Pre-clinical testing of two serologically distinct chimpanzee-origin adenovirus vectors expressing spike of SARS-CoV-2

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   ScreenIT

   Feb 27, 2022

   SciScore for 10.1101/2022.02.23.481620: (What is this?)

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

   Table 1: Rigor

   Ethics	not detected.
   Sex as a biological variable	Mice and mouse procedures: Female C57Bl/6 mice (6–8 weeks of age) were purchased from the Jackson Laboratories (Bar Harbor, ME).
   Randomization	not detected.
   Blinding	not detected.
   Power Analysis	not detected.
   Cell Line Authentication	not detected.

   Table 2: Resources

   Antibodies
   Sentences	Resources
   Expression of ACE-2: Cells diluted to 1 × 106 per microtiter plate well were incubated for 45 min at room temperature with a 1 in 100 dilution of a mouse IgG2a antibody to human/hamster ACE-2 (clone 171606 R&D Systems, Minneapolis, MN)	mouse IgG2a suggested: (Millipore Cat# 171606-100UG, RRID:AB_10690063)
   In parallel the membrane was probed with a mouse monoclonal IgG …

   SciScore for 10.1101/2022.02.23.481620: (What is this?)

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

   Table 1: Rigor

   Ethics	not detected.
   Sex as a biological variable	Mice and mouse procedures: Female C57Bl/6 mice (6–8 weeks of age) were purchased from the Jackson Laboratories (Bar Harbor, ME).
   Randomization	not detected.
   Blinding	not detected.
   Power Analysis	not detected.
   Cell Line Authentication	not detected.

   Table 2: Resources

   Antibodies
   Sentences	Resources
   Expression of ACE-2: Cells diluted to 1 × 106 per microtiter plate well were incubated for 45 min at room temperature with a 1 in 100 dilution of a mouse IgG2a antibody to human/hamster ACE-2 (clone 171606 R&D Systems, Minneapolis, MN)	mouse IgG2a suggested: (Millipore Cat# 171606-100UG, RRID:AB_10690063)
   In parallel the membrane was probed with a mouse monoclonal IgG antibody to ß-actin (Sc-47778, Santa Cruz Biotechnology, Dallas, TX) as a loading control for 1 hr at room temperature.	ß-actin suggested: (RayBiotech Cat# 168-10656, RRID:AB_2885189) Sc-47778 suggested: None
   The loading control antibody was probed with HRP-conjugated goat anti-mouse secondary IgG (SAB3701047, Sigma, St. Louis, MO) for 1 hr at room temperature.	anti-mouse secondary IgG suggested: None
   An alkaline-phosphate-conjugated goat anti-mouse IgG antibody (Sigma-Aldrich, St Louis, MO) diluted to 1:1500 in 3%BAS-PBS was added at 60 μl/well for 1 hr at room temperature.	An alkaline-phosphate-conjugated goat anti-mouse IgG antibody suggested: None anti-mouse IgG suggested: None
   ACE binding inhibition assay: Sera were tested for inhibition of ACE binding to the RBD of S by the Anti-SARS-CoV-2 Neutralizing Antibody Titer Serologic Assay Kit from Acro Biosystems (Newark, DE) following the manufacturer’s instructions.	Anti-SARS-CoV-2 suggested: None
   A positive standard with a known concentration provided by the kit was used to extrapolated anti-S antibody concentrations in μg in the mouse sera.	anti-S suggested: None
   Experimental Models: Cell Lines
   Sentences	Resources
   Cell lines: HEK 293 cells and VeroE6 cells were grown in Dulbecco’s Modified Eagles medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and antibiotics in a 5% CO2 incubator.	HEK 293 suggested: None VeroE6 suggested: JCRB Cat# JCRB1819, RRID:CVCL_YQ49)
   66 BHK-21/WI-2 cells were plated into a 6 well plate, the next day cells were first transfected with 1 μg/well of the paT7 plasmids using polyethyleneimine (PEI) (Polysciences, Inc. Warrington, PA); 2 hours later, the cells were treated with a predetermined optimal amount of ΔG-GFP (G*ΔG-GFP) rVSV.	BHK-21/WI-2 suggested: RRID:CVCL_HB78)
   Experimental Models: Organisms/Strains
   Sentences	Resources
   Mice and mouse procedures: Female C57Bl/6 mice (6–8 weeks of age) were purchased from the Jackson Laboratories (Bar Harbor, ME).	C57Bl/6 suggested: None
   Recombinant DNA
   Sentences	Resources
   66 BHK-21/WI-2 cells were plated into a 6 well plate, the next day cells were first transfected with 1 μg/well of the paT7 plasmids using polyethyleneimine (PEI) (Polysciences, Inc. Warrington, PA); 2 hours later, the cells were treated with a predetermined optimal amount of ΔG-GFP (G*ΔG-GFP) rVSV.	paT7 suggested: None
   VNA assay with VSV-S: VSV-S vectors was initially titrated on BHK-21/WI-2 cells.	VSV-S suggested: None

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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:

   VNAs to the vaccine vectors will likely pose limitations to repeated booster immunization using the same Ad vector by not only reducing, but also, as we reported previously, modifying transgene product-specific immune responses.56 To avoid interference by Ad vector-specific VNAs, we developed a vaccine regimen composed of two serologically distinct E1-deleted AdC vectors. We selected AdC vectors as pre-existing VNAs to these chimpanzee viruses are rare in humans and individuals who have VNAs in general have low titers.44 We developed two serologically distinct AdC vectors to prevent blunting of booster immunizations by VNAs induced by the AdC vector used for priming. The two AdC vectors express the S protein of an isolate from Sweden, which is identical to that of the original Wuhan virus. We did not codon-optimize the S sequence, nor did we incorporate the K986P and V987P stabilizing mutations into the S2 sequence which both were used for the S gene expressed by the RNA vaccines,57 mutations of the S1/S2 furin cleavage site which, in addition to the stabilizing mutations, were incorporated into the S gene carried by the J&J COVID-19 vaccine,58 or AstraZeneca’s engineered leader sequence.59 Phase III trial results of the different Ad vector vaccines fail to indicate that any of these modifications have a major impact on vaccine efficacy. In our study, as expected, the magnitude of S protein-specific antibody responses after a single immunization increased with higher vaccine ...

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

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2. 

   Version published to 10.1101/2022.02.23.481620v1 on bioRxiv

   Feb 24, 2022