key: cord-0683729-0mructd7
authors: Zost, Seth J.; Gilchuk, Pavlo; Chen, Rita E.; Case, James Brett; Reidy, Joseph X.; Trivette, Andrew; Nargi, Rachel S.; Sutton, Rachel E.; Suryadevara, Naveenchandra; Chen, Elaine C.; Binshtein, Elad; Shrihari, Swathi; Ostrowski, Mario; Chu, Helen Y.; Didier, Jonathan E.; MacRenaris, Keith W.; Jones, Taylor; Day, Samuel; Myers, Luke; Lee, F. Eun-Hyung; Nguyen, Doan C.; Sanz, Ignacio; Martinez, David R.; Baric, Ralph S.; Thackray, Larissa B.; Diamond, Michael S.; Carnahan, Robert H.; Crowe, James E.
title: Rapid isolation and profiling of a diverse panel of human monoclonal antibodies targeting the SARS-CoV-2 spike protein
date: 2020-05-13
journal: bioRxiv
DOI: 10.1101/2020.05.12.091462
sha: c9628d9ac274d744dd89c708503923943400880a
doc_id: 683729
cord_uid: 0mructd7

Antibodies are a principal determinant of immunity for most RNA viruses and have promise to reduce infection or disease during major epidemics. The novel coronavirus SARS-CoV-2 has caused a global pandemic with millions of infections and hundreds of thousands of deaths to date1,2. In response, we used a rapid antibody discovery platform to isolate hundreds of human monoclonal antibodies (mAbs) against the SARS-CoV-2 spike (S) protein. We stratify these mAbs into five major classes based on their reactivity to subdomains of S protein as well as their cross-reactivity to SARS-CoV. Many of these mAbs inhibit infection of authentic SARS-CoV-2 virus, with most neutralizing mAbs recognizing the receptor-binding domain (RBD) of S. This work defines sites of vulnerability on SARS-CoV-2 S and demonstrates the speed and robustness of new antibody discovery methodologies.

IDBiologics, Inc. Vanderbilt University has applied for patents concerning SARS-CoV-2 231 antibodies that are related to this work. Emory University has applied for a patent concerning 

healthy subject (subject 6). Plots show CD19 + IgD -IgMpopulation using gating strategy 264 as in b. Cells labeled with biotinylated S2P ecto or RBD-mFc antigens were detected using 265 phycoerythrin (PE)-conjugated streptavidin. 266 d. Plasma or serum neutralizing activity against the WA1/2020 strain SARS-CoV-2 for 267 subjects 1 to 4 or a healthy donor (subject 6). % neutralization is reported. white indicates neutralizing activity was not detected. Based on both binding and 296 neutralization, we grouped the mAbs into classes. Class I mAbs bind to both S2P ecto and 297 S RBD proteins and are SARS-CoV-2 specific; Class II mAbs also bind to both S2P ecto and 298 S RBD proteins and cross-react with SARS-CoV; Class III mAbs bind to both S2P ecto and 299 S NTD proteins and are mostly SARS-CoV-2 specific; Class IV mAbs bind only to S2P ecto 300 protein and are SARS-CoV-2 specific; Class V mAbs bind only to S2P ecto protein and 301 cross-react with SARS-CoV. The frequency counts are derived from the total number of unique sequences with the 306 corresponding V and J genes. The V/J frequency counts then were transformed into a z-307 score by first subtracting the average frequency, then normalizing by the standard 308 deviation of each subject. Red denotes more common gene usage, while blue denotes 309 less common gene usage. e. CDR3 amino acid length distribution. The CDR3 of each sequence was determined 311

using PyIR software. The amino acid length of each CDR3 was counted. The 312 distribution of CDR3 amino acid lengths for heavy or light chains then was plotted as a 313 histogram and fitted using kernel density estimation for the curves. h. Quantitative test for neutralizing activity against SARS-CoV using a nano-327 luciferase virus. A representative purified mAb that exhibited cross-reactive binding to 328 SARS-CoV S2P ecto protein in (b) above and that also exhibited full neutralizing activity 329

in the screening neutralization test in (c) above was tested in a serial dilution series in a 330 neutralization test with a recombinant, reverse-genetics-derived SARS-CoV encoding a 331 nano-luciferase reporter gene, and reduction of luciferase activity was used to calculate 332 % neutralization. Values shown are the mean of two technical replicates, and error bars 333 denote the standard deviation for each point. coronavirus by a human mAb to S1 protein that blocks receptor association. Proc 395 Natl Acad Sci U S A 101, 2536-2541 (2004) . 10 -5 10 -4 10 -3 10 -2 10 -1 10 -5 10 -4 10 -3 10 -2 10 -1 10 -5 10 -4 10 -3 10 -2 10 -1 10 -5 10 -4 10 -3 10 -2 10 -1 Research subjects. We studied four subjects in North America with recent laboratory-3 confirmed symptomatic SARS-CoV-2 infection that were acquired in China ( Table S1) on January 19, 2020 1 , a blood sample was obtained for study on February 19, 2020. Subject 2 10 (52-year-old female) was infected following close exposure in Beijing, China to an infected 11 person from Wuhan, China during the period between January 23 to January 29, 2020. She 12 presented with mild respiratory disease symptoms from February 1 to 4, 2020 that occurred presented to a hospital in Toronto, January 23, 2020 complaining of fever, cough and 21 shortness of breath; a nasopharyngeal swab was positive by PCR testing for SARS-CoV-2. His 22 chest x-ray at that time was abnormal, and he was admitted for non-ICU impatient care. He 23 improved gradually with supportive care, was discharged January 30, 2020 and rapidly 24 became asymptomatic except for a residual dry cough that persisted for a month. He had a 25 negative nasopharyngeal swab PCR test on February 19, 2020. Subject 4 is the wife of subject 3 26 who traveled with her husband from Wuhan. She was never symptomatic with respiratory 27 symptoms or fever but was tested because of her exposure. Her nasopharyngeal swab was 28 positive for SARS-CoV-2 by PCR, on January 24, 2020; repeat testing in followup on February 29 21, 2020 was negative. PBMCs were obtained by leukopheresis from subjects 3 and 4 on 30

March 10, 2020, which was 50 days since the symptom onset of subject 3. Samples were 31 Disease Control and Prevention (a gift from Natalie Thornburg). Virus was passaged in Vero 50

CoV-2 was approved by the Washington University School of Medicine or UNC-Chapel Hill 52

Institutional Biosafety Committees and conducted in approved BSL3 facilities using 53 appropriate powered air purifying respirators and personal protective equipment. 54 55 Recombinant antigens and proteins. A gene encoding the ectodomain of a prefusion 56 conformation-stabilized SARS-CoV-2 spike (S2P ecto ) protein was synthesized and cloned into a 57 DNA plasmid expression vector for mammalian cells. A similarly designed S protein antigen 58 with two prolines and removal of the furin cleavage site for stabilization of the prefusion form 59 of S was reported previously 3 . Briefly, this gene includes the ectodomain of SARS-CoV-2 (to 60 residue 1,208), a T4 fibritin trimerization domain, an AviTag site-specific biotinylation 61 sequence, and a C-terminal 8x-His tag. To stabilize the construct in the prefusion 62 conformation, we included substitutions K968P and V969P and mutated the furin cleavage 63 site at residues 682-685 from RRAR to ASVG. This recombinant spike 2P-stabilized protein 64 (designated here as S2P ecto ) was isolated by metal affinity chromatography on HisTrap Excel 65 columns (GE Healthcare), and protein preparations were purified further by size-exclusion 66 chromatography on a Superose 6 Increase 10/300 column (GE Healthcare). The presence of 67 trimeric, prefusion conformation S protein was verified by negative-stain electron microscopy 68 ( Figure S1 ). To express the S RBD subdomain of SARS-CoV-2 S protein, residues 319-541 were 69 cloned into a mammalian expression vector downstream of an IL-2 signal peptide and 70 upstream of a thrombin cleavage site, an AviTag, and a 6x-His tag. RBD protein fused to 71 mouse IgG1 Fc domain (designated RBD-mFc), was purchased from Sino Biological (40592-72 V05H). For B cell labeling and sorting, RBD-mFc and S2P ecto proteins were biotinylated using 73 the EZ-Link™ Sulfo-NHS-LC-Biotinylation Kit and vendor's protocol (ThermoFisher 74 Scientific, 21435). 75

First case of 2019 novel coronavirus in the United States

Enhancing dengue virus maturation using a stable furin 348 over-expressing cell line

Cryo-EM structure of the 2019-nCoV spike in the prefusion 351 conformation

Negative staining and image classification -354

Powerful tools in modern electron microscopy

Automated electron microscope tomography using robust 357 prediction of specimen movements

cryoSPARC: algorithms 360 for rapid unsupervised cryo-EM structure determination

Topaz-Denoise: general deep denoising 364 models for cryoEM

UCSF Chimera--a visualization system for exploratory 367 research and analysis

Analysis of a therapeutic antibody cocktail reveals determinants 370 for cooperative and broad ebolavirus neutralization

Factors of the bone marrow microniche that support human 374 plasma cell survival and immunoglobulin secretion

An efficient 377 method to generate monoclonal antibodies from human B cells

PyIR: A scalable wrapper for processing billions of 382 immunoglobulin and T cell receptor sequences using IgBLAST

Cleavage efficient 2A peptides for high level monoclonal antibody 385 expression in CHO cells

Integrated technology platform for accelerated discovery of 393 antiviral antibody therapeutics

A highly conserved cryptic epitope in the receptor binding 396 domains of SARS-CoV-2 and SARS-CoV

Reverse genetics with a full-length infectious cDNA of the 399

Middle East respiratory syndrome coronavirus

Reverse genetics with a full-length infectious cDNA of severe 403 acute respiratory syndrome coronavirus

protein. For screening and imaging of negatively-stained (NS) SARS-CoV-2 S2P ecto 78 protein, approximately 3 µL of the sample at concentrations of about 10 to 15 µg/mL 79 was applied to a glow discharged grid with continuous carbon film on 400 square mesh 80 copper EM grids (Electron Microscopy Sciences, Hatfield, PA). The grids were stained 81 with 0.75% uranyl formate (UF) 4 . Images were recorded on a Gatan US4000 4k × 4k 82 CCD camera using an FEI TF20 (TFS) transmission electron microscope operated at 200 83 keV and control with SerialEM 5 . All images were taken at 50,000´ magnification with a 84 pixel size of 2.18 Å/pix in low-dose mode at a defocus of 1.5 to 1.8 μm. Total dose for 85 the micrographs was ~25 e − /Å 2 . Image processing was performed using the 86 cryoSPARC software package 6 . Images were imported, and particles were CTF 87 estimated. The images then were denoised and picked with Topaz 7 . The particles were 88 extracted with a box size of 256 pixels and binned to 128 pixels. 2D class averages were 89 performed and good classes selected for ab-initio model and refinement without 90 symmetry (see also 

SARS-CoV-2 in PBMCs from a cohort of four subjects with documented previous infection 96 with the virus were analyzed for antigen specificity, and PBMCs were used for SARS-CoV-2-97 specific B cell enrichment. The frequency of SARS-CoV-2 S protein-specific B cells was 98 identified by antigen-specific staining with either biotinylated S2P ecto or RBD-mFc protein. Briefly, B cells were purified magnetically (STEMCELL Technologies) and stained with anti-100 CD19-APC (clone HIB19, 982406), -IgD-FITC (clone LA6-2, 348206), and -IgM-FITC (clone 101 MNM-88, 314506) phenotyping antibodies (BioLegend) and biotinylated antigen. A 4′,6-102 diamidino-2-phenylindole (DAPI) stain was used as a viability dye to discriminate dead cells. 103Antigen-labeled class-switched memory B cell-antigen complexes (CD19 + IgM -IgD -Ag + DAPI -) 104were detected with a R-phycoerythrin (PE)-labeled streptavidin conjugate (ThermoFisher 105 Scientific, S866). After identification of the two subjects with the highest B cell response 106 against SARS-CoV-2 (subjects 3 and 4), target-specific memory B cells were isolated by flow 107 cytometric sorting using an SH800 cell sorter (Sony) from pooled PBMCs of these two 108 subjects, after labeling of B cells with either biotinylated S2P ecto or RBD-mFc proteins. 109 110 Overall, from > 4 x 10 8 PBMCs, 2,126 RBD-mFc-reactive and 5,544 S2P ecto protein-reactive B 111 cells were sorted and subjected to further analysis. Several methods were implemented for the 112 preparation of sorted B cells for sequencing. Approximately 4,500 sorted cells were subjected 113 to direct sequencing immediately after flow cytometric sorting. The remaining cells were 114 expanded in culture for eight days in the presence of irradiated 3T3 feeder cells that were 115 engineered to express human CD40L, IL-21, and BAFF, as described previously 9 . The 116 expanded lymphoblastoid cell lines (LCLs) secreted high levels of S protein-specific 117 antibodies, as confirmed by ELISA to detect antigen-specific human antibodies in culture 118supernatants. Approximately 40,000 expanded LCLs were sequenced using the Chromium 119 sequencing method (10x Genomics). light is used to transfer B cells into individual nanoliter-volume chambers 127 (NanoPens™). Using this light-based manipulation, thousands of LCLs were 128 transferred into pens across multiple chips in each workflow. We performed an on-chip, 129fluorescence-based assay to identify antibodies that bound SARS-CoV-2 S2P ecto or RBD-130 mFc protein. We prepared 6-to 8-micron and 10-to 14-micron RBD-mFc-conjugated 131 beads by coupling biotinylated RBD-mFc protein to streptavidin-coated polystyrene 132 particles (Spherotech Inc.). We prepared 6-to 8-micron S2P ecto protein-conjugated beads 133 by coupling full-length S2P ecto protein to streptavidin-coated polystyrene particles. 134Assays consisted of mixing beads conjugated with the RBD-mFc or S2P ecto proteins with 135 fluorescently-labeled anti-human secondary antibodies (AF568, Thermo Fisher 136 Scientific) and importing this assay mixture into OptoSelect 11k chips. Antigen-specific 137 antibodies bound the antigen-conjugated beads, which then sequestered the fluorescent 138 secondary antibody. Cells secreting antigen-specific antibodies were identified by 139 locating the NanoPens immediately adjacent to the fluorescent beads. Antigen-specific 140 cells of interest were exported from specific NanoPen chambers to individual wells of 141 96-well RT-PCR plates containing lysis buffer. 142 143 Sequencing and cloning of single antigen-specific B cells. After export from the Beacon, 144 antibody heavy and light chain sequences for B cells secreting antibodies with RBD-mFc-or 145 S2P ecto -binding antibodies were amplified and recovered using components of the 146Opto™ Plasma B Discovery cDNA Synthesis Kit (Berkeley Lights). Antibody heavy and light 147 chain sequences were amplified through a 5'RACE approach using the kit's included "BCR 148Primer 2" forward primer and isotype-specific reverse primers. The 5'-RACE amplified cDNA 149 was sequenced using the Pacific Biosciences Sequel platform using the SMRTbell Barcoded 150Adapt Complete Prep-96 kit (Pacific Biosciences) and a 6-hour movie time. In a redundant 151 sequencing approach, heavy and light chain sequences were amplified using a cocktail of 152 custom V and J gene-specific primers (similar to previously described human Ig gene-specific 153 primers 11 ) from the original 5'-RACE-amplified cDNA while the products of the gene-specific 154 amplification were sent for Sanger sequencing (GENEWIZ). The sequences generated by these 155 two approaches were analyzed using our Python-based antibody variable gene analysis tool 156 (PyIR; https://github.com/crowelab/PyIR) 12 to identify which V and J genes most closely 157 matched the nucleotide sequence. Heavy and light chain sequences were then amplified from 158 the original cDNA using cherry-picked V and J gene-specific primers most closely 159 corresponding to the V and J gene calls made by PyIR. These primers include adapter 160 sequences which allow Gibson-based cloning into a monocistronic IgG1 expression vector 161 (pMCis_G1). Similar to a vector described below, this vector contains an enhanced 2A 162 sequence and GSG linker that allows simultaneous expression of mAb heavy and light chain 163 genes from a single construct upon transfection 13 . The pMCis_G1 vector was digested using 164 the New England BioLabs restriction enzyme FspI, and the amplified paired heavy and light 165 chain sequences were cloned through Gibson assembly using NEBuilder HiFi DNA Assembly 166Master Mix. After recovered sequences were cloned into pMCis_G1 expression constructs, 167 recombinant antibodies were expressed in Chinese hamster ovary (CHO) cells and purified 168 by affinity chromatography as detailed below. Antigen-binding activity was confirmed using 169 plate-based ELISA. Biotech) and TMB (3,3¢,5,5¢-tetramethylbenzidine) substrate (Thermo Fisher Scientific). 247Color development was monitored, 1N hydrochloric acid was added to stop the reaction, 248 and the absorbance was measured at 450 nm using a spectrophotometer (Biotek). For dose-249 response assays, serial dilutions of purified mAbs were applied to the wells in triplicate, 250and mAb binding was detected as detailed above. Half-maximal effective concentration 251 were 324 unique nucleotide sequences that were analyzed for V/D/J gene usage, CDR3 289 length, and somatic mutation. First, the number of sequences with corresponding V and 290 J genes were counted. The V/J frequency counts were then transformed into a z-score 291 by first subtracting away the average frequency then normalizing by the standard 292 deviation of each subject. The z-score was then plotted as a heatmap using python 293 seaborn library. The amino acid length of each CDR3 was counted. The distribution of 294 CDR3 amino acid lengths were then plotted as histograms and fitted using kernel 295 density estimation for the curves using python seaborn library. The number of 296 mutations from each inferred germ-line sequence starting from FR-1 to FR4 was 297 counted up for each chain. This number was then transformed into a percentage value. 298These values are then plotted as a categorical distribution plot as a violin plot using the 299 python seaborn.catplot library. NanoLuc virus was mixed with serially diluted antibodies. Antibody-virus complexes 321were incubated at 37°C in 5% CO 2 for 1 hr. Following incubation, growth medium was 322 removed and virus-antibody dilution complexes were added to the cells in duplicate. 323Virus-only and cell-only controls were included in each neutralization assay plate. 324Following infection, plates were incubated at 37°C in 5% CO 2 for 48 hours. After the 48-325 hour incubation, cells were lysed and luciferase activity was measured using the Nano-326Glo Luciferase Assay System (Promega), according to the manufacturer's specifications. 327 328 High-throughput mAb quantification. High-throughput quantification of micro-scale 329 produced mAbs was performed from CHO culture supernatants or micro-scale purified 330 mAbs in a 96-well plate format using the Cy-Clone Plus Kit and an iQue Plus Screener flow 331 cytometer (IntelliCyt Corp), according to the vendor's protocol. Purified mAbs were assessed 332 at a single dilution (1:10 final, using 2 μL of purified mAb per reaction), and a control human 333IgG solution with known concentration was used to generate a calibration curve. Data were 334 analyzed using ForeCyt software version 6.2 (IntelliCyt Corp).