Heteologous saRNA-Prime, DNA Dual-Antigen-Boost SARS-CoV-2 Vaccination Elicits Robust Cellular Immunogenicity and Cross-Variant Neutralizing Antibodies

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1. 

   Version published to 10.1101/2021.11.29.470440v2 on bioRxiv

   Mar 12, 2022
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   ScreenIT

   Dec 3, 2021

   SciScore for 10.1101/2021.11.29.470440: (What is this?)

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

   Table 1: Rigor

   Ethics	Field Sample Permit: All in vivo experiments described were carried out in strict accordance with good animal practice according to NIH recommendations. IACUC: All procedures for animal use were approved by the IACUC Committee at Omeros, Inc. (Seattle, WA, USA) and under an approved protocol.
   Sex as a biological variable	CD-1 female mice (Charles River Laboratories) 6-8 weeks of age were used for immunological studies performed at the vivarium facilities of Omeros Inc. (Seattle, WA).
   Randomization	not detected.
   Blinding	not detected.
   Power Analysis	not detected.
   Cell Line Authentication	not detected.

   Table 2: Resources

   Antibodies
   Sentences	Resources
   Fluorescent-conjugated anti-mouse antibodies used for …

   SciScore for 10.1101/2021.11.29.470440: (What is this?)

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

   Table 1: Rigor

   Ethics	Field Sample Permit: All in vivo experiments described were carried out in strict accordance with good animal practice according to NIH recommendations. IACUC: All procedures for animal use were approved by the IACUC Committee at Omeros, Inc. (Seattle, WA, USA) and under an approved protocol.
   Sex as a biological variable	CD-1 female mice (Charles River Laboratories) 6-8 weeks of age were used for immunological studies performed at the vivarium facilities of Omeros Inc. (Seattle, WA).
   Randomization	not detected.
   Blinding	not detected.
   Power Analysis	not detected.
   Cell Line Authentication	not detected.

   Table 2: Resources

   Antibodies
   Sentences	Resources
   Fluorescent-conjugated anti-mouse antibodies used for labeling included CD8β antibody (clone H35-17.2, ThermoFisher), CD4 (clone RM4-5, BD), IFN-γ (clone XMG1.2, BD), TNF-α (clone MP6-XT22, BD) and IL-2 (clone JES6-5H4; BD), and staining was performed in the presence of unlabeled anti-CD16/CD32 antibody (clone 2.4G2; BD).	anti-mouse suggested: None CD4 suggested: (BioLegend Cat# 391503, RRID:AB_2721611) IFN-γ suggested: None TNF-α suggested: (Leinco Technologies Cat# T698, RRID:AB_2737572) IL-2 suggested: None anti-CD16/CD32 suggested: None
   The cells (2-4 × 105 cells per well of a 96-well plate) were added to the ELISpot plate containing an immobilized primary antibody to either IFN-γ or IL-4 (BD Cat# 551881 and BD Cat# 551878, respectively), and were exposed to various stimuli (e.g. control peptides SIV and ConA, S-WT and N peptides pools – see catalog numbers above) at a concentration of 1-2 μg/mL peptide pools for 36-40 hours.	IL-4 suggested: (BD Biosciences Cat# 551878, RRID:AB_2336921)
   ELISA for detection of antibodies: For IgG antibody detection in inoculated mouse sera and lung homogenates, ELISAs for spike-binding (including S1 Delta) and nucleocapsid-binding antibodies and IgG subclasses (IgG1, IgG2a, IgG2b, and IgG3) were used.	IgG1, IgG2a suggested: None IgG3 suggested: None
   After incubation, the wells were washed with PBST and 100 μL of a 1/5000 dilution of anti-mouse IgG HRP (GE Health Care; Cat # NA9310V), anti-mouse IgG1 HRP (Sigma; Cat # SAB3701171), anti-mouse IgG2a HRP (Sigma; Cat # SAB3701178), anti-mouse IgG2b HRP (Sigma; catalog# SAB3701185), anti-mouse IgG3 HRP conjugated antibody (Sigma; Cat # SAB3701192), or anti-mouse IgA HRP conjugated antibody (Sigma; Cat # A4789) was added to wells.	anti-mouse IgG suggested: None anti-mouse IgG1 suggested: None anti-mouse IgG2a suggested: None anti-mouse IgG2b suggested: None anti-mouse IgG3 suggested: None anti-mouse IgA HRP suggested: None
   Calculation of relative ng amounts of antibodies and the Th1/Th2 IgG subclass bias: A standard curve of IgG for OD vs. ng mouse IgG was generated using purified mouse IgG (Sigma Cat #15381; absorbance values were converted into mass equivalents for both anti-S and anti-N antibodies.	anti-S suggested: None anti-N suggested: None
   Experimental Models: Cell Lines
   Sentences	Resources
   Serum-virus mix was then added in duplicate to seeded hACE2 expressing HEK293 cells (BEI Resources) and incubated at 37°C, 5% CO2 for 72 hours.	HEK293 suggested: None
   Experimental Models: Organisms/Strains
   Sentences	Resources
   Murine immunization and blood/tissue collection: The design of vaccination study performed using CD-1 mice is shown in Figure 2.	CD-1 suggested: None
   Software and Algorithms
   Sentences	Resources
   Fluorescent-conjugated anti-mouse antibodies used for labeling included CD8β antibody (clone H35-17.2, ThermoFisher), CD4 (clone RM4-5, BD), IFN-γ (clone XMG1.2, BD), TNF-α (clone MP6-XT22, BD) and IL-2 (clone JES6-5H4; BD), and staining was performed in the presence of unlabeled anti-CD16/CD32 antibody (clone 2.4G2; BD).	ThermoFisher suggested: (ThermoFisher; SL 8; Centrifuge, RRID:SCR_020809)
   Flow cytometry was performed using a Beckman-Coulter Cytoflex S flow cytometer and analyzed using Flowjo Software.	Flowjo suggested: (FlowJo, RRID:SCR_008520)
   Statistical analyses and graph generation: All statistical analyses were performed and graphs generated used in figures were generated using GraphPad Prism software.	GraphPad Prism 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: 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.
   • 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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3. 

   Version published to 10.1101/2021.11.29.470440v1 on bioRxiv

   Nov 30, 2021