Targeted isolation of diverse human protective broadly neutralizing antibodies against SARS-like viruses

Version published to 10.1038/s41590-022-01222-1

Jun 1, 2022

SciScore for 10.1101/2021.09.08.459480: (What is this?)

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

Table 1: Rigor

Ethics	Field Sample Permit: Convalescent COVID-19 and human vaccinee sera: Sera from convalescent COVID-19 donors 34, spike-mRNA-vaccinated humans, and from COVID-19-recovered vaccinated donors, were provided through the “Collection of Biospecimens from Persons Under Investigation for 2019-Novel Coronavirus Infection to Understand Viral Shedding and Immune Response Study” UCSD IRB# 200236. Consent: All human donors were assessed for medical decision-making capacity using a standardized, approved assessment, and voluntarily gave informed consent prior to being enrolled in the
Sex as a biological variable	not detected.
Randomization	Biotinylation of proteins: To randomly biotinylate the proteins described in this paper, we used an EZ-Link NHS-PEG Solid-Phase Biotinylation Kit (Thermo Scientific #21440).
Blinding	not detected.
Power Analysis	not detected.
Cell Line Authentication	not detected.

Table 2: Resources

Antibodies
Sentences	Resources
The plate was coated with 2 µg/mL mouse anti-His antibody (Invitrogen cat. #MA1-21315-1MG	anti-His suggested: None
Following washes, secondary antibody (AffiniPure Goat anti-human IgG Fc fragment specific, Jackson ImmunoResearch Laboratories cat. #109-055-008) was added for an additional 1h.	AffiniPure Goat anti-human IgG Fc fragment specific , Jackson ImmunoResearch Laboratories suggested: None anti-human IgG suggested: (Jackson ImmunoResearch Labs Cat# 109-055-008, RRID:AB_2337601)
The curves were fitted by nonlinear regression and the 50% pseudovirus neutralizing (IC50) or binding (ID50) antibody titer was calculated.	ID50 suggested: (bNAber Cat# bNAberID_50, RRID:AB_2491067)
Experimental Models: Cell Lines
Sentences	Resources
Cell lines: HEK293F cells (Life Technologies) and Expi293F cells (Life Technologies) were maintained using 293FreeStyle expression medium (Life Technologies) and Expi293 Expression Medium (Life Technologies), respectively.	HEK293F suggested: None Expi293F suggested: RRID:CVCL_D615)
Transfection for protein expression: For expression of mAbs, HC and LC gene segments that were cloned into corresponding expression vectors were transfected into Expi293 cells (Life Technologies) (	Expi293 suggested: RRID:CVCL_D615)
Pseudovirus production: To generate pseudoviruses, plasmids encoding the SARS-CoV-1, SARS-CoV-2 or other variants spike proteins with the ER retrieval signal removed were co-transfected with MLV-gag/pol and MLV-CMV-Luciferase plasmids into HEK293T cells.	HEK293T suggested: CCLV Cat# CCLV-RIE 1018, RRID:CVCL_0063)
Pseudovirus entry and serum neutralization assays: To generate hACE2-expressing stable cell lines for the pseudovirus infection test, we used lentivirus to transduce the hACE2 into HeLa cells.	HeLa suggested: CLS Cat# 300194/p772_HeLa, RRID:CVCL_0030)
Just before the end of the incubation, HeLa-hACE2 cells were suspended with culture medium at a concentration of 2 x 105/mL.	HeLa-hACE2 suggested: None
The truncated heavy chains were co-transfected with the corresponding light chains in 293Expi cells to produce the Fabs.	293Expi suggested: None
Recombinant DNA
Sentences	Resources
To produce truncated proteins of SARS-CoV-1 and SARS-CoV-2 spike, the PCR amplifications of the gene fragments encoding SARS-CoV-1 RBD (residue 307-513), SARS-CoV-2 NTD (residue 1-290), RBD (residue 320-527), RBD-SD1 (residue 320-591), and RBD-SD1-2 (residue 320-681) subdomains were carried out using the SARS-CoV-1 and SARS-CoV-2 plasmids as templates.	SARS-CoV-2 suggested: RRID:Addgene_164583)
The spike encoding genes of Pang17 (residues 1-1249, GenBank: QIA48632.1), WIV1 (residues 1-1238, GenBank: KF367457) and SHC014 (residue 1-1238, GenBank: AGZ48806.1) were constructed into the phCMV3 vector (Genlantis, USA) using the Gibson assembly (NEB, E2621L) according to the manufacturer’s instructions.	phCMV3 suggested: None
Briefly, the pBOB-hACE2 plasmid and lentiviral packaging plasmids (	pBOB-hACE2 suggested: None
pMDL, pREV, and pVSV-G (Addgene #12251, #12253, #8454)) were co-transfected into HEK293T cells using the Lipofectamine 2000 reagent (ThermoFisher Scientific, 11668019)	pMDL suggested: None pREV suggested: None pVSV-G suggested: RRID:Addgene_138479)
Software and Algorithms
Sentences	Resources
The protocol was approved by the UCSD Human Research Protection Program.	UCSD Human Research Protection Program suggested: None
The solution was then loaded into an Econo-Pac column (BioRad #7321010), washed with 1 column volume of PBS, and mAbs were eluted with 0.2 M citric acid (pH 2.67).	BioRad suggested: None
All proteins were evaluated by BioLayer Interferometry after biotinylation.	BioLayer suggested: (Harvard Medical School Center for Macromolecular Interactions Core Facility, RRID:SCR_018270)
4k x 4k) camera at 52,000 X magnification using Leginon automated image collection software 74.	Leginon suggested: (Leginon, RRID:SCR_016731)
Particles were picked using DogPicker 75 and data was processed using Relion 3.0 76.	Relion suggested: (RELION, RRID:SCR_016274)

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: Please consider improving the rainbow (“jet”) colormap(s) used on page 37. At least one figure is not accessible to readers with colorblindness and/or is not true to the data, i.e. not perceptually uniform.

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.

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

Read the original source

Targeted isolation of diverse human protective broadly neutralizing antibodies against SARS-like viruses

This article has been Reviewed by the following groups

Listed in

Abstract

Article activity feed

SARS-CoV-2 Fusion Peptide-Directed Antibodies Elicited by Natural Infection Mediate Broad Sarbecovirus Neutralization

COVID-19 vaccination induces cross-neutralisation of sarbecoviruses related to SARS-CoV-2

Development of a thermostable and broadly neutralizing pan-sarbecovirus vaccine candidate