Structural basis of a shared antibody response to SARS-CoV-2

A common theme in antibody responses In the fight against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), antibodies are a key tool, both as potential therapeutics and to guide vaccine development. Yuan et al. focused on finding shared antibody responses, in which multiple individuals develop antibodies against the same antigen using the same genetic elements and modes of recognition. The authors identified the immunoglobulin heavy-chain variable region 3-53 gene as the most frequently used among 294 antibodies that target the receptor-binding domain (RBD) of the viral spike protein. These antibodies have few somatic mutations, and crystal structures of two neutralizing antibodies bound to the RBD show that mostly germline-encoded residues are involved in binding. The minimal affinity maturation and high potency of these antibodies is promising for vaccine design. Science, this issue p. 1119

T he ongoing coronavirus disease 2019 (COVID-19) pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has resulted in enormous global health and socioeconomic damage and requires urgent development of an effective vaccine (1). Although multiple vaccine candidates have entered clinical trials (2), the molecular features that contribute to an effective antibody response are not clear. Shared antibody responses to specific microbial pathogens have been found in which the same genetic elements and modes of recognition are observed in multiple individuals against a given antigen. Such responses to microbial pathogens have been observed against influenza (3), dengue (4), malaria (5), and HIV (6). Characterization of the molecular interactions between pathogens and cognate antigen can provide insight into how the immune repertoire is able to quickly respond to novel microbial pathogens and will facilitate the rational design of vaccines against them (7,8).
To understand the molecular features that endow IGHV3-53 with favorable properties for RBD recognition, we determined the crystal structures of two IGHV3-53-neutralizing antibodies, CC12.1 and CC12.3, in complex with the SARS-CoV-2 RBD and with the cross-reactive Fab CR3022 to SARS-like CoVs (17). CC12.1 and CC12.3 were previously isolated from a SARS-CoV-2-infected patient and were shown to be specific for the RBD (27). CC12.1 and CC12.3 [median inhibitory concentration (IC 50 ), 20 ng/ml] were among the top four highly potent neutralizing antibodies in the panel of antibodies assayed against live replicating SARS-CoV-2 virus and pseudovirus (27). Although CC12.1 and CC12.3 are both encoded by IGHV3-53, CC12.1 uses IGHJ6, IGKV1-9, and IGKJ3, whereas CC12.3 uses IGHJ4, IGKV3-20, and IGKJ1. This variation in IGHJ, IGKV, and IGKJ usage indicates that CC12.1 and CC12.3 belong to different clonotypes but are encoded by a common IGHV3-53 germline gene ( fig. S2). IgBlast analysis (32) showed that IGHV and IGKV of CC12.1 have acquired only four amino acid changes (somatic mutations) during affinity maturation from the original germline antibody sequence ( fig. S2, A and B). Similarly, CC12.3 is also minimally somatically mutated, with three amino acid changes in IGHV and a single amino acid deletion in IGKV  (23). Epitope residues contacting the heavy chain are shown in orange and those contacting the light chain are shown in yellow. CDR loops are labeled in the left panels; epitope residues are labeled in the right panels. For clarity, only representative epitope residues are labeled. Epitope residues that are also involved in ACE2 binding are shown in red.
(D) ACE2-binding residues are shown in blue. ACE2 is shown in green in the left panel in a semitransparent cartoon representation. ACE2-binding residues are labeled in the right panel. A total of 17 residues were used for ACE2 binding (12), but only 15 are labeled here because the other two are at the back of the structure in this view and do not interact with the antibodies here. (E) Epitope residues for CC12.1, CC12.3, and B38 were identified by PISA (41) and annotated on the SARS-CoV-2 RBD sequence, which is aligned to the SARS-CoV RBD sequence with nonconserved residues highlighted. The 17 ACE2-binding residues were identified from a SARS-CoV-2 RBD-ACE2 complex structure as described previously (12).
To understand why IGHV3-53 is elicited as a shared antibody response, the molecular interactions between the RBD and the heavy chains of CC12.1, CC12.3, and B38 were analyzed. The complementarity-determining regions (CDRs) H1 and H2 of these antibodies interact extensively with the RBD mainly through specific hydrogen bonds (Fig. 3, A and B). All residues on CDR H1 and H2 that hydrogen bond with the RBD are encoded by the germ line IGHV3-53 ( fig. S2 and table S3). These interactions are almost identical among CC12.1, CC12.3, and B38, with the only difference at the variable region of immunoglobulin heavy chain (V H ) residue 58. A somatic mutation V H Y58F in CC12.1 and CC12.3, but not in B38 (Fig. 3, A to C, boxed residues, and fig. S9), results in similar van der Waals interactions, with only a loss of a single hydrogen bond from the hydroxyl of the germ line Tyr in B38 to the RBD (Fig. 3C).
Our structural analysis reveals two key motifs in the IGHV3-53 germline sequence that are important for RBD binding: an NY motif at V H residues 32 and 33 in the CDR H1 and an SGGS motif at V H residues 53 to 56 in the CDR H2 ( Fig. 3 and fig. S10). The side chain of V H N32 in the NY motif hydrogen bonds with the backbone carbonyl of A475 on the RBD, and this interaction is stabilized by an extensive network of hydrogen bonds with other antibody residues as well as a bound water molecule (Fig. 4A). V H N32 also hydrogen bonds with V H R94, which in turn hydrogen bonds with N487 and Y489 on the RBD (Fig. 4A).
These polar contacts not only enhance the RBD-Fab interaction but also stabilize the CDR conformations with the surrounding residues (framework). V H Y33 in the NY motif inserts into a hydrophobic cage formed by RBD residues Y421, F456, and L455 and the aliphatic component of K417 (Fig. 4B). A hydrogen bond between V H Y33 and the carbonyl oxygen of L455 on the RBD further strengthens the interaction. The second key motif, SGGS, in CDR H2 forms an extensive hydrogen bond network with the RBD (Fig. 4C), including four hydrogen bonds that involve the hydroxyl side chains of V H S53 and V H S56 and four water-mediated hydrogen bonds to the backbone carbonyl of V H G54, the backbone amide of V H S56, and the 3 of 5 side chain of V H S56. Along with V H Y52, the SGGS motif takes part in a type I beta turn, with a positive F angle for V H G55 at the end of the turn. In addition, the Ca of V H G54 is only 4 Å away from the RBD, indicating that side chains of other amino acids would clash with the RBD if they were present at this position.
The NY and SGGS motifs, important for RBD binding, are both encoded in the IGHV3-53 germline gene. In addition to IGHV3-53, only the closely related IGHV3-66 contains an NY motif in CDR H1 and an SGGS motif in CDR H2 (33). IGHV3-66 is commonly observed (19-22, 24, 26) in antibodies in SARS-CoV-2 patients (Fig. 1A) and is also well represented in the repertoire of healthy individuals (0.3 to 1.7% of total antibodies) (29). Overall, our structural analysis has identified two germline-encoded binding motifs that enable IGHV3-53 to target the SARS-CoV-2 RBD, with mutations apparently not required from affinity maturation.
Although the binding mode of CDR H1 and H2 to RBD is highly similar among CC12.1, CC12.3, and B38, the interaction of CDR H3 with the RBD varies (Fig. 3, A to C) because of differences in the CDR H3 sequences and conformations ( fig. S1 and Fig. 4D). For example, whereas CDR H3 of CC12.1 interacts with RBD Y453 through a hydrogen bond, CDR H3 of CC12.3 and B38 do not form such a bond (Fig. 3, A to C). Similarly, because of the dif-ference in light-chain gene usage, light-chain interactions with the RBD can vary substantially in IGHV3-53 antibodies ( fig. S11). Overall, our structural analysis demonstrates that IGHV3-53 provides a versatile framework with which to target the ACE2-binding site in SARS-CoV-2 RBD.
In addition to IGHV3-53, several other IGHV genes, such as IGHV1-2, IGHV3-9, and IGHV3-30, are also more frequently observed than other germ lines in SARS-CoV-2 RBD-targeting antibodies (Fig. 1A). Future work will investigate the molecular mechanisms of these IGHV responses to SARS-CoV-2, as well as whether other germline gene segments, including IGHD and the light chain, contribute in recurring motifs to the SARS-CoV-2 antibody response. The characterization of these IGHV3-53 antibodies to SARS-CoV-2 is a promising starting point for rational vaccine design (39), given that limited to no affinity maturation is required to achieve a highly potent neutralizing antibody response to the RBD. Because IGHV3-53 is found at a reasonable frequency in healthy individuals (29,30), this particular antibody response could be commonly elicited during vaccination (40).  View/request a protocol for this paper from Bio-protocol.