An alpaca nanobody neutralizes SARS-CoV-2 by blocking receptor interaction

Abstract

SARS-CoV-2 enters host cells through an interaction between the spike glycoprotein and the angiotensin converting enzyme 2 (ACE2) receptor. Directly preventing this interaction presents an attractive possibility for suppressing SARS-CoV-2 replication. Here, we report the isolation and characterization of an alpaca-derived single domain antibody fragment, Ty1, that specifically targets the receptor binding domain (RBD) of the SARS-CoV-2 spike, directly preventing ACE2 engagement. Ty1 binds the RBD with high affinity, occluding ACE2. A cryo-electron microscopy structure of the bound complex at 2.9 Å resolution reveals that Ty1 binds to an epitope on the RBD accessible in both the ‘up’ and ‘down’ conformations, sterically hindering RBD-ACE2 binding. While fusion to an Fc domain renders Ty1 extremely potent, Ty1 neutralizes SARS-CoV-2 spike pseudovirus as a 12.8 kDa nanobody, which can be expressed in high quantities in bacteria, presenting opportunities for manufacturing at scale. Ty1 is therefore an excellent candidate as an intervention against COVID-19.

SciScore for 10.1101/2020.06.02.130161: (What is this?)

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

Table 1: Rigor

Institutional Review Board Statement	not detected.
Randomization	not detected.
Blinding	not detected.
Power Analysis	not detected.
Sex as a biological variable	In brief, the adult male alpaca Tyson at PreClinics, Germany, was immunized 4 times in a 60-day immunization schedule.
Cell Line Authentication	Contamination: All cell lines used for experiments were negative for Mycoplasma as determined by PCR.

Table 2: Resources

Antibodies
Sentences	Resources
Bound nanobodies were detected with anti-E tag (Bethyl laboratories) secondary antibody.	anti-E tag suggested: None
Cells were incubated with anti-dsRNA antibody (1:2000, J2 Scicons) for 1 hour at room temperature followed by 1 hour staining with the secondary antibody …

SciScore for 10.1101/2020.06.02.130161: (What is this?)

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

Table 1: Rigor

Institutional Review Board Statement	not detected.
Randomization	not detected.
Blinding	not detected.
Power Analysis	not detected.
Sex as a biological variable	In brief, the adult male alpaca Tyson at PreClinics, Germany, was immunized 4 times in a 60-day immunization schedule.
Cell Line Authentication	Contamination: All cell lines used for experiments were negative for Mycoplasma as determined by PCR.

Table 2: Resources

Antibodies
Sentences	Resources
Bound nanobodies were detected with anti-E tag (Bethyl laboratories) secondary antibody.	anti-E tag suggested: None
Cells were incubated with anti-dsRNA antibody (1:2000, J2 Scicons) for 1 hour at room temperature followed by 1 hour staining with the secondary antibody anti-mouse-Alexa Fluor 488 (1:2000, Thermo Fisher Scientific), Hoechst (1:1000, Invitrogen) and Ty1-AS635P (0.05 μg/mL).	anti-dsRNA suggested: (Millipore Cat# MABE1134, RRID:AB_2819101) anti-mouse-Alexa suggested: None
Experimental Models: Cell Lines
Sentences	Resources
A HEK293T cell line engineered to overexpress human ACE2 (HEK293T-ACE2) was generated by the lentiviral transduction of HEK293T cells.	HEK293T suggested: NCBI_Iran Cat# C498, RRID:CVCL_0063)
TG1 cells (Lucigen) were transformed with this library by electroporation.	TG1 suggested: RRID:CVCL_0P34)
Approximately 15,000 HEK293T-ACE2 cells were then added to each well and the plates were incubated at 37°C for 48 hours.	HEK293T-ACE2 suggested: None
Immunofluorescence: Vero E6 cells were seeded onto coverslips in a 24-well plate and incubated overnight at 37°C/5% CO2.	Vero E6 suggested: None
Software and Algorithms
Sentences	Resources
Fluorescence was quantified using a BD FACSCelesta and the FlowJo software package.	FlowJo suggested: (FlowJo, RRID:SCR_008520)
Coverslips were mounted in mounting media and images were obtained using Zeiss Axiovert microscope and processed using Adobe Photoshop.	Adobe Photoshop suggested: (Adobe Photoshop, RRID:SCR_014199)
Extracted particles were imported into cryoSPARC v2.15.036 for 2D classification, 3D classification and non-uniform 3D refinement.	cryoSPARC suggested: (cryoSPARC, RRID:SCR_016501)
Structure refinement and manual model building were performed using COOT and PHENIX44 in interspersed cycles with secondary structure and geometry restrained.	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:

Thank you for including a conflict of interest statement. Authors are encouraged to include this statement when submitting to a journal.
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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An alpaca nanobody neutralizes SARS-CoV-2 by blocking receptor interaction

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