T Cell Predominant Response to AAV-Spike Protects hACE2 Mice from SARS-CoV-2 Pneumonia

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   ScreenIT

   Aug 20, 2021

   SciScore for 10.1101/2021.08.16.456441: (What is this?)

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

   Table 1: Rigor

   Ethics	IACUC: Mice: Murine experiments were executed in compliance with approval from the Animal Care and Use Committees of the University of Pennsylvania and University of Wisconsin at Madison. c57BL/10SnJ and B6.Cg-Tg(K18-ACE2)2Prlmn/J mice were purchased from the Jackson Laboratory. Euthanasia Agents: Toe pinch was performed to ensure mice were deeply anesthetized before mice were euthanized by cervical dislocation.
   Sex as a biological variable	not detected.
   Randomization	Mixed gender 2-5-month-old C57BL/10 mice or 6–9-week-old k18-hACE2 mice were randomly assigned to groups and anesthetized with isoflurane before receiving an intra gastrocnemius injection of either 50 ul AAV or 50 ul formulation …

   SciScore for 10.1101/2021.08.16.456441: (What is this?)

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

   Table 1: Rigor

   Ethics	IACUC: Mice: Murine experiments were executed in compliance with approval from the Animal Care and Use Committees of the University of Pennsylvania and University of Wisconsin at Madison. c57BL/10SnJ and B6.Cg-Tg(K18-ACE2)2Prlmn/J mice were purchased from the Jackson Laboratory. Euthanasia Agents: Toe pinch was performed to ensure mice were deeply anesthetized before mice were euthanized by cervical dislocation.
   Sex as a biological variable	not detected.
   Randomization	Mixed gender 2-5-month-old C57BL/10 mice or 6–9-week-old k18-hACE2 mice were randomly assigned to groups and anesthetized with isoflurane before receiving an intra gastrocnemius injection of either 50 ul AAV or 50 ul formulation buffer using a custom 100 uL Hamilton syringe with a 32-gauge needle (475-41182, Lot 81008).
   Blinding	Additionally, k18-hACE2 mice were ear tagged as to keep the team performing the challenge experiments blinded to the animal’s treatment status. c57BL/10 muscle procurement, sectioning, and storage for immunohistochemistry: At pre-determined timepoints post-vaccination, mice were anesthetized with 4% isoflurane in an anesthesia chamber before being transferred to a nose cone where they were maintained on 4% isoflurane in 100% O2 at a flow rate of 1 L/minute.
   Power Analysis	not detected.
   Cell Line Authentication	not detected.

   Table 2: Resources

   Antibodies
   Sentences	Resources
   The membrane was then washed 3×15 minutes in PBST and then incubated in secondary antibody solution (PBST + 5% BSA + Goat a-Rabbit-HRP 1:5000) (Abcam, ab205718) for one hour at room temperature.	a-Rabbit-HRP suggested: None
   Also included in this mixture to neutralize any potential VSV-G carryover virus was 1E9F9, a mouse anti-VSV Indiana G, at a concentration of 600 ng/ml (Absolute Antibody, Ab01402-2.0).	anti-VSV suggested: None
   Plates were then incubated in ddH2O for 2 minutes and then washed 5× with PBS before incubating with biotinylated anti-cytokine antibody at the manufacture’s recommended concentration in PBS + 1% BSA for 2 hr at RT.	anti-cytokine suggested: None
   Plates were then incubated in ddH2O for 2 minutes and then washed 5× with PBS before incubating with ALP-conjugated anti-IFNγ antibody at the manufacture’s recommended concentration in PBS + 1% BSA for 2 hr at RT.	anti-IFNγ suggested: None
   Experimental Models: Cell Lines
   Sentences	Resources
   AAV6/9 vector generation: AAV6 and AAV9 were individually generated and purified by the University of Pennsylvania preclinical vector core using the triple transfection method in HEK293 cells as previously described12.	HEK293 suggested: None
   Transfection and Immunocytochemistry of C2C12 cells: 3.0E4 C2C12 cells were plated into each well of an 8 well chamber slide (Millipore, PEZGS0816) and cultured for 24 hours in growth media (DMEM high glucose supplemented with 10% FBS, 1× anti-anti, 1× GlutaMAX, 1× NEAA).	C2C12 suggested: None
   Neutralization Assay: Production of VSV pseudotyped with SARS-CoV-2 S: 293T cells plated 24 hours previously at 5 × 106 cells per 10 cm dish were transfected using calcium phosphate with 35 μg of pCG1 SARS-CoV-2 S D614G delta18 expression plasmid encoding a codon optimized SARS-CoV2 S gene with an 18-residue truncation in the cytoplasmic tail (kindly provided by Stefan Pohlmann).	293T suggested: None
   Vero E6 cells stably expressing TMPRSS2 were seeded in 100 μl at 2.5×104 cells/well in a 96 well collagen coated plate.	Vero E6 suggested: None
   The serum-virus mixture was then used to replace the media on VeroE6 TMPRSS2 cells.	VeroE6 TMPRSS2 suggested: JCRB Cat# JCRB1819, RRID:CVCL_YQ49)
   Experimental Models: Organisms/Strains
   Sentences	Resources
   Mice: Murine experiments were executed in compliance with approval from the Animal Care and Use Committees of the University of Pennsylvania and University of Wisconsin at Madison. c57BL/10SnJ and B6.Cg-Tg(K18-ACE2)2Prlmn/J mice were purchased from the Jackson Laboratory.	B6.Cg-Tg(K18-ACE2)2Prlmn/J suggested: RRID:IMSR_JAX:034860)
   Mixed gender 2-5-month-old C57BL/10 mice or 6–9-week-old k18-hACE2 mice were randomly assigned to groups and anesthetized with isoflurane before receiving an intra gastrocnemius injection of either 50 ul AAV or 50 ul formulation buffer using a custom 100 uL Hamilton syringe with a 32-gauge needle (475-41182, Lot 81008).	C57BL/10 suggested: None
   Recombinant DNA
   Sentences	Resources
   M8/M8B were cloned into the AAV transfer plasmid pZac2.1 along with (or flanked by) a CMV promoter and SV40 PolyA sequence.	pZac2.1 suggested: None
   Western Blot of transfected HEK293: M8/M8B-pZac2.1 plasmid transfected HEK293 cell pellets were lysed in RIPA lysis buffer (Santa Cruz Biotechnology, sc-24948) supplemented with protease inhibitor cocktail (Roche, 39802300).	M8/M8B-pZac2.1 suggested: None
   Neutralization Assay: Production of VSV pseudotyped with SARS-CoV-2 S: 293T cells plated 24 hours previously at 5 × 106 cells per 10 cm dish were transfected using calcium phosphate with 35 μg of pCG1 SARS-CoV-2 S D614G delta18 expression plasmid encoding a codon optimized SARS-CoV2 S gene with an 18-residue truncation in the cytoplasmic tail (kindly provided by Stefan Pohlmann).	pCG1 SARS-CoV-2 suggested: None
   Software and Algorithms
   Sentences	Resources
   Red blood cells were lysed with ACK lysis buffer (Quality Biological Inc.,	Quality Biological 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: 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: Please consider improving the rainbow (“jet”) colormap(s) used on page 3. 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.
   • 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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   Version published to 10.1101/2021.08.16.456441v1 on bioRxiv

   Aug 17, 2021