Immunisation of ferrets and mice with recombinant SARS-CoV-2 spike protein formulated with Advax-SM adjuvant protects against COVID-19 infection

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1. Version published to 10.1016/j.vaccine.2021.07.087

   Sep 1, 2021
2. 

   ScreenIT

   Jul 7, 2021

   SciScore for 10.1101/2021.07.03.451026: (What is this?)

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

   Table 1: Rigor

   Ethics	IACUC: The protocol was approved by the Animal Welfare Committee of Flinders University and carried out in strict accordance with the Australian Code of Practice for the Care and Use of Animals for Scientific Purposes (2013)
   Sex as a biological variable	Mouse Immunisation Protocol: Female, BALB/c and C57BL/6 (BL6) mice (6-10 weeks old) were supplied by the central animal facility of Flinders University.
   Randomization	not detected.
   Blinding	Lung lesions were scored by a board-certified veterinary pathologist blinded to the study groups as follows: Alveolar (ALV) score: 1 = focal, 2 = multifocal, 3 = multifocal to coalescing, 4 = majority of section infiltrated by leukocytes; Perivascular cuffing …

   SciScore for 10.1101/2021.07.03.451026: (What is this?)

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

   Table 1: Rigor

   Ethics	IACUC: The protocol was approved by the Animal Welfare Committee of Flinders University and carried out in strict accordance with the Australian Code of Practice for the Care and Use of Animals for Scientific Purposes (2013)
   Sex as a biological variable	Mouse Immunisation Protocol: Female, BALB/c and C57BL/6 (BL6) mice (6-10 weeks old) were supplied by the central animal facility of Flinders University.
   Randomization	not detected.
   Blinding	Lung lesions were scored by a board-certified veterinary pathologist blinded to the study groups as follows: Alveolar (ALV) score: 1 = focal, 2 = multifocal, 3 = multifocal to coalescing, 4 = majority of section infiltrated by leukocytes; Perivascular cuffing (PVC) score: 1 = 1 layer of leukocytes surrounding blood vessel, 2 = 2-5 layers, 3 = 6 – 10 layers, 4 = greater than 10 cells thick; Interstitial Pneumonia (IP) score: 1 = alveolar septa thickened by 1 leukocyte, 2 = 2 leukocytes thick, 3 = 3 leukocytes, 4 = 4 leukocytes.
   Power Analysis	not detected.
   Cell Line Authentication	not detected.

   Table 2: Resources

   Antibodies
   Sentences	Resources
   After blocking, 100 μl of diluted serum samples were added followed by biotinylated anti-mouse IgG (Sigma-Aldrich), IgG1, IgG2a/c, IgG2b, IgG3 and IgM antibodies (all from Abcam) with horseradish peroxidase(HRP)-conjugated Streptavidin (BD Biosciences) for 1 hour (h).	anti-mouse IgG suggested: None IgG1 suggested: None IgG2b , IgG3 suggested: None IgM suggested: None
   Briefly, single-cell suspensions were prepared from spleens of mice and plated in Millipore MultiScreen-HA 96-well filter plates (Millipore) pre-coated with anti-mouse IL-2, IL-4, IL-17 or IFN-γ antibodies overnight at 4°C and blocked by RPMI-1640 containing 10% FBS.	IL-17 suggested: None
   Wells were washed and incubated with biotinylated labelled anti-mouse IL-2, IL-4, IL-17 or IFN-γ antibody at room temperature (RT).	anti-mouse IL-2 suggested: None IL-4 suggested: None IFN-γ suggested: None
   The following day, plates were washed 5 times with 0.05% PBST, and IgG antibodies were detected using horseradish peroxidase (HRP)-conjugated goat anti-ferret polyclonal IgG detection antibody (Abcam, Cambridge, UK) at a 1:4,000 dilution for a 90 min incubation at 37°C.	IgG suggested: None anti-ferret polyclonal IgG suggested: None
   The lung sections were blocked with 5% horse serum in PBS for 1 h at RT, incubated with SARS-CoV-2 Nucleoprotein polyclonal antibody at 1:500 dilution (Invitrogen, Carlsbad, CA, USA) overnight at 4°C, and then incubated with biotinylated goat-antibody Rabbit IgG H&L (Abcam, Waltham, MA, USA) at 1:1000 dilution for 1 h at RT.	Rabbit IgG suggested: None
   Experimental Models: Cell Lines
   Sentences	Resources
   Recombinant baculovirus was expanded in Sf9 cells to P3 and then used for infection of High Five cells for protein expression.	Sf9 suggested: CLS Cat# 604328/p700_Sf9, RRID:CVCL_0549)
   Lentiviral particles pseudotyped with SARS-COV2 Spike envelope were produced by co-transfecting HEK 293T cells with a GFP encoding 3rd generation lentiviral plasmid HRSIN-CSGW (a gift from Camille Frecha [26]), psPAX2 and plasmid expressing codon optimized but C-terminal truncated SARS COV2 S protein (pCG1-SARS-2-S Delta18 [27], herein Spike Delta18) courtesy of Professor Stefan Polhman using polyethylenimine as described previously [25].	HEK 293T suggested: None
   Virus-serum was then added onto ACE2-HEK 293T cells seeded at 2,500 cells per well in a 384-well tissue culture plate a day before.	293T suggested: None
   Determination of Virus Titres in Ferret Nasal Washes: Nasal washes were titrated in quadruplicates in Vero E6 cells.	Vero E6 suggested: RRID:CVCL_XD71)
   Experimental Models: Organisms/Strains
   Sentences	Resources
   Mouse Immunisation Protocol: Female, BALB/c and C57BL/6 (BL6) mice (6-10 weeks old) were supplied by the central animal facility of Flinders University.	BALB/c suggested: None C57BL/6 suggested: None
   Recombinant DNA
   Sentences	Resources
   The insect cell codon-optimised expression cassette was cloned into pFASTBac1, and baculovirus was generated following standard Bac-to-Bac	pFASTBac1 suggested: RRID:Addgene_1956)
   The hACE2 open reading frame (Addgene# 1786) was cloned into a 3rd generation lentiviral expression vector pRRLSIN.cPPT.PGK-GFP.WPRE (Addgene# 122053), and clonal HEK 293T cells stably expressing ACE2 were generated by lentiviral transductions as described previously [25], followed by single cell sorting into 50% HEK 293T conditioned media (media conditioned from 50% confluent HEK 293T cultures)	pRRLSIN.cPPT.PGK-GFP.WPRE suggested: RRID:Addgene_12252)
   Lentiviral particles pseudotyped with SARS-COV2 Spike envelope were produced by co-transfecting HEK 293T cells with a GFP encoding 3rd generation lentiviral plasmid HRSIN-CSGW (a gift from Camille Frecha [26]), psPAX2 and plasmid expressing codon optimized but C-terminal truncated SARS COV2 S protein (pCG1-SARS-2-S Delta18 [27], herein Spike Delta18) courtesy of Professor Stefan Polhman using polyethylenimine as described previously [25].	HRSIN-CSGW suggested: None psPAX2 suggested: RRID:Addgene_12260) pCG1-SARS-2-S suggested: None
   Software and Algorithms
   Sentences	Resources
   The docked model was optimized using AMBER99SB-ILDN force field in Gromacs2020 (https://www.gromacs.org/).	https://www.gromacs.org/ suggested: (GROMACS, RRID:SCR_014565)
   Statistical analysis: GraphPad Prism 8.3.1 for Windows was used for drawing graphs and statistical analysis (GraphPad Software, San Diego, CA, USA).	GraphPad Prism suggested: (GraphPad Prism, RRID:SCR_002798) GraphPad 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:

   A limitation of the current study was that ferrets do not exhibit weight loss or other signs of SARS-CoV-2 clinical infection [51], with no animal models fully reproducing the features of severe SARS-CoV-2 clinical infection in humans. Ongoing studies are testing our Covax-19 vaccine in other species including hamsters and non-human primates to see whether the effects of the vaccine on inhibition of nasal virus replication extends to other species. The current study also only assessed protection soon after immunisation and it will also be important to assess the durability of vaccine protection. Conclusion: The COVID-19 pandemic represents a significant evolving global health crisis. The key to overcoming the pandemic lies in the development of an effective vaccine against SARS-CoV-2 that ideally prevents transmission as well as serious disease. Recombinant protein-based approaches to COVID-19 offer benefits over other technologies including a 40-year record of safety and effectiveness including in very young infants, together with reliable large scale manufacture and high stability under typical refrigerated conditions [52]. By contrast, other available technologies, including nucleic acid and adenoviral vector platforms have a high level of reactogenicity and pose cold chain and other distribution challenges [53, 54]. This study showed that an Advax-SM adjuvanted rSp vaccine (Covax-19 vaccine) when administered as two sequential intramuscular doses several weeks apart induc...

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   Results from TrialIdentifier: No clinical trial numbers were referenced.

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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: 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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3. 

   Version published to 10.1101/2021.07.03.451026v1 on bioRxiv

   Jul 5, 2021