A natural mutation between SARS-CoV-2 and SARS-CoV determines neutralization by a cross-reactive antibody

Epitopes that are conserved among SARS-like coronaviruses are attractive targets for design of cross-reactive vaccines and therapeutics. CR3022 is a SARS-CoV neutralizing antibody to a highly conserved epitope on the receptor binding domain (RBD) on the spike protein that can cross-react with SARS-CoV-2, but with lower affinity. Using x-ray crystallography, mutagenesis, and binding experiments, we illustrate that of four amino acid differences in the CR3022 epitope between SARS-CoV-2 and SARS-CoV, a single mutation P384A fully determines the affinity difference. CR3022 does not neutralize SARS-CoV-2, but the increased affinity to SARS-CoV-2 P384A mutant now enables neutralization with a similar potency to SARS-CoV. We further investigated CR3022 interaction with the SARS-CoV spike protein by negative-stain EM and cryo-EM. Three CR3022 Fabs bind per trimer with the RBD observed in different up-conformations due to considerable flexibility of the RBD. In one of these conformations, quaternary interactions are made by CR3022 to the N-terminal domain (NTD) of an adjacent subunit. Overall, this study provides insights into antigenic variation and potential for cross-neutralizing epitopes on SARS-like viruses.


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The ongoing COVID-19 pandemic, which is caused by the new coronavirus SARS-CoVthe RBD structures from SARS-CoV and SAR-CoV-2 when bound with CR3022. The RBDs have a Cα RMSD of only 0.6 Å (0.7 Å for CR3022 epitope residues). At residue 148 384, the backbone of SARS-CoV-2 is further from CR3022, as compared to that of SARS- proposed [20], the CR3022-bound RBD was indeed rotated compared to that in the 173 unliganded S protein [26][27][28], such that, in this conformation, steric hinderance between 174 CR3022 and the N-terminal domain (NTD) is minimized.

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While our results here demonstrate that CR3022 Fab could form a stable complex with 177 SARS-CoV S protein in a prefusion conformation, a recent study reported that prefusion [29]. It should be noted that the three-up conformation is much more rarely observed than 180 the other RBD conformations (all-down, one-up, and two-up) in SARS-CoV by cryo-EM 181 [26][27][28], or SARS-CoV-2 by cryo-EM [30][31][32] and cryo-electron tomography [33,34], and 182 could relate to differences in the stability of S trimers in SARS-CoV versus SARS CoV-2 183 when CR3022 is bound. Further studies will be required to investigate whether such a 184 difference between SARS-CoV-2 and SARS-CoV is related to stability differences in the 185 recombinant spike proteins, or to different dynamics of the RBD on the virus or infected 186 cells.

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To address some of these issues, we performed cryo-EM analysis to interrogate the 9 the total particle number for classes 2 and 4 together exceed that for class 3 195 (Supplementary Figure 4). In contrast, class 1 is the least represented. In classes 2 and of the CR3022 light chain in classes 2 and 4 is in close proximity to a loop region in NTD 200 corresponding to residues 106-110. In addition, the constant region of CR3022 appears 201 to contact residue D23 of NTD. Another important observation is that the Fab in class 2 202 and 4 would clash with the adjacent RBD if it were in the "down" conformation. So, for the 203 Fab to exist in this quaternary conformation, the adjacent RBD has to be in the "up"

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bound RBD, bivalent binding of CR3022 to S protein does not seem to occur on the virus 218 surface since an IgG avidity effect was not observed in the neutralization assay (see 219 above, Figure 2). Overall, these structural analyses indicate that RBD rotational flexibility 220 . CC-BY 4.0 International license (which was not certified by peer review) is the author/funder. It is made available under a   neutralize SARS-CoV-2 at an IC50 of ~20 ng/ml [3,37]. Of note, the KD and IC50 of CC12.1 244 and CC12.3 were measured in the same manner as this study. The lack of correlation 245 between affinity and neutralizing activity is therefore not due to the difference in the assays 246 . CC-BY 4.0 International license (which was not certified by peer review) is the author/funder. It is made available under a The copyright holder for this preprint this version posted September 21, 2020. . https://doi.org/10. 1101 between studies. In fact, a previous study also demonstrated a lack of correlation between 247 RBD binding and neutralization for monoclonal antibodies [3]. Together, these 248 observations suggest that the affinity threshold for SARS-CoV-2 neutralization by RBD-249 targeting antibodies may be epitope dependent. The difference in affinity threshold for 250 different epitopes is also likely to be related not only in the ability to block ACE2-binding 251 [3,38], but also in antibody avidity where bivalent binding can cross-link different RBD

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. CC-BY 4.0 International license (which was not certified by peer review) is the author/funder. It is made available under a The copyright holder for this preprint this version posted September 21, 2020. . https://doi.org/10.1101/2020.09.21.305441 doi: bioRxiv preprint Negative-stain electron microscopy application. Grids were imaged on Tecnai T12 Spirit at 120 keV with a 4k x 4k Eagle CCD.

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Micrographs were collected using Leginon [48] and images were transferred to Appion

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[49] for particle picking using a difference-of-Gaussians picker (DoG-picker) [50] and 355 generation of particle stacks. Particle stacks were further transferred to Relion [51] for 2D 356 classification followed by 3D classification to select good classes. Select 3D classes were 357 auto-refined on Relion and used for making figures using UCSF Chimera [52].

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Cryo-EM sample preparation glycol (LMNG) solution immediately before sample deposition onto a 1.2/1.3 300-Gold grid 363 (EMS). The grids were plasma cleaned for 7 seconds using a Gatan Solarus 950 Plasma 364 system prior to sample deposition. Following sample application, grids were blotted for 3 365 seconds before being vitrified in liquid ethane using a Vitrobot Mark IV (Thermo Fisher).

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Cryo-EM data collection and processing 368 Data collection was performed using a Talos Arctica TEM at 200 kV with a Gatan K2 [48,53]. Micrographs were transferred to CryoSPARC 2.9 for further processing [54]. CTF 374 estimations were performed using GCTF and micrographs were selected using the Curate

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Exposures tool in CryoSPARC based on their CTF resolution estimates (cutoff 5 Å) for downstream particle picking, extraction and iterative rounds of 2D classification and selection [55]. Particles selected from 2D classes were transferred to Relion 3.1 for direct 378 C3 refinement, symmetry expansion of particles and iterative rounds of 3D focused

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