Attenuated Influenza Virions Expressing the SARS-CoV-2 Receptor-Binding Domain Induce Neutralizing Antibodies in Mice

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1. Version published to 10.3390/v12090987

   Sep 5, 2020
2. 

   Version published to 10.1101/2020.08.12.248823v2 on bioRxiv

   Sep 3, 2020
3. 

   ScreenIT

   Aug 14, 2020

   SciScore for 10.1101/2020.08.12.248823: (What is this?)

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

   Table 1: Rigor

   Institutional Review Board Statement	IACUC: Animal work was approved under Fred Hutchinson Cancer Research Center IACUC protocol (1893). 2.5. Spike Pseudotyped Lentivirus Neutralization assays: SARS-CoV-2
   Randomization	not detected.
   Blinding	not detected.
   Power Analysis	not detected.
   Sex as a biological variable	Animal studies: Seven week old female BALB/c mice (Jackson Labs) anesthetized intraperitonially with 100mg/kg of ketamine + 10mg/kg of xylazine in PBS and infected intranasally with 50ul of either a high (8 × 105 TCID50) or low (8 × 104 TCID50) dose of ΔNA(RBD)-Flu, or with ΔNA(GFP)-Flu virus (8 × 104 TCID50), or mock infected with OptiMEM.
   Cell Line Authentication	not detected.

   Table 2: Resources

   …

   SciScore for 10.1101/2020.08.12.248823: (What is this?)

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

   Table 1: Rigor

   Institutional Review Board Statement	IACUC: Animal work was approved under Fred Hutchinson Cancer Research Center IACUC protocol (1893). 2.5. Spike Pseudotyped Lentivirus Neutralization assays: SARS-CoV-2
   Randomization	not detected.
   Blinding	not detected.
   Power Analysis	not detected.
   Sex as a biological variable	Animal studies: Seven week old female BALB/c mice (Jackson Labs) anesthetized intraperitonially with 100mg/kg of ketamine + 10mg/kg of xylazine in PBS and infected intranasally with 50ul of either a high (8 × 105 TCID50) or low (8 × 104 TCID50) dose of ΔNA(RBD)-Flu, or with ΔNA(GFP)-Flu virus (8 × 104 TCID50), or mock infected with OptiMEM.
   Cell Line Authentication	not detected.

   Table 2: Resources

   Antibodies
   Sentences	Resources
   Cells were stained with either recombinant biotinylated ACE2 ectodomain (ACROBiosystems, AC2-H82E6) or CR3022 antibody (kindly provided by Neil King and Mike Murphy, University of Washington, Institute for Protein Design) for 1 hour at room temperature, washed with FACS buffer, resuspended in secondary stain, a 1:200 dilution of PE-conjugated streptavidin (Thermo Fisher, S866) or PE-conjugated Goat Anti-Human IgG (Jackson Labs, 109-115-098), and incubated on ice for 1 hour.	AC2-H82E6 suggested: None CR3022 suggested: (Imported from the IEDB Cat# CR3022, RRID:AB_2848080) Anti-Human IgG suggested: (Jackson ImmunoResearch Labs Cat# 109-115-098, RRID:AB_2337675)
   Experimental Models: Cell Lines
   Sentences	Resources
   Briefly, we transfected a co-culture of 293T cells and MDCK-SIAT1-TMPRSS2 cells [34] with reverse genetics plasmids encoding the internal segments from the A/WSN/33 virus (PB1, PB2, PA, NP, M, NS), the hemagglutinin (HA) segment from the A/Aichi/2/1968 virus with an amino acid mutation in the receptor binding site (Y98F), and either the ΔNA(RBD) or ΔNA(GFP) segment described above, or the NA segment from A/WSN/33 virus.	293T suggested: None MDCK-SIAT1-TMPRSS2 suggested: None
   We infected MDCK-SIAT1-TMPRSS-2 cells at a low multiplicity of infection (MOI = 0.02 TCID50).	MDCK-SIAT1-TMPRSS-2 suggested: None
   Infections of 293T-ACE2 cells were performed in poly-L-lysine coated plates.	293T-ACE2 suggested: RRID:CVCL_YZ65)
   The neutralization assays were performed in MDCK-SIAT1-CMV-PB1 cells using this GFP-expressing virus as described previously [36–39].	MDCK-SIAT1-CMV-PB1 suggested: None
   Experimental Models: Organisms/Strains
   Sentences	Resources
   Animal studies: Seven week old female BALB/c mice (Jackson Labs) anesthetized intraperitonially with 100mg/kg of ketamine + 10mg/kg of xylazine in PBS and infected intranasally with 50ul of either a high (8 × 105 TCID50) or low (8 × 104 TCID50) dose of ΔNA(RBD)-Flu, or with ΔNA(GFP)-Flu virus (8 × 104 TCID50), or mock infected with OptiMEM.	BALB/c suggested: None
   Software and Algorithms
   Sentences	Resources
   Analysis and compensation were performed using FlowJo v10.7.	FlowJo suggested: (FlowJo, RRID:SCR_008520)
   As with Spike pseudotyped lentivirus neutralization assays, curves were plotted and IC50s were calculated using the neutcurve Python package.	Python suggested: (IPython, RRID:SCR_001658)

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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: We detected the following sentences addressing limitations in the study:

   Our study also has several important limitations. First, we examined the effect of ΔNA(RBD)-Flu vaccination only in influenza-naïve mice. For other live attenuated vaccines, pre-existing immunity against the vaccine vector (influenza virus, in this case) can reduce effectiveness [46,47]. This limitation could potentially be overcome by creating variants of ΔNA(RBD)-Flu that contain HA subtypes to which the human population is naïve, although we did not attempt that here. Second, we performed experiments using only in a single strain of mice (BALB/cJ) and have not examined the results of vaccination with ΔNA(RBD)-Flu in non-human primates or other animal models. Third, although the ΔNA(RBD)-Flu grew to reasonably high titers that were sufficient for our experiments, the titers were still lower than those obtained using virus with an intact NA segment. Fourth, although we demonstrated high SARS-CoV-2 neutralizing antibody titers at three weeks post vaccination, we did not perform experiments to examine the durability of these titers over longer timeframes. Finally, while the presence of neutralizing titers against SARS-CoV-2 is correlated with protection against infection in animal models [48,49] and preliminarily in human studies [50], we did not directly test if the neutralizing antibodies elicited by ΔNA(RBD)-Flu are protective against viral challenge. Despite these limitations, vaccines based on ΔNA(RBD)-Flu have several possible advantages. First, the vaccine induces neutr...

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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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4. 

   Version published to 10.1101/2020.08.12.248823v1 on bioRxiv

   Aug 13, 2020