Structural definition of a neutralization epitope on the N-terminal domain of MERS-CoV spike glycoprotein

Most neutralizing antibodies against Middle East respiratory syndrome coronavirus (MERS-CoV) target the receptor-binding domain (RBD) of the spike glycoprotein and block its binding to the cellular receptor dipeptidyl peptidase 4 (DPP4). The epitopes and mechanisms of mAbs targeting non-RBD regions have not been well characterized yet. Here we report the monoclonal antibody 7D10 that binds to the N-terminal domain (NTD) of the spike glycoprotein and inhibits the cell entry of MERS-CoV with high potency. Structure determination and mutagenesis experiments reveal the epitope and critical residues on the NTD for 7D10 binding and neutralization. Further experiments indicate that the neutralization by 7D10 is not solely dependent on the inhibition of DPP4 binding, but also acts after viral cell attachment, inhibiting the pre-fusion to post-fusion conformational change of the spike. These properties give 7D10 a wide neutralization breadth and help explain its synergistic effects with several RBD-targeting antibodies.


Statistics
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Software and code
Policy information about availability of computer code Data collection NCBI was used for downloading the published MERS-Spike sequences to do the mutation site analysis. Data analysis HKL2000, CCP4, COOT and PHNIX were used at the determination of complex structure for data processing, model building and refinement. PyMOL was used to generate the structural figures. GraphPad was used to do data analysis and make some of the figures.
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Data
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Life sciences study design
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Sample size
The total number of R26-hDPP4 mouse model was 17. Protection with 7D10-H or MERS-4 each had 5 mice. Control with 3C11 or PBS had 3 and 4 mice, respectively.

Mycoplasma contamination
All cell lines were tested negative for mycoplasma contamination.

Commonly misidentified lines (See ICLAC register)
Not applicable

Animals and other organisms
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Laboratory animals
Female BALB/c mice aged six to eight weeks were used for mAb production. Genetically-modified R26-hDPP4 mice aged four weeks were used for protection assay.

Wild animals
This study didn't involve any wild anmals.

Field-collected samples
This study didn't collect any samples from the field.

Ethics oversight
All studies were performed in compliance with animal protocols (#2017 Note that full information on the approval of the study protocol must also be provided in the manuscript.

Flow Cytometry
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Sample preparation
The samples were Huh7 cells alone or incubation with the mix of soluble MERS-CoV S trimer and antibodies (7D10-H , MERS-4 and their Fabs, scFvs), then stained the cell surface with the Streptavidin-APC to run by flow cytometry.

Software
FlowJo V10 was used to analyze the data.
Cell population abundance In this assay, in total 20000 cells of each sample were collected.

Gating strategy
We used SSC-A/FSC-A, FSC-H/FSC-A and SSC-H/SSC-A to select single cells for each sample. No other gating strategy used in the cell surface staining assay.
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