Structures of SARS-CoV-2 B.1.351 neutralizing antibodies provide insights into cocktail design against concerning variants

The spread of the SARS-CoV-2 variants could seriously dampen the global effort to tackle the COVID-19 pandemic. Recently, we investigated the humoral antibody responses of SARS-CoV-2 convalescent patients and vaccinees towards circulating variants, and identified a panel of monoclonal antibodies (mAbs) that could efficiently neutralize the B.1.351 (Beta) variant. Here we investigate how these mAbs target the B.1.351 spike protein using cryo-electron microscopy. In particular, we show that two superpotent mAbs, BD-812 and BD-836, have non-overlapping epitopes on the receptor-binding domain (RBD) of spike. Both block the interaction between RBD and the ACE2 receptor; and importantly, both remain fully efficacious towards the B.1.617.1 (Kappa) and B.1.617.2 (Delta) variants. The BD-812/BD-836 pair could thus serve as an ideal antibody cocktail against the SARS-CoV-2 VOCs.

information, Figs. S1-S3 and Table S1). In addition, local refinements were performed to further improve the densities around the Fab and RBD regions, yielding local maps of 3.3, 3.4, and 3.2 Å, respectively, which enabled us to construct the structure models unambiguously. We also solved the cryo-EM structures of two binary complexes: BD-667/S6P(B.  Table S1). Together, these cryo-EM structures provide detailed structural information for eight SARS-CoV-2 B.1.351 neutralizing antibodies (Supplementary information, Video S1).
Dejnirattisai et al. 8 dissected the antigenic anatomy of RBD by analogizing it to a human torso, and divided the antibody-binding sites into six clusters, namely left shoulder, neck, right shoulder, left flank, chest, and right flank (Fig. 1b). We adopt this nomenclature here for ease of description. The two superpotent mAbs, BD-812 and BD-836, bind to the right and left shoulders of RBD in a non-interfering manner (Fig. 1c), and bury 788 Å 2 and 799 Å 2 surface areas on RBD, respectively. Among the prominent interactions between RBD and BD-812, RBD residues Leu441, Val445, Pro499, and Thr500 are targeted by BD-812 via hydrophobic and van der Waals interactions; whereas Arg346, Lys444, as well as the main chain groups of Val445 and Thr500 are probed by salt bridge and hydrogen bond interactions (Supplementary information, Fig. S6a). As for the interaction between RBD and BD-836, RBD residues Phe456, Ala475, Phe486, and Tyr489 mediate strong nonpolar interactions with BD-836; whereas Tyr473, Thr478, Asn487, Gln493, and the main chain group of Val483 are engaged in hydrogen bond contacts (Supplementary information, Fig. S6b). Notably, the epitope of BD-812 involves neither Leu452 nor Thr478. As expected, BD-812 effectively neutralized both the B.1.617.1 and B.1.617.2 variants that contain the L452R mutation (Fig. 1a). Leu452 is not targeted by BD-836 either, whereas Thr478 interacts with an acid residue in the CDRH3 of BD-836 (Asp108; Supplementary information, Fig. S6b). Due to the presence of this negatively charged residue in BD-836, the T478K mutation did not adversely affect its activity. Indeed, BD-836 also potently neutralized both B.1.617.1 and B.1.617.2 (Fig. 1a).
RBD-directed SARS-CoV-2 neutralizing antibodies can be classified into four classes depending on whether they structurally compete with ACE2 and whether they can bind to the 'down' RBDs in the prefusion state of the spike protein: class 1 and 2 antibodies directly block ACE2, whereas class 3 and 4 bind outside of ACE2 site; class 2 and 3 can bind to RBDs regardless of their 'up' and 'down' conformations, whereas class 1 and 4 can only gain access to the 'up' RBDs. 9 BD-812 and BD-836 would both clash with ACE2 (Supplementary information, Fig. S6c). Structural comparisons with the spike prefusion state (PDB ID: 6VSB) 10 suggest that BD-812 can engage both the 'up' and 'down' RBDs, whereas BD-836's binding to a 'down' RBD will be sterically impeded by an adjacent RBD in the spike trimer (Supplementary information, Fig. S6d, e). Therefore, BD-812 and BD-836 belong to class 2 and 1 RBD-neutralizing antibodies, respectively (Fig. 1a).
pair, BD-821 appears to be able to interact with both the 'up' and 'down' RBDs, whereas BD-771's binding to a 'down' RBD is sterically prohibited (Supplementary information, Fig. S7d, e).
Unlike the five antibodies described above, the epitope of BD-744 has no overlap with the ACE2-binding site ( Fig. 1e and Supplementary information, Fig. S8a). BD-744 occupies the front chest of RBD, and mainly uses its VH domain to target RBD. Several β-strands in the VH domain, in addition to the CDR loops, are uniquely employed to interact with RBD, which allows BD-744 to pack on RBD in an almost parallel fashion and attack a rather flat surface area. Major RBD residues that are targeted by BD-744 include Glu340, Thr345, Arg346, Asn354, Tyr449, Asn450, Leu452, Arg466, Ile468, Thr470, Phe490, and Leu492 (Supplementary information, Fig. S8d). Arg346 also forms a salt bridge with an Asp (Asp94) in the VL domain, representing the only interaction between RBD and the light chain of BD-744. If bound to a 'down' RBD, BD-744 would clash with a neighboring NTD slightly, and it is unclear whether this structural arrangement is compatible with the prefusion state of the spike trimer (Supplementary information, Fig. S8e). BD-744 does not interact with Thr478; however, Leu452 is located in the center of BD-744's epitope and is involved in hydrophobic interaction with several BD-744 residues (Supplementary information, Fig. S8d). Consequently, BD-744 displayed significantly reduced activity towards the B.1.617.1 and B.1.617.2 variants due to the L452R mutation (Fig. 1a).
We also characterized two other mAbs that do not compete with ACE2: BD-667 and BD-804. Like BD-744, BD-667 engages the front chest of RBD ( Fig. 1f and Supplementary information,  Fig. S9a); but unlike BD-744 that runs parallel to RBD, BD-667 docks on RBD in a perpendicular manner. The epitope of BD-667 largely overlaps with that of BD-744, mainly consisting of RBD residues Glu340, Thr345, Arg346, Asn354, Arg355, Arg357, Tyr449, Asn450, Leu452, Arg466, Thr470, Lys484, and Phe490 (Supplementary information, Fig. S9b). The unusually long CDRH3 of BD-667 (22 residues) forms a long hairpin to attach to the lower chest of RBD, and at the same time contacts a neighboring NTD (Supplementary information, Fig. S9c). It is also unclear whether this interaction can occur on a 'down' RBD without disrupting the prefusion state of the spike trimer (Supplementary information,  Fig. S9d). BD-804, on the other hand, targets the upper chest of RBD ( Fig. 1g and Supplementary information, Fig. S10a), and its epitope is fully exposed irrespective of the 'up' or 'down' states of RBD (Supplementary information, Fig. S10b). RBD residues that BD-804 recognizes include Thr345, Arg346, Tyr351, Leu441, Lys444, Val445, Tyr449, Asn450, Leu452, Thr470, Gly482, Lys484, and Phe490 (Supplementary information, Fig. S10c). The light chain of BD-804 contributes more than its heavy chain to interact with RBD, burying 660 Å 2 RBD surface compared to 459 Å 2 buried by VH. Furthermore, the VL domain also uniquely interacts with a glycan moiety attached on Asn165 in an adjacent NTD to gain additional binding strength (Supplementary information,  Fig. S10d). Similar to BD-744, the epitopes of both BD-667 and BD-804 involve Leu452; and the B.1.617.1 and B.1.617.2 variants showed substantial escapes from these mAbs (Fig. 1a). Obviously, the L452R mutation would nullify the neutralization effect of many mAbs that target the RBD front chest.
In summary, we have characterized eight SARS-CoV-2 B.1.351 neutralizing antibodies using cryo-EM. Structural analyses suggest that five of them directly antagonize the binding of ACE2, whereas the neutralization mechanisms of the other three do not depend on ACE2 blocking. Together with BD-515 and BD-623 that we described previously, 4 we have collected ten neutralizing antibodies that can potentially serve as prophylactics and therapeutics for SARS-CoV-2 B.1.351 (Fig. 1h). Many of these antibodies have non-overlapping epitopes, and multiple combination strategies can be designed based on their structural information (Fig. 1i)