A trimeric NTD and RBD SARS-CoV-2 subunit vaccine induced protective immunity in CAG-hACE2 transgenic mice and rhesus macaques

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   ScreenIT

   Nov 19, 2021

   SciScore for 10.1101/2021.11.03.467182: (What is this?)

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

   Table 1: Rigor

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   Ethics	IACUC: Ethics statement: The protocol and procedures used in the studies with animals were reviewed and approved by the Laboratory Animal Welfare and Ethics Committee in Institute of Medical Biology, Chinese Academy of Medical Sciences, and Center of Laboratory Animal Sciences, Wuhan University (Wuhan, China), respectively.
   Sex as a biological variable	36 female CAG-hACE2 transgenic mice, 5-6 weeks old, were divided into 4 groups, namely the negative control group(n=6), adjuvant control group(n=6), low-dose vaccine group (n=12) (4μg/dose) and high-dose vaccine group(n=12) (16μg/dose).
   Randomization	Twelve rhesus macaques were randomly divided into two groups, with 6 animals in each group.
   Blinding

   SciScore for 10.1101/2021.11.03.467182: (What is this?)

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

   Table 1: Rigor

   Ethics	IACUC: Ethics statement: The protocol and procedures used in the studies with animals were reviewed and approved by the Laboratory Animal Welfare and Ethics Committee in Institute of Medical Biology, Chinese Academy of Medical Sciences, and Center of Laboratory Animal Sciences, Wuhan University (Wuhan, China), respectively.
   Sex as a biological variable	36 female CAG-hACE2 transgenic mice, 5-6 weeks old, were divided into 4 groups, namely the negative control group(n=6), adjuvant control group(n=6), low-dose vaccine group (n=12) (4μg/dose) and high-dose vaccine group(n=12) (16μg/dose).
   Randomization	Twelve rhesus macaques were randomly divided into two groups, with 6 animals in each group.
   Blinding	not detected.
   Power Analysis	not detected.
   Cell Line Authentication	not detected.

   Table 2: Resources

   Antibodies
   Sentences	Resources
   Following blocking and incubation with serial dilutions of sera, anti-mouse IgG, IgG1, IgG2a HRP-conjugated antibody were used as secondary Abs and incubated for 1 h at RT.	anti-mouse IgG suggested: None IgG1 suggested: None
   Immunogenicity analysis of ReCOV in mice: Two groups of female BALB/c mice (n=10) were intramuscularly administrated 4µg or 8µg RECOV with BFA03 adjuvant in a two-dose regimen (D0/D21 interval), and two weeks after second dose, the levels of antigen specific IgG antibody, neutralizing antibody, cross-neutralization against the main prevalent variants and IgG2a/IgG1 ratio, were evaluated.	antigen specific IgG suggested: None
   Experimental Models: Cell Lines
   Sentences	Resources
   Construction, expression and purification of SARS-CoV-2 NTD-RBD-foldon: To construct recombinant vector for expression of SARS-CoV-2 NTD-RBD-foldon in CHO-K1 cell, the fragment 1-541 of SARS-CoV-2 spike protein (strain Wuhan-1/2020) fused with foldon was codon-optimized and synthesized.	CHO-K1 suggested: None
   For expression of NTD-RBD-foldon in CHO cell, the NTD-RBD-foldon gene was PCR amplifized and cloned separately into pWX039 and pWX040 vectors (WuXi Biologics), yielding expression plasmid pWX039-PR-Z-7323B and pWX040-PR-B-7323B.	CHO suggested: None
   Vero cells were added after 1 h and allowed to incubate for 24 h.	Vero suggested: None
   Experimental Models: Organisms/Strains
   Sentences	Resources
   Immunogenicity analysis of ReCOV in mice: Two groups of female BALB/c mice (n=10) were intramuscularly administrated 4µg or 8µg RECOV with BFA03 adjuvant in a two-dose regimen (D0/D21 interval), and two weeks after second dose, the levels of antigen specific IgG antibody, neutralizing antibody, cross-neutralization against the main prevalent variants and IgG2a/IgG1 ratio, were evaluated.	BALB/c suggested: None
   Recombinant DNA
   Sentences	Resources
   The fragment 1-541 of spike protein and foldon were fused together with a GSGSG linker and inserted into the backbone vector pWX4.1(WuXi Biologics), yielding plasmid pWX4.1-Pr-7323-2.	pWX4.1 suggested: None pWX4.1-Pr-7323-2 suggested: None
   For expression of NTD-RBD-foldon in CHO cell, the NTD-RBD-foldon gene was PCR amplifized and cloned separately into pWX039 and pWX040 vectors (WuXi Biologics), yielding expression plasmid pWX039-PR-Z-7323B and pWX040-PR-B-7323B.	pWX039 suggested: None pWX040 suggested: None pWX039-PR-Z-7323B suggested: None pWX040-PR-B-7323B suggested: None
   Software and Algorithms
   Sentences	Resources
   Statistical analysis: Statistical analyses were performed using GraphPad Prism 8.0 (GraphPad Software) and comparison between groups was performed using a two-tailed nonparametric Mann-Whitney U t test.	GraphPad suggested: (GraphPad Prism, RRID:SCR_002798)

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   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).

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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: We found the following clinical trial numbers in your paper:
   

   Identifier	Status	Title
   NCT04818801	Recruiting	Safety, Reactogenicity and Immunogenicity Study of ReCOV

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   Results from Barzooka: We found bar graphs of continuous data. We recommend replacing bar graphs with more informative graphics, as many different datasets can lead to the same bar graph. The actual data may suggest different conclusions from the summary statistics. For more information, please see Weissgerber et al (2015).

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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.
   • No funding statement was detected.
   • No protocol registration statement was detected.

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

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   Version published to 10.1101/2021.11.03.467182 on bioRxiv

   Nov 5, 2021