A prefusion SARS-CoV-2 spike RNA vaccine is highly immunogenic and prevents lung infection in non-human primates

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1. 

   ScreenIT

   Mar 1, 2021

   SciScore for 10.1101/2020.09.08.280818: (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	not detected.
   Power Analysis	not detected.
   Sex as a biological variable	BNT162b2-immunised (n=6) and age-matched saline control-immunised (n=3) 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 described26.
   Cell Line Authentication	Contami…

   SciScore for 10.1101/2020.09.08.280818: (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	not detected.
   Power Analysis	not detected.
   Sex as a biological variable	BNT162b2-immunised (n=6) and age-matched saline control-immunised (n=3) 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 described26.
   Cell Line Authentication	Contamination: Cell lines were tested for mycoplasma contamination after receipt, before expansion and cryopreservation.

   Table 2: Resources

   Antibodies
   Sentences	Resources
   Avi-tagged ACE2-PD was captured on streptavidin coated sensors and B38 antibody was captured on sensors coated with protein G.	B38 suggested: None
   Bound rhesus macaque or human anti-S1 antibodies present in the serum were detected with a fluorescently labelled goat anti-human polyclonal secondary antibody (Jackson ImmunoResearch).	anti-S1 suggested: None anti-human polyclonal secondary suggested: None
   Binding analysis of captured murine IgG antibodies to S1-His or RBD-His (Sino Biological) 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 hour at 37 °C, the inoculum 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
   Transfected HEK293T/17 cells were stained with Fixable Viability Dye (eBioscience).	HEK293T/17 suggested: ATCC Cat# CRL-11268, RRID:CVCL_1926)
   The TwinStrep-tagged P2 S was expressed in Expi293 cells.	Expi293 suggested: RRID:CVCL_D615)
   Serial dilutions of mouse serum samples were prepared and pre-incubated for 10 minutes at room temperature with VSV/SARS-CoV-2 pseudovirus suspension (4.8 × 103 infectious units [IU]/mL) before transferring the mix to Vero 76 cells.	Vero 76 suggested: IZSLER Cat# BS CL 101, RRID:CVCL_0603)
   Inoculated Vero-76 cells were incubated for 20 hours at 37 °C.	Vero-76 suggested: None
   Serial dilutions of heat-inactivated sera were incubated with the reporter virus (2 × 104 PFU per well) to yield approximately a 10-30% infection rate of the Vero CCL81 monolayer for 1 hour 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
   Software and Algorithms
   Sentences	Resources
   RNA was purified using magnetic particles36, integrity assessed by microfluidic capillary electrophoresis (Agilent Fragment Analyser), and concentration, pH, osmolality, endotoxin level and bioburden determined.	Agilent Fragment Analyser suggested: (OMICtools, RRID:SCR_002250)
   FlowJo LLC, BD Biosciences)	FlowJo suggested: (FlowJo, RRID:SCR_008520)
   All subsequent processing was performed in RELION 3.1-beta39.	RELION suggested: (RELION, RRID:SCR_016274)
   Spot counts denoted too numerous to count by the software were set to 1,500.	Spot suggested: (Spot, RRID:SCR_018915)
   Mouse cells were acquired on a BD Symphony A3 or BD Celesta (B-cell subtyping) flow cytometer (BD Bioscience) using BD FACSDiva software version 9.1 or 8.0.1.1, respectively, and analysed with FlowJo 10.6 (FlowJo LLC, BD Biosciences).	BD FACSDiva suggested: (BD FACSDiva Software, RRID:SCR_001456)
   Cell counts were enumerated by nuclear stain (Hoechst 33342) and fluorescent virally 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)
   Titers were calculated in GraphPad Prism version 8.4.2 by generating a 4-parameter (4PL) logistical fit of the percent neutralisation at each serial serum dilution.	GraphPad Prism suggested: (GraphPad Prism, RRID:SCR_002798)
   PROC RANK and PROC GLM from SAS® 9.4 were used to calculate the p-values.	SAS® suggested: (SASqPCR, RRID:SCR_003056)

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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).

   ---

   Results from LimitationRecognizer: We detected the following sentences addressing limitations in the study:

   Limitation and clearance of virus infection is promoted by the interplay of neutralising antibodies with CD8+ T cells that eliminate 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 presentation28,29. The contributions of the immune effector systems to human protection from SARS-CoV-2 is not yet understood. Therefore, it appears prudent to develop COVID-19 vaccines that enlist concomitant cognate B cell, CD4+ T cell, and CD8+ T-cell responses. The immunogenicity of BNT162b2 in rhesus macaques paralleled its immunogenicity in mice. Seven days after Dose 2 of 100 μg, the neutralising GMT reached 18-times that of a human SARS-CoV-2 convalescent serum panel and remained 3.3-times higher than this benchmark five weeks after the last immunisation. The strongly TH1-biased CD4+ T-cell response and IFNγ+ CD8+ T-cell response to BNT162b2 is a pattern favoured for vaccine safety and efficacy, providing added reassurance for clinical translation30. BNT162b2 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 and with no evidence of clinical exacerbation. Strong RT-qPCR evidence for lower respiratory tract protection was demonstrated by the absence of detectable SARS-CoV-2 RNA in serial BAL samples obtained starting 3 days ...

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

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

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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.

   ---

   Read the original source
2. 

   ScreenIT

   Sep 9, 2020

   SciScore for 10.1101/2020.09.08.280818: (What is this?)

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

   Table 1: Rigor

   Institutional Review Board Statement

   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

   Female BALB/c mice (Janvier; 8-12 weeks) and were randomly allocated to groups.

   Blinding

   All personnel performing clinical, radiological, histopathological, or RT-qPCR evaluations were blinded to the group assignments of the macaques.

   Power Analysis

   not detected.

   Sex as a biological variable

   To assess BNT162b2-mediated protection in non-human primates, groups of six male, 2-4 year old rhesus macaques were immunised IM with 30 or 100 µg …

   SciScore for 10.1101/2020.09.08.280818: (What is this?)

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

   Table 1: Rigor

   Institutional Review Board Statement

   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

   Female BALB/c mice (Janvier; 8-12 weeks) and were randomly allocated to groups.

   Blinding

   All personnel performing clinical, radiological, histopathological, or RT-qPCR evaluations were blinded to the group assignments of the macaques.

   Power Analysis

   not detected.

   Sex as a biological variable

   To assess BNT162b2-mediated protection in non-human primates, groups of six male, 2-4 year old rhesus macaques were immunised IM with 30 or 100 µg of BNT162b2 or saline control on Days 0 and 21.

   Cell Line Authentication

   Cell lines were tested for mycoplasma contamination after receipt, before expansion and cryopreservation.

   Table 2: Resources

   Antibodies
   Sentences	Resources
   Avi-tagged ACE2-PD was captured on streptavidin coated sensors and B38 antibody was captured on sensors coated with protein G.	B38 suggested: None
   Bound rhesus macaque or human anti-S1 antibodies present in the serum were detected with a fluorescently labelled goat anti-human polyclonal secondary antibody (Jackson ImmunoResearch).	anti-S1 suggested: None anti-human polyclonal secondary suggested: None
   Binding analysis of captured murine IgG antibodies to S1-His or RBD-His (Sino Biological) 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 hour at 37 °C, the inoculum 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: None
   Experimental Models: Cell Lines
   Sentences	Resources
   Transfected HEK293T/17 cells were stained with Fixable Viability Dye (eBioscience).	HEK293T/17 suggested: ATCC Cat# CRL-11268, RRID:CVCL_1926)
   The TwinStrep-tagged P2 S was expressed in Expi293 cells.	Expi293 suggested: RRID:CVCL_D615)
   Serial dilutions of mouse serum samples were prepared and pre-incubated for 10 minutes at room temperature with VSV/SARS-CoV-2 pseudovirus suspension (4.8 × 103 infectious units [IU]/mL) before transferring the mix to Vero 76 cells.	Vero 76 suggested: ECACC Cat# 85020205, RRID:CVCL_0603)
   Inoculated Vero-76 cells were incubated for 20 hours at 37 °C.	Vero-76 suggested: None
   Serial dilutions Page 17 of heat-inactivated sera were incubated with the reporter virus (2 x 104 PFU per well) to yield approximately a 10-30% infection rate of the Vero CCL81 monolayer for 1 hour 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
   Experimental Models: Organisms/Strains
   Sentences	Resources
   To characterise BNT162b2-elicited B- and T-cell responses, BALB/c mice were immunized intramuscularly (IM) once with 0.2, 1, or 5 µg BNT162b2 or received a buffer control.	BALB/c suggested: None
   Software and Algorithms
   Sentences	Resources
   Immunisations for the non-human primate (NHP) study were performed at the University of Louisiana at Lafayette-New Iberia Research Center (NIRC), which is accredited by the Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC, Animal Assurance #: 000452).	NIRC suggested: None
   RNA was purified using magnetic particles36, integrity assessed by microfluidic capillary electrophoresis (Agilent Fragment Analyser), and concentration, pH, osmolality, endotoxin level and bioburden determined.	Agilent Fragment Analyser suggested: (OMICtools, RRID:SCR_002250)
   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	BD FACSDiva suggested: (BD FACSDiva Software, RRID:SCR_001456)
   FlowJo LLC, BD Biosciences)	FlowJo suggested: (FlowJo, RRID:SCR_008520)
   All subsequent processing was performed in RELION 3.1-beta39.	RELION suggested: (RELION, RRID:SCR_016274)
   Spot counts denoted too numerous to count by the software were set to 1,500.	Spot suggested: (Spot, RRID:SCR_018915)
   Cell counts were enumerated by nuclear stain (Hoechst 33342) and fluorescent virally 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)
   PROC RANK and PROC GLM from SAS® 9.4 were used to calculate the pvalues.	SAS® suggested: (SASqPCR, RRID:SCR_003056)
   All remaining analyses were carried out using GraphPad Prism 8.4.	GraphPad Prism suggested: (GraphPad Prism, RRID:SCR_002798)

   ---

   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).

   ---

   Results from LimitationRecognizer: We detected the following sentences addressing limitations in the study:

   Limitation and clearance of virus infection is promoted by the interplay of neutralising antibodies with CD8+ T cells that eliminate 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 presentation28,29. The contributions of the immune effector systems to human protection from Page 7 SARS-CoV-2 is not yet understood. Therefore, it appears prudent to develop COVID-19 vaccines that enlist concomitant cognate B cell, CD4+ T cell, and CD8+ T-cell responses. The immunogenicity of BNT162b2 in rhesus macaques paralleled its immunogenicity in mice. Seven days after Dose 2 of 100 µg, the neutralising GMT reached 18-times that of a human SARS-CoV-2 convalescent serum panel and remained 3.3-times higher than this benchmark five weeks after the last immunisation. The strongly TH1-biased CD4+ T-cell response and IFNγ+ CD8+ T-cell response to BNT162b2 is a pattern favoured for vaccine safety and efficacy, providing added reassurance for clinical translation30. BNT162b2 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 and with no evidence of clinical exacerbation. Strong RT-qPCR evidence for lower respiratory tract protection was demonstrated by the absence of detectable SARS-CoV-2 RNA in serial BAL samples obtained starting ...

   ---

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

   Identifier	Status	Title
   NCT04368728	Recruiting	Study to Describe the Safety, Tolerability, Immunogenicity, ...
   NCT04380701	Recruiting	A Trial Investigating the Safety and Effects of Four BNT162 ...

   ---

   Results from Barzooka: We did not find any issues relating to the usage of bar graphs.

   ---

   Results from JetFighter: We did not find any issues relating to colormaps.

   ---

   About SciScore

   SciScore is an automated tool that is designed to assist expert reviewers by finding and presenting formulaic information scattered throughout a paper in a standard, easy to digest format. SciScore checks for the presence and correctness of RRIDs (research resource identifiers), and for rigor criteria such as sex and investigator blinding. For details on the theoretical underpinning of rigor criteria and the tools shown here, including references cited, please follow this link.

   Read the original source
3. 

   Version published to 10.1101/2020.09.08.280818v1 on bioRxiv

   Sep 8, 2020