Cross-reactive coronavirus antibodies with diverse epitope specificities and Fc effector functions

Lead contact

Further information and requests for resources and reagents should be directed to the Lead Contact, Ivelin Georgiev (Ivelin.Georgiev@Vanderbilt.edu).

Materials availability

All unique/stable reagents generated in this study are available from the Lead Contact with a completed Materials Transfer Agreement. Please direct resource and reagent requests to the Lead Contact specified above, Ivelin Georgiev.

Data and code availability

Sequences for antibodies identified and characterized in this study have been deposited to GenBank under GenBank accession numbers MZ126644-MZ126658 (heavy chain) and MZ126659-MZ126673 (light chain). Raw sequencing data used in this study are available on the Sequence Read Archive under BioProject accession number PRJNA727275. Custom scripts used to analyze data in this manuscript are available upon request to the corresponding author.

Human subjects

The donor had prior SARS-CoV infection during the 2004 outbreak in Hong Kong, and the PBMC sample was collected over 10 years post infection (20 million PBMCs). Additional information about the donor is not available.

Cell lines

A variety of cell lines were utilized for various assays in this study.

Expi293F mammalian cells (ThermoFisher) were maintained in FreeStyle F17 expression medium supplemented at final concentrations of 0.1% Pluronic Acid F-68 and 20% 4mM L-Glutamine. These cells were cultured at 37°C with 8% CO₂ saturation and shaking.

FreeStyle293F cells were grown while shaking at 37 C in 8% CO2 and 80% humidity. Freestyle293F cells are derived from female human embryonic kidney epithelial cells.

THP-1 cells obtained from the AIDS Reagent Program (Division of AIDS, NIAID, NIH contributed by Dr. Li Wu and Vineet N. KewalRamani) were used for both the ADCP and ADCT assays. Cells were cultured at 37°C, 5% CO2 in RPMI containing 10% heat-inactivated fetal bovine serum (GIBCO, Gaithersburg, MD), 1% Penicillin Streptomycin (GIBCO, Gaithersburg, MD) and 2-mercaptoethanol to a final concentration of 0.05 mM. These cells were not allowed to exceed 4 × 105 cells/ml to prevent differentiation and are from a male donor.

HEK293T cells were obtained from Dr George Shaw and were used for the ADCT assay. These adherent cell lines were cultured at 37°C, 5% CO2, in DMEM containing 10% heat-inactivated fetal bovine serum (GIBCO BRL Life Technologies) and supplemented with 50 μg/ml gentamicin (Sigma). Cells were disrupted at confluence with 0.25% trypsin in 1 mM EDTA (Sigma) every 48–72 hours. HEK293F suspension cells were cultured in 293Freestyle media (GIBCO BRL Life Technologies) and grown in a shaking incubator at 37°C, 5% CO2, 70% humidity at 125rpm. Cells were diluted twice a week to between 0.2 and 0.5 million cells/ml. Both HEK293 derived cell lines are from female donors.

Murine model

12-month old female BALB/c mice (BALB/cAnHsd; Envigo, stock number 047) were used in a murine infection model for SARS-CoV-2 with a mouse adapted strain.

Eleven to twelve-month old female BALB/c mice (BALB/c AnNHsd, Envigo, stock# 047) were used for mouse-adapted SARS-CoV-2 (SARS-CoV-2 MA10) in vivo protection experiments as described previously.³¹ All mouse studies were performed at the University of North Carolina (Animal Welfare Assurance #A3410-01) using protocols (19-168) approved by the UNC Institutional Animal Care and Use Committee (IACUC) and were performed in a BSL3 facility at UNC.

Antigen purification

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

Plasmids encoding residues 1–1208 of the SARS-CoV-2 spike with a mutated S1/S2 cleavage site, proline substitutions at positions 986 and 987, and a C-terminal T4-fibritin trimerization motif, an 8x HisTag, and a TwinStrepTag (SARS-CoV-2 S-2P); residues 1-1190 of the SARS-CoV spike with proline substitutions at positions 968 and 969, and a C-terminal T4-fibritin trimerization motif, an 8x HisTag, and a TwinStrepTag (SARS-CoV S-2P); residues 1-1291 of the MERS-CoV spike with a mutated S1/S2 cleavage site, proline substitutions at positions 1060 and 1061, and a C-terminal T4-fibritin trimerization motif, an AviTag, an 8x HisTag, and a TwinStrepTag (MERS-CoV S-2P Avi); residues 1-751 of the MERS-CoV spike with a C-terminal T4-fibritin trimerization motif, 8x HisTag, and a TwinStrepTag (MERS-CoV S1); residues 1-1277 of the HCoV-HKU1 spike with a mutated S1/S2 cleavage site, proline substitutions at positions 1067 and 1068, and a C-terminal T4-fibritin trimerization motif, an 8x HisTag, and a TwinStrepTag (HCoV-HKU1 S-2P); residues 1-1278 of the HCoV-OC43 spike with proline substitutions at positions 1070 and 1071, and a C-terminal T4-fibritin trimerization motif, an 8x HisTag, and a TwinStrepTag (HCoV-OC43 S-2P); or residues 319–591 of SARS-CoV-2 S with a C-terminal monomeric human IgG Fc-tag and an 8x HisTag (SARS-CoV-2 RBD-SD1) were transiently transfected into FreeStyle293F cells (Thermo Fisher) using polyethylenimine. The coronavirus trimer spike antigens were in a prefusion-stabilized (S-2P) conformation that better represents neutralization-sensitive epitopes in comparison to their wild-type forms⁵¹. Two hours post-transfection, cells were treated with kifunensine to ensure uniform glycosylation. Transfected supernatants were harvested after 6 days of expression. SARS-CoV-2 RBD-SD1 was purified using Protein A resin (Pierce), SARS-CoV-2 S-2P, SARS-CoV S-2P, MERS-CoV S-2P Avi, MERS-CoV S1, HCoV-HKU1 S-2P and HCoV-OC43 S-2P were purified using StrepTactin resin (IBA). Affinity-purified SARS-CoV-2 RBD-SD1 was further purified over a Superdex75 column (GE Life Sciences). MERS-CoV S1 was purified over a Superdex200 Increase column (GE Life Sciences). SARS-CoV-2 S-2P, SARS-CoV S-2P, MERS-CoV S-2P Avi, HCoV-HKU1 S-2P and HCoV-OC43 S-2P were purified over a Superose6 Increase column (GE Life Sciences).

For the HIV-1 gp140 SOSIP variant from strain ZM197 (clade C) and CZA97 (clade C), recombinant, soluble antigens contained an AviTag and were expressed in Expi293F cells using polyethylenimine transfection reagent and cultured. FreeStyle F17 expression medium supplemented with pluronic acid and glutamine was used. The cells were cultured at 37°C with 8% CO₂ saturation and shaking. After 5-7 days, cultures were centrifuged and supernatant was filtered and run over an affinity column of agarose bound Galanthus nivalis lectin. The column was washed with PBS and antigens were eluted with 30 mL of 1M methyl-a-D-mannopyranoside. Protein elutions were buffer exchanged into PBS, concentrated, and run on a Superdex 200 Increase 10/300 GL Sizing column on the AKTA FPLC system. Fractions corresponding to correctly folded protein were collected, analyzed by SDS-PAGE and antigenicity was characterized by ELISA using known monoclonal antibodies specific to each antigen. Avi-tagged antigens were biotinylated using BirA biotin ligase (Avidity LLC). Non-Avi-tagged antigens were biotinylated using the EZ-Link Sulfo-NHS-Biotin kits using a 50:1 biotin to protein molar ratio.

For binding studies, SARS-CoV-2 HexaPro S, SARS-CoV S, SARS-CoV-2 RBD, SARS-CoV RBD, and MERS-CoV RBD constructs were expressed in the transient expression system previously mentioned. S proteins were purified using StrepTrap HP columns and RBD constructs were purified over protein A resin, respectively. Each resulting protein was further purified to homogeneity by size-exclusion chromatography on a Superose 6 10/300 GL column.

SARS-CoV-2 S1, SARS-CoV-2 S1 D614G, SARS-CoV-2 S2, and SARS-CoV-2 NTD truncated proteins were purchased from the commercial vendor, Sino Biological.

DNA-barcoding of antigens

We used oligos that possess 15 bp antigen barcode, a sequence capable of annealing to the template switch oligo that is part of the 10X bead-delivered oligos, and contain truncated TruSeq small RNA read 1 sequences in the following structure: 5′-CCTTGGCACCCGAGAATTCCANNNNNNNNNNNNNCCCATATAAGA∗A∗A-3′, where Ns represent the antigen barcode. We used the following antigen barcodes: GCTCCTTTACACGTA (SARS-CoV-2 S), TGACCTTCCTCTCCT (SARS-CoV S), ACAATTTGTCTGCGA (MERS-CoV S), TCCTTTCCTGATAGG (MERS-CoV S1), CAGGTCCCTTATTTC (HCoV-HKU1 S), TAACTCAGGGCCTAT (HCoV-OC43 S), CAGCCCACTGCAATA (CZA97), and ATCGTCGAGAGCTAG (ZM197). Oligos were ordered from IDT with a 5′ amino modification and HPLC purified.

For each antigen, a unique DNA barcode was directly conjugated to the antigen itself. In particular, 5′amino-oligonucleotides were conjugated directly to each antigen using the Solulink Protein-Oligonucleotide Conjugation Kit (TriLink cat no. S-9011) according to manufacturer’s instructions. Briefly, the oligo and protein were desalted, and then the amino-oligo was modified 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 between the protein and the oligonucleotide. The concentration of the antigen-oligo conjugates was determined by a BCA assay, and the HyNic molar substitution ratio of the antigen-oligo conjugates was analyzed using the NanoDrop according to the Solulink protocol guidelines. AKTA FPLC was used to remove excess oligonucleotide from the protein-oligo conjugates, which were also verified using SDS-PAGE with a silver stain. Antigen-oligo conjugates were also used in flow cytometry titration experiments.

Antigen specific B cell sorting

Cells were stained and mixed with DNA-barcoded antigens and other antibodies, and then sorted using fluorescence activated cell sorting (FACS). First, cells were counted and viability was assessed using Trypan Blue. Then, cells were washed three times with DPBS supplemented with 0.1% bovine serum albumin (BSA). Cells were resuspended in DPBS-BSA and stained with cell markers including viability dye (Ghost Red 780), CD14-APC-Cy7, CD3-FITC, CD19-BV711, and IgG-PE-Cy5. Additionally, antigen-oligo conjugates were added to the stain. After staining in the dark for 30 minutes at room temperature, cells were washed three times with DPBS-BSA at 300 g for five minutes. Cells were then incubated for 15 minutes at room temperature with Streptavidin-PE to label cells with bound antigen. Cells were washed three times with DPBS-BSA, resuspended in DPBS, and sorted by FACS. Antigen positive cells were bulk sorted and delivered to the Vanderbilt Technologies for Advanced Genomics (VANTAGE) sequencing core at an appropriate target concentration for 10X Genomics library preparation and subsequent sequencing. FACS data were analyzed using FlowJo.

Sample and library preparation, and sequencing

Single-cell suspensions were loaded onto the Chromium Controller microfluidics device (10X Genomics) and processed using the B cell Single Cell V(D)J solution according to manufacturer’s suggestions for a target capture of 10,000 B cells per 1/8 10X cassette, with minor modifications in order to intercept, amplify and purify the antigen barcode libraries as previously described.²⁷

Sequence processing and bioinformatic analysis

We utilized and modified our previously described pipeline to use paired-end FASTQ files of oligo libraries as input, process and annotate reads for cell barcode, UMI, and antigen barcode, and generate a cell barcode - antigen barcode UMI count matrix.²⁷ BCR contigs were processed using Cell Ranger (10X Genomics) using GRCh38 as reference. Antigen barcode libraries were also processed using Cell Ranger (10X Genomics). The overlapping cell barcodes between the two libraries were used as the basis of the subsequent analysis. We removed cell barcodes that had only non-functional heavy chain sequences as well as cells with multiple functional heavy chain sequences and/or multiple functional light chain sequences, reasoning that these may be multiplets. Additionally, we aligned the BCR contigs (filtered_contigs.fasta file output by Cell Ranger, 10X Genomics) to IMGT reference genes using HighV-Quest.⁴⁷ The output of HighV-Quest was parsed using ChangeO⁴⁸ and merged with an antigen barcode UMI count matrix. Finally, we determined the LIBRA-seq score for each antigen in the library for every cell as previously described.²⁷

Antibody expression and purification

For each antibody, variable genes were inserted into custom plasmids encoding the constant region for the IgG1 heavy chain as well as respective lambda and kappa light chains (pTwist CMV BetaGlobin WPRE Neo vector, Twist Bioscience). Antibodies were expressed in Expi293F mammalian cells (ThermoFisher) by co-transfecting heavy chain and light chain expressing plasmids using polyethylenimine transfection reagent and cultured for 5-7 days. Cells were maintained in FreeStyle F17 expression medium supplemented at final concentrations of 0.1% Pluronic Acid F-68 and 20% 4mM L-Glutamine. These cells were cultured at 37°C with 8% CO₂ saturation and shaking. After transfection and 5-7 days of culture, cell cultures were centrifuged and supernatant was 0.45 μm filtered with Nalgene Rapid Flow Disposable Filter Units with PES membrane. Filtered supernatant was run over a column containing Protein A agarose resin equilibrated with PBS. The column was washed with PBS, and then antibodies were eluted with 100 mM Glycine HCl at 2.7 pH directly into a 1:10 volume of 1M Tris-HCl pH 8.0. Eluted antibodies were buffer exchanged into PBS 3 times using Amicon Ultra centrifugal filter units and concentrated. Antibodies were analyzed by SDS-PAGE. Additionally, antibodies 46472-1, 46472-2, 46472-3, 46472-4, 46472-6 and 46472-12 were assessed by size exclusion chromatography on a Superdex 200 Increase 10/300 GL Sizing column with the AKTA FPLC system.

High-throughput antibody expression

For high-throughput production of recombinant antibodies, approaches were used that are designated as microscale. For antibody expression, microscale transfection were performed (∼1cml per antibody) of CHO cell cultures using the GIBCO ExpiCHO Expression System and a protocol for deep 96-well blocks (Thermo Fisher Scientific). In brief, synthesized antibody-encoding DNA (∼2cμg per transfection) was added to OptiPro serum free medium (OptiPro SFM), incubated with ExpiFectamine CHO Reagent and added to 800cμl of ExpiCHO cell cultures into 96-deep-well blocks using a ViaFlo 384 liquid handler (Integra Biosciences). The plates were incubated on an orbital shaker at 1,000crpm with an orbital diameter of 3cmm at 37c°C in 8% CO₂. The next day after transfection, ExpiFectamine CHO Enhancer and ExpiCHO Feed reagents (Thermo Fisher Scientific) were added to the cells, followed by 4cd incubation for a total of 5cd at 37c°C in 8% CO₂. Culture supernatants were collected after centrifuging the blocks at 450g for 5cmin and were stored at 4°C until use. For high-throughput microscale antibody purification, fritted deep-well plates were used containing 25cμl of settled protein G resin (GE Healthcare Life Sciences) per well. Clarified culture supernatants were incubated with protein G resin for mAb capturing, washed with PBS using a 96-well plate manifold base (QIAGEN) connected to the vacuum and eluted into 96-well PCR plates using 86cμl of 0.1cM glycine-HCL buffer pHc2.7. After neutralization with 14cμl of 1cM Tris-HCl pHc8.0, purified mAbs were buffer-exchanged into PBS using Zeba Spin Desalting Plates (Thermo Fisher Scientific) and stored at 4°C until use.

ELISA

To assess antibody binding, soluble protein was plated at 2 μg/ml overnight at 4°C. The next day, plates were washed three times with PBS supplemented with 0.05% Tween-20 (PBS-T) and coated with 5% milk powder in PBS-T. Plates were incubated for one hour at room temperature and then washed three times with PBS-T. Primary antibodies were diluted in 1% milk in PBS-T, starting at 10 μg/ml with a serial 1:5 dilution and then added to the plate. The plates were incubated at room temperature for one hour and then washed three times in PBS-T. The secondary antibody, goat anti-human IgG conjugated to peroxidase, was added at 1:10,000 dilution in 1% milk in PBS-T to the plates, which were incubated for one hour at room temperature. Goat anti-mouse secondary was used for SARS-CoV specific control antibody 240CD (BEI Resources). Plates were washed three times with PBS-T and then developed by adding TMB substrate to each well. The plates were incubated at room temperature for ten minutes, and then 1N sulfuric acid was added to stop the reaction. Plates were read at 450 nm.

Data are represented as mean ± SEM for one ELISA experiment. ELISAs were repeated 2 or more times. The area under the curve (AUC) was calculated using GraphPad Prism 8.0.0. For antibody 240CD, the following reagent was obtained through BEI Resources, NIAID, NIH: Monoclonal Anti-SARS-CoV S Protein (Similar to 240C), NR-616.

Competition ELISA

Competition ELISAs were performed as described above, with some modifications. After coating with antigen and blocking, 25 μL of non-biotinylated competitor antibody was added to each well at 10 μg/ml and incubated at 37°C for 10 minutes. Then, without washing, 75 μL biotinylated antibody (final concentration of 0.1 μg/ml) was added and incubated at 37°C for 1 hour. After washing three times with PBS-T, streptavidin-HRP was added at 1:10,000 dilution in 1% milk in PBS-T and incubated for 1 hour at room temperature. Plates were washed and substrate and sulfuric acid were added as described above. ELISAs were repeated at least 2 times. Data is shown as the % decrease in binding.

Autoreactivity

Monoclonal antibody reactivity to nine autoantigens (SSA/Ro, SS-B/La, Sm, ribonucleoprotein (RNP), Scl 70, Jo-1, dsDNA, centromere B, and histone) was measured using the AtheNA Multi-Lyte® ANA-II Plus test kit (Zeus scientific, Inc, #A21101). Antibodies were incubated with AtheNA beads for 30min at concentrations of 50, 25, 12.5 and 6.25 μg/mL. Beads were washed, incubated with secondary and read on the Luminex platform as specified in the kit protocol. Data were analyzed using AtheNA software. Positive (+) specimens received a score > 120, and negative (-) specimens received a score < 100. Samples between 100-120 were considered indeterminate.

Mannose competition

Mannose competition ELISAs were performed as described above with minor modifications. After antigen coating and washing, nonspecific binding was blocked by incubation with 5% FBS diluted in PBS for 1 hour at RT. Primary antibodies were diluted in 5% FBS-PBST ± 1M D-(+)-Mannose starting at 10 μg/ml with a serial 1:5 dilution and then added to the plate for 1 hour at RT. After washing, antibody binding was detected with goat anti-human IgG conjugated to peroxidase and added at 1:10,000 dilution in 5% FBS in PBS-T to the plates. After 1 hour incubation, plates were washed and substrate and sulfuric acid were added as described above. Data shown is representative of three replicates.

Epitope mapping visualization

SARS-CoV-2 Spike (PDB-6VSB) was visualized using PyMOL software. Antibody epitopes were visualized on the SARS-CoV-2 spike using a structure of the pre-fusion stabilized SARS-CoV-2 S-2P construct⁵ modeled in the molecular graphics software PyMOL (The PyMOL Molecular Graphics System, Version 2.3.5 Schrödinger, LLC).

RTCA neutralization assay

To assess for neutralizing activity against SARS-CoV-2 strain 2019 n-CoV/USA_WA1/2020 (obtained from the Centers for Disease Control and Prevention, a gift from N. Thornburg), we used the high-throughput RTCA assay and xCelligence RTCA HT Analyzer (ACEA Biosciences) that has been described previously.¹¹ After obtaining a background reading of a 384-well E-plate, 6,000 Vero-furin cells⁵² were seeded per well. Sensograms were visualized using RTCA HT software version 1.0.1 (ACEA Biosciences). One day later, equal volumes of virus were added to antibody samples and incubated for 1ch at 37°C in 5%cCO₂. mAbs were tested in triplicate with a single (1:20) dilution. Virus–mAb mixtures were then added to Vero-furin cells in 384-well E-plates. Controls were included that had Vero-furin cells with virus only (no mAb) and media only (no virus or mAb). E-plates were read every 8–12 h for 72 h to monitor virus neutralization. At 32 h after virus-mAb mixtures were added to the E-plates, cell index values of antibody samples were compared to those of virus only and media only to determine presence of neutralization.

Nano-luciferase neutralization assay

A full-length SARS-CoV-2 virus based on the Seattle Washington isolate and a full-length SARS-CoV virus based on the Urbani isolate were designed to express luciferase and was recovered via reverse genetics and described previously.^53,54 Viruses were titered in Vero E6 USAMRID cells to obtain a relative light units (RLU) signal of at least 10X the cell only control background. Vero E6 USAMRID cells were plated at 20,000 cells per well the day prior in clear bottom black walled 96-well plates (Corning 3904). Neutralizing antibody serum samples were tested at a starting dilution of 1:40 and were serially diluted 4-fold up to eight dilution spots. Antibody-virus complexes were incubated at 37C with 5% CO2 for 1 hour. Following incubation, growth media was removed and virus-antibody dilution complexes were added to the cells in duplicate. Virus-only controls and cell-only controls were included in each neutralization assay plate. Following infection, plates were incubated at 37C with 5% CO2 for 48 hours. After the 48 hour incubation, cells were lysed and luciferase activity was measured via Nano-Glo Luciferase Assay System (Promega) according to the manufacturer specifications. SARS-CoV and SARS-CoV-2 neutralization titers were defined as the sample dilution at which a 50% reduction in RLU was observed relative to the average of the virus control wells.

SPR

His-tagged SARS-CoV-2 RBD-SD1 was immobilized to a NiNTA sensorchip to a level of ∼150 RUs using a Biacore X100. Serial dilutions of purified Fab 46472-12 were evaluated for binding, ranging in concentration from 1 to 0.25 μM. The resulting data were fit to a 1:1 binding model using Biacore Evaluation Software.

Fc effector function assays

Antibody prophylaxis - murine model of infection

For evaluating the prophylactic efficacy of mAbs, 12-month old female BALB/c mice (BALB/cAnHsd; Envigo, stock number 047) were treated with 200 μg mAb intraperitoneally (i.p.) 12 hours prior to virus inoculation. The next day, mice were administered intranasally with 1x10³ PFU or 1x10⁴PFU of SARS-CoV-2 MA10, respectively. Mice were monitored daily for weight loss, morbidity, and mortality, and after four days, mice were sacrificed and lung tissue was harvested for viral titer as measured by plaque assays. One lung lobe was taken for pathological analysis and the other lobe was processed for qPCR and viral load determination as previously described.³¹ For viral plaque assays, the caudal lobe of the right lung was homogenized in PBS, and the tissue homogenate was then serial-diluted onto confluent monolayers of Vero E6 cells, followed by agarose overlay. Plaques were visualized with overlay of Neutral Red dye on day 2 post infection. Gross pulmonary hemorrhage was observed at time of tissue harvest and scored on a scale of 0 (no hemorrhage in any lobe, normal pink healthy lung) to 4 (complete hemorrhage in all lobes of the lung, completely dark red lung).

For viral titer and hemorrhage score comparisons, an ordinary one-way ANOVA test with multiple comparisons was performed using Prism software, GraphPad Prism version 8.0.

ACE2 binding inhibition assay

Wells of 384-well microtiter plates were coated with purified recombinant SARS-CoV-2 S-2P ectoprotein at 4°C overnight. Plates were blocked with 2% non-fat dry milk and 2% normal goat serum in DPBS-T for 1 hr. Purified mAbs were diluted two-fold in blocking buffer starting from 10 μg/mL in triplicate, added to the wells (20 μL/well), and incubated at ambient temperature. Recombinant human ACE2 with a C-terminal FLAG tag protein was added to wells at 2 μg/mL in a 5 μL/well volume (final 0.4 μg/mL concentration of ACE2) without washing of antibody and then incubated for 40 min at ambient temperature. Plates were washed, and bound ACE2 was detected using HRP-conjugated anti-FLAG antibody and TMB substrate. ACE2 binding without antibody served as a control. Experiment was done in biological replicate and technical triplicates, shown is representative of one replicate with positive control mAb COV2-2196.¹¹

Identification of residue-level mutants

Potential cross-reactive epitopes were identified based on sequence and structural homology. Reference sequences for each Coronavirus S were obtained either from NCBI for SARS-CoV-2 (YP_009724390.1) and MERS-CoV (YP_009047204.1) or from Uniprot for SARS-CoV (P59594) of the spikes was then obtained using MUSCLE⁴⁹ and the amino acid similarity to SARS-CoV-2 at each residue position was calculated using the BLOSUM-62 scoring matrix.⁵⁰ These scores were then used to color each residue position on the SARS-CoV-2 S structure (PDB ID: 6VSB) in PyMOL (Schrodinger, version 2.3.5) in order to visualize surface patches and linear epitopes with structural homology. These conserved regions were then visualized on the other human coronavirus spike structures by retrieving them from the Protein Databank (SARS-CoV: 5X5B, MERS-CoV: 5W9I) and aligning them to the SARS-CoV-2 S structure. Finally, the residue N165 was part of a conserved surface patches and was mutated to alanine and tested for binding with antibodies. The N709A mutant tested was previously described in Acharya et al., BioRxiv (2020).