydrogen bonds are not shown for C121 because of limited resolution (3.9 Å).

We previously isolated another potent IGHV1-2 antibody, CV05-163, targeting the SARS-CoV-2 RBD (fig. S6) from a COVID-19 patient (17). CV05-163 likely represents a shared antibody response for IGHV1-2 RBD antibodies across patients (fig. S7). Negative-stain electron microscopy (nsEM) of CV05-163 in complex with the SARS-CoV-2 S trimer illustrates that it can bind in various stoichiometries, including molar ratios of 1:1, 2:1, and 3:1 (Fab to S protein trimer), and can accommodate RBDs in both up and down conformations (fig. S8). We also determined a crystal structure of Fab CV05-163 with SARS-CoV-2 RBD and Fab CR3022 to 2.25 Å resolution (Fig. 3B, figs. S9 to S11, and tables S1 and S2) and found that it does indeed bind RBS-B (Fig. 3) and makes extensive interactions with Glu484 through H-bonds (VH Trp50 and VH Asn58) and a salt bridge (VL Arg91) (Fig. 3B); these interactions explain why CV05-163 binding and neutralization were diminished with E484K (Fig. 1, C and D, and fig. S2). However, CV05-163 is rotated 90° (Fig. 3B) relative to IGHV1-2 antibodies 2-4, S2M11, and C121 (Fig. 3A). Thus, IGHV1-2 antibodies, akin to IGHV3-53/66 (75), can engage the RBD in two different binding modes, both of which are susceptible to escape by E484K but not by K417N (Fig. 1, C and D).

A further group of antibodies target the back side of the RBS ridge (RBS-C) (9). To date, five nAbs isolated from COVID-19 patients are known to bind RBS-C: CV07-270 (17), BD-368-2 (38), P2B-2F6 (18), C104 (10), and P17 (78). These RBS-C nAbs also interact with Glu484 (Fig. 4A), mainly through an arginine in CDR H3, which suggests that E484K may have an adverse impact on RBS-C antibodies. Indeed, binding and neutralization by CV07-270 was abrogated by E484K (Fig. 1C and fig. S2). Intriguingly, these five RBS-C antibodies are encoded by five different IGHV genes (79), but they all target a similar epitope with similar angles of approach. In addition, neutralization by REGN10933, a potent antibody used for therapeutic treatment, was reduced to a lesser extent by K417N and E484K (Fig. 1C) (28). REGN10933 binds at a slightly different angle from RBS-A antibodies and other RBS-B antibodies. Lys417 then interacts with CDRs H1 and H3 of REGN10933, whereas Glu484 contacts CDR H2 (fig. S12). Overall, our results demonstrate that RBS mutations K417N and E484K can either abolish or extensively reduce the binding and neutralization of several major classes of SARS-CoV-2 RBD antibodies.

Fig. 4 Antibodies targeting other major antigenic sites are differentially affected by mutations in recent variants.

(A) Interactions between RBS-C antibodies and SARS-CoV-2 RBD. The RBD is shown in white with Glu484 and Lys417 represented as red and blue spheres, respectively. The various antibodies illustrated are in different colors. Only the variable domains are shown for clarity. Hydrogen bonds and salt bridges to Glu484 are represented by dashed lines. Published structures [PDB 6XKP (17), 7CHF (38), 7BWJ (18), 7K8U (10), and 7CWN (78)] are used to depict structures of SARS-CoV-2 RBD with CV07-270, BD-368-2, P2B-2F6, C104, and P17, respectively. The electron density for the full side chain of VH Asn52 was not well resolved in the 3.8-Å structure of C104 in complex with SARS-CoV-2 S. The full side chain is modeled here and shown as transparent sticks to illustrate a possible interaction with Glu484. (B) Cross-neutralizing antibodies to the RBD are not affected by Glu484 and Lys417 mutations. COVA1-16 targets the CR3022 cryptic site (yellow) (80) and CV38-142 targets the S309 proteoglycan site (blue) (81) to the RBD. Glycans at the Asn343 glycosylation site are represented by sticks. The RBS surface is shown in green. Glu484 and Lys417 are highlighted as red and blue spheres, respectively. (C) Neutralization of CV38-142 and COVA1-16 against SARS-CoV-2 wild type, K417N, or E484K pseudoviruses.

” data-icon-position=”” data-hide-link-title=”0″>

Fig. 4 Antibodies targeting other major antigenic sites are differentially affected by mutations in recent variants.

(A) Interactions between RBS-C antibodies and SARS-CoV-2 RBD. The RBD is shown in white with Glu484 and Lys417 represented as red and blue spheres, respectively. The various antibodies illustrated are in different colors. Only the variable domains are shown for clarity. Hydrogen bonds and salt bridges to Glu484 are represented by dashed lines. Published structures [PDB 6XKP (17), 7CHF (38), 7BWJ (18), 7K8U (10), and 7CWN (78)] are used to depict structures of SARS-CoV-2 RBD with CV07-270, BD-368-2, P2B-2F6, C104, and P17, respectively. The electron density for the full side chain of VH Asn52 was not well resolved in the 3.8-Å structure of C104 in complex with SARS-CoV-2 S. The full side chain is modeled here and shown as transparent sticks to illustrate a possible interaction with Glu484. (B) Cross-neutralizing antibodies to the RBD are not affected by Glu484 and Lys417 mutations. COVA1-16 targets the CR3022 cryptic site (yellow) (80) and CV38-142 targets the S309 proteoglycan site (blue) (81) to the RBD. Glycans at the Asn343 glycosylation site are represented by sticks. The RBS surface is shown in green. Glu484 and Lys417 are highlighted as red and blue spheres, respectively. (C) Neutralization of CV38-142 and COVA1-16 against SARS-CoV-2 wild type, K417N, or E484K pseudoviruses.

Two other non-RBS sites that are distant from Lys417 and Glu484 have been repeatedly shown to be neutralizing sites on the SARS-CoV-2 RBD, namely the CR3022 cryptic site and the S309 proteoglycan site (9) (Fig. 1C and Fig. 4B). Antibodies from COVID-19 patients can neutralize SARS-CoV-2 by targeting the CR3022 site, including COVA1-16 (80), S304, S2A4 (31), and DH1047 (41). Recently, we isolated antibody CV38-142 that targets the S309 site (81). Antibodies targeting these two epitopes are often cross-reactive with other sarbecoviruses, as these sites are more evolutionarily conserved than the RBS. To test the effect of the K417N and E484K mutations on nAbs that target the S309 and CR3022 sites, we assessed binding and neutralization by CV38-142 and COVA1-16 to SARS-CoV-2. Both mutations have minimal effect on these antibodies (Fig. 1C and Fig. 4C).

The most potent neutralizing antibodies to SARS-CoV-2 generally tend to target the RBS (table S3), as they directly compete with receptor binding. Such RBS antibodies often interact with Lys417, Glu484, or Asn501 and are therefore sensitive to RBS mutations at these positions in the VOCs. On the other hand, antibodies targeting the CR3022 and S309 sites are often less potent but are less affected by the VOCs, as their epitopes do not contain mutated residues. In fact, recent studies have shown that sera from convalescent or vaccinated individuals can retain neutralization activity, albeit reduced, against the mutated variants (48, 50, 82), which is possibly due to antibodies targeting other epitopes including the CR3022 and S309 sites. Thus, the CR3022 and S309 sites are promising targets to avoid interference by SARS-CoV-2 mutations observed to date.

As SARS-CoV-2 continues to circulate in humans and increasing numbers of COVID-19 vaccines are administered, herd immunity to SARS-CoV-2 should be approached locally and globally. However, as with other RNA viruses, such as influenza and HIV (83), further antigenic drift is anticipated in SARS-CoV-2. Within-host antigenic drift has also been observed in an immunosuppressed COVID-19 patient who had low titers of neutralizing antibodies that allowed the emergence of N501Y and E484K mutations (84). Whereas antibody responses elicited by the wild-type lineage that initiated the COVID-19 pandemic have been well characterized in natural infection (11, 14–18, 20, 23, 26, 27, 29) and vaccination (50, 85–87), data on the immune response to VOCs are now only starting to emerge (88) and will clarify the similarity and differences in the antibodies elicited. Because SARS-CoV-2 is likely to become endemic (89), the findings here and in other recent studies can be used to fast-track the development of more broadly effective vaccines and therapeutics.

References and Notes

  1. ↵
  2. ↵
  3. ↵
  4. ↵

    V. Tchesnokova, H. Kulakesara, L. Larson, V. Bowers, E. Rechkina, D. Kisiela, Y. Sledneva, D. Choudhury, I. Maslova, K. Deng, K. Kutumbaka, H. Geng, C. Fowler, D. Greene, J. Ralston, M. Samadpour, E. Sokurenko, Acquisition of the L452R mutation in the ACE2-binding interface of Spike protein triggers recent massive expansion of SARS-Cov-2 variants. bioRxiv 432189 [preprint]. 11 March 2021.pmid:432189

  5. ↵
  6. ↵
  7. ↵
  8. ↵
  9. ↵
  10. ↵
  11. ↵
  12. ↵
  13. ↵
  14. ↵
  16. ↵
  17. ↵
  18. ↵
  20. ↵
  23. ↵
  26. ↵
  27. ↵
  28. ↵
  29. ↵
  30. ↵
  31. ↵
  32. ↵
  38. ↵
  41. ↵

  42. B. B. Banach, G. Cerutti, A. S. Fahad, C. H. Shen, M. O. de Souza, P. S. Katsamba, Y. Tsybovsky, P. Wang, M. S. Nair, Y. Huang, I. M. F. Urdániz, P. J. Steiner, M. Gutiérrez-González, L. Liu, S. N. López Acevedo, A. Nazzari, J. R. Wolfe, Y. Luo, A. S. Olia, I. T. Teng, J. Yu, T. Zhou, E. R. Reddem, J. Bimela, X. Pan, B. Madan, A. D. Laflin, R. Nimrania, K. T. Yuen, T. A. Whitehead, D. D. Ho, P. D. Kwong, L. Shapiro, B. J. DeKosky, Paired heavy and light chain signatures contribute to potent SARS-CoV-2 neutralization in public antibody responses. bioRxiv 424987 [preprint]. 3 January 2021.pmid:424987

  44. ↵
  45. ↵

  47. E. Andreano, G. Piccini, D. Licastro, L. Casalino, N. V. Johnson, I. Paciello, S. D. Monego, E. Pantano, N. Manganaro, A. Manenti, R. Manna, E. Casa, I. Hyseni, L. Benincasa, E. Montomoli, R. E. Amaro, J. S. McLellan, R. Rappuoli, SARS-CoV-2 escape in vitro from a highly neutralizing COVID-19 convalescent plasma. bioRxiv 424451 [preprint]. 28 December 2020.pmid:424451

  48. ↵
  50. ↵
  57. ↵
  62. ↵
  63. ↵
  66. ↵
  68. ↵
  69. ↵
  70. ↵
  71. ↵Binding and neutralization of IGHV3-66 antibodies were also abolished by K417N, as shown by (62), where IGHV3-66 antibody CB6, which binds to RBD in IGHV3-53/3-66 binding mode 1 (fig. S4), was not able to bind or neutralize K417N and B.1.351 or P.1.
  72. ↵The epitopes have been assigned on their interaction with a single RBD. Quaternary epitopes are not considered in these assignments.
  73. ↵Although the antibodies structurally characterized to date do not represent all of the antibodies in a polyclonal response, they do represent major families of antibodies that have been found in the sera of convalescent SARS-CoV-2 patients.
  74. ↵We originally referred these IGHV3-53/3-66 binding modes as “A” and “B” in (9) and (75). To distinguish from “RBS-A” and “RBS-B”, these IGHV3-53/3-66 binding modes are referred to as “IGHV3-53/3-66 binding mode 1” and “2” in this study. Likewise, IGHV1-2 antibodies approach the RBD in two binding modes, which are referred as “IGHV1-2 binding mode 1” and “2”. The definitions of the binding modes in these two germline-encoded antibodies differ from “class 1” and “class 2” in (10), which were defined as ACE2-blocking antibodies that bind to “up-RBD only” and to “up/down RBD”, respectively.
  75. ↵
  76. ↵
  77. ↵
  78. ↵
  79. ↵RBS-C antibodies CV07-270, BD-368-2, P2B-2F6, C104, and P17 are encoded by IGHV3-11, IGHV3-23, IGHV4-38-2, IGHV4-34, and IGHV3-30, respectively.
  80. ↵
  81. ↵
  82. ↵

    K. Wu, A. P. Werner, J. I. Moliva, M. Koch, A. Choi, G. B. E. Stewart-Jones, H. Bennett, S. Boyoglu-Barnum, W. Shi, B. S. Graham, A. Carfi, K. S. Corbett, R. A. Seder, D. K. Edwards, mRNA-1273 vaccine induces neutralizing antibodies against spike mutants from global SARS-CoV-2 variants. bioRxiv 427948 [preprint]. 25 January 2021.pmid:427948

  83. ↵
  84. ↵
  85. ↵
  87. ↵
  88. ↵
  89. ↵
  90. ↵
  91. ↵Structures used for the analysis: CC12.1 (PDB: 6XC3), CC12.3 (6XC4), COVA2-04 (7JMO), B38 (7BZ5), CB6 (7C01), CV30 (6XE1), C105 (6XCN), BD-236 (7CHB), BD-604 (7CH4), BD-629 (7CH5), C102 (7K8M), C1A-B3 (7KFW), C1A-C2 (7KFX), C1A-B12 (7KFV), C1A-F10 (7KFY), P4A1 (7CJF), P2C-1F11 (7CDI), LY-CoV481 (7KMI), LY-CoV488 (7KM8), 910-30 (7KS9), 222 (7NX6), S2H14 (7JX3), COVA2-39 (7JMP), C144 (7K90), BD23 (7BYR), 2-4 (6XEY), CV07-250 (6XKQ), REGN10933 (6XDG), C121 (7K8X), C002 (7K8S), P2C-1A3 (7CDJ), S2E12 (7K4N), S2M11 (7K43), S2H13 (7JV2), CT-P59 (7CM4), LY-CoV555 (7L3N), BD-368-2 (7CHH), P2B-2F6 (7BWJ), CV07-270 (6XKP), C104 (7K8U), P17 (7CWO), C110 (7K8V), C119 (7K8W), REGN10987 (6XDG), CR3022 (6W41), COVA1-16 (7JMW), EY6A (6ZDG), S304 (7JW0), S2A4 (7JVA), DH1047 (7LD1), S309 (6WPS), C135 (7K8Z), and CV38-142 (7LM8). The paratope residues of the C104 3.8-Å structure that were truncated due to weak electron density were modeled as full side chains before performing calculations.
  92. ↵

  108. Y. Guo et al., A SARS-CoV-2 neutralizing antibody with exceptional spike binding coverage and optimized therapeutic potentials. Research Square [preprint]. 14 November 2020.pmid:78945

  110. ↵
Acknowledgments: We thank H. Tien for technical support with the crystallization robot, J. Matteson and Y. Hua for their contributions to mammalian cell culture, W. Yu for insect cell culture, and R. Stanfield for assistance in data collection. Funding: Supported by Bill and Melinda Gates Foundation grants OPP1170236 and INV-004923 INV (I.A.W., A.B.W., and D.R.B.); NIH grants R00 AI139445 (N.C.W.), NIH P01 AI110657 (I.A.W., A.B.W., and R.W.S.), R01 AI132317 (D.N. and D.H.), and R01 AI142945 (L.P.); German Research Foundation grants PR 1274/3-1 and PR 1274/5-1, Helmholtz Association grants HIL-A03 and SO-097, and German Federal Ministry of Education and Research Connect-Generate grant 01GM1908D (H.P.); and a Vici fellowship from the Netherlands Organisation for Scientific Research (NWO) (R.W.S.). This research used resources of the Advanced Photon Source, a US Department of Energy (DOE) Office of Science User Facility, operated for the DOE Office of Science by Argonne National Laboratory under contract DE-AC02-06CH11357. Extraordinary facility operations were supported in part by the DOE Office of Science through the National Virtual Biotechnology Laboratory, a consortium of DOE national laboratories focused on the response to COVID-19, with funding provided by the Coronavirus CARES Act. Author contributions: M.Y., D.H., C.-C.D.L., N.C.W., and I.A.W. conceived and designed the study; M.Y., C.-C.D.L., N.C.W., and H.L. expressed and purified the proteins for crystallization; S.M.R., H.P., and J.K. provided CV05-163 and other antibody clones and sequences; M.J.v.G., R.W.S., and D.R.B. provided plasmids for some of the antibodies reported in (11, 20), respectively; M.Y. and X.Z. performed the crystallization and x-ray data collection and determined and refined the x-ray structures; D.H., L.P., and D.N. performed the neutralization assays; M.Y. and C.-C.D.L. carried out the binding assays; A.M.J. and A.B.W. provided nsEM data and performed reconstructions; M.Y., C.-C.D.L., N.C.W., and I.A.W. wrote the paper; and all authors reviewed and/or edited the paper. Competing interests: Related to this work, the German Center for Neurodegenerative Diseases (DZNE) and Charité–Universitätsmedizin Berlin previously filed a patent application that included anti-SARS-CoV-2 antibody CV05-163 first reported in (17). Data and materials availability: The x-ray coordinates and structure factors have been deposited to the RCSB Protein Data Bank under accession code 7LOP. The EM maps have been deposited in the Electron Microscopy Data Bank (EMDB) under accession codes EMD-23466 (one bound), EMD-23467 (two bound), and EMD-23468 (three bound). This work is licensed under a Creative Commons Attribution 4.0 International (CC BY 4.0) license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. To view a copy of this license, visit https://creativecommons.org/licenses/by/4.0/. This license does not apply to figures/photos/artwork or other content included in the article that is credited to a third party; obtain authorization from the rights holder before using such material.

antigenicdriftfunctionalramificationsSARSCoV2Structuralvariants
Share
Related Posts

New insights into acoustic bubbles give boost to future applications

T cells’ capability to fully prevent acute viral infections opens new avenues for vaccine development

‘What is that?’ Scientists explain white patch that appears near northern lights

A fast-moving belly flop: Researchers unveil the unique skills of cricket frogs

Harnessing microwave flow reaction to convert biomass into useful sugars

Cleopatra’s sister remains missing | ScienceDaily

11 January 2025 Horoscope: What Is the Zodiac Sign of People Celebrating Birthday Today? Know the Sun Sign, Lucky Colour and Number Prediction
Comments (0)
Add Comment