Design of a highly thermotolerant, immunogenic SARS-CoV-2 spike fragment

Version published to 10.1074/jbc.ra120.016284

Jan 1, 2021

SciScore for 10.1101/2020.08.15.252437: (What is this?)

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

Table 1: Rigor

Institutional Review Board Statement	IACUC: All animal studies were approved by the Institutional Animal Ethics committee (IAEC) No. RR/IAEC/72-2019, Invivo/GP/084.
Randomization	not detected.
Blinding	not detected.
Power Analysis	not detected.
Sex as a biological variable	Guinea Pig Immunizations: Groups of four, female, Hartley strain guinea pigs, (6-8 weeks old, approximately weighing 300 g) were immunized with 20 μg of purified antigen protein diluted in 50 μl PBS, (pH 7.4), and mixed with 50 μl of AddaVax™ adjuvant (vac-adx-10) (1:1 v/v Antigen: AddaVax™ ratio per animal/dose) (InvivoGen, USA).
Cell Line Authentication	not detected.

Table 2: Resources

Antibodies
Sentences	Resources
Genes for the heavy and …

SciScore for 10.1101/2020.08.15.252437: (What is this?)

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

Table 1: Rigor

Institutional Review Board Statement	IACUC: All animal studies were approved by the Institutional Animal Ethics committee (IAEC) No. RR/IAEC/72-2019, Invivo/GP/084.
Randomization	not detected.
Blinding	not detected.
Power Analysis	not detected.
Sex as a biological variable	Guinea Pig Immunizations: Groups of four, female, Hartley strain guinea pigs, (6-8 weeks old, approximately weighing 300 g) were immunized with 20 μg of purified antigen protein diluted in 50 μl PBS, (pH 7.4), and mixed with 50 μl of AddaVax™ adjuvant (vac-adx-10) (1:1 v/v Antigen: AddaVax™ ratio per animal/dose) (InvivoGen, USA).
Cell Line Authentication	not detected.

Table 2: Resources

Antibodies
Sentences	Resources
Genes for the heavy and light chain of the CR3022 antibody were obtained from Genscript (USA) and cloned into the pcDNA3.4 vector Purification of recombinant proteins expressed in Expi293F cells: Transfections were performed according to the manufacturer’s guidelines (Gibco, Thermofisher).	CR3022 suggested: (Imported from the IEDB Cat# CR3022, RRID:AB_2848080)
The expression levels were monitored by dot blot analysis with anti-His tag antibodies.	anti-His tag antibodies . suggested: None
Following this blot was washed and incubated with α-guinea pig ALP conjugated antibody (Sigma) at 1:5000.	α-guinea pig ALP suggested: None
Following this, Rabbit ALP enzyme conjugated to anti-Guinea Pig IgG secondary antibody (diluted 1:5000 in blocking buffer) (50 μL/well) was added and incubated for 1 hour at 25 °C, 300 rpm (Sigma-Aldrich).	anti-Guinea Pig IgG suggested: (LSBio (LifeSpan Cat# LS-C56297-5000, RRID:AB_10379625)
Next, rabbit ALP enzyme conjugated to anti-Human IgG secondary antibody (diluted 1:5000 in blocking buffer) (50 μl/well) was added and samples incubated for 1 hour at 25°C, 300 rpm (Sigma-Aldrich).	anti-Human IgG secondary antibody suggested: None anti-Human IgG suggested: None
The wells were incubated with anti-His Antibody (1:10000 dilution) conjugated with Horseradish peroxidase (HRP) enzyme for 1 hr at RT following which the reaction was visualized by adding 50μL of the chromogenic substrate, TMB (Thermo Fisher).	anti-His suggested: (LSBio (LifeSpan Cat# LS-C67878-100, RRID:AB_1815148)
Experimental Models: Cell Lines
Sentences	Resources
Briefly, the plasmids pCAGGS-SARS2-S and pCAGGS-SARS-S were transiently expressed on HEK 293T cells using Polyethylenimine (PEI) (Polysciences, USA).	HEK 293T suggested: NCBI_Iran Cat# C498, RRID:CVCL_0063)
Then the viruses were titrated in Vero E6 cells and stored at −80 °C.	Vero E6 suggested: RRID:CVCL_XD71)
Software and Algorithms
Sentences	Resources
The unbound tag-free protein was collected and protein concentration was determined by absorbance (A280) using NanoDrop™2000c with the theoretical molar extinction coefficient calculated using the ProtParam tool (ExPASy).	ProtParam suggested: (ProtParam Tool, RRID:SCR_018087)
Reference-free 2D classification using single-particle analysis: The evaluation of micrographs was done with EMAN 2.1 (56).	EMAN suggested: (EMAN, RRID:SCR_016867)
Reference free 2D classification of different projections of particle were performed using simple_prime2D of SIMPLE 2.1 software (57	SIMPLE suggested: (SIMPLE, RRID:SCR_009389)
Production of Pseudotyped SARS-CoV-2 and pseudovirus neutralisation assay: The full-length synthetic construct of Spike glycoprotein of SARS-CoV-2 (GenBank: MN908947) was synthesized from Genewiz, UK.	Genewiz suggested: (GENEWIZ, RRID:SCR_003177)

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:

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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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Design of a highly thermotolerant, immunogenic SARS-CoV-2 spike fragment

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