A self-amplifying mRNA COVID-19 vaccine drives potent and broad immune responses at low doses that protects non-human primates against SARS-CoV-2

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   ScreenIT

   Nov 19, 2021

   SciScore for 10.1101/2021.11.08.467773: (What is this?)

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

   Table 1: Rigor

   Ethics	Field Sample Permit: Mouse studies: Mouse studies were conducted at Murigenics (Vallejo, CA) under IACUC approved protocols. IACUC: Mouse studies: Mouse studies were conducted at Murigenics (Vallejo, CA) under IACUC approved protocols.
   Sex as a biological variable	Female Balb/c mice (Envigo), 6–8 weeks old were used for all studies.
   Randomization	not detected.
   Blinding	not detected.
   Power Analysis	not detected.
   Cell Line Authentication	not detected.

   Table 2: Resources

   Antibodies
   Sentences	Resources
   Membranes were blocked for 1h in 5% skim milk in TBST and then probed with an anti-S2 mouse monoclonal antibody (GeneTex) at a 1:1000 dilution for 2h-overnight.	anti-S2 suggested: None
   Membranes were washed (0.05% …

   SciScore for 10.1101/2021.11.08.467773: (What is this?)

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

   Table 1: Rigor

   Ethics	Field Sample Permit: Mouse studies: Mouse studies were conducted at Murigenics (Vallejo, CA) under IACUC approved protocols. IACUC: Mouse studies: Mouse studies were conducted at Murigenics (Vallejo, CA) under IACUC approved protocols.
   Sex as a biological variable	Female Balb/c mice (Envigo), 6–8 weeks old were used for all studies.
   Randomization	not detected.
   Blinding	not detected.
   Power Analysis	not detected.
   Cell Line Authentication	not detected.

   Table 2: Resources

   Antibodies
   Sentences	Resources
   Membranes were blocked for 1h in 5% skim milk in TBST and then probed with an anti-S2 mouse monoclonal antibody (GeneTex) at a 1:1000 dilution for 2h-overnight.	anti-S2 suggested: None
   Membranes were washed (0.05% Tween 20 in 1X TBS) and then probed with a Rabbit anti-Mouse HRP antibody (Bethyl labs) for 1h before washing and detection with a SuperSignal West Femto Maximum Sensitivity Substrate (Pierce).	anti-Mouse HRP suggested: None
   A parallel blot using a mouse anti-actin antibody (Thermo Fisher) was used to ensure equivalent protein amounts per well.	anti-actin suggested: None
   Extracellular staining was performed in FACS buffer (PBS + 2% FBS + 2mM EDTA) with the following antibodies: CD4 (GK1.5, Biolegend), CD8 (53-6.7, BD).	CD4 suggested: None CD8 suggested: None
   Intracellular staining was then performed in permeabilization buffer with the following antibodies: IFNγ (XMG21.2, Invitrogen), TNFα (MP6-XT22, eBiosciences), IL2 (JES6-5H4, eBiosciences), IL4 (11B11, Biolegend), IL10 (JES5-16E3, Biolegend).	IL2 suggested: None IL4 suggested: None IL10 suggested: None JES5-16E3 suggested: None
   The intensity of the light being emitted is inversely proportional to the amount of anti-SARS-CoV-2 neutralizing Spike antibodies bound to the VSVΔG – Spike ΔCT particles.	anti-SARS-CoV-2 neutralizing Spike suggested: (Creative Diagnostics Cat# CABT-CS064, RRID:AB_2891088)
   Plates were incubated with anti-nucleocapsid protein primary antibody cocktail (clones HM1056 and HM1057) (EastCoast Bio, North Berwick, ME) for 60 minutes at 37°C (Battelle Memorial Institute, Patent Number 63/041,551 Pending, 2020).	anti-nucleocapsid protein suggested: None
   The plates were washed and the secondary antibody (goat anti-mouse IgG Horse Radish Peroxidase (HRP) conjugate; Fitzgerald, North Acton, MA) was added to the wells, and the plates were incubated for 60 minutes at 37°C1.	anti-mouse IgG suggested: None
   Wells were washed and incubated with 25 μL of 1 μg/mL SULFO-TAG labeled anti-species antibody (MSD), diluted in DPBS + 1% BSA (Sigma-Aldrich, St. Louis, MO), for 1 hour at room temperature on an orbital shaker.	MSD suggested: None
   Experimental Models: Cell Lines
   Sentences	Resources
   Western analysis: HEK293F cells seeded at 5e5 cells/mL were infected with an MOI of 1 IU/cell.	HEK293F suggested: RRID:CVCL_6642)
   The VSVΔG virus was transduced in HEK293T cells previously transfected with the spike glycoprotein of the SARS-CoV-2 coronavirus (Wuhan strain) for which the last 19 amino acids of the cytoplasmic tail were removed (ΔCT).	HEK293T suggested: None
   After the incubation of the serum/plasma-pseudotyped virus complex, the serum/plasma-pseudotyped virus complex was transferred to the plate containing Vero E6 cells (ATCC).	Vero E6 suggested: RRID:CVCL_XD71)
   Experimental Models: Organisms/Strains
   Sentences	Resources
   Female Balb/c mice (Envigo), 6–8 weeks old were used for all studies.	Balb/c suggested: RRID:IMSR_ORNL:BALB/cRl)
   Recombinant DNA
   Sentences	Resources
   The sequence of a E1 (578-3404 bp)/E3 deleted virus (2,125-31,825 bp) was assembled into pUC19 from VR-594-derived and synthetic (SGI-DNA) fragments.	pUC19 suggested: RRID:Addgene_50005)
   The pA68-E4d-Spike plasmids were linearized, purified using a Nucleospin kit (Machery-Nagel) and transfected into 2 mL of 293F cells (0.5 mL/mL) using TransIT-Lenti (Mirus bio).	pA68-E4d-Spike suggested: None
   Spike sequences were PCR amplified and cloned into PacI/BstBI sites of a pUC02-VEE vector.	pUC02-VEE suggested: None
   Software and Algorithms
   Sentences	Resources
   Vector generation: The ChAd68 nucleotide sequence was based on the wild-type sequence obtained by MiSeq (Ilumina sequencing) of virus obtained from the ATCC (VR-594).	MiSeq suggested: (A5-miseq, RRID:SCR_012148)
   Analysis of flow cytometry data was performed using FlowJo software.	FlowJo suggested: (FlowJo, RRID:SCR_008520)
   Data processing was performed using the R programming language and graphed using GraphPad Prism.	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: We found the following clinical trial numbers in your paper:
   

   Identifier	Status	Title
   NCT03639714	Active, not recruiting	A Study of a Personalized Neoantigen Cancer Vaccine
   NCT03953235	Recruiting	A Study of a Personalized Cancer Vaccine Targeting Shared Ne…
   NCT04776317	Recruiting	Chimpanzee Adenovirus and Self-Amplifying mRNA Prime-Boost P…

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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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   Read the original source
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   Version published to 10.1101/2021.11.08.467773 on bioRxiv

   Nov 9, 2021