Molecular basis of SARS-CoV-2 Omicron variant evasion from shared neutralizing antibody response

A detailed understanding of the molecular features of the neutralizing epitopes developed by viral escape mutants is important for predicting and developing vaccines or therapeutic antibodies against continuously emerging SARS-CoV-2 variants. Here, we report three human monoclonal antibodies (mAbs) generated from COVID-19 recovered individuals during first wave of pandemic in India. These mAbs had publicly shared near germline gene usage and potently neutralized Alpha and Delta, but poorly neutralized Beta and completely failed to neutralize Omicron BA.1 SARS-CoV-2 variants. Structural analysis of these three mAbs in complex with trimeric spike protein showed that all three mAbs are involved in bivalent spike binding with two mAbs targeting class-1 and one targeting class-4 Receptor Binding Domain (RBD) epitope. Comparison of immunogenetic makeup, structure, and function of these three mAbs with our recently reported class-3 RBD binding mAb that potently neutralized all SARS-CoV-2 variants revealed precise antibody footprint, specific molecular interactions associated with the most potent multi-variant binding / neutralization efficacy. This knowledge has timely significance for understanding how a combination of certain mutations affect the binding or neutralization of an antibody and thus have implications for predicting structural features of emerging SARS-CoV-2 escape variants and to develop vaccines or therapeutic antibodies against these.


Introduction 53
SARS-CoV-2 Omicron subvariants are continuously emerging and escaping therapeutic 54 monoclonal antibodies (mAbs) and vaccines (1)(2)(3). Mutations acquired in the spike 55 protein of SARS-CoV-2 variants, a target for neutralizing antibodies (nAbs), are primarily 56 responsible for this immune escape (1,4). Identifying nAbs / non-nAbs to these variants 57 and determining their prevalence in human population allows us to understand the 58 shared mechanisms of immune protection among diverse populations (5, 6). Since the 59 emergence of COVID-19, >11,000 SARS-CoV-2 mAbs have been identified (7). Among 60 these, nAbs encoded by human antibody heavy chain variable germline genes such as 61 IGHV3-53/3-66, IGHV1-58, IGHV3-30 and IGHV1-69 are commonly observed in many 62 individuals across the globe (7). These related rearrangements, known as a public 63 antibody response, suggest a shared immune response with a similar genetic makeup and 64 recognizes adjacent RBDs in a spike trimer (Figure 3A and S4). 002-13 mAb, belongs to 127 the public clonotype encoded by IGHV3-30 and IGLV6-57 that have not been structurally 128 characterized before. Notably, the 22-residue long CDRH3 region encoded by IGHD2-8 129 gene of 002-13 mAb contains a CxGGxC motif which is shared by other 81 RBD-specific 130 mAbs ( Figure S1A) documented in CoV-AbDab database (7). Like other class-4 131 antibodies, 002-13 RBD binding is dominated by the heavy chain contributing ~76% of 132 total interaction with a total buried surface area of ~887 Å 2 ( Figure 3B) (16,17). Most of 133 the heavy chain interactions are mediated through the CDR3 region that forms a foot-like 134 loop, stabilized by an intra-loop disulfide bond between residues C105 and C110 of 135 CxGGxC motif (Figure 3C and 3D). We observe that multiple interactions involving the 136 residues in RBD region S371-C379 and the heavy chain CDR3 loop are responsible for 137 epitope recognition ( Figure 3C). Heavy chain CDR2 residues D57 and S56 engage RBD We then marked key mutations present in Beta (yellow), Delta (red) and Omicron (green) 150 variants within the 002-13 epitope surface and observed that while all VOC except for 151 Omicron carry no mutations, Omicron carries three mutations (S371L, S373P and S375F) 152 within the 002-13 epitope ( Figure 3I). This suggests that while the binding and 153 neutralization of 002-13 mAb will be preserved for most SARS-CoV-2 variants, it might 154 be impacted towards Omicron as these mutations are known to induce a local 155 conformational change in the Omicron RBD structure and thus, could exclusively evade 156 Omicron (18, 19). mAb 002-02 and 034-32: Antibodies 002-02 and 034-32 were isolated from two 158 different individuals and are encoded by public clonotype genes IGHV3-53/3-66 ( Figure  159 1A). They both show very similar properties with high binding specificity towards SARS-160 CoV-2 RBD, effectively compete ACE2 and potently neutralize WA.1 (14) (Figure 2). To 161 define the details of epitope recognition, we determined the Cryo-EM structure of each 162 002-02 (Figure 4 and S5) and 034-32 ( Figure S6 and S7) in complex with WA.1 Spike-163 6P at a resolution of 3.8 and 4.3 Å, respectively. For both complexes, we observe intra-164 spike bivalent binding, where each Fab region of IgG binds two neighboring RBDs in the 165 spike trimer in the "up" conformation. The RBD that does not engage in binding Fab 166 remains in the "down" conformation ( Figure 4A). Both mAb structures recognize 167 epitopes in the top RBD pocket that aligns with the RBM surface suggesting direct ACE2 168 competition and based on this they are classified as RBD-2/class-1 antibodies. Since 002-169 02 and 034-32 recognize the RBD in a very similar manner, we focus our structural 170 analysis on mAb-RBD recognition in the locally refined map for 002-02. 171 While all CDR loops are involved in epitope recognition ( Figure 4A and 4B), most RBD 172 contacts are dominated by the heavy chain, contributing ~70% of the total of 1058 Å 2 of 173 buried surface between mAb and RBD ( Figure 4B). Primary interactions in the heavy 174 chain are mediated by CDR1 and CDR2 regions. Most mAbs that belong to class-1 175 antibodies are encoded by public clonotype genes IGHV3-53/IGHV3-66 (5, 13). The 176 common features among these mAbs include a conserved NY and SGGS motif in CDR1 and 177 CDR2 regions, respectively, that contribute significantly toward RBD binding (12). We 178 also observed a network of hydrogen bonds with the RBD through the CDR2 SGGS motif. 179 The side chain of S53 and S56 in the CDR2 heavy chain engages in a hydrogen bond with 180 side chains of Y421 and D420 in RBD, respectively ( Figure 4C). However, the CDR3 loop 181 heavy chain residues in this mAb class varies more. In 002-02, the heavy chain CDR3 182 residue D101 forms a hydrogen bond with K417 and Y453 in the RBD (Figure 4D). In the 183 light chain, CDR1 and CDR3 make some contact with the inner left side of the RBD. The 184 S30 and Y32 residue in the CDR1 region of the light chain makes a hydrogen bond with 185 Q498 and R403 in RBD, respectively ( Figure 4E). Also, the S93 in the CDR3 region of the 186 light chain interacts with Y505 and D405 ( Figure 4F). 187 Like 002-13, we also mapped mutations found in Beta (yellow), Delta (red) and Omicron 188 (green) variants onto the 002-02 / 034-32 epitope ( Figure 4G). While Delta carries no 189 . CC-BY-NC-ND 4.0 International license perpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in neutralization. Collectively, based on these observations both 002-02 and 034-32 mAbs 195 will be less or ineffective towards both Beta and Omicron variants. 196

Assessing binding and neutralization breadth towards SARS-CoV-2 variants 197
To link the paratope mutation landscape in VOC to the antibody function, we tested 198 binding and neutralization of these three mAbs against SARS-CoV-2 variants. In  Altogether, this data catalogues the epitope class-specific antibody susceptibility towards 219 existing SARS-CoV-2 variant and can inform their action on a newly emerging variant. 220 . CC-BY-NC-ND 4.0 International license perpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in These mutations must be tracked to find effective solutions to combat emerging variants. 276 Further, the structure guided prediction made for three SARS-CoV-2 shared nAbs that 277 potently neutralized the WA.1 strain holds true towards the functional efficacy of these 278 mAbs against SARS-CoV-2 variants, including Omicron. 279 In summary, this study vastly improves our understanding of how Omicron escaped from 280 shared antibody responses to SARS-CoV-2 elicited during the natural infection and has 281 implications towards concepts for fast-tracking effective broad range therapeutics 282 against continuously emerging SARS-CoV-2 variants. 283

Materials and Methods 284
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SARS-CoV-2 RBD-specific ELISA binding assays 285
The recombinant SARS-CoV-2 RBD gene was cloned, expressed, purified and ELISAs were 286  RBD binding signals was next purified using Protein A/G beads (Thermo Scientific), 334 concentrated using a 30 kDa or 100 kDa cut-off concentrator (Vivaspin, Sartorius) and 335 stored at 4°C for further use. 336

Electrochemiluminescence antibody binding assay 337
Binding analysis of SARS-CoV-2 mAb to spike protein was performed using an 338 electrochemiluminescence assay as described earlier (14). Briefly, V-PLEX COVID-19 339 Panel 24 (Meso Scale Discovery) was used to measure the IgG1 mAb binding to SARS-340 CoV-2 spike antigens following the manufacturer's recommendations. antigen coated 341 plates were blocked with 150 μl/well of 5% BSA in PBS for 30 minutes. Plates were 342 washed 3x with 150 μl/well of PBS with 0.05% Tween between each incubation step. 343 mAbs were serially diluted for concentrations ranging from 10 μg/ml to 0.1 pg/ml and 344 . CC-BY-NC-ND 4.0 International license perpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in added to each plate immediately before reading on an MSD QuickPlex plate reader. 349

Octet BLI analysis 350
Octet biolayer interferometry (BLI) was performed using an Octet Red96 instrument 351 (ForteBio, Inc.) as described earlier (14). A 5 μg/ml concentration of each mAb was 352 captured on a protein A sensor and its binding kinetics were tested with serial 2-fold 353 diluted RBD (600 nM to 37.5 nM) and spike hexapro protein (100 nM to 6.25 nM). The

Spike protein expression and purification 362
SARS-CoV-2 Spike-6P trimer protein carrying WA.1 was expressed and purified by 363 transfecting expi293F cells using WA.1-spike-6P plasmids as described previously (14). 364 Transfections were performed as per the manufacturer's protocol (Thermo Fisher). 365 Briefly, expi293F cells (2.5x10 6 cells/ml) were transfected using ExpiFectamine TM 293 366 transfection reagent (ThermoFisher, cat. no. A14524). The cells were harvested 4-5 days 367 post-transfection. The spike protein was purified using His-Pur Ni-NTA affinity 368 purification method. Column was washed with Buffer containing 25 mM Imidazole, 6.7 369 mM NaH2PO4.H2O and 300 mM NaCl in PBS followed by spike protein elution in elution 370 buffer containing 235 mM Imidazole, 6.7 mM NaH2PO4.H2O and 300 mM NaCl in PBS. 371 Eluted protein was dialyzed against PBS and concentrated. The concentrated protein was 372 loaded onto a Superose-6 Increase 10/300 column and protein eluted as trimeric spike 373 collected. Protein quality was evaluated by SDS-PAGE and by Negative Stain-EM. 374 . CC-BY-NC-ND 4.0 International license perpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for this this version posted October 24, 2022. ; https://doi.org/10.1101/2022.10.24.513517 doi: bioRxiv preprint

Sample preparation for Cryo-EM 382
SARS-CoV-2 spike-6P trimer incubated with the mAb (full-length IgG) at 0.7 mg/ml 383 concentration. The complex was prepared at a 0.4 sub-molar ratio of mAb to prevent 384 inter-spike crosslinking, mediated by bi-valent binding of intact antibody. The complex 385 was incubated at room temperature for ~5 min before vitrification. Three µL of the 386 complex was applied onto a freshly glow-discharged (PLECO easiGLOW) 400 mesh, 387

Cryo-EM data acquisition 391
Single-particle Cryo-EM data for WA.1 spike-IgG complexes of mAb 002-02, 002-13 and 392 034-32 were collected on a 300 kV Titan Krios transmission electron microscope 393 (ThermoFisher Scientific) equipped with Gatan K3 direct electron detector behind 30 eV 394 slit width energy filter. Multi-frame movies were collected at a pixel size of 1.0691 Å per 395 pixel with a total dose of 63 e/Å 2 at defocus range of -0.7 to -2.7 µm. 396

Cryo-EM data analysis and model building 397
Cryo-EM movies were motion-corrected by Patch motion correction implemented in 398 Cryosparc v3.3.1 (28). Motion-corrected micrographs were corrected for contrast 399 transfer function using Cryosparc's implementation of Patch CTF estimation. 400 Micrographs with poor CTF fits were discarded using CTF fit resolution cutoff to ~6.0 Å. 401 Particles were picked using a Blob picker, extracted and subjected to an iterative round 402 of 2D classification. Particles belonging to the best 2D classes with secondary structure 403 . CC-BY-NC-ND 4.0 International license perpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in 3D class were refined in non-uniform 3D refinement with per particle CTF and higher-406 order aberration correction turned on. To further improve the resolution of the RBD-IgG 407 binding interface a soft mask was created covering one RBD and interacting Fab region 408 of IgG and refined locally in Cryosparc using Local Refinement on signal subtracted 409 particles. All maps were density modified in Phenix (29) using Resolve Cryo-EM. The 410 combined Focused Map tool in Phenix was used to integrate high resolution locally 411 refined maps into an overall map. Additional data processing details are summarized in 412 the PME method [24] with a real space cut-off of 10 Å. Van der Waals interactions were 428 modelled using Lennard-Jones 6-12 potentials with a 14 Å cut-off. The temperature was 429 maintained at 300 K using V-rescale; hydrogen bonds were constrained using the LINCS 430 algorithm [25]. Energy minimization was carried out to reach a maximum force of no 431 more than 10 kJ/mol using steepest descent algorithm. The time step in all molecular 432 dynamics simulations was set to 2 fs. Prior to the production run, the minimized systems 433 were equilibrated for 5ns with NVT and followed with NPT at 300 K. 434 . CC-BY-NC-ND 4.0 International license perpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in Light chain VJ-gene bar plot of SARS-CoV-2 RBD-specific human mAbs documented in 608 CoV-AbDab dataset. 609   Omicron (green) mutations on RBD relative to the 002-02 epitope site (black outline). 640 concern (VOCs). Curves shown are the best fit one-site binding curves calculated by Prism 644 9.0 (D-F) Live virus neutralization curves and FRNT50 values of three potent mAbs for 645 . CC-BY-NC-ND 4.0 International license perpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in

RBD Inner Face
. CC-BY-NC-ND 4.0 International license perpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for this this version posted October 24, 2022. ; https://doi.org/10.1101/2022.10.24.513517 doi: bioRxiv preprint  . CC-BY-NC-ND 4.0 International license perpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for this this version posted October 24, 2022. ; https://doi.org/10.1101/2022.10.24.513517 doi: bioRxiv preprint