Synthetic repertoires derived from convalescent COVID-19 patients enable discovery of SARS-CoV-2 neutralizing antibodies and a novel quaternary binding modality

Abstract

The ongoing evolution of SARS-CoV-2 into more easily transmissible and infectious variants has sparked concern over the continued effectiveness of existing therapeutic antibodies and vaccines. Hence, together with increased genomic surveillance, methods to rapidly develop and assess effective interventions are critically needed. Here we report the discovery of SARS-CoV-2 neutralizing antibodies isolated from COVID-19 patients using a high-throughput platform. Antibodies were identified from unpaired donor B-cell and serum repertoires using yeast surface display, proteomics, and public light chain screening. Cryo-EM and functional characterization of the antibodies identified N3-1, an antibody that binds avidly (K _d,app = 68 pM) to the receptor binding domain (RBD) of the spike protein and robustly neutralizes the virus in vitro . This antibody likely binds all three RBDs of the trimeric spike protein with a single IgG. Importantly, N3-1 equivalently binds spike proteins from emerging SARS-CoV-2 variants of concern, neutralizes UK variant B.1.1.7, and binds SARS-CoV spike with nanomolar affinity. Taken together, the strategies described herein will prove broadly applicable in interrogating adaptive immunity and developing rapid response biological countermeasures to emerging pathogens.

SciScore for 10.1101/2021.04.07.438849: (What is this?)

Please note, not all rigor criteria are appropriate for all manuscripts.

Table 1: Rigor

Institutional Review Board Statement	IACUC: All experiments with SARS-CoV-2 were performed in the Eva J Pell BSL-3 laboratory at Penn State and were approved by the Penn State Institutional Biosafety Committee (IBC # 48625).
Randomization	not detected.
Blinding	not detected.
Power Analysis	not detected.
Sex as a biological variable	not detected.
Cell Line Authentication	not detected.

Table 2: Resources

Antibodies
Sentences	Resources
Strains and media: Yeast strain EBY100 (MATa AGA1::GAL1-AGA1::URA3 ura3-52 trp1 leu2-delta200 his3-delta200 pep4::HIS3 prbd1.6R can1 GAL) was acquired from ATCC (cat. no. MYA-4941) and used for antibody expression and selection.	ura3-52 suggested: None trp1 suggested: None
To improve …

SciScore for 10.1101/2021.04.07.438849: (What is this?)

Please note, not all rigor criteria are appropriate for all manuscripts.

Table 1: Rigor

Institutional Review Board Statement	IACUC: All experiments with SARS-CoV-2 were performed in the Eva J Pell BSL-3 laboratory at Penn State and were approved by the Penn State Institutional Biosafety Committee (IBC # 48625).
Randomization	not detected.
Blinding	not detected.
Power Analysis	not detected.
Sex as a biological variable	not detected.
Cell Line Authentication	not detected.

Table 2: Resources

Antibodies
Sentences	Resources
Strains and media: Yeast strain EBY100 (MATa AGA1::GAL1-AGA1::URA3 ura3-52 trp1 leu2-delta200 his3-delta200 pep4::HIS3 prbd1.6R can1 GAL) was acquired from ATCC (cat. no. MYA-4941) and used for antibody expression and selection.	ura3-52 suggested: None trp1 suggested: None
To improve antibody expression the human chaperones BIP (binding immunoglobulin protein) and PDI (protein disulfide isomerase) were genomically integrated as an expression cassette in the HO locus.	binding immunoglobulin protein suggested: None PDI (protein disulfide isomerase) suggested: None
Antigens and antibodies: Spike antigen was biotinylated using the EZ-link kit (Thermo Scientific, cat. no. 21435) and labeled with streptavidin-AF647 (Invitrogen, cat. no. S32357).	Antigens suggested: None
Antibody variable chain sequencing: Peripheral blood mononuclear cells (PBMCs) from processed donor samples were provided in Trizol as a kind gift from Dr. Gregory C. Ippolito34.	Ippolito34 suggested: None
Antigen was incubated with cells in 1 mL in PBSA at 200 nM at RT for one hour, washed with PBSA at 4C, and labeled with secondary antibodies (mouse anti-human FITC, 1:100; streptavidin-AF647, 1:100; mouse anti-human Fc-AF647, 1:50).	anti-human FITC suggested: None anti-human Fc-AF647 suggested: None
Cells were washed 3X with PBS-BSA, resuspended in __ mL and incubated with Alexa Fluor 488 (anti-mouse) and Alexa Fluor 647 (anti-human) antibodies for 30 min, shaking at 4°C.	anti-mouse suggested: None anti-human suggested: None
Experimental Models: Cell Lines
Sentences	Resources
Briefly, plasmids expressing full-length SARS-CoV-2 spike proteins (Spike-Linker-3XFLAG-TM), WT (HexaPro-D614G) and variants, were transfected into HEK293T cells using Lipofectamine 2000 according to manufacturer’s instructions.	HEK293T suggested: None
Diluted antibodies were mixed with the SARS-CoV-2 WA1 strain, incubated at 37°C for 1 h, then added to Vero-E6 cells at target MOI of 0.4.	Vero-E6 suggested: None
After incubating for 1 hour at 37°C, the virus and mAb mixture was then added to Vero E6 cells (ATCC CRL-1586) in a 96-well microtiter plate and incubated at 37°C.	Vero E6 suggested: None
Experimental Models: Organisms/Strains
Sentences	Resources
Surface plasmon resonance: To investigate the binding kinetics of mAb N3-1 binding to the spikes, purified His-tagged spike variants (SARS-CoV Tor2 S-2P, SARS-CoV-2 Wuhan-Hu-1 S-HexaPro, SARS-CoV-2 B.1.1.7 S-Hexapro and SARS-CoV-2 B.	SARS-CoV-2 Wuhan-Hu-1 S-HexaPro suggested: None
Software and Algorithms
Sentences	Resources
Ig-Seq MS data analysis: Mass spectra were analyzed using Proteome Discoverer 2.2 software (Thermo Scientific).	Proteome Discoverer suggested: (Proteome Discoverer, RRID:SCR_014477)
False discovery rates for peptide-spectrum matches (PSMs) were estimated by decoy-based error modelling through the Percolator node.	Percolator suggested: (OMSSAPercolator, RRID:SCR_000287)
MinION R10.3).	MinION suggested: (MinION, RRID:SCR_017985)
All data was analyzed with FlowJo (BD Bioscience).	FlowJo suggested: (FlowJo, RRID:SCR_008520)
Cells were fixed 24 h post-infection, and the number of infected cells was determined using SARS-CoV-S specific mAb (Sino Biological, cat. no. 401430-R001) and fluorescently-labeled secondary antibody.	SARS-CoV-S suggested: None
Cryo-EM data processing: Gain reference- and motion-corrected micrographs processed by Warp48 were imported into cryoSPARC v2.15.049, which was used to perform CTF correction, micrograph curation, particle picking, and particle curation via iterative rounds of 2D classification.	cryoSPARC suggested: (cryoSPARC, RRID:SCR_016501)

Results from OddPub: We did not detect open data. We also did not detect open code. Researchers are encouraged to share open data when possible (see Nature blog).

Results from LimitationRecognizer: An explicit section about the limitations of the techniques employed in this study was not found. We encourage authors to address study limitations.

Results from TrialIdentifier: No clinical trial numbers were referenced.

Results from Barzooka: We did not find any issues relating to the usage of bar graphs.

Results from JetFighter: We did not find any issues relating to colormaps.

Results from rtransparent:

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Thank you for including a funding statement. Authors are encouraged to include this statement when submitting to a journal.
No protocol registration statement was detected.

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Synthetic repertoires derived from convalescent COVID-19 patients enable discovery of SARS-CoV-2 neutralizing antibodies and a novel quaternary binding modality

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