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

Molecular-level understanding of human neutralizing antibody responses to SARS-CoV-2 could accelerate vaccine design and facilitate drug discovery. We analyzed 294 SARS-CoV-2 antibodies and found that IGHV3–53 is the most frequently used IGHV gene for targeting the receptor binding domain (RBD) of the spike (S) protein. We determined crystal structures of two IGHV3–53 neutralizing antibodies +/− Fab CR3022 ranging from 2.33 to 3.11 Å resolution. The germline-encoded residues of IGHV3–53 dominate binding to the ACE2 binding site epitope with no overlap with the CR3022 epitope. Moreover, IGHV3–53 is used in combination with a very short CDR H3 and different light chains. Overall, IGHV3–53 represents a versatile public VH in neutralizing SARS-CoV-2 antibodies, where their specific germline features and minimal affinity maturation provide important insights for vaccine design and assessing outcomes.


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Similar to the ACE2 binding site (11), the epitopes of these antibodies can only be 93 accessed when the RBD is in the "up" conformation ( Fig. S5). Among 16 ACE2 binding 94 residues on RBD, 10 are within the epitopes of CC12.1 and B38, and 6 are in the 95 epitope of CC12.3 ( Fig. 2A-D). Many of the epitope residues are not conserved between 103 Given that CC12.3 (80% BSA from the heavy chain) binds the RBD with similar affinity to 104 CC12.1 (56% BSA from heavy chain) (Fig. S2), the light-chain identity seems not to be 105 as critical as the heavy chain. In fact, among the RBD-targeting IGHV3-53 antibodies, 106 nine different light chains are observed, although IGKV1-9 and IGKV3-20 are the most 107 frequently found to date (Fig. S6).

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To understand why IGHV3-53 is elicited as a public antibody response, the molecular 110 interactions between the RBD and the heavy chains of CC12.1, CC12.3, and B38 were 111 analyzed. The complementarity-determining regions (CDR) H1 and H2 of these 112 antibodies interact extensively with the RBD mainly through specific hydrogen bonds 113 ( Fig. 3A-B). Interestingly, all residues on CDR H1 and H2 that hydrogen bond with the 114 RBD are encoded by the germline IGHV3-53 ( Fig. S1 and S7, Table S3). These 115 . CC-BY 4.0 International license was not certified by peer review) is the author/funder. It is made available under a The copyright holder for this preprint (which this version posted June 9, 2020. . https://doi.org/10.1101/2020.06.08.141267 doi: bioRxiv preprint 6 interactions are almost identical among CC12.1, CC12.3, and B38 with the only 116 difference at VH residue 58. A somatic mutation VH Y58F is present in both CC12.1 and 117 CC12.3, but not in B38 ( Fig. 3A-C, boxed residues). Nevertheless, this somatic mutation 118 is unlikely to be essential for IGHV3-53 to engage the RBD, since VH residue 58 in B38 119 still interacts with the RBD through an additional hydrogen bond (Fig. 3C). Of note, none 120 of these antibody interactions mimic ACE2 binding (Fig. 3D).

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Our structural analysis reveals two key motifs in the IGHV3-53 germline sequence that 123 are important for RBD binding, namely an NY motif at VH residues 32 and 33 in the CDR 124 H1, and an SGGS motif at VH residues 53 to 56 in the CDR H2 (Fig. S8). The side chain 125 of VH N32 in the NY motif forms a hydrogen bond with the backbone carbonyl of A475 on 126 the RBD, and this interaction is stabilized by an extensive network of hydrogen bonds 127 with other antibody residues as well as a bound water molecule (Fig. 4A). VH N32 also 128 hydrogen bonds with VH R94, which in turn hydrogen bonds with N487 and Y489 on the 129 RBD (Fig. 4A). These interactions enhance not only RBD-Fab interaction, but also 130 stabilize CDR and framework residues and conformations. VH Y33 in the NY motif 131 inserts into a hydrophobic cage formed by RBD residues Y421, F456, L455 and the 132 aliphatic component of K417 (Fig. 4B). A hydrogen bond between VH Y33 and the 133 carbonyl oxygen of L455 on RBD further strengthens the interaction. The second key 134 motif SGGS in CDR H2 forms extensive hydrogen bond network with the RBD (Fig. 4C),

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including four hydrogen bonds that involve the hydroxyl side chains of VH S53 and VH 136 S56, and four water-mediated hydrogen bonds to the backbone carbonyl of VH G54, the 137 backbone amide of VH S56, and the side chain of VH S56. Along with VH Y52, the SGGS 138 motif takes part in a type I beta turn, with a positive Φ-angle for VH G55 at the end of the 139 turn. In addition, the Cα of VH G54 is only 4 Å away from the RBD, indicating that side 140 chains of other amino acids would clash with the RBD if they were present at this 141 . CC-BY 4.0 International license was not certified by peer review) is the author/funder. It is made available under a The copyright holder for this preprint (which this version posted June 9, 2020. . https://doi.org/10.1101/2020.06.08.141267 doi: bioRxiv preprint 7 position. As a result, the SGGS motif is a perfect fit for interacting with the RBD at this 142 location.

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Overall, these observations demonstrate the importance of the NY and SGGS motifs, 145 which are both encoded in the IGHV3-53 germline, for engaging the RBD. In fact, 146 besides IGHV3-53, the only other IGHV gene that contains an NY motif in CDR H1 and 147 an SGGS motif in CDR H2 is IGHV3-66, which is a closely-related IGHV gene to IGHV3-148 53 (32). As compared to IGHV3-53, IGHV3-66 has a lower occurrence frequency in the 149 repertoire of healthy individuals (0.3% to 1.7%) (29), which may explain why IGHV3-66 150 is less prevalent than IGHV3-53, but yet is still quite commonly observed (19-22, 24, 26) 151 in antibodies in SARS-CoV-2 patients (Fig. 1A). Overall, our structural analysis has 152 identified two germline-encoded binding motifs that enable IGHV3-53 to act as a public 153 antibody and target the SARS CoV-2 RBD with no mutations required from affinity 154 maturation.

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While the binding mode of CDR H1 and H2 to RBD is highly similar among CC12.1, 157 CC12.3, and B38, CDR H3 interaction with the RBD is different ( Fig. 3A-C) due to 158 differences in the CDR H3 sequences and conformations ( Fig. 5A 170 residues) on average that seem to be required for many broadly neutralizing antibodies 171 to HIV-1 (34). Similarly, antibody B38 has a very short CDR H3 length of seven residues 172 (23). It is unlikely that longer CDR H3's can be accommodated in these antibodies since 173 their epitopes are relatively flat with no large pocket to insert a protruding CDR ( Fig. 2A-

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C). This conclusion was also arrived in a recent study that also reported SARS-CoV-2 175 RBD-targeting antibodies that are encoded by either IGHV3-53 or IGHV3-66 tend to

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Besides IGHV3-53, several other IGHV genes such as IGHV1-2, IGHV3-9, and IGHV3-190 30 are also more frequently observed than other germlines in SARS-CoV-2 RBD-191 targeting antibodies (Fig. 1A). The molecular mechanisms of these antibody responses 192 . CC-BY 4.0 International license was not certified by peer review) is the author/funder. It is made available under a The copyright holder for this preprint (which this version posted June 9, 2020. . https://doi.org/10.1101/2020.06.08.141267 doi: bioRxiv preprint 9 to SARS-CoV-2 will need to be characterized in the future. In addition, whether other 193 antibody germline gene segments, including the IGHD and the light chain, contribute to 194 public antibody responses to SARS-CoV-2 will also need to be further addressed.

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Notwithstanding, the detailed characterization of this public antibody response to SARS

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CoV-2 is already be a promising starting point for rational vaccine design (36), especially 197 given limited to no affinity maturation is required from the germline to achieve a high

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. CC-BY 4.0 International license was not certified by peer review) is the author/funder. It is made available under a The copyright holder for this preprint (which this version posted June 9, 2020. . https://doi.org/10.1101/2020.06.08.141267 doi: bioRxiv preprint

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. CC-BY 4.0 International license was not certified by peer review) is the author/funder. It is made available under a The copyright holder for this preprint (which this version posted June 9, 2020.

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. CC-BY 4.0 International license was not certified by peer review) is the author/funder. It is made available under a The copyright holder for this preprint (which this version posted June 9, 2020.     16 ACE2-binding residues were as described previously (12).    The copyright holder for this preprint (which this version posted June 9, 2020. . https://doi.org/10.1101/2020.06.08.141267 doi: bioRxiv preprint

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The N-glycan observed at SARS-CoV-2 RBD N343 is shown in red.

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. CC-BY 4.0 International license was not certified by peer review) is the author/funder. It is made available under a The copyright holder for this preprint (which this version posted June 9, 2020.

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. CC-BY 4.0 International license was not certified by peer review) is the author/funder. It is made available under a The copyright holder for this preprint (which this version posted June 9, 2020. . https://doi.org/10.1101/2020.06.08.141267 doi: bioRxiv preprint 32 515 . CC-BY 4.0 International license was not certified by peer review) is the author/funder. It is made available under a The copyright holder for this preprint (which this version posted June 9, 2020. . https://doi.org/10.1101/2020.06.08.141267 doi: bioRxiv preprint CC-BY 4.0 International license was not certified by peer review) is the author/funder. It is made available under a The copyright holder for this preprint (which this version posted June 9, 2020. . https://doi.org/10.1101/2020.06.08.141267 doi: bioRxiv preprint

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e Rfree was calculated as for Rcryst, but on a test set comprising 5% of the data excluded from refinement.

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. CC-BY 4.0 International license was not certified by peer review) is the author/funder. It is made available under a The copyright holder for this preprint (which this version posted June 9, 2020. . https://doi.org/10.1101/2020.06.08.141267 doi: bioRxiv preprint

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. CC-BY 4.0 International license was not certified by peer review) is the author/funder. It is made available under a The copyright holder for this preprint (which this version posted June 9, 2020. . https://doi.org/10.1101/2020.06.08.141267 doi: bioRxiv preprint