key: cord-0689754-1j4hh2uu
authors: Yuan, Meng; Huang, Deli; Lee, Chang-Chun D.; Wu, Nicholas C.; Jackson, Abigail M.; Zhu, Xueyong; Liu, Hejun; Peng, Linghang; van Gils, Marit J.; Sanders, Rogier W.; Burton, Dennis R.; Reincke, S. Momsen; Prüss, Harald; Kreye, Jakob; Nemazee, David; Ward, Andrew B.; Wilson, Ian A.
title: Structural and functional ramifications of antigenic drift in recent SARS-CoV-2 variants
date: 2021-05-20
journal: Science
DOI: 10.1126/science.abh1139
sha: f27700eebbe3d4e2ccf24c46f68ea2f7c1dee453
doc_id: 689754
cord_uid: 1j4hh2uu

Neutralizing antibodies (nAbs) elicited against the receptor-binding site (RBS) of the spike protein of wild-type SARS-CoV-2 are generally less effective against recent variants of concern. RBS residues E484, K417 and N501 are mutated in variants first described in South Africa (B.1.351) and Brazil (P.1). We analyzed their effects on ACE2 binding and K417N and E484K mutations 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 alternate 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.

urally circulating SARS-CoV-2. Among 585,054 SARS-CoV-2 genome sequences in the GISAID database (March 5, 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) (10) (11) (12) . Certain IGHV genes are highly enriched in the antibody response to SARS-CoV-2 infection, with IGHV3-53 (11, (13) (14) (15) (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 1,593 RBD-targeting antibodies from 32 studies (11, (Fig. 1B) . We investigated the effects of the prevalent SARS-CoV-2 mutations on neutralization by these multi-donor class antibodies, 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 isolated from COVID-19 convalescent plasma and vaccinated individuals (45) (46) (47) (48) (49) (50) (51) (52) (53) (54) (55) (56) (57) (58) (59) (60) (61) (62) . B.1.351 is ~8-14 fold and P.1 is ~2.6-5 fold more resistant to neutralization by convalescent plasma and mRNA vaccinee sera (63) (64) (65) (66) (67) (68) . Some variants are able to escape neutralization by some nAbs (e.g., LY-CoV555, 910-30, COVOX-384, S2H58, C671, etc.) while 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 related to the four classes assigned in (10) (Fig. 1C ). Twenty one of 23 IGHV3-53/3-66 antibodies target RBS-A ( Fig. 2A) . All IGHV1-2 antibodies with structures to date bind to the RBS-B epitope. A large fraction of antibodies in these two main families make contact with K417, E484 or N501 in their epitopes ( Fig. 2A) (73) . Almost all RBS-A antibodies interact extensively with K417 and N501, whereas most RBS-B and RBS-C antibodies contact E484, and most RBS-C antibodies interact with L452. We also examined the buried surface area (BSA) of K417, E484, and N501 upon interaction with these RBD-targeting antibodies ( fig. S3C ). The extensive BSA confirmed why mutations at 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 ).

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 CDR H3 of <15 amino acids and bind RBS-A (13, 16, 32) , while 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 ). K417 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 Y33 and Y52 make hydrophobic interactions with the aliphatic moiety of K417, and its ε-amino group interacts with CDR H3 through a salt bridge (D97 or E97), hydrogen bond (Hbond), or cation-π interaction (F99) (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 ( ). 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, suggesting 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 (77), further highlighting structural convergence of IGHV1-2 antibodies in targeting the same RBD epitope. Importantly, all IGHV1-2 antibodies to date form extensive interactions with E484 ( Fig. 3A and fig. S3C ). In particular, germline-encoded VH Y33, N52 (somatically mutated to S52 in C121) and S54 are involved in polar interactions with the RBD-E484 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 ).

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 downconformations ( 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 E484 through H-bonds (VH W50 and V H N58) and a salt bridge (V L R91) (Fig. 3B ) that explains 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 ) compared to other 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 E484 (Fig. 4A) , mainly through an arginine in CDRH3, suggesting that E484K may adversely impact 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 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 less extent by K417N and E484K (Fig. 1C) (28) . REGN10933 binds at a slightly different angle from RBS-A antibodies and other RBS-B antibodies. K417 then interacts with CDRs H1 and H3 of REGN10933, whereas E484 contacts CDRH2 (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.

Two other non-RBS sites that are distant from K417 and E484 have been repeatedly shown to be neutralizing sites on the SARS-CoV-2 RBD, namely the CR3022 cryptic site and 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 compared to 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 K417, E484, or N501, which are located in the RBS, and are therefore sensitive to RBS mutations 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. Intra-host antigenic drift has also been observed in an immunosuppressed COVID-19 patient who had low titers of neutralizing antibodies that allowed emergence of N501Y and E484K mutations (84) . While 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) (86) (87) , data on the immune response to VOCs are now only starting to emerge (88) and will inform on the similarity and differences in the antibodies elicited. Since SARS-CoV-2 is likely to become endemic (89) 

Investigation of Novel SARS-CoV-2 Variant: Variant of Concern 202012/01. Technical Briefing 5

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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. S4A), was not able to bind or neutralize K417N and B

The epitopes have been assigned on their interaction with a single RBD. Quaternary epitopes are not considered in these assignments

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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

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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 as solid bars. The frequently used IGHV3-53 and IGHV3-66 genes are highlighted in blue, and IGHV1-2 in orange. The IGHV gene usage in 1,593 SARS-CoV-2 RBD-targeting antibodies (11, 14-44) compared to healthy individuals (baseline) (76) (fold-enrichment) is shown as black lines. # : IGHV gene frequencies in healthy individuals that were not reported in (76) are shown with hashtags ( # ). *: IGHV genes that are significantly enriched over the baseline repertoire (76) (p < 0.05, one-sample proportion test with Bonferroni correction) are shown with an asterisk (*). A fold-enrichment of one (red dashed line) represents no difference over baseline. (C) Effects of single mutations on the neutralization activity and binding affinity of each neutralizing antibody

We thank Henry Tien for technical support with the crystallization robot, Jeanne Matteson and Yuanzi Hua for their contributions to mammalian cell culture, Wenli Yu for insect cell culture, and Robyn Stanfield for assistance in data collection. 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.