A live attenuated virus-based intranasal COVID-19 vaccine provides rapid, prolonged, and broad protection against SARS-CoV-2

SciScore for 10.1101/2021.11.13.468472: (What is this?)

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

Table 1: Rigor

Ethics	IACUC: Animals that lost more than 25% of their initial body weight were euthanized in accordance with our animal ethics protocol.
Sex as a biological variable	Two groups of ferrets (5 female and 5 male ferrets in the vaccine group and 3 female and 3 male ferrets in the control group) were immunized intranasally with a single-dose 1×106 PFU of dNS1-RBD and CA04-WT virus respectively diluted in 1640 media to a final 500 μL volume.
Randomization	not detected.
Blinding	not detected.
Power Analysis	not detected.
Cell Line Authentication	not detected.

Table 2: Resources

Antibodies
Sentences	Resources
Proteins were separated on a 10% gel, and then following transfer, blots were incubated with an anti-influenza A …

SciScore for 10.1101/2021.11.13.468472: (What is this?)

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

Table 1: Rigor

Ethics	IACUC: Animals that lost more than 25% of their initial body weight were euthanized in accordance with our animal ethics protocol.
Sex as a biological variable	Two groups of ferrets (5 female and 5 male ferrets in the vaccine group and 3 female and 3 male ferrets in the control group) were immunized intranasally with a single-dose 1×106 PFU of dNS1-RBD and CA04-WT virus respectively diluted in 1640 media to a final 500 μL volume.
Randomization	not detected.
Blinding	not detected.
Power Analysis	not detected.
Cell Line Authentication	not detected.

Table 2: Resources

Antibodies
Sentences	Resources
Proteins were separated on a 10% gel, and then following transfer, blots were incubated with an anti-influenza A NP protein antibody 19C10 generated by our laboratory (1:1000)	anti-influenza A NP protein suggested: None
and anti-V5 tag antibody (Thermo,1:5000) and visualized with horseradish peroxidase (HRP)-conjugated anti-mouse IgG (Invitrogen, 1:5000)	anti-V5 suggested: None anti-mouse IgG suggested: (LSBio (LifeSpan Cat# LS-C69682-5000, RRID:AB_1653096)
Based on the ELISA results using a sandwich assay with anti-RBD monoclonal antibodies on both sides (Wantai, Beijing, China) and plaque assay results, serial passages 1 to 10 of purified vaccines were confirmed to be stable under current vaccine manufacturing conditions.	anti-RBD suggested: None
Anti-RBD IgG measurements: RBD-specific antibody titers in serum samples collected from immunized animals with 1×106 PFU of vaccine were determined by indirect ELISA.	Anti-RBD IgG suggested: None
Diluted sera (1:100) were successively diluted in a 2-fold series and applied to each well for 1 h at 37°C, followed by incubation with goat anti-mouse, anti-hamster or anti-human antibodies conjugated with HRP for 1 h at 37°C after 3 washes.	anti-mouse , suggested: None anti-human suggested: None
The cells were stained with murine antibodies for phenotype and activation (CD4 [clone GK1.5, APC/Cy7], CD8 [clone 53-6.7, PerCP/Cy5.5], CD11b [clone M1/70, PE], CD11c [clone N418, BV421],	CD4 suggested: (Miltenyi Biotec Cat# 130-109-536, RRID:AB_2657974) CD8 suggested: None CD11b suggested: None CD11c suggested: None BV421 suggested: None
Immunohistochemical staining was performed by using a mouse monoclonal anti-SARS-CoV-2 N protein antibody.	anti-SARS-CoV-2 N protein suggested: None
Experimental Models: Cell Lines
Sentences	Resources
Human embryonic kidney cells (293T), African green monkey kidney epithelial cells (Vero E6), and Madin-Darby canine kidney cells (MDCK) were maintained in DMEM-high glucose (Sigma Aldrich, USA) supplemented with 10% low endotoxin FBS (Cegrogen Biotech, Germany) and penicillin-streptomycin.	MDCK suggested: None
Generation and passage of dNS1-RBD viruses: Eight pHW2000 plasmids containing the DelNS1 segment and the other seven influenza virus genomic segments, together with an NS1 expression plasmid, pCX-CA04-NS1-Flag, which derived from the parental influenza virus A/California/04/2009(H1N1) (GenBank: MN371610.1-371617.1), were transfected into 293T cells and incubated overnight at 37°C.	293T suggested: None
SARS-CoV-2 titration assay: Live virus titers in homogenized lung tissues and cell cultures were measured by the standard TCID50 method in Vero E6 cells seeded in 96-well plates.	Vero E6 suggested: None
Experimental Models: Organisms/Strains
Sentences	Resources
Immunization and infection of mice: BALB/c mice were immunized intranasally with 50 μL containing 1×106 PFU of the vaccine prepared as indicated above under isoflurane anesthesia, while the control group was administered CA04-WT or CA04-dNS1 virus.	BALB/c suggested: None
For cellular immune response analyses of PBMCs, splenic lymphocytes, pulmonary lymphocytes and lymph node cells, C57BL/6 mice (6-8 weeks old) were immunized intranasally with 1×106 PFU of the vaccine by the one-dose or two-dose regimen as described above (10 animals in each group).	C57BL/6 suggested: None
Immunization and infection of hACE2-KI/NIFDC mice: hACE2-KI/NIFDC mice (8-10 weeks old) were divided into three groups and treated intranasally with 1×106 PFU of the vaccine by gently adding 50 μL droplets of virus stock for the vaccine-immunized group (5 animals) at two time points (days 0 and 14), and then, the vaccine-immunized group and unvaccinated group (3 animals each) were challenged with 1×104 PFU of SARS-CoV-2 by the intranasal route 30 days post immunization.	hACE2-KI/NIFDC suggested: None
Recombinant DNA
Sentences	Resources
The sequence encoding the RBD segment was then cloned into the NS1 deletion plasmid pHW2000-DelNS1 as described previously.	pHW2000-DelNS1 suggested: None
Generation and passage of dNS1-RBD viruses: Eight pHW2000 plasmids containing the DelNS1 segment and the other seven influenza virus genomic segments, together with an NS1 expression plasmid, pCX-CA04-NS1-Flag, which derived from the parental influenza virus A/California/04/2009(H1N1) (GenBank: MN371610.1-371617.1), were transfected into 293T cells and incubated overnight at 37°C.	pHW2000 suggested: None pCX-CA04-NS1-Flag suggested: None
Software and Algorithms
Sentences	Resources
The data were analyzed by FlowJo V10.6.0 and GraphPad Prism 9.	FlowJo suggested: (FlowJo, RRID:SCR_008520)
Statistical analysis: Statistical significance was assigned when P values were < 0.05 using GraphPad Prism 8.0 (GraphPad Software, Inc.)	GraphPad suggested: (GraphPad Prism, RRID:SCR_002798)

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 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).

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.

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

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A live attenuated virus-based intranasal COVID-19 vaccine provides rapid, prolonged, and broad protection against SARS-CoV-2

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