Thermodynamically coupled biosensors for detecting neutralizing antibodies against SARS-CoV-2 variants

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1. Version published to 10.1038/s41587-022-01280-8

   Apr 28, 2022
2. 

   ScreenIT

   Jul 1, 2021

   SciScore for 10.1101/2021.06.22.449355: (What is this?)

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

   Table 1: Rigor

   Ethics	Euthanasia Agents: Mice were injected intramuscularly into the gastroc nemius muscle of each hind leg using a 27-gauge needle (BD, San Diego, CA) with 50 mL per injection site (100 mL total) of immunogen under isoflurane anesthesia.
   Sex as a biological variable	HEK293F is a female human embryonic kidney cell line transformed and adapted to grow in suspension (Life Technologies).
   Randomization	De-identified clinical serum samples were randomly used for spiking in target proteins.
   Blinding	No sample was excluded from data analysis, and no blinding was used.
   Power Analysis	not detected.
   Cell Line Authentication	not detected.

   Table 2: Resources

   Antibodies
   Sentences	Resources
   TMPRSS2 expression was confirmed …

   SciScore for 10.1101/2021.06.22.449355: (What is this?)

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

   Table 1: Rigor

   Ethics	Euthanasia Agents: Mice were injected intramuscularly into the gastroc nemius muscle of each hind leg using a 27-gauge needle (BD, San Diego, CA) with 50 mL per injection site (100 mL total) of immunogen under isoflurane anesthesia.
   Sex as a biological variable	HEK293F is a female human embryonic kidney cell line transformed and adapted to grow in suspension (Life Technologies).
   Randomization	De-identified clinical serum samples were randomly used for spiking in target proteins.
   Blinding	No sample was excluded from data analysis, and no blinding was used.
   Power Analysis	not detected.
   Cell Line Authentication	not detected.

   Table 2: Resources

   Antibodies
   Sentences	Resources
   TMPRSS2 expression was confirmed using an anti-V5 antibody (Thermo Fisher Scientific, 2F11F7) or anti-TMPRSS2 mAb (Abnova, Clone 2F4) and APC-conjugated goat anti-mouse IgG (BioLegend, 405308).	anti-V5 suggested: (Thermo Fisher Scientific Cat# 37-7500, RRID:AB_2533339) anti-TMPRSS2 suggested: None anti-mouse IgG suggested: (BioLegend Cat# 405308, RRID:AB_315011)
   One day post-transfection, cells were infected with VSV(G*ΔG-luciferase) and after 2 h were washed five times with DMEM before adding medium supplemented with anti-VSV-G antibody (I1-mouse hybridoma supernatant, CRL-2700, ATCC)	anti-VSV-G suggested: None
   Antibody levels were quantified by conversion of the optical density to a z-score relative to pre-pandemic serum anti-S1 IgG concentrations, as previously described23,33.	anti-S1 IgG suggested: (Imported from the IEDB Cat# 2E10, RRID:AB_2848047)
   Experimental Models: Cell Lines
   Sentences	Resources
   Expi293F cells are derived from the HEK293F cell line (Life Technologies).	HEK293F suggested: RRID:CVCL_6642)
   Vero E6 (CRL-1586, American Type Culture Collection), Vero-TMPRSS2 (a gift of S. Ding, Washington University) and Vero-hACE2-TMPRSS2 (a gift of A. Creanga and B. Graham, National Institutes of Health (NIH)) cells were cultured at 37°C in Dulbecco’s modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 10 mM HEPES (pH 7.3), 1 mM sodium pyruvate, 1× nonessential amino acids and 100 U mL-1of penicillin-streptomycin.	Vero E6 suggested: None
   Vero-hACE2-TMPRSS2 cell cultures were supplemented with 10 μg ml-1of puromycin.	Vero-hACE2-TMPRSS2 suggested: None
   Briefly for MLV, HEK293T cells were co-transfected using Lipofectamine 2000 (Life Technologies) with an S-encoding plasmid, an MLV Gag-Pol packaging construct, and the MLV transfer vector encoding a luciferase reporter according to the manufacturer’s instructions.	HEK293T suggested: None
   Briefly, 293T cells in DMEM supplemented with 10% FBS, 1% PenStrep seeded in 10-cm dishes were transfected with the plasmid encoding for the corresponding S glycoprotein using lipofectamine 2000 (Life Technologies) following manufacturer’s indications.	293T suggested: None
   Live and pseudovirus entry and serum neutralization assays: SARS2-02 and SARS2-38 were assayed for neutralization potency by focus-reduction neutralization test (FRNT) as described previously34, and using Vero-TMPRSS2 cells.	Vero-TMPRSS2 suggested: JCRB Cat# JCRB1818, RRID:CVCL_YQ48)
   Immune complexes were then added to Vero-TMRPSS2 cell monolayers in a 96-well plate and incubated for 1 h at 37°C prior to the addition of 1% (w/v) methylcellulose in MEM.	Vero-TMRPSS2 suggested: None
   For the mAbs CV30, B38, and CR3022 and for the vaccinated human serum samples, HEK-hACE2 cells were cultured in DMEM with 10% FBS (Hyclone) and 1% PenStrep with 8% CO2 in a 37C incubator (ThermoFisher).	HEK-hACE2 suggested: None
   Experimental Models: Organisms/Strains
   Sentences	Resources
   Monoclonal antibodies: The murine mAbs SARS2-02 and SARS2-38 studied were isolated from BALB/c mice immunized with SARS-CoV-2 spike and RBD proteins and have been described previously25.	BALB/c suggested: None
   Recombinant DNA
   Sentences	Resources
   Genes encoding CR302226, B3827, and CV3018 heavy and light chains were ordered from GenScript and cloned into pCMV/R.	pCMV/R suggested: None
   General procedures for bacterial protein production and purification: The E. coli Lemo21(DE3) strain (NEB) was transformed with a pET29b+ plasmid encoding the synthesized gene of interest.	pET29b+ suggested: None
   Plasmid construction for RBD: The SARS-CoV-2 RBD (BEI NR-52422) construct was synthesized by GenScript into pcDNA3.1-with an N-terminal mu-phosphatase signal peptide and a C-terminal octa-histidine tag (GHHHHHHHH).	pcDNA3.1-with suggested: None
   Software and Algorithms
   Sentences	Resources
   Relative luciferase units were plotted and normalized in Prism (GraphPad) using a zero value of cells alone and a 100% value of 1:2 virus alone.	Prism suggested: (PRISM, RRID:SCR_005375) GraphPad suggested: (GraphPad Prism, RRID:SCR_002798)
   Systems of ordinary differential equations describing the kinetics of the interactions between the species involved in each sensor were numerically integrated using integrate.odeint() as implemented in Scipy (version 1.6.3)35.	Scipy suggested: (SciPy, RRID:SCR_008058)
   The python code for running these simulations is provided as a Jupyter notebook: https://github.com/bwicky/covid_nAb_sensor_simulation.	python suggested: (IPython, RRID:SCR_001658)
   All data were analyzed and plotted using GraphPad Prism 8.	GraphPad Prism suggested: (GraphPad Prism, RRID:SCR_002798)

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   Results from OddPub: Thank you for sharing your code.

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   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.

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   Results from TrialIdentifier: No clinical trial numbers were referenced.

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   Results from Barzooka: We did not find any issues relating to the usage of bar graphs.

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   Results from JetFighter: We did not find any issues relating to colormaps.

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

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3. 

   Version published to 10.1101/2021.06.22.449355v1 on bioRxiv

   Jun 22, 2021