SARS-CoV-2 Evolution and Immune Escape in Immunocompromised Patients

Version published to 10.1056/nejmc2202861

Jun 23, 2022

SciScore for 10.1101/2022.04.12.22273675: (What is this?)

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

Table 1: Rigor

Ethics	IRB: This study was approved by the institutional review board at Emory University under protocols STUDY00000260, 00022371, and 00045821.
Sex as a biological variable	not detected.
Randomization	not detected.
Blinding	not detected.
Power Analysis	not detected.
Cell Line Authentication	not detected.

Table 2: Resources

Antibodies
Sentences	Resources
anti-SARS monoclonal antibody CR302240 was generously provided by Jens Wrammert	anti-SARS suggested: None
Spike Trimer Capture ELISA: The following ELISA was adapted from previously published methods17: 96-well half area, high binding plates (Corning #3690) were coated with anti-6x-His-tag monoclonal antibody (#MA1-21315MG, ThermoFisher) at 2 µg /mL in PBS at 4°C …

SciScore for 10.1101/2022.04.12.22273675: (What is this?)

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

Table 1: Rigor

Ethics	IRB: This study was approved by the institutional review board at Emory University under protocols STUDY00000260, 00022371, and 00045821.
Sex as a biological variable	not detected.
Randomization	not detected.
Blinding	not detected.
Power Analysis	not detected.
Cell Line Authentication	not detected.

Table 2: Resources

Antibodies
Sentences	Resources
anti-SARS monoclonal antibody CR302240 was generously provided by Jens Wrammert	anti-SARS suggested: None
Spike Trimer Capture ELISA: The following ELISA was adapted from previously published methods17: 96-well half area, high binding plates (Corning #3690) were coated with anti-6x-His-tag monoclonal antibody (#MA1-21315MG, ThermoFisher) at 2 µg /mL in PBS at 4°C overnight.	anti-6x-His-tag suggested: None
Approximately one million viable PBMCs were stained with Zombie aqua fixable cell viability dye (BioLegend) to exclude dead cells; washed with PBS containing 2% FBS, referred to as FACS buffer; surface-stained with the following fluorescent monoclonal antibodies: CD3 (clone SK7, BioLegend),	CD3 suggested: None
After washing with FACS buffer and fixing and permeabilizing cells with Cytofix/Cytoperm (BD Biosciences), the cells were stained intracellularly with the following fluorescent monoclonal antibodies: CD154 (clone CD40L 24-31, BioLegend),	CD154 suggested: None
After washing with FACS buffer and fixing and permeabilizing cells with Cytofix/Cytoperm (BD Biosciences), the cells were stained intracellularly with the following fluorescent monoclonal antibodies: CD154 (clone CD40L 24-31, BioLegend), IL-2 (clone MQ1-17H12, BD Biosciences), IFN-γ (clone 4S.B3, eBioscience), TNF (clone Mab11, BD Biosciences).	IL-2 suggested: None IFN-γ suggested: None TNF suggested: None
IFN-γ spots were detected with biotinylated murine anti-human IFN-γ antibody (clone 7-B6-1, Mabtech), followed by incubation with streptavidin-HRP (BD) and then developed using AEC substrate (EMD Millipore).	anti-human IFN-γ suggested: None
Experimental Models: Cell Lines
Sentences	Resources
A HeLa cell line transduced to stably express the human ACE2 receptor (ACE2-HeLa) was generously provided by David Nemazee17.	HeLa suggested: None
Wuhan-Hu-1 spike trimer protein expression: Spike trimer plasmids were transiently transfected into Expi293 cells (ThermoFisher) with 5 mM kifunensine (Mfr), purified with His-Trap columns (Cytiva), trimers selected with a Superdex 200 gel filtration column (Mfr), and finished product dialyzed into 20 mM Tris pH 8.0, 200 mM sodium chloride, 0.02% sodium azide by the BioExpression and Fermentation Facility at the University of Georgia.	Expi293 suggested: RRID:CVCL_D615)
Pseudovirus production: Pseudoviruses were produced by seeding 16 million 293T cells (ATCC CRL-3216) into DMEM with 10% heat-inactivated FBS and 1% GlutaMAX (ThermoFisher) (DMEM-10) in a T-150 flask the night prior to transfection and incubating at 37°C in a humidified 5% CO2 incubator.	293T suggested: None
DMEM-10 media was then removed from plates with cells and 50 µl pseudovirus dilutions added onto ACE2-HeLa cells and incubated for two hours at 37°C.	ACE2-HeLa suggested: None
Recombinant DNA
Sentences	Resources
Plasmids pCMV ΔR8.2 (	pCMV ΔR8.2 suggested: None
Plasmid nCoV-2P-F3CH2S43 expressing a His-tagged, pre-fusion stabilized SARS-CoV-2 spike trimer from Wuhan-Hu-1 isolate was generously provided by Jason McLellan.	nCoV-2P-F3CH2S43 suggested: None
On the day of transfection, the HIV-1 lentiviral packaging plasmid, pCMV R8.2 (17.5	pCMV suggested: RRID:Addgene_16459)
Software and Algorithms
Sentences	Resources
Sequences from immunocompromised patients were aligned with 301 reference sequences collected from patients within the Emory Healthcare System between 1/1/2021 and 4/30/2021 using MAFFT as implemented in geneious (geneious.com).	MAFFT suggested: (MAFFT, RRID:SCR_011811)
A maximum-likelihood tree was constructed using a general time reversible model with empirical base frequencies and a 3 rate model in IQ-TREE version 2.0 with 1,000 ultrafast boostraps38 and visualized in FigTree (http://tree.bio.ed.ac.uk/software/figtree).	IQ-TREE suggested: (IQ-TREE, RRID:SCR_017254) FigTree suggested: (FigTree, RRID:SCR_008515)
To identify iSNVs, reads were mapped to reference sequence NC_045512.1 using minimap2, variants were called using vphaser2 with maximum strand bias of 5, and variants annotated with SNPeff, all as implemented in viral-ngs version 2.1.19.0-rc119.	SNPeff suggested: (SnpEff, RRID:SCR_005191)
To ascertain a precise endpoint titer (ET), curve data (best fit values for the bottom, top, logEC50, and hill slope) were processed by a MATLAB program designed to determine the sample dilution at which each regression curve intersected the healthy control cutoff value.	MATLAB suggested: None
After washing with FACS buffer and fixing and permeabilizing cells with Cytofix/Cytoperm (BD Biosciences), the cells were stained intracellularly with the following fluorescent monoclonal antibodies: CD154 (clone CD40L 24-31, BioLegend), IL-2 (clone MQ1-17H12, BD Biosciences), IFN-γ (clone 4S.B3, eBioscience), TNF (clone Mab11, BD Biosciences).	BD Biosciences suggested: None
Flow cytometry data were collected on an LSR Fortessa (BD Biosciences) and analyzed using FlowJo software V10 (Tree Star).	FlowJo suggested: (FlowJo, RRID:SCR_008520)

Results from OddPub: Thank you for sharing your data.

Results from LimitationRecognizer: We detected the following sentences addressing limitations in the study:

Limitations to our study include a small number of patients and the use of convenience samples. Larger clinical studies in immunocompromised populations are needed, including serial sampling to further elucidate therapies that promote immune evasion. Our work and others’ emphasize the need to both protect immunocompromised patients from acquiring infection, and to prevent the forward spread of viruses with immune escape mutations. Such needs might be met with broad spectrum monoclonal antibodies and next generation SARS-CoV-2 vaccines that induce potent neutralizing antibody responses to prevent infection and memory CD8+ T cell responses to control breakthrough.

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.

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

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SARS-CoV-2 Evolution and Immune Escape in Immunocompromised Patients

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