Potent neutralizing nanobodies resist convergent circulating variants of SARS-CoV-2 by targeting diverse and conserved epitopes

Abstract

Interventions against variants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are urgently needed. Stable and potent nanobodies (Nbs) that target the receptor binding domain (RBD) of SARS-CoV-2 spike are promising therapeutics. However, it is unknown if Nbs broadly neutralize circulating variants. We found that RBD Nbs are highly resistant to variants of concern (VOCs). High-resolution cryoelectron microscopy determination of eight Nb-bound structures reveals multiple potent neutralizing epitopes clustered into three classes: Class I targets ACE2-binding sites and disrupts host receptor binding. Class II binds highly conserved epitopes and retains activity against VOCs and RBD _SARS-CoV . Cass III recognizes unique epitopes that are likely inaccessible to antibodies. Systematic comparisons of neutralizing antibodies and Nbs provided insights into how Nbs target the spike to achieve high-affinity and broadly neutralizing activity. Structure-function analysis of Nbs indicates a variety of antiviral mechanisms. Our study may guide the rational design of pan-coronavirus vaccines and therapeutics.

SciScore for 10.1101/2021.03.09.434592: (What is this?)

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

Table 1: Rigor

NIH rigor criteria are not applicable to paper type.

Table 2: Resources

Antibodies
Sentences	Resources
Anti-T7 tag HRP-conjugated secondary antibodies were diluted at 1:5000 and incubated at room temperature for 1 hour.	Anti-T7 tag suggested: None
Experimental Models: Cell Lines
Sentences	Resources
To express the S protein, HEK293-ES cells were transiently transfected with the plasmid using polyethyleneimine and 3.5 mM valproic acid sodium salt to enhance protein production.	HEK293-ES suggested: None
Pseudovirus neutralization assay: The 293T-hsACE2 stable cell line and the pseudotyped SARS-CoV-2 particles (wild-type and mutants) with luciferase reporters were purchased from the Integral Molecular.	293T-hsACE2 sug…

SciScore for 10.1101/2021.03.09.434592: (What is this?)

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

Table 1: Rigor

NIH rigor criteria are not applicable to paper type.

Table 2: Resources

Antibodies
Sentences	Resources
Anti-T7 tag HRP-conjugated secondary antibodies were diluted at 1:5000 and incubated at room temperature for 1 hour.	Anti-T7 tag suggested: None
Experimental Models: Cell Lines
Sentences	Resources
To express the S protein, HEK293-ES cells were transiently transfected with the plasmid using polyethyleneimine and 3.5 mM valproic acid sodium salt to enhance protein production.	HEK293-ES suggested: None
Pseudovirus neutralization assay: The 293T-hsACE2 stable cell line and the pseudotyped SARS-CoV-2 particles (wild-type and mutants) with luciferase reporters were purchased from the Integral Molecular.	293T-hsACE2 suggested: None
Software and Algorithms
Sentences	Resources
Movies of the specimen were recorded with a nominal defocus setting in the range of −0.5 to −2.5 μm using SerialEM with beam-tilt image-shift data collection strategy with a 3 x 3 pattern and 3 shot per hole.	SerialEM suggested: (SerialEM, RRID:SCR_017293)
Beam-induced motion correction was performed using the motion correction program implemented in Relion to generate average micrographs and dose-weighted micrographs from all frames.	Relion suggested: (RELION, RRID:SCR_016274)
For other structures, each movie stack was processed on-the-fly using CryoSPARC live (version 3.0.0) 44,45.	CryoSPARC suggested: (cryoSPARC, RRID:SCR_016501)
After refinement, each residue of the sequence-updated models was manually checked and refined iteratively in Coot and Phenix.	Coot suggested: (Coot, RRID:SCR_014222) Phenix suggested: (Phenix, RRID:SCR_014224)
Structural models were validated by MolProbity.	MolProbity suggested: (MolProbity, RRID:SCR_014226)
The Nb-RBD complex structure was superimposed on its best matched Fab-RBD structure and protein volume was calculated using ProteinVolume51.	ProteinVolume51 suggested: None
The data was then processed by Prism GraphPad 9.0 to fit into a 4PL curve and to calculate the logIC50 (half-maximal inhibitory concentration).	GraphPad suggested: (GraphPad Prism, RRID:SCR_002798)
Analysis of the data was performed using Excel.	Excel suggested: None

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:

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.

Read the original source

Potent neutralizing nanobodies resist convergent circulating variants of SARS-CoV-2 by targeting diverse and conserved epitopes

This article has been Reviewed by the following groups

Listed in

Abstract

Article activity feed