Immunological and pathological outcomes of SARS-CoV-2 challenge following formalin-inactivated vaccine in ferrets and rhesus macaques

Version published to 10.1126/sciadv.abg7996

Sep 10, 2021

SciScore for 10.1101/2020.12.21.423746: (What is this?)

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

Table 1: Rigor

Institutional Review Board Statement	IACUC: All experimental work was conducted under the authority of a UK Home Office approved project licence that had been subject to local ethical review at PHE Porton Down by the Animal Welfare and Ethical Review Body (AWERB).
Randomization	Vaccinations: Animals were randomly assigned to control (Ad-GFP for ferrets, no vaccine for rhesus macaques) and FIV-vaccinated groups.
Blinding	Three qualified veterinary pathologists examined the tissues independently and were blinded to treatment and group details and the slides randomised prior to examination in order to prevent bias.
Power Analysis	not detected.
Sex as a biological variable	Animals. Ferrets: Ten healthy, female ferrets (
Cell Line Authentication	not detected.

Table 2: Resources

Antibodies
Sentences	Resources
Immunophenotyping: Whole blood immunophenotyping assays were performed using 50 μl of heparinised blood incubated for 30 min at room temperature with optimal dilutions of the following antibodies: anti-CD3-AF700, anti-CD4-APC-H7, anti-CD8-PerCP-Cy5.5, anti-CD95-Pe-Cy7, anti-CD14-PE, anti-HLA-DR-BUV395, anti-CD25-FITC (all from BD Biosciences, Oxford, UK); anti-CD127-APC (eBioscience); anti-γδ-TCR-BV421, anti-CD16-BV786, anti-PD-1-BV711, anti-CD20-PE-Dazzle (all from BioLegend); and amine reactive fixable viability stain red (Life Technologies); all prepared in brilliant stain buffer (BD Biosciences).	anti-CD3-AF700 suggested: None anti-CD4-APC-H7 suggested: None anti-CD8-PerCP-Cy5.5 suggested: None anti-CD95-Pe-Cy7 suggested: None anti-CD14-PE suggested: (Sigma-Aldrich Cat# P5435, RRID:AB_477370) anti-HLA-DR-BUV395 suggested: None anti-CD25-FITC suggested: (Millipore Cat# FCMAB189F, RRID:AB_10807951) anti-CD127-APC suggested: None anti-γδ-TCR-BV421 suggested: None anti-CD16-BV786 suggested: None anti-PD-1-BV711 suggested: None anti-CD20-PE-Dazzle suggested: None
A polyclonal rabbit anti-human CD3 antibody (1:200; Agilent Technologies Inc, CA) was applied for 15 min and used with Leica Polymer Refine Detection kit to complete the staining.	anti-human CD3 suggested: (LSBio (LifeSpan Cat# LS-C51896-200, RRID:AB_1272857)
For S trimer capture ELISA, high protein binding ELISA plates (PerkinElmer) were coated overnight at 4°C with anti-myc antibody 9E10 at 4 μg/mL.	anti-myc suggested: None
Experimental Models: Cell Lines
Sentences	Resources
Viruses and cells: SARS-CoV-2 Victoria/01/202026 was provided by The Doherty Institute, Melbourne, Australia at P1 and passaged twice in Vero/hSLAM cells [ECACC 04091501].	Vero/hSLAM suggested: ECACC Cat# 04091501, RRID:CVCL_L037)
Virus titre was determined by plaque assay on Vero/E6 cells [ECACC 85020206].	Vero/E6 suggested: JCRB Cat# JCRB1819, RRID:CVCL_YQ49)
Proteins were expressed in 293F cells and purified by nickel column (Thermofisher) for S trimer and protein A column (Thermofisher) for RBD-Fc.	293F suggested: RRID:CVCL_D615)
Software and Algorithms
Sentences	Resources
Whole genome sequencing was performed, on the challenge isolate, using both Nanopore and Illumina as described previously24.	Nanopore suggested: None
Immunophenotyping: Whole blood immunophenotyping assays were performed using 50 μl of heparinised blood incubated for 30 min at room temperature with optimal dilutions of the following antibodies: anti-CD3-AF700, anti-CD4-APC-H7, anti-CD8-PerCP-Cy5.5, anti-CD95-Pe-Cy7, anti-CD14-PE, anti-HLA-DR-BUV395, anti-CD25-FITC (all from BD Biosciences, Oxford, UK); anti-CD127-APC (eBioscience); anti-γδ-TCR-BV421, anti-CD16-BV786, anti-PD-1-BV711, anti-CD20-PE-Dazzle (all from BioLegend); and amine reactive fixable viability stain red (Life Technologies); all prepared in brilliant stain buffer (BD Biosciences).	BioLegend) suggested: (BioLegend, RRID:SCR_001134)
Cells were analysed using a five laser LSRII Fortessa instrument (BD Biosciences) and data were analysed using FlowJo (version 10, Treestar, Ashland, US).	FlowJo suggested: (FlowJo, RRID:SCR_008520)
Lymphocyte sub populations including T-cells, NK-cells, NKT-cells and B-cells were delineated by the expression pattern of CD3, CD20, CD95, CD4, CD8, CD127, CD25, CD16 and the activation and inhibitory markers HLA-DR and PD-1. GraphPad Prism (version 8.0.1) was used to generate graphical representations of flow cytometry data.	GraphPad suggested: (GraphPad Prism, RRID:SCR_002798)
Absorbance at 450 and 570 nm was read on a SpectraMax M5 plate reader (Molecular Devices) and data analysed in GraphPad Prism v7.	GraphPad Prism suggested: (GraphPad Prism, RRID:SCR_002798)
Molecular modelling: The structure shown is PDB 6lzg, and the views shown were generated in Pymol 2.3.5 (Schrodinger LLC).	Pymol suggested: (PyMOL, RRID:SCR_000305)

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: We detected the following sentences addressing limitations in the study:

There are limitations to the current study, the principal of which is the small number of ferrets in the early culled group. The transient nature of the pathology meant that these differences resolved by the second necropsy time point. Some insight into the weak anti-S neutralising response induced by the FIV was gained using a capture ELISA which preserved the conformation of the S trimer on the solid phase, unlike direct coating onto the ELISA plate which modified antigenicity (data not shown), as has been observed for soluble forms of the HIV-1 envelope glycoprotein trimer52. Formaldehyde treatment of S trimer in this format was the same as that used to inactivate the vaccine, allowing extrapolation of ligand binding to the ELISA-captured formaldehyde-treated S trimer to that on the virus. Formaldehyde cross-linking resulted in a 2-fold reduction in binding of ACE2-Fc and two RBD-specific MAbs (CR3022 and EY6A) to formaldehyde-treated compared to untreated S trimer. By contrast, formaldehyde treatment of recombinant RBD did not affect binding of ACE2-Fc or these MAbs, implying that formaldehyde treatment cross-linked a proportion of S trimer into a non-ligand binding conformation. These results are consistent with the location of lysine and arginine residues, and suggest that RBD exposure may be limited by cross-linking, reducing exposure of neutralising antibody epitopes on the RBD. This result may be of more general interest, since other viral envelope glycoproteins, suc...

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

Immunological and pathological outcomes of SARS-CoV-2 challenge following formalin-inactivated vaccine in ferrets and rhesus macaques

This article has been Reviewed by the following groups

Listed in

Abstract

Article activity feed