key: cord-0309219-7jma3jjt
authors: Moyo-Gwete, T.; Madzivhandila, M.; Mkhize, N. N.; Kgagudi, P.; Ayres, F.; Lambson, B. E.; Manamela, N. P.; Richardson, S. I.; Makhado, Z.; van der Mescht, M.; de Beer, Z.; de Villiers, T. R.; Burgers, W. A.; Ntusi, N. A.; Rossouw, T.; Ueckermann, V.; Boswell, M. T.; Moore, P. L.
title: Shared N417-dependent epitope on the SARS-CoV-2 Omicron, Beta and Delta-plus variants
date: 2022-04-26
journal: nan
DOI: 10.1101/2022.04.24.22273395
sha: b2c85509e41d8b7cda6115c4b780a188e2a30b20
doc_id: 309219
cord_uid: 7jma3jjt

As SARS-CoV-2 continues to evolve, several variants of concern (VOCs) have arisen which are defined by multiple mutations in their spike proteins. These mutations have resulted in variable escape from antibody responses and the elicitation of qualitatively different antibody responses during infection. By studying plasma from individuals infected with either the original D614G, Beta or Delta variants, we showed that the Beta and Delta variants elicit antibody responses that are overall more cross-reactive than those triggered by D614G. Patterns of cross-reactivity varied, and the Beta and Delta variants did not elicit cross-reactive responses to each other. However, Beta-elicited plasma was highly cross-reactive against Delta plus (Delta+) which differs from Delta by a single K417N mutation in the receptor binding domain, suggesting the plasma response targets the N417 residue. To probe this further, we isolated monoclonal antibodies from a Beta-infected individual with plasma responses against Beta, Delta+ and Omicron, which all possess the N417 residue. We isolated a N417-dependent antibody, 084-7D, which showed similar neutralization breadth to the plasma. The 084-7D mAb utilized the IGHV3*23*01 germline gene and had similar somatic hypermutations compared to previously described public antibodies which target the 417 residue. Thus, we have identified a novel antibody which targets a shared epitope found on three distinct VOCs. Understanding the antibody response towards escape mutations such as K417N, which repeatedly emerge through convergent evolution in SARS-CoV-2 variants, may aid in the development of next-generation antibody therapeutics and vaccines.

trigger qualitatively different antibody responses during infection. By studying plasma 34 from individuals infected with either the original D614G, Beta or Delta variants, we 35 show that the Beta and Delta variants elicit antibody responses that are overall more 36 cross-reactive than those triggered by D614G. Patterns of cross-reactivity varied, 37 and the Beta and Delta variants did not elicit cross-reactive responses to each other. 38 However, Beta-elicited plasma was highly cross-reactive against Delta plus (Delta+) 39 which differs from Delta by a single K417N mutation in the receptor binding domain, 40 suggesting the plasma response targets the N417 residue. To probe this further, we 41 isolated monoclonal antibodies from a Beta-infected individual with plasma 42 responses against Beta, Delta+ and Omicron, which all possess the N417 residue. 43 We isolated a N417-dependent antibody, 084-7D, which showed similar CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) Importance 57 58

The evolution of SARS-CoV-2 has resulted in variants of concern (VOCs) with 59 distinct spike mutations conferring varying immune escape profiles. These variable 60 mutations also influence the cross-reactivity of the antibody response mounted by 61 individuals infected with each of these variants. This study sought to understand the 62 antibody responses elicited by different SARS-CoV-2 variants, and to define shared 63 epitopes. We show that Beta and Delta infection resulted in antibody responses that 64 were more cross-reactive compared to the original D614G variant, but each with 65 differing patterns of cross-reactivity. We further isolated an antibody from Beta 66 infection, which targeted the N417 site, enabling cross-neutralization of Beta, Delta+ 67 and Omicron, all of which possess this residue. The discovery of antibodies which 68 target escape mutations common to multiple variants highlights conserved epitopes 69 to target in future vaccines and therapeutics. CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. Since the emergence of SARS-CoV-2 in 2019, several variants of concern (VOCs) 90 and interest (VOIs) have emerged. Many of these variants contain mutations in the 91 spike protein that confer escape from neutralizing antibodies (1) . The first 92 neutralization-resistant VOC identified, Beta, contained receptor binding domain 93 (RBD) mutations at positions K417N and E484K, which confer escape from Class I 94 and Class II neutralization antibodies, respectively (2, 3) . These mutations have 95 since been observed in multiple variants. One such example is the charge-altering 96 E484K/Q/A mutation, which occurs in several variants including Gamma, the Delta 97 lineage, C.1.2 and A.VOI.V2 (1, (4) (5) (6) . The E484K mutation is also present in Omicron CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)

The copyright holder for this preprint this version posted April 26, 2022. ; https://doi.org/10.1101 https://doi.org/10. /2022 levels of cross-reactivity towards the D614G and the Gamma variant (18) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)

The copyright holder for this preprint this version posted April 26, 2022. ; https://doi.org/10.1101 https://doi.org/10. /2022 

Neutralizing responses triggered by variants of concern possess differential 156 patterns of cross-reactivity 157 158 We first compared antibody responses in unvaccinated individuals infected by VOCs 159 circulating in three distinct SARS-CoV-2 epidemiological waves in South Africa from 160 May 2020 to July 2021. In each wave, sampling occurred at the time when each Figure 1A ). In contrast, as we previously reported, plasma from Beta infections 169 was highly cross-reactive against the D614G variant (2.9 fold reduction) (18) but 170 showed less cross-reactivity against other VOCs, with reductions against Delta, 171 Omicron and SARS-CoV-1 of 11.3-fold, 9.4-fold and 9.3-fold, respectively ( Figure   172 1A). We also tested plasma from Delta-infected individuals and found that although CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted April 26, 2022. ; https://doi.org/10.1101 https://doi.org/10. /2022 breadth, by assessing the cumulative area under the curve (AUC) value for all 188 variants, normalized relative to the GMT of the autologous virus to account for 189 differential potency of responses ( Figure 1B) . were both approximately 2-fold higher (AUC: 114 and 103) than D614G (AUC: 52) 191 ( Figure 1B) , indicating that the neutralizing responses triggered by these two 192 variants were more cross-reactive than those elicited by the early circulating CoV -2 variant. 194 195 Although other sites, such as the RBD Class III antibody binding site, have CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted April 26, 2022. ; https://doi.org/10.1101/2022.04.24.22273395 doi: medRxiv preprint reactivity of the plasma towards Delta+ was similar to what we observed with other 222 Beta-elicited plasma ( Figure 1C ). 223 We performed single cell sorting of the participant's peripheral blood mononuclear 224 cells (PBMCs). Using Beta and D614G spike proteins as cell sorting baits, we sorted 225 a total of 68 cells, with approximately 58 of the cells being Beta-specific and the rest 226 of the cells being double-positive ( Figure 2C ). We identified an IgG1 mAb, 084-7D, 227 which bound to the Beta spike antigen (Figure 2) . We tested the functionality of mAb CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted April 26, 2022. ; https://doi.org/10.1101/2022.04.24.22273395 doi: medRxiv preprint 256 We have previously shown that, in addition to neutralization, the Beta variant triggers 257 cross-reactive Fc functionality compared to D614G (26). We therefore analyzed the 258 ability of the 084-7D mAb to mediate antibody-dependent cellular phagocytosis 259 (ADCP) and measured the ability of mAb 084-7D to cross-link CD16 and SARS- (Figure 4) . The 084-7D antibody was isolated as an IgG1, IgA1 286 and IgM but was cloned and tested as an IgG1 for this study ( Figure 1A) . The mAb 287 uses the 3-23*01 variable heavy (VH) chain gene, exhibiting 5.9% somatic 288 hypermutation in the variable gene region, despite being isolated early in infection 289 . CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted April 26, 2022. ; https://doi.org/10.1101/2022.04.24.22273395 doi: medRxiv preprint ( Figure 4A ). The 1-5*03 variable kappa (VK) chain gene is used by this mAb, and 290 we observed less somatic hypermutation in the VK gene (2.9%) compared to the 291 heavy chain. We compared the 084-7D mAb to a previously reported SARS-CoV-2 292 RBD-specific antibody, CAB-A17, that utilizes the VH3-53*01 germline gene, which 293 is similar to VH3-23*01 with 91% identity. The CAB-A17 lineage developed VOC 294 cross-neutralization through interactions with the N417 residue, suggesting similar 295 epitope targeting compared to mAb 084-7D (28). Furthermore, the light chain of Class I K417-targeting antibodies has been shown to 306 play a role in epitope interactions through the CDRL1 28 VSSS 31 -motif (Kabat 307 numbering) (13, 28). We noted that the 084-7D mAb possessed a germline encoded 308 ISS-motif, which is similar to the CAB-A17 mAb which also had a deletion in this 309 region, resulting in an LST-motif ( Figure 4B) . Altogether, these data show that the 310 084-7D mAb is similar to a highly broad and potent mAb which targets a similar 311 epitope. In this study, we investigated the cross-reactive potential of plasma elicited by three 334 different variants that circulated in South Africa during three sequential waves of CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted April 26, 2022. ; https://doi.org/10.1101/2022.04.24.22273395 doi: medRxiv preprint across VOCs and is also more potent against Omicron than the 084-7D mAb (28). 395 Exploring the differences between the two mAbs may suggest a mechanism required 396 to achieve high levels of breadth and potency in this class of antibody. The CAB-A17 397 mAb contains key mutations that resulted in increased breadth towards the Omicron 398 variant -G26E, T28I, S53P and Y58F. The 084-7D mAb contains 2/4 of these 399 mutations although the residue changes differ -T28S and S53G. The changes in 400 mAb CAB-A17 at these sites are much more substantial, especially the serine to 401 proline change at position 53 that may alter the conformation of that region and 402 therefore affect epitope binding. These differences may contribute to the disparity in 403 breadth and potency between these two mAbs. CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted April 26, 2022. ; https://doi.org/10.1101/2022.04.24.22273395 doi: medRxiv preprint 429 . CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted April 26, 2022. ; https://doi.org/10.1101/2022.04.24.22273395 doi: medRxiv preprint CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted April 26, 2022. ; https://doi.org/10.1101 https://doi.org/10. /2022 CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted April 26, 2022. ; https://doi.org/10.1101 https://doi.org/10. /2022 Single cell PCR amplification of heavy and light chain variable genes 494 Genes encoding IGHV and IGLV chains were amplified from sorted cells by reverse 495 real-time and nested PCR. Reverse transcription was performed using Superscript III 496 reverse transcriptase (Invitrogen) and random hexamer primers (36, 37). Following 497 cDNA synthesis VH, Vκ, and Vλ antibody genes were amplified as previously 498 described (38, 39). Determination of gene usage was done using IMGT/V-QUEST 499 (https://www.imgt.org/IMGT_vquest/analysis). Antibody-dependent cellular cytotoxicity (ADCC) assay 521 . CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted April 26, 2022. ; https://doi.org/10.1101 https://doi.org/10. /2022 The ability of the mAb to cross-link between FcγRIIIa (CD16) and spike expressed 522 on cells was used as a proxy for ADCC. To express cell-surface spike, HEK 293T 523 cells were transfected with SARS-CoV-2 614G WT, Beta or Omicron expressing 524 plasmids, with incubation at 37°C over 2 days. Spike expressing cells were 525 incubated with 100 µg/ml of 084-7D or control mAbs titrated four times 1 in 5 in 526 RPMI media, 10% FBS, 1% penicillin-streptomycin for an hour at 37°C. Following CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted April 26, 2022. ; https://doi.org/10.1101 https://doi.org/10. /2022 CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted April 26, 2022. ; https://doi.org/10.1101 https://doi.org/10. /2022 was tested for neutralization activity in a pseudovirus-based neutralization assay 594 against the same VOC/VOIs tested with the plasma. mAb neutralization was 595 measured as an IC 50 (ug/ml). The threshold of detection for the neutralization assay 596 is IC 50 >20 ug/ml (E) A SARS-CoV-2 ELISA was used to map the specificity of the 597 084-7D mAb. The mAb was tested against the D614G and Beta N-terminal domain, 598 . CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted April 26, 2022. ; https://doi.org/10.1101 https://doi.org/10. /2022 spike and RBD antigens, as well as SBD (subdomain 1) proteins mutated to include 599 Beta-specific SBD single mutants, K417N, E484K and N501Y. The starting 600 concentration of the mAb was 5ug/ml. The threshold of detection for the binding 601 assay is OD450nm>0.04. CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted April 26, 2022. is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted April 26, 2022. is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted April 26, 2022. is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted April 26, 2022. 

Curi 629 R, Durigon EL. 2022. SARS-COV-2 Variants: Differences and Potential of

Immune Evasion. Frontiers in Cellular and Infection Microbiology

Detection of a SARS-CoV-2 633 variant of concern in South Africa

CoV-2 501Y. V2 escapes neutralization by South African COVID-19 donor 637 plasma

Emergence and phenotypic characterization of C.1.2, a 646 globally detected lineage that rapidly accumulated mutations of concern

Rapid epidemic expansion of the SARS-657

CoV-2 Omicron variant in southern

A novel variant of interest of SARS-CoV-2 with 664 multiple spike mutations detected through travel surveillance in Africa

Variable loss of 668 antibody potency against SARS-CoV-2 B.1.1.529 (Omicron). bioRxiv 669

COVID-19 vaccine boosters induce neutralizing immunity against SARS-CoV

Molecular rationale 682 for SARS-CoV-2 spike circulating mutations able to escape bamlanivimab and 683 etesevimab monoclonal antibodies

Delta variant (B. 1.617. 2) 686 sublineages do not show increased neutralization resistance

Omicron (BA.1) and Sub-689

Variants (BA.1, BA.2 and BA.3) of SARS-CoV-2 Spike Infectivity and 690 Pathogenicity: A Comparative Sequence and Structural-based Computational

SARS-CoV-2 694 neutralizing antibody structures inform therapeutic strategies

A SARS-CoV-2 variant elicits an antibody 697 response with a shifted immunodominance hierarchy

The SARS-CoV-2 Delta variant 701 induces an antibody response largely focused on class 1 and 2 antibody 702 epitopes

Mapping Neutralizing and 711

Immunodominant Sites on the SARS-CoV-2 Spike Receptor-Binding Domain 712 by Structure-Guided High-Resolution Serology

Antibodies elicited by mRNA-1273 vaccination bind 715 more broadly to the receptor binding domain than do those from SARS-CoV-2 716 infection

Antibody Responses Elicited by SARS-CoV-2 501Y.V2 (B.1.351). New

the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted

SARS-CoV-2 prolonged infection 726 during advanced HIV disease evolves extensive immune escape. Cell host & 727 microbe

.617 by vaccine and convalescent serum

Neutralizing antibody activity in convalescent sera from 737 infection in humans with SARS-CoV-2 and variants of concern

Omicron triggers cross-reactive neutralization and Fc effector 742 functions in previously vaccinated, but not unvaccinated individuals

The Delta SARS-CoV-2 746 variant has a higher viral load than the Beta and the historical variants in 747 nasopharyngeal samples from newly diagnosed COVID-19 patients

Mapping SARS-CoV-2 antigenic 754 relationships and serological responses

SARS-CoV-2 Beta variant 758 infection elicits potent lineage-specific and cross-reactive antibodies

SARS-CoV-2 Beta 762 and Delta variants trigger Fc effector function with increased cross-reactivity

Compromised humoral functional evolution 766 tracks with SARS-CoV-2 mortality

Structural basis of Omicron 769 neutralization by affinity-matured public antibodies. bioRxiv

Human neutralizing antibodies 777 elicited by SARS-CoV-2 infection

Structure-based analyses of neutralization antibodies 780 interacting with naturally occurring SARS-CoV-2 RBD variants

Geng 783 C. 2020. Potent neutralizing antibodies against SARS-CoV-2 identified by 784 high-throughput single-cell sequencing of convalescent patients' B cells

Affinity 788 maturation of SARS-CoV-2 neutralizing antibodies confers potency, breadth, 789 and resilience to viral escape mutations

Somatic Mutations of the Immunoglobulin 797 Framework Are Generally Required for Broad and Potent HIV-1

Efficient generation of monoclonal antibodies from single human B cells 801 by single cell RT-PCR and expression vector cloning

Sequence and structural 805 convergence of broad and potent HIV antibodies that mimic CD4 binding

New Member of the V1V2-Directed CAP256-VRC26 Lineage That 813 Shows Increased Breadth and Exceptional Potency