BNT162b vaccines are immunogenic and protect non-human primates against SARS-CoV-2

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1. 

   ScreenIT

   Dec 12, 2020

   SciScore for 10.1101/2020.12.11.421008: (What is this?)

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

   Table 1: Rigor

   Institutional Review Board Statement	IACUC: All procedures performed on these animals were in accordance with regulations and established guidelines and were reviewed and approved by an Institutional Animal Care and Use Committee or through an ethical review process.
   Randomization	not detected.
   Blinding	Images were interpreted by a board-certified veterinary radiologist blinded to treatment groups.
   Power Analysis	not detected.
   Sex as a biological variable	BNT162b1-immunised (n=6), BNT162b2-immunised (n=6), and age-matched saline-immunised (n=9) male rhesus macaques (control) were challenged with 1.05 × 106 plaque forming units of SARS-CoV-2 USA-WA1/2020 isolate, split equally between the intranasal (IN; …

   SciScore for 10.1101/2020.12.11.421008: (What is this?)

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

   Table 1: Rigor

   Institutional Review Board Statement	IACUC: All procedures performed on these animals were in accordance with regulations and established guidelines and were reviewed and approved by an Institutional Animal Care and Use Committee or through an ethical review process.
   Randomization	not detected.
   Blinding	Images were interpreted by a board-certified veterinary radiologist blinded to treatment groups.
   Power Analysis	not detected.
   Sex as a biological variable	BNT162b1-immunised (n=6), BNT162b2-immunised (n=6), and age-matched saline-immunised (n=9) male rhesus macaques (control) were challenged with 1.05 × 106 plaque forming units of SARS-CoV-2 USA-WA1/2020 isolate, split equally between the intranasal (IN; 0.25 mL) and intratracheal (IT; 0.25 mL) routes as previously described28.
   Cell Line Authentication	Contamination: Cell lines were tested for mycoplasma contamination after receipt, before expansion and cryopreservation.

   Table 2: Resources

   Antibodies
   Sentences	Resources
   Blotted proteins were detected with a monoclonal antibody that recognizes SARS-CoV-2 S1 (SinoBiological) and a secondary anti-rabbit horse radish peroxidase (HRP)-conjugated antibody (Sigma Aldrich).	anti-rabbit suggested: None
   Free binding sites were blocked and cells incubated with a rabbit monoclonal antibody that recognizes the SARS-CoV-2 S1 subunit (SinoBiological), an anti-rabbit IgG secondary antibody (Jackson ImmunoResearch), labelled lectin HPA (Thermo Fisher Scientific) and concanavalin A (Fisher Scientific GmbH).	anti-rabbit IgG suggested: None labelled lectin HPA suggested: None
   Binding analysis of captured murine IgG antibodies to S1-His or RBD-His (Sino Biological Inc.) was performed using a multi-cycle kinetic method with concentrations ranging from 25 to 400 nM or 1.5625 to 50 nM, respectively.	murine IgG suggested: (Novus Cat# NB120-5625, RRID:AB_2292017) S1-His suggested: None
   After incubation for 1 h at 37 °C, the inoculums was removed, and cells were washed with PBS before medium supplemented with anti-VSV-G antibody (clone 8G5F11, Kerafast Inc.) was added to neutralise residual input virus.	anti-VSV-G suggested: (Absolute Antibody Cat# Ab01401-2.0, RRID:AB_2883992)
   Experimental Models: Cell Lines
   Sentences	Resources
   Cell culture: Human embryonic kidney (HEK)293T and Vero 76 cells (both ATCC) were cultured in Dulbecco’s modified Eagle’s medium (DMEM) with GlutaMAX™ (Gibco) supplemented with 10% fetal bovine serum (FBS [Sigma-Aldrich]).	HEK)293T suggested: None Vero 76 suggested: None
   Transfection of HEK cells: HEK293T cells were transfected with 1 µg RiboJuice transfection reagent-mixed BNT162b1 RNA or BNT162b2 RNA or with the vaccine candidates BNT162b1 (LNP-formulated BNT162b1 RNA) or BNT162b2 (LNP-formulated BNT162b2 RNA) by incubation for 18 hours.	HEK suggested: None
   Western blot analysis of size fractions of the medium of BNT162b1 RNA transfected cells: Medium from cultured HEK293T cells were collected.	HEK293T suggested: NCBI_Iran Cat# C498, RRID:CVCL_0063)
   Vero-76 cells were seeded in 96-well plates.	Vero-76 suggested: None
   Serial dilutions of heat-inactivated sera were incubated with the reporter virus (2 x 104 plaque forming units [PFU] per well) to yield an approximately 10-30% infection rate of the Vero CCL81 monolayer for 1 h at 37 °C before inoculating Vero CCL81 cell monolayers (targeted to have 8,000 to 15,000 cells in the central field of each well at the time of seeding, one day before infection) in 96-well plates to allow accurate quantification of infected cells.	Vero CCL81 suggested: None
   The working virus stock was generated from two passages of the SARS-CoV-2 USA-WA1/2020 isolate (a 4th passage seed stock purchased from BEI Resources; NR-52281) in Vero E6 cells.	Vero E6 suggested: RRID:CVCL_XD71)
   Software and Algorithms
   Sentences	Resources
   Blots were developed with Clarity Western ECL Substrate (Bio-Rad) and imaged with a Fusion FX Imager (Vilber) using the Image Lab software version 6.0.	Image Lab suggested: (Image Lab Software, RRID:SCR_014210)
   Cells were acquired on a FACSCanto II flow cytometer (BD Biosciences) using BD FACSDiva software version 8.0.1 and analysed by FlowJo software version 10.6.2 (FlowJo LLC, BD Biosciences).	BD FACSDiva suggested: (BD FACSDiva Software, RRID:SCR_001456) FlowJo suggested: (FlowJo, RRID:SCR_008520)
   The resulting particle set was subjected to 2D classification in RELION 3.0.644.	RELION suggested: (RELION, RRID:SCR_016274)
   The microscope was operated for image acquisition using SerialEM software version 3.8.0 beta48.	SerialEM suggested: (SerialEM, RRID:SCR_017293)
   Cell counts were enumerated by nuclear stain (Hoechst 33342), and fluorescent virus-infected foci were detected 16-24 hours after inoculation with a Cytation 7 Cell Imaging Multi-Mode Reader (BioTek) with Gen5 Image Prime version 3.09.	Gen5 suggested: (Gen5, RRID:SCR_017317)
   Twelve to nineteen days prior to challenge, animals were moved from the NIRC, in New Iberia, LA, where they had been immunised, to the animal biosafety level 3 facility at SNPRC (in San Antonio, TX).	NIRC suggested: None SNPRC suggested: (Southwest National Primate Research Center, RRID:SCR_008292)
   PROC RANK and PROC GLM from SAS® 9.4 were used to calculate the p-values.	SAS® suggested: (SASqPCR, RRID:SCR_003056)
   All remaining analyses were two-tailed and carried out using GraphPad Prism 8.4.	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: We detected the following sentences addressing limitations in the study:

   Limitation and clearance of virus infections is promoted by the interplay of neutralising antibodies that eliminate infectious particles with CD8+ T cells that target intracellular virus reservoirs. CD8+ T cells may also reduce the influx of monocytes into infected lung tissue, which can be associated with undesirable IL-6 and TNF production and impaired antigen presentation35, 36. The responses elicited by the vaccine candidates reflect a pattern favourable for vaccine safety and efficacy, providing added reassurance for clinical translation37. The contributions of the individual immune effector systems to human protection from SARS-CoV-2 are not yet understood. Therefore, it appears prudent to develop COVID-19 vaccines that enlist concomitant cognate B cells, CD4+ T cells, and CD8+ T-cell responses. Both candidates protected 2-4 year old rhesus macaques from infectious SARS-CoV-2 challenge, with reduced detection of viral RNA in immunised animals compared to those that received saline. Immunisation with BNT162b2 provided particularly strong RT-qPCR evidence for lower respiratory tract protection, as demonstrated by the absence of detectable SARS-CoV-2 RNA in serial BAL samples obtained starting 3 days after challenge. The lack of serological response to the SARS-CoV-2 challenge in BNT162b1- or BNT162b2-immunised macaques, despite a neutralising response to challenge in control-immunised macaques, suggests suppression of infection by the vaccine candidates. Clinical signs of...

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   Results from TrialIdentifier: We found the following clinical trial numbers in your paper:
   

   Identifier	Status	Title
   NCT04380701	Recruiting	A Trial Investigating the Safety and Effects of Four BNT162 …
   NCT04368728	Active, not recruiting	Study to Describe the Safety, Tolerability, Immunogenicity, …

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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 pages 66, 67, 68, 69, 70 and 71. 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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   Read the original source
2. 

   Version published to 10.1101/2020.12.11.421008v1 on bioRxiv

   Dec 11, 2020