key: cord-0916450-e5g8pqzz
authors: Kramer, Kevin J.; Johnson, Nicole V.; Shiakolas, Andrea R.; Suryadevara, Naveenchandra; Periasamy, Sivakumar; Raju, Nagarajan; Williams, Jazmean K.; Wrapp, Daniel; Zost, Seth J.; Holt, Clinton M.; Hsieh, Ching-Lin; Sutton, Rachel E.; Paulo, Ariana; Davidson, Edgar; Doranz, Benjamin J.; Crowe, James E.; Bukreyev, Alexander; Carnahan, Robert H.; McLellan, Jason S.; Georgiev, Ivelin S.
title: Potent neutralization of SARS-CoV-2 variants of concern by an antibody with a unique genetic signature and structural mode of spike recognition
date: 2021-05-16
journal: bioRxiv
DOI: 10.1101/2021.05.16.444004
sha: e6cb9239151f48fe64ca9e200e6c2bbeb4897bdd
doc_id: 916450
cord_uid: e5g8pqzz

The emergence of novel SARS-CoV-2 lineages that are more transmissible and resistant to currently approved antibody therapies poses a considerable challenge to the clinical treatment of COVID-19. Therefore, the need for ongoing discovery efforts to identify broadly reactive monoclonal antibodies to SARS-CoV-2 is of utmost importance. Here, we report a panel of SARS-CoV-2 antibodies isolated using the LIBRA-seq technology from an individual who recovered from COVID-19. Of these antibodies, 54042-4 showed potent neutralization against authentic SARS-CoV-2 viruses, including variants of concern (VOCs). A cryo-EM structure of 54042-4 in complex with the SARS-CoV-2 spike revealed an epitope composed of residues that are highly conserved in currently circulating SARS-CoV-2 lineages. Further, 54042-4 possesses unique genetic and structural characteristics that distinguish it from other potently neutralizing SARS-CoV-2 antibodies. Together, these findings motivate 54042-4 as a lead candidate for clinical development to counteract current and future SARS-CoV-2 VOCs.

The COVID-19 pandemic caused by a novel coronavirus from the Sarbecovirus genus, 47 Figure 3A ) are highly conserved in circulating SARS-CoV-2 lineages ( Figure 5C ). The only 242 exception is residue N439, which has a substitution frequency of 2.1% ( Figure 5C) ; however, 243 this residue makes only minimal contacts with antibody 54042-4 (Supplemental Figure 3A) , 244 suggesting that residue N439 may not be critical for 54042-4 recognition of the SARS-CoV-2 245 spike. 246

To investigate the ability of antibody 54042-4 to recognize current SARS-CoV-2 VOCs, 247 we next performed ELISAs to test binding of 54042-4 to RBD proteins containing substitutions 248 found in one or more VOCs. These substitutions included K417N (Wadman, 2021) . These observations underscore the ongoing need for genomic 291 surveillance to monitor the emergence and spread of new SARS-CoV-2 variants and their 292 effects on population immunity. 293

In addition to vaccines, antibody therapeutics can play an important role for treating 294 SARS-CoV-2 infections. Given the unknown future trajectory of the pandemic and the potential 295 for emergence of VOCs that may escape neutralization by vaccine-elicited immunity, the 296 development of a wide array of candidate antibody therapeutics that are insensitive to 297 substitutions found in major VOCs may prove critical in the fight against COVID-19. However, 298 current VOCs have already shown an ability to escape neutralization by a number of antibodies 299 in clinical development (Chen et al., 2021; Wang et al., 2021) . In contrast, our binding, 300 neutralization, and structural data suggest that antibody 54042-4 is capable of avoiding all of the 301 current major substitutions in circulating VOCs. Combined with those observations, the unique 302 features of 54042-4 in comparison to other SARS-CoV-2 antibodies motivate further clinical 303 development of this antibody to complement the existing pool of therapeutic countermeasures. 304

As SARS-CoV-2 virus evolution continues due to various factors, such as a lack of vaccine 305 access and the associated delayed vaccine rollout to underserved parts of the world, new VOCs 306 are likely to keep emerging, with the potential to decrease or even abrogate protection induced 307 by current vaccines. Antibody therapeutic development, especially focusing on broad protection 308 against diverse SARS-CoV-2 variants, is therefore of continued significance.

The donor had previous laboratory-confirmed COVID-19, 3 months prior to blood collection. The 312 studies were reviewed and approved by the Institutional Review Board of Vanderbilt University 313

Medical Center. The sample was obtained after written informed consent was obtained. 314 315

A variety of recombinant soluble protein antigens were used in the LIBRA-seq experiment and 317 other experimental assays. 318

Plasmids encoding residues 1-1208 of the SARS-CoV-2 spike with a mutated S1/S2 319 cleavage site, proline substitutions at positions 817, 892, 899, 942, 986 and 987, and a C-320 terminal T4-fibritin trimerization motif, an 8x HisTag, and a TwinStrepTag (SARS-CoV-2 spike 321 HP); 1-1208 of the SARS-CoV-2 spike with a mutated S1/S2 cleavage site, proline substitutions 322 at positions 817, 892, 899, 942, 986 and 987, a glycine mutation at 614, and a C-terminal T4-323 fibritin trimerization motif, an 8x HisTag, and a TwinStrepTag (SARS-CoV-2 spike HP D614G) 324 1-1208 of the SARS-CoV-2 spike with a mutated S1/S2 cleavage site, proline substitutions at For each antigen, a unique DNA barcode was directly conjugated to the antigen itself. In 403 particular, 5'amino-oligonucleotides were conjugated directly to each antigen using the Solulink 404

Protein-Oligonucleotide Conjugation Kit (TriLink cat no. S-9011) according to manufacturer's 405 instructions. Briefly, the oligo and protein were desalted, and then the amino-oligo was modified 406 with the 4FB crosslinker, and the biotinylated antigen protein was modified with S-HyNic. Then, the 4FB-oligo and the HyNic-antigen were mixed together. This causes a stable bond to form 408 between the protein and the oligonucleotide. The concentration of the antigen-oligo conjugates 409 was determined by a BCA assay, and the HyNic molar substitution ratio of the antigen-oligo 410 conjugates was analyzed using the NanoDrop according to the Solulink protocol guidelines. 411 AKTA FPLC was used to remove excess oligonucleotide from the protein-oligo conjugates, 412 which were also verified using SDS-PAGE with a silver stain. Antigen-oligo conjugates were 413 also used in flow cytometry titration experiments. The secondary antibody, goat anti-human IgG conjugated to peroxidase, was added at 1:10,000 498 dilution in 1% milk in PBS-T to the plates, which were incubated for one hour at room 499 temperature. Plates were washed three times with PBS-T and then developed by adding 500 3,3′,5,5′-tetramethylbenzidine (TMB) substrate to each well. The plates were incubated at room 501 temperature for ten minutes, and then 1N sulfuric acid was added to stop the reaction. Plates 502 were read at 450 nm. Data are represented as mean ± SEM for one ELISA experiment. ELISAs reduced the reference antibody binding by more than 60% and non-competing if the signal was 527 reduced by less than 30%. 528

To screen for neutralizing activity in the panel of recombinantly expressed antibodies, we used a 531 high-throughput and quantitative RTCA assay and xCelligence RTCA HT Analyzer (ACEA 532 Epitope mapping was performed essentially as described previously (Davidson, 2014) 

CoV-2 (strain Wuhan-Hu-1) spike protein RBD shotgun mutagenesis mutation library, made using an 576 expression construct for full-length spike protein. 184 residues of the RBD (between spike residues 577 335 and 526) were mutated individually to alanine, and alanine residues to serine and clones arrayed 578 in 384-well plates, one mutant per well. Antibody binding to each mutant clone was determined, in 579 duplicate, by high-throughput flow cytometry. Each spike protein mutant was transfected into HEK-580 293T cells and allowed to express for 22 hrs. Cells were fixed in 4% (v/v) paraformaldehyde (Electron 581 Microscopy Sciences), and permeabilized with 0.1% (w/v) saponin (Sigma-Aldrich) in PBS plus 582 calcium and magnesium (PBS++) before incubation with antibodies diluted in PBS++, 10% normal 583 goat serum (Sigma), and 0.1% saponin. Antibody screening concentrations were determined using 584 an independent immunofluorescence titration curve against cells expressing wild-type spike protein 585 to ensure that signals were within the linear range of detection. Antibodies were detected using 3.75 586 μg/mL of AlexaFluor488-conjugated secondary antibody (Jackson ImmunoResearch Laboratories) in 587 10% normal goat serum with 0.1% saponin. Cells were washed three times with PBS++/0.1% saponin 588 followed by two washes in PBS, and mean cellular fluorescence was detected using a high-589 throughput Intellicyte iQue flow cytometer (Sartorius). Antibody reactivity against each mutant spike 590 protein clone was calculated relative to wild-type spike protein reactivity by subtracting the signal from 591 mock-transfected controls and normalizing to the signal from wild-type S-transfected controls. 592

Mutations within clones were identified as critical to antibody binding if they did not support reactivity 593 of the test antibody, but supported reactivity of other SARS-CoV-2 antibodies. This counter-screen 594 strategy facilitates the exclusion of spike mutants that are locally misfolded or have an expression 595 defect. 596 597

The virus neutralization with live authentic SARS-CoV-2 virus (USA-WA1) was performed in the 599 BSL-3 facility of the Galveston National Laboratory using Vero E6 cells (ATCC CRL-1586) 600

following the standard procedure. Briefly, Vero E6 cells were cultured in 96-well plates (10 4 601 cells/well). Next day, 4-fold serial dilutions of antibodies were made using MEM-2% FBS, as to get an initial concentration of 100 µg/ml. Equal volume of diluted antibodies (60 µl) were mixed 603 gently with original SARS-CoV-2 or B.1.1.7 variant or B.1.351 variant (60 µl containing 200 pfu) 604 and incubated for 1 h at 37°C/5% CO 2 atmosphere. The virus-serum mixture (100 µl) was 605 added to cell monolayer in duplicates and incubated for 1 at h 37°C/5% CO2 atmosphere. Later, 606 virus-serum mixture was discarded gently, and cell monolayer was overlaid with 0.6% 607 methylcellulose and incubated for 2 days. The overlay was removed, and the plates were fixed 608 in 4% paraformaldehyde twice following BSL-3 protocol. The plates were stained with 1% 609 crystal violet and virus-induced plaques were counted. The percent neutralization and/or NT 50 of The next day, plates were washed three times with PBS supplemented with 0.05% Tween-20 627 (PBS-T) and coated with 5% milk powder in PBS-T. Plates were incubated for one hour at room 628 temperature and then washed three times with PBS-T. Purified antibodies were diluted in 629 blocking buffer at 10 μg/mL in triplicate, added to the wells, and incubated at room temperature. 630

Without washing, recombinant human ACE2 protein with a mouse Fc tag was added to wells for 631 a final 0.4 μg/mL concentration of ACE2 and incubated for 40 minutes at room temperature. 632

Plates were washed three times with PBS-T, and bound ACE2 was detected using HRP-633 conjugated anti-mouse Fc antibody and TMB substrate. The plates were incubated at room 634 temperature for ten minutes, and then 1N sulfuric acid was added to stop the reaction. Plates 635 were read at 450 nm. ACE2 binding without antibody served as a control. Experiment was done 636 in biological replicate and technical triplicates. Additional details about data collection parameters can be found in Supplemental Table 1 . 667 668

Motion correction, CTF estimation, particle picking, and preliminary 2D classification were 670 performed using cryoSPARC v3.2.0 live processing (Punjani et al., 2017) . The final iteration of 671 2D class averaging distributed 374,669 particles into 60 classes using an uncertainty factor of 2. 672

From that, 241,732 particles were used to perform an ab inito reconstruction with four classes 673 followed by heterogeneous refinement of those four classes. Particles from the two highest quality classes were used for homogenous refinement of the best volume with applied C3 

PHENIX: building new 911 software for automated crystallographic structure determination

IMGT((R)) tools for the 914 nucleotide analysis of immunoglobulin (IG) and T cell receptor (TR) V-(D)-J repertoires, 915 polymorphisms, and IG mutations: IMGT/V-QUEST and IMGT/HighV-QUEST for NGS

Early introductions and 919 transmission of SARS-CoV-2 variant B.1.1.7 in the United States

SARS-CoV-2 neutralizing 922 antibody structures inform therapeutic strategies

Potent neutralizing 925 antibodies from COVID-19 patients define multiple targets of vulnerability

Neutralizing Antibody and Soluble ACE2 Inhibition of a 929

Replication-Competent VSV-SARS-CoV-2 and a Clinical Isolate of SARS-CoV-2

Resistance of SARS-CoV-2 variants to neutralization by 933 monoclonal and serum-derived polyclonal antibodies

A neutralizing human antibody binds to the N-terminal domain of the Spike 936 protein of SARS-CoV-2

COSMIC2 -A Science gateway for 938 cryo-electron microscopy

COSMIC2: A Science Gateway for Cryo-Electron Microscopy Structure Determination in 941 Proceedings of the Practice and Experience in Advanced Research Computing 2017 on 942 Sustainability, Success, and Impact 1-5

ISOLDE: a physically realistic environment for model building into low-944 resolution electron-density maps

A high-throughput shotgun mutagenesis approach to mapping B-946 cell antibody epitopes

The antigenic anatomy of SARS-CoV-2 949 receptor binding domain

Genetic and structural basis for recognition of 952 SARS-CoV-2 spike protein by a two-antibody cocktail

SAbPred: a structure-based antibody prediction server. 955

Data, disease and diplomacy: GISAID's innovative contribution to 957 global health

Coot: model-building tools for molecular graphics

Mutational escape from 964 the polyclonal antibody response to SARS-CoV-2 infection is largely shaped by a single class of 965 antibodies

Change-O: a toolkit for analyzing large-scale B cell immunoglobulin repertoire 968 sequencing data

Studies in humanized mice and convalescent humans yield a 971 SARS-CoV-2 antibody cocktail

Controlling the SARS-CoV-2 spike glycoprotein 974 conformation

SARS-CoV-2 variants B.1.351 and P.1 977 escape from neutralizing antibodies

Structure-based design of prefusion-stabilized 980 SARS-CoV-2 spikes

The neutralizing 983 antibody, LY-CoV555, protects against SARS-CoV-2 infection in non-human primates

Shemer-986

SARS-CoV-2 spike variants exhibit differential 987 infectivity and neutralization resistance to convalescent or post-vaccination sera

Structure, Function, and Evolution of Coronavirus Spike Proteins

Potent neutralizing antibodies against multiple epitopes on SARS-993 CoV-2 spike

Convergent Antibody Responses to SARS-CoV-2

UCSF ChimeraX: Structure visualization for researchers, educators, 999 and developers

Mapping Neutralizing and

Immunodominant Sites on the SARS-CoV-2 Spike Receptor-Binding Domain by Structure-1003 Guided High-Resolution Serology

Cross-neutralization of SARS-CoV-2 by a human 1006 monoclonal SARS-CoV antibody

cryoSPARC: algorithms for 1008 rapid unsupervised cryo-EM structure determination

Non-uniform refinement: adaptive regularization 1010 improves single-particle cryo-EM reconstruction

CoV-AbDab: the 1012 coronavirus antibody database

Alignment of cryo-EM movies of individual particles 1014 by optimization of image translations

Resurgence 1017 of COVID-19 in Manaus, Brazil, despite high seroprevalence

DeepEMhancer: a deep learning solution for cryo-EM volume post-processing

High-Throughput Mapping of B Cell 1022 Receptor Sequences to Antigen Specificity

Structural basis of receptor recognition by SARS-CoV-2

Cross-reactive coronavirus antibodies 1027 with diverse epitope specificities and extra-neutralization functions

Deep Mutational Scanning

Receptor Binding Domain Reveals Constraints on Folding and ACE2 Binding

Neutralizing and protective human 1034 monoclonal antibodies recognizing the N-terminal domain of the SARS-CoV-2 spike protein

Detection of a SARS-CoV-2 variant of concern 1038 in South Africa

Circulating SARS-CoV-2 spike 1041 N439K variants maintain fitness while evading antibody-mediated immunity

Structural insights into coronavirus entry

Novavax vaccine delivers 89% efficacy against COVID-19 in U.K.-but is 1046 less potent in South Africa

Antibody resistance of SARS-CoV-2 variants B.1.351 and B.1.1.7

Broad sarbecovirus neutralizing antibodies 1052 define a key site of vulnerability on the SARS-CoV-2 spike protein

REGN-COV2, a Neutralizing Antibody Cocktail, in Outpatients 1055 with Covid-19

Cryo-EM structure of the 2019-nCoV spike in the prefusion conformation

Neutralization of SARS-CoV-2 spike 69/70 deletion, E484K and 1061 N501Y variants by BNT162b2 vaccine-elicited sera

Structural basis of a shared antibody response to SARS-CoV-2

1066 (2020b). A highly conserved cryptic epitope in the receptor binding domains of SARS-CoV-2 1067 and SARS-CoV

Rapid isolation and profiling of a diverse panel 1070 of human monoclonal antibodies targeting the SARS-CoV-2 spike protein

symmetry. Non-uniform refinement was performed on the resulting volume using per-particle 676 defocus and per-group CTF optimizations applied (Punjani et al., 2020; Rubinstein and 677 Brubaker, 2015) . To improve the 54042-4 Fab-RBD density, C3 symmetry expansion was 678 performed followed by local refinement using a mask created in ChimeraX that encompassed 679 the entire 54042-4 Fab and RBD (Pettersen et al., 2021) . Local refinement was performed using 680 a pose/shift gaussian prior during alignment, 3° standard deviation of prior over rotation and 1 Å 681 standard deviation of prior over shifts. Additionally, maximum alignment resolution was limited to 682 2.8 Å resolution to avoid over-refining. To improve map quality, the focused refinement volumes 683 were processed using the DeepEMhancer(Sanchez-Garcia, 2021) tool via COSMIC 2 science 684 gateway, which included the use of our refinement mask to help define noise while sharpening 685 (Cianfrocco, 2017a; Cianfrocco, 2017b ). An initial model was generated by docking PDBID: To evaluate the conservation of 54042-4 epitope residues, we utilized the GISAID database 695 (Elbe, 2017) comprising sequences from 1229459 SARS-CoV-2 variants (as of May 6th, 2021). 696The spike glycoprotein sequences were extracted and translated, and pairwise sequence 697 alignment with the reference sequence hCoV-19/Wuhan/WIV04/2019 was then performed. After 698 removing incomplete sequences and sequences with alignment errors, the pairwise alignments 699 for the remaining 1,148,887 spike protein sequences were combined to compute the 700 conservation of each residue position using in-house perl scripts.