Conserved Neutralizing Epitopes on the N-Terminal Domain of Variant SARS-CoV-2 Spike Proteins

Abstract

SARS-CoV-2 infection or vaccination produces neutralizing antibody responses that contribute to better clinical outcomes. The receptor binding domain (RBD) and the N-terminal domain (NTD) of the spike trimer (S) constitute the two major neutralizing targets for the antibody system. Neutralizing antibodies targeting the RBD bind to several different sites on this domain. In contrast, most neutralizing antibodies to NTD characterized to date bind to a single supersite, however these antibodies were obtained by methods that were not NTD specific. Here we use NTD specific probes to focus on anti-NTD memory B cells in a cohort of pre-omicron infected individuals some of which were also vaccinated. Of 275 NTD binding antibodies tested 103 neutralized at least one of three tested strains: Wuhan-Hu-1, Gamma, or PMS20, a synthetic variant which is extensively mutated in the NTD supersite. Among the 43 neutralizing antibodies that were further characterized, we found 6 complementation groups based on competition binding experiments. 58% targeted epitopes outside the NTD supersite, and 58% neutralized either Gamma or Omicron, but only 14% were broad neutralizers. Three of the broad neutralizers were characterized structurally. C1520 and C1791 recognize epitopes on opposite faces of the NTD with a distinct binding pose relative to previously described antibodies allowing for greater potency and cross-reactivity with 7 different variants including Beta, Delta, Gamma and Omicron. Antibody C1717 represents a previously uncharacterized class of NTD-directed antibodies that recognizes the viral membrane proximal side of the NTD and SD2 domain, leading to cross-neutralization of Beta, Gamma and Omicron. We conclude SARS-CoV-2 infection and/or Wuhan-Hu-1 mRNA vaccination produces a diverse collection of memory B cells that produce anti-NTD antibodies some of which can neutralize variants of concern. Rapid recruitment of these cells into the antibody secreting plasma cell compartment upon re-infection likely contributes to the relatively benign course of subsequent infections with SARS-CoV-2 variants including omicron.

SciScore for 10.1101/2022.02.01.478695: (What is this?)

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

Table 1: Rigor

Ethics	Consent: All participants provided written informed consent before participation in the study, and the study was conducted in accordance with Good Clinical Practice.
Sex as a biological variable	Cell Lines: 293T cells (Homo sapiens; sex: female, embryonic kidney) obtained from the ATCC (CRL-3216) and HT1080Ace2 cl14 cells (parental HT1080: homo sapiens; sex: male, fibrosarcoma) (Schmidt et al., 2020) were cultured in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) at 37 °C and 5% CO2.
Randomization	not detected.
Blinding	not detected.
Power Analysis	not detected.
Cell Line Authentication	Contamination: All cell lines have been tested negative for contamination with mycoplasma.

Table 2: Resources

Antibodies
Sentences	Resources
Plates were washed 6 times with washing buffer and then incubated with anti-human IgG, IgM or IgA secondary antibody conjugated to horseradish peroxidase (HRP) (Jackson Immuno Research	anti-human IgG suggested: None IgA suggested: None
The antibody-virus-mix was then directly applied to each well (n = 3 per dilution) and incubated for 24 h at 37 °C.	antibody-virus-mix suggested: None
A rabbit polyclonal anti-SARS-CoV-2 nucleocapsid antibody (GeneTex, catalog no. GTX135357) was added to the cells at 1:1,000 dilution in blocking solution and incubated overnight at 4 °C.	anti-SARS-CoV-2 suggested: None
Next, goat anti-rabbit AlexaFluor 594 (Life Technologies, catalog no. A-11012) was used as a secondary antibody at a dilution of 1:2,000 and incubated overnight at 4 °C.	anti-rabbit suggested: None
The enriched B cells were incubated in FACS buffer (1× PBS, 2% FCS, 1 mM EDTA) with the following anti-human antibodies (all at 1:200 dilution): anti-CD20-PECy7 (BD Biosciences, 335793), anti-CD3-APC-eFluro 780 (Invitrogen, 47-0037-41)	anti-human suggested: (GenWay Biotech Inc. Cat# 18-202-335793-0.1 mg, RRID:AB_1981874) anti-CD20-PECy7 suggested: None anti-CD3-APC-eFluro 780 suggested: None
Experimental Models: Cell Lines
Sentences	Resources
Cell Lines: 293T cells (Homo sapiens; sex: female, embryonic kidney) obtained from the ATCC (CRL-3216) and HT1080Ace2 cl14 cells (parental HT1080: homo sapiens; sex: male, fibrosarcoma) (Schmidt et al., 2020) were cultured in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) at 37 °C and 5% CO2.	293T suggested: None HT1080Ace2 cl14 suggested: None
VeroE6 cells (Chlorocebus sabaeus; sex: female, kidney epithelial) obtained from the ATCC (CRL-1586™) and from Ralph Baric (University of North Carolina at Chapel Hill), and Caco-2 cells (Homo sapiens; sex: male, colon epithelial) obtained from the ATCC (HTB-37™) were cultured in DMEM supplemented with 1% nonessential amino acids (NEAA) and 10% FBS at 37 °C and 5% CO2.	VeroE6 suggested: None
The original WT virus was amplified in Caco-2 cells, which were infected at a multiplicity of infection (MOI) of 0.05 plaque forming units (PFU)/cell and incubated for 6 days at 37 °C.	Caco-2 suggested: None
VeroE6TMPRSS2 cells were infected at a MOI = 0.1 PFU/cell and incubated for 4 days at 33 °C.	VeroE6TMPRSS2 suggested: JCRB Cat# JCRB1819, RRID:CVCL_YQ49)
The infectious dose for each virus was pre-determined on VeroE6UNC cells to yield 50-60% antigen-positive cells upon this incubation period (USA-WA1/2020: 1,250 PFU/well and B.1.351: 175 PFU/well).	VeroE6UNC suggested: None
Recombinant DNA
Sentences	Resources
(R683G) and pSARS-CoV-SΔ19 expressing C-terminally truncated SARS-CoV-2 and SARS-CoV spike proteins and the polymutant PMS20 spike were as described before (Schmidt et al., 2021c).	pSARS-CoV-SΔ19 suggested: None
A panel of plasmids expressing spike proteins from SARS-CoV-2 variants were based on pSARS-CoV-2-SΔ19(R683G) and contain the following substitutions/deletions: Alpha (B.1.1.7): ΔH69/V70, ΔY144, N501Y, A470D, D614G, P681H, T761I, S982A,	pSARS-CoV-2-SΔ19 suggested: None
Briefly, 293T cells were transfected with pNL4-3ΔEnv-nanoluc(Robbiani et al., 2020; Schmidt et al., 2020) and either spike plasmid.	pNL4-3ΔEnv-nanoluc(Robbiani suggested: None
Software and Algorithms
Sentences	Resources
The average of its signal was used for normalization of all of the other values on the same plate with Excel software before calculating the area under the curve using Prism V9.1(GraphPad).	Excel suggested: None Prism suggested: (PRISM, RRID:SCR_005375)
For monoclonal antibodies, the EC50 was determined using four-parameter nonlinear regression (GraphPad Prism V9.1).	GraphPad Prism suggested: (GraphPad Prism, RRID:SCR_002798)
All statistical analyses were done using Prism 8 software (Graphpad).	Graphpad suggested: (GraphPad, RRID:SCR_000306)
NTD-AF647+ B cells were sorted into individual wells of 96-well plates containing 4 μl of lysis buffer (0.5× PBS, 10 mM DTT, 3,000 units/ml RNasin Ribonuclease Inhibitors (Promega, N2615) per well using a FACS Aria III and FACSDiva software (Becton Dickinson) for acquisition and FlowJo for analysis.	FACSDiva suggested: (BD FACSDiva Software, RRID:SCR_001456) FlowJo suggested: (FlowJo, RRID:SCR_008520)
Sequence analysis was performed using MacVector.	MacVector suggested: (MacVector, RRID:SCR_015700)
Cryo-EM data collection and processing: Single-particle cryo-EM data were collected on a Titan Krios transmission electron microscope (Thermo Fisher) equipped with a Gatan K3 direct detector, operating at 300 kV and controlled using SerialEM automated data collection software(Mastronarde, 2005).	SerialEM suggested: (SerialEM, RRID:SCR_017293)
A subset of 4x-downsampled particles were used to generate ab initio models, which were then used for heterogeneous refinement of the entire dataset in cryoSPARC.	cryoSPARC suggested: (cryoSPARC, RRID:SCR_016501)
3D classifications (k=6, tau_fudge=4) were carried out using Relion v3.1.1(Fernandez-Leiro and Scheres, 2017) without imposing symmetry and a soft mask.	Relion suggested: (RELION, RRID:SCR_016274)
Sequence-updated models were built manually in Coot(Emsley et al., 2010) and then refined using iterative rounds of refinement in Coot and Phenix.	Phenix suggested: (Phenix, RRID:SCR_014224)
Glycans were modeled at potential N-linked glycosylation sites (PNGSs) in Coot.	Coot suggested: (Coot, RRID:SCR_014222)

Results from OddPub: Thank you for sharing your code and data.

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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Results from scite Reference Check: We found no unreliable references.

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Conserved Neutralizing Epitopes on the N-Terminal Domain of Variant SARS-CoV-2 Spike Proteins

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