key: cord-0827009-y2739974
authors: Chen, Elaine C.; Gilchuk, Pavlo; Zost, Seth J.; Suryadevara, Naveenchandra; Winkler, Emma S.; Cabel, Carly R.; Binshtein, Elad; Chen, Rita; Sutton, Rachel E.; Rodriguez, Jessica; Day, Samuel; Myers, Luke; Trivette, Andrew; Williams, Jazmean K.; Davidson, Edgar; Li, Shuaizhi; Doranz, Benjamin J.; Campos, Samuel K.; Carnahan, Robert H.; Thorne, Curtis A.; Diamond, Michael S.; Crowe, James E.
title: Convergent antibody responses to the SARS-CoV-2 spike protein in convalescent and vaccinated individuals
date: 2021-08-10
journal: Cell Rep
DOI: 10.1016/j.celrep.2021.109604
sha: d0dae22170534925b28121db85f36f6c9d8f97cd
doc_id: 827009
cord_uid: y2739974

Unrelated individuals can produce genetically similar clones of antibodies, known as public clonotypes, which have been seen in responses to different infectious diseases as well as healthy individuals. Here we identify 37 public clonotypes in memory B cells from convalescent survivors of SARS-CoV-2 infection or in plasmablasts from an individual after vaccination with mRNA-encoded spike protein. We identified 29 public clonotypes, including clones recognizing the receptor-binding domain (RBD) in the spike protein S1 subunit (including a neutralizing, ACE2-blocking clone that protects in vivo), and others recognizing non-RBD epitopes that bound the S2 domain. Germline-revertant forms of some public clonotypes bound efficiently to spike protein, suggesting these common germline-encoded antibodies are preconfigured for avid recognition. Identification of large numbers of public clonotypes provides insight into the molecular basis of efficacy of SARS-CoV-2 vaccines and sheds light on the immune pressures driving the selection of common viral escape mutants.

exhibited lower binding avidity (higher EC 50 values) than its matured counterparts COV2-2164 289 or CnC2t1p1_B10. The Group 3 germline revertant maintained binding to SARS-CoV-2 S6P ecto 290 and RBD proteins (Fig. 4a, d) . Each germline revertant also bound to the surface of virus-291 infected cells (Fig. 4b,d) . While none of the germline revertants exhibited neutralizing capacity 292 ( Fig. 4c, d) , the Group 3 germline revertant showed a low level of ACE2 blocking (Fig. 4d,e) . 293

binding of some mAbs to the surface of virus-infected cells (Fig. 2e , h) suggested that these 295 antibodies also might act through Fc-mediated functions. Therefore, it was important to test 296 some public clonotypes in vivo. We tested the efficacy of these antibodies against SARS-CoV-297 2 in vivo. We used K18-hACE2 transgenic mice, which develop severe lung infection and 298 disease after intranasal inoculation (Golden et al., 2020; Winkler et al., 2020; Zheng et al., 2021) . 299 K18-hACE2 transgenic mice received either one antibody from Group 2 (COV2-2164), one 300 antibody from Group 3 (COV2-2531), or an isotype-control antibody (DENV 2D22) via 301 intraperitoneal injection (200 µg, 10 mg/kg) one day prior to intranasal inoculation with 10 3 PFU 302 of SARS-CoV-2 (WA1/2020). Mice treated with COV2-2531 were protected completely from 303 weight loss (Fig. 5a) and showed reduced viral infection in the lung, nasal wash, heart, and brain 304 ( Fig. 5b, c, d) compared to the isotype-control antibody-treated group. However, mice treated 305 with COV2-2164 were not protected from weight loss yet showed a reduction in viral load in the 306 lung and brain (Fig. 5b, e) but not in the nasal wash and heart (Fig 5c, d) . Thus, antibodies that 307 compete for binding with the SARS-CoV-1 mAb rCR3022 can be elicited after SARS-CoV-2 308 infection, some of which can confer protection. 309

CoV-2 S protein. We hypothesized that SARS-CoV-2 mRNA vaccines might induce public 311 clonotypes that are shared with those seen in convalescent individuals after natural infection. We 312 obtained peripheral blood mononuclear cells from a volunteer 10 days after first vaccine dose 313 and 7 days after second vaccine dose with the Pfizer-BioNTech vaccine. Circulating 314 plasmablasts were enriched directly from blood by negative selection using paramagnetic beads 315 and purified further by flow cytometric sorting (Fig. 6a, b) . Sorted plasmablasts were loaded on 316 a Beacon microfluidics instrument for single-cell secreted antibody binding screening and 317 antibody gene sequencing or in a Chromium single-cell microfluidics device (10X Genomics) 318 followed by reverse transcription with PCR and sequence analysis to obtain paired antibody 319 sequences, as described (Zost et al., 2020b) . Enzyme-linked immunospot (ELISpot) assay 320 analysis revealed a large increase in the frequency of S-reactive cells in the enriched plasmablast 321 cell fraction on day 7 after the second vaccination compared to that on day 10 after the first 322 vaccine dose, confirming induction of target-specific responses in this individual. SARS-CoV-2 323 S6P ecto -specific secreted antibodies were of IgG and IgA classes and accounted for >10% of total 324 plasmablasts ( Fig. 6c) . Further, single-cell antibody secretion analysis of a total of 4,797 purified 325 plasmablasts revealed that a large fraction of SARS-CoV-2-reactive clones (included S6P ecto -326 and/or RBD-reactive clones) secreted RBD-specific IgG (Fig. 6d) . 327 We also analyzed antibody reactivity and neutralization in serum collected on the day 328 before vaccination (day 0), on day 10 after the first vaccine dose, on day 7 after the second 329 vaccine dose, and on day 28 after the second vaccine dose. The reactivity of serum antibodies to 330 both SARS-CoV-2 S6P ecto and SARS-CoV-2 RBD was measured by ELISA for binding (Fig. 6f ) 331 and by VSV-SARS-CoV-2 neutralizing assay (Fig. 6g) . Binding and neutralizing activities 332 steadily increased over time, with maximal activity detected on day 28 after the second vaccine 333 dose. 334 We obtained 725 paired heavy and light chain sequences from plasmablasts following 335 primary immunization and 8,298 paired sequences from plasmablasts following the second dose 336 of vaccine. The same procedure was carried out on a sample collected 35 days after onset of 337 symptoms from a convalescent individual with confirmed SARS-CoV-2 infection. This 338 individual's serum had been determined previously to contain neutralizing antibodies (Zost et al., 339 2020b ). Single-cell antibody secretion analysis revealed that a minor fraction (0.5%) of total 340 plasmablasts produced S-protein-reactive antibodies. We identified 1,883 paired heavy and light 341 chain antibody sequences for this specimen. 342

Antibody sequences identified in these new studies and sequences we collected from 343 previous SARS-CoV-2 antibody discovery studies were clustered as described in Fig. 1 . We 344 identified a total of 37 public clonotypes, 26 of which represented clonotypes shared between 345 antibodies isolated from the vaccinee and individuals with exposure history to natural SARS-346

CoV-2 infection (Fig. 6h) . The antigen-binding specificity of each group was inferred through 347 review of data in each respective publication in which the antibodies were reported. 14 of the 26 348 newly-identified shared clonotypes encoded antibodies specific to the SARS-CoV-2 S protein. 349

Within that panel of mAbs, 8 of 26 clonotypes reacted with SARS-CoV-2 RBD protein, and 6 of 350 the 26 public clonotypes cross-reacted with both SARS-CoV-1 and SARS-CoV-2 (Fig. 7) . Most 351 antibodies shared in public clonotypes were IgG, with a subset of IgAs noted. This finding shows 352 that the Pfizer-BioNTech vaccine induces many antibodies that are genetically similar to ones 353 elicited through natural SARS-CoV-2 infection, including multiple public clonotypes in 354 convalescent donors encoded by commonly used V H genes such as IGHV3-53, IGHV3-66,  355  IGHV1-58, IGHV3-30, and IGHV3-30-3. Additionally, of the 37 total public clonotypes, 16  356 bound to RBD, and of these, 11 of 16 were neutralizing. All neutralizing public clonotypes 357 recognized RBD. However, of the 37 public clonotypes identified, 21 are directed to antigenic 358 sites other than the RBD, including ones described here directed to the S2 domain. Overall, these 359 results suggest that many of the public clonotypes observed in previously infected individuals 360 likely are found in vaccinated individuals. 361

The high number of identified public B cell clonotypes in the response to SARS-CoV-2 364 infection or vaccination is striking. Many public clonotypes are shared between both infected and 365 vaccinated individuals. Public clonotypes were induced by each of the currently known antigenic 366 sites on the S protein and are found in both the neutralizing and non-neutralizing repertoires. 367 Some clonotypes in the shared SARS-CoV-2 response appear preconfigured in the germline state 368 to recognize particular S epitopes, and this recognition likely is driven by particular structural 369 features on S. In this study we compared the sequences of more than 14,000 paired B cell 370 sequences encoding antibodies to S protein of SARS-CoV-2. Likely, the availability of large 371 numbers of paired antibody gene sequences from a multitude of donors contributed to our ability 372 to identify such a high number of paired-sequence public clonotypes. 373

Several neutralizing public clonotypes have be identified previously, most commonly 374 clonotypes encoded by the closely related heavy chain genes IGHV3-53, IGHV3-66 (Tan et al., 375 2021; Yuan et al., 2020a) , IGHV1-2 (Micah Rapp, 2021), and IGHV3-30 (Robbiani et al., 2020) . 376

Structural features of these public clonotypes likely drive the frequent selection of such clones, 377 such as the canonical configuration of aromatic residues in the public clonotype IGHV1-58 + 378 IGHJ3 and IGKV3-20 + IGKJ1 that engages the SARS-CoV-2 RBD F486 residue (Dong et al., 379 2021) . Members of this public clonotype, such as COV2-2196, engage the RBD using 380 predominantly germline-encoded residues in both the heavy and light chain (Dong et al., 2021; 381 Kreer et al., 2020; Nielsen et al., 2020; Robbiani et al., 2020; Tortorici et al., 2020) . 382

Identification of public clonotypes from multiple donors suggest these antibodies could 383 contribute to humoral responses that mediate protection if they appear not only in memory B 384 cells but also as antibodies from plasma cells secreted into the serum (Voss et al., 2020) . The 385 high prevalence of public clonotypes elicited to the SARS-CoV-2 S trimer may contribute to the 386 high efficacy of S-encoding mRNA vaccines in large populations. 387

If diverse individuals independently make the same antibody in response to an antigen, 388 there could be a constant and collective selective pressure on that epitope, resulting in a high 389 potential for escape variants at that site. For example, while IGHV3-53-and IGHV3-66-encoded 390 public clonotypes have been described in numerous individuals, neutralization of these 391 antibodies is impacted adversely by the K417N or K417T substitutions present in the B.1.351 or 392 P.1 SARS-CoV-2 variants of concern, respectively (Yuan et al., 2021). A similar case was 393 described for IGHV1-2-encoded antibodies that target the RBD and IGHV1-24-encoded 394 antibodies that target the NTD. These antibodies are found in the serum of convalescent 395 individuals (Voss et al., 2020) , but neutralization of these antibodies is negatively affected for 396 501Y.V2 variant viruses (Wibmer et al., 2021) . A possible explanation for the selective pressure 397 that led to the emergence and propagation of these variants is the humoral immunity mediated by 398 these public clonotypes. 399

The new Group 3 public clonotype neutralizing and protective antibodies described here 400 bind to the cryptic face of the RBD and compete with the SARS-CoV-2 non-neutralizing mAb 401 CR3022. Neutralizing antibodies that bind to the more conserved base of the RBD are of interest, 402 as these sites are largely unaffected by common mutations in the variants of concern such as 403 E484K, K417N, and N501Y (Yuan et al., 2021) . Importantly, recent work has identified a 404 B.1.1.7 variant with a deletion of RBD residues 375-377. This deletion disrupts the epitope of 405

CR3022, yet appears to be functionally tolerated (Rennick et al., 2021) . As Group 3 antibodies 406 share a similar epitope, with critical residues of COV2-2531 and C126 being K378 and A372, 407 but with additional critical residues of Y369, N370, F374, and P384 identified for C126, this 408 deletion might abrogate binding of antibodies from this public clonotype. Group 3 antibody 409 characterization reveals additional insights on antibodies binding to epitopes similar to that of 410 CR3022. Recently published literature suggests that there is an avidity threshold that affects 411 antibody neutralization at the cryptic face of the RBD . We hypothesize that affinity 412 improvements via somatic hypermutation play an important role in the ability of COV2-2531 to 413 neutralize, particularly since neither C126 nor the germline revertant of Group 3 neutralize virus. 414

To our knowledge, public clonotypes specific to the S2 domain have not been described. 415

In this study, we identified two public clonotypes that target the S2 domain of the S trimer. 416

These mAbs do not neutralize, but they react with S proteins of both SARS-CoV-2 and SARS-417

CoV-1. It is likely that these S2 epitopes are the target of non-neutralizing antibodies in multiple 418 individuals following infection or vaccination. Previous studies have identified broadly 419 immunogenic epitopes that are conserved in the functional domains of the SARS-CoV-2 S trimer 420 S2 domain, including cross-reactivity to endemic coronaviruses, and therefore these findings 421 have important implications for antibody and vaccine design (Ladner et al., 2021) . The S2 region 422 of the S trimer may be more capable of recruiting preexisting memory B cells for diverse 423 coronaviruses, since the S2 domain is more conserved for functionally important sites such as the 424 heptad repeat regions and fusion loop (Anderson et al., 2021) . 425

We propose that there are essentially four classes of public clonotypes: (1) neutralizing 426 public clonotypes that bind to relatively invariant sites on S, (2) neutralizing public clonotypes 427 that bind to sites that tolerate high sequence variability, (3) non-neutralizing public clonotypes 428 that target relatively invariant sites, and (4) non-neutralizing public antibodies that target variable 429 sites. The first class of antibodies is likely the most protective class in a population, as these 430 mAbs neutralize and recognize residues unlikely to be sustained with mutations due to loss of 431 viral fitness. An example of this class would be IGHV1-58-encoded antibodies as described 432

previously (Dong et al., 2021) . Examples of this group were discussed here, such as IGHV3-53-433 and IGHV3-66-encoded antibodies that target the RBD (Yuan et al., 2021) . Here, we described 434 three new public clonotypes following natural infection (Groups 1, 2, and 3) and a total of 29 435 new clonotypes after mRNA vaccination. Public clonotype Groups 1 and 2 fall into the third 436 class of antibodies described here (non-neutralizing antibodies that target invariant sites), and 437 public clonotype Group 3 antibodies falls into the second class (neutralizing public clonotypes 438 that bind to variable sites). Future public clonotypes to SARS-CoV-2 could be binned with this 439 four-quadrant scheme to better understand how public clonotypes contribute to humoral 440 immunity against COVID-19. 441

Understanding the antibody response that is shared between convalescent and vaccinated 442 individuals also will be of continued interest as the percentage of vaccinated individuals 443 increases in the facing of emergence of new viral variants of concern. The understanding of viral 444 epitopes that induce protective antibodies in multiple individuals has implications for predicting 445 the most common responses to new vaccines in large populations. The emergence of SARS-446

CoV-2 variants with acquired mutations in epitopes for neutralizing antibodies, including 447 antibody regimens currently authorized for EUA, is a cause for concern (Collier et Christopher Gainza for support in antibody purifications. We also thank Berkeley Lights and 459

Jonathan Didier for expert technical support, and STEMCELL Technologies and Aida Mayhew 460 for supplying crucial B cell enrichment reagents. We thank Jem Uhrlaub, Brendan Larson, and 461

Dr. Mike Worobey (University of Arizona) for preparation and sequencing of early-passage 462 SARS-CoV-2 stocks. We thank Eun-Hyung Lee, Doan C. Nguyen, and Ignacio Sanz from 463 Emory University for sharing the plasmablast survival medium that promotes antibody secretion. 464

We thank the anonymous donors of the plasma samples for their consent that allowed the WHO 465

International standard for anti-SARS-CoV-2 human immunoglobulin to be prepared; we express 466 our gratitude to those who have coordinated the collection of the convalescent plasma clonotypes that are shared between the individuals listed in the key to the right side. 504

The heat map is color coded so that red represents a higher number of sequences 505 using the corresponding genes, and blue represents a lower number of sequences 506 using the corresponding genes. 507 b. CDR3 sequences of the heavy and light chains of each of the remaining eight public 508 clonotypes are shown. Dashes represent amino acids that differed in the public 509 clonotype. Each box color correlates to the public clonotypes in Fig. 1a and Fig. 1c . were inoculated with SARS-CoV-2. Antibody concentrations started at 10 µg/mL and 535 were titrated three-fold. 536 d. Antibody binding to full-length S (grey) or S protein C-terminus S2 region (red) 537 expressed on the surface of HEK-293T cells that were fixed and permeabilized. 538

Antibodies were screened at 1 µg/mL. Antibody reactivity was measured by flow 539 cytometry and cellular florescence values were determined. COV2-2490, an NTD-540 directed antibody, was used as a control. 541 e. Binding to VSV-SARS-CoV-2-infected Vero cells (SARS-CoV-2 WT) was measured 542 using flow cytometry and median florescence intensity values were determined for dose-543 response binding curves. Antibody was diluted 3-fold staring from 10 µg/mL 544 f. Binding to S protein C-terminus S2 region expressed on HEK-293T cells (SARS-CoV-2 545 WT S2) was measured using flow cytometry and mean fluorescence intensity values were 546 determined for dose-response binding curves. Antibody was diluted 3-fold starting from 547 10 µg/mL. shown. 600 f. Alanine scanning mutagenesis results for Group 1, 2 or 3 antibodies. S2 Epitope residues 601 are shown (green spheres or blue spheres) on the S protein structure (PDB 6XR8), S1 is 602 colored yellow, S2 red. RBD epitopes are shown in red on the RBD structure (PDB 603 6XR8). Primary data shown in Fig. S5 . ....................GN..................A........S........................................F..G.............  .......................G...................A.....N........................................... 

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Reagents kits (CG000086_REV C). Amplicons were sequenced on an Illumina Novaseq 6000, 1015 and data were processed using the CellRanger software v3.1.0 (10X Genomics). 1016Single-cell antibody secretion analysis using Beacon instrument. FACS-purified 1017 plasmablasts were resuspended in plasmablast survival medium that promotes antibody secretion 1018and assessed for reactivity of secreted antibodies using the 11k chip on Beacon optofluidic 1019 instrument (Berkley Lights) as previously described (Zost et al., 2020b) . Single cell-antibody 1020 secretion binding assay was performed as previously described (Zost et al., 2020b) and then PBS containing 0.05% Tween, and then incubated with either goat anti-human IgG-HRP 1032 conjugated antibodies (Southern Biotech), goat anti-human IgA-HRP conjugated antibodies 1033 (Southern Biotech), or goat anti-human IgM-HRP conjugated antibodies (Southern Biotech) for 2 h 1034 at room temperature. After washing three times with PBS containing 0.05% Tween/1% BSA, plates 1035 were developed using 3-amino-9-ethyl-carbazole (AEC) substrate (Sigma). The developed plates 1036 were scanned, and spots were analyzed using an automated ELISpot counter (Cellular Technologies 1037Ltd.). Plasmablasts or sorted plasmablasts from PBMCs were added to the plates and incubated 18-