A Next Generation Bivalent Human Ad5 COVID-19 Vaccine Delivering Both Spike and Nucleocapsid Antigens Elicits Th1 Dominant CD4+, CD8+ T-cell and Neutralizing Antibody Responses

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   ScreenIT

   Aug 2, 2020

   SciScore for 10.1101/2020.07.29.227595: (What is this?)

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

   Table 1: Rigor

   Institutional Review Board Statement	not detected.
   Randomization	not detected.
   Blinding	not detected.
   Power Analysis	not detected.
   Sex as a biological variable	Vaccination of CD-1 mice with the hAd5 S-Fusion + N-ETSD vaccine candidate: CD-1 female mice (Charles River Laboratories) 7 weeks of age were used for immunological studies performed at the vivarium facilities of Omeros Inc. (Seattle, WA).
   Cell Line Authentication	not detected.

   Table 2: Resources

   Antibodies
   Sentences	Resources
   The constructs created included: Transfection of HEK 293T cells with hAd5 constructs: To determine surface expression of the RBD epitope by vaccine candidate constructs, we transfected HEK 293T cells with hAd5 construct DNA and …

   SciScore for 10.1101/2020.07.29.227595: (What is this?)

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

   Table 1: Rigor

   Institutional Review Board Statement	not detected.
   Randomization	not detected.
   Blinding	not detected.
   Power Analysis	not detected.
   Sex as a biological variable	Vaccination of CD-1 mice with the hAd5 S-Fusion + N-ETSD vaccine candidate: CD-1 female mice (Charles River Laboratories) 7 weeks of age were used for immunological studies performed at the vivarium facilities of Omeros Inc. (Seattle, WA).
   Cell Line Authentication	not detected.

   Table 2: Resources

   Antibodies
   Sentences	Resources
   The constructs created included: Transfection of HEK 293T cells with hAd5 constructs: To determine surface expression of the RBD epitope by vaccine candidate constructs, we transfected HEK 293T cells with hAd5 construct DNA and quantified surface RBD by flow cytometric detection using anti-RBD antibodies.	anti-RBD suggested: None
   Cells were harvested 1, 2, 3, and 7 days post transfection by gently pipetting cells into medium and labeled with an anti-RBD monoclonal antibody (clone D003 Sino Biological Catalog # 40150-D003) and F(ab’)2-Goat anti-Human IgG-Fc secondary antibody conjugated with R-phycoerythrin (ThermoFisher Catalog # H10104).	anti-Human IgG-Fc suggested: (Thermo Fisher Scientific Cat# H10104, RRID:AB_2536546) R-phycoerythrin suggested: (Thermo Fisher Scientific Cat# H10104, RRID:AB_2536546)
   To label N, cells were then incubated with an anti-flag monoclonal (Anti-Flag M2 produced in mouse, Sigma cat# F1804) antibody at 1:1000 in phosphate buffered saline with 3% BSA overnight at 4°C, followed by washes in PBS and a 1 hour incubation with a goat anti-Mouse IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor Plus 555 (Life Technologies, Cat# A32727) at 1:500.	Anti-Flag suggested: (Sigma-Aldrich Cat# F1804, RRID:AB_262044) anti-Mouse IgG suggested: (Thermo Fisher Scientific Cat# A32727, RRID:AB_2633276)
   For co-localization studies, cells were also incubated overnight at 4°C with a sheep anti-Lamp1 Alexa Fluor 488-conjugated (lysosomal marker) antibody (R&D systems, Cat# IC7985G) at 1:10 or a rabbit anti-CD71 (transferrin receptor, endosomal marker) antibody (ThermoFisher Cat# PA5-83022) at 1:200.	anti-Lamp1 suggested: (Rockland Cat# 200-301-G09, RRID:AB_2611215) anti-CD71 (transferrin receptor, endosomal marker) suggested: None
   After removal of the primary antibody, two washes in PBS and three 3 washes in PBS with 3% BSA, cells were incubated with fluor-conjugated secondary antibodies when applicable at 1:500 (Goat anti-Rabbit IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor 488, Life technologies, A-11034) for 1 hour at room temperature.	anti-Rabbit IgG suggested: (Thermo Fisher Scientific Cat# A-11034, RRID:AB_2576217)
   Anti-Spike S2 (SinoBiological Cat #40590-T62) was used as the primary antibody and IRDye® 800CW Goat anti-Rabbit IgG (H + L) (Li-Cor, 925-32211) as the secondary antibody using the Ibind Flex platform.	Anti-Spike S2 suggested: (Imported from the IEDB Cat# S2, RRID:AB_2833224)
   Cells were incubated with ACE2-Fc for 20 minutes and, after a washing step, were then labeled with a PE conjugated F(ab’)2-goat anti-human IgG Fc secondary antibody at a 1:100 dilution, incubated for 20 minutes, washed and acquired on flow cytometer.	anti-human IgG suggested: None
   Fluorescent-conjugated antibodies against mouse CD8β antibody (clone H35-17.2, ThermoFisher), CD4 (clone RM4-5, BD), IFN-γ (clone XMG1.2, BD), and TNF-α (clone MP6-XT22, BD) and staining was performed in the presence of unlabeled anti-CD16/CD32 antibody (clone 2.4G2).	mouse CD8β suggested: (Bio X Cell Cat# BE0223, RRID:AB_2687706) CD4 suggested: (BioLegend Cat# 391503, RRID:AB_2721611) IFN-γ suggested: None TNF-α suggested: (Leinco Technologies Cat# T798, RRID:AB_2832121) 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 antibodies to either IFN-γ or IL-4 (BD), and were exposed to various stimuli (e.g. control peptides, target peptide pools/proteins) comprising 2 μg/mL peptide pools or 10 μg/mL protein for 36-40 hours.	IL-4 (BD) suggested: None
   ELISA for detection of antibodies: For antibody detection in sera from inoculated mice, ELISAs specific for spike and nucleocapsid antibodies, as well as for IgG subtype (IgG1, IgG2a, IgG2b, and IgG3) antibodies were used.	IgG subtype (IgG1 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), or anti-mouse IgG1 HRP (Sigma; Cat # SAB3701171), or anti-mouse IgG2a HRP (Sigma; Cat # SAB3701178), or anti-mouse IgG2b HRP (Sigma; catalog# SAB3701185), or anti-mouse IgG3 HRP conjugated antibody (Sigma; Cat # SAB3701192) was added to wells.	anti-mouse IgG1 suggested: None anti-mouse IgG2a suggested: None anti-mouse IgG2b suggested: None anti-mouse IgG3 suggested: None
   Calculation of relative μg amounts of antibodies: A standard curve of IgG was generated and 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
   The constructs created included: Transfection of HEK 293T cells with hAd5 constructs: To determine surface expression of the RBD epitope by vaccine candidate constructs, we transfected HEK 293T cells with hAd5 construct DNA and quantified surface RBD by flow cytometric detection using anti-RBD antibodies.	HEK 293T suggested: None
   Immunocytochemical labeling of hAd5 infected HeLa cells: To determine subcellular localization of N after infection or transfection of HeLa cells with hAd5 N-wild type (WT) or hAd5 N-ETSD (each with a flag tag to allow labeling), 48 hours after infection or transfection cells were fixed with 4% paraformaldehyde (PFA) and permeabilized with 0.4% Triton X100, in PBS) for 15 min.	HeLa suggested: None
   Recombinant ACE2-IgG1Fc protein was produced using Maxcyte transfection in CHO-S cells that were cultured for 14 days.	CHO-S suggested: None
   Vero E6 cell neutralization assay: All aspects of the assay utilizing virus were performed in a BSL3 containment facility according to the ISMMS Conventional Biocontainment Facility SOPs for SARS-CoV-2 cell culture studies.	Vero E6 suggested: RRID:CVCL_XD71)
   Experimental Models: Organisms/Strains
   Sentences	Resources
   Vaccination of CD-1 mice with the hAd5 S-Fusion + N-ETSD vaccine candidate: CD-1 female mice (Charles River Laboratories) 7 weeks of age were used for immunological studies performed at the vivarium facilities of Omeros Inc. (Seattle, WA).	CD-1 suggested: RRID:MGI:2686808)
   Software and Algorithms
   Sentences	Resources
   Cells were harvested 1, 2, 3, and 7 days post transfection by gently pipetting cells into medium and labeled with an anti-RBD monoclonal antibody (clone D003 Sino Biological Catalog # 40150-D003) and F(ab’)2-Goat anti-Human IgG-Fc secondary antibody conjugated with R-phycoerythrin (ThermoFisher Catalog # H10104).	ThermoFisher Catalog suggested: None
   Fluorescent-conjugated antibodies against mouse CD8β antibody (clone H35-17.2, ThermoFisher), CD4 (clone RM4-5, BD), IFN-γ (clone XMG1.2, BD), and TNF-α (clone MP6-XT22, BD) and staining was performed in the presence of unlabeled anti-CD16/CD32 antibody (clone 2.4G2).	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)

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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 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: Please consider improving the rainbow (“jet”) colormap(s) used on page 14. At least one figure is not accessible to readers with colorblindness and/or is not true to the data, i.e. not perceptually uniform.

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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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   Version published to 10.1101/2020.07.29.227595v1 on bioRxiv

   Jul 30, 2020