Impact of glycosylation on a broad-spectrum vaccine against SARS-CoV-2

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1. 

   Version published to 10.1101/2021.05.25.445523v2 on bioRxiv

   Aug 26, 2021
2. 

   ScreenIT

   May 27, 2021

   SciScore for 10.1101/2021.05.25.445523: (What is this?)

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

   Table 1: Rigor

   Ethics	Euthanasia Agents: On day 3 after challenge, hamsters were euthanized by carbon dioxide. IACUC: All animal experiments were evaluated and approved by the Institutional Animal Care and Use Committee of Academia Sinica.
   Sex as a biological variable	Glycans categorization follows previous studies according to the composition detected and visualized by Graphpad Prism 9.0.0.13 Animal vaccination and virus challenge: For mice vaccination study, female 6- to 8-week-old BALB/c mice (n= 5) were immunized intramuscularly with 20ug purified Sfg or Smg proteins mixed with aluminum hydroxide (20ug) at day 0, day 14 and day 56.
   Randomization	not detected.
   Blinding	not detected.
   Power Analysis	not detected.
   Cell …

   SciScore for 10.1101/2021.05.25.445523: (What is this?)

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

   Table 1: Rigor

   Ethics	Euthanasia Agents: On day 3 after challenge, hamsters were euthanized by carbon dioxide. IACUC: All animal experiments were evaluated and approved by the Institutional Animal Care and Use Committee of Academia Sinica.
   Sex as a biological variable	Glycans categorization follows previous studies according to the composition detected and visualized by Graphpad Prism 9.0.0.13 Animal vaccination and virus challenge: For mice vaccination study, female 6- to 8-week-old BALB/c mice (n= 5) were immunized intramuscularly with 20ug purified Sfg or Smg proteins mixed with aluminum hydroxide (20ug) at day 0, day 14 and day 56.
   Randomization	not detected.
   Blinding	not detected.
   Power Analysis	not detected.
   Cell Line Authentication	not detected.

   Table 2: Resources

   Antibodies
   Sentences	Resources
   The probe radius in the FreeSASA program was set to 7.2Å to approximate the average size of the hypervariable loops in the CDR of an antibody9.	antibody9 suggested: None
   A monoclonal antibody specific for HIV-1 Gag p24 was pre-coated onto a microplate.	HIV-1 suggested: None
   Standards and samples are dispensing into appropriately labeled duplicate wells and any HIV-1 Gag p24 present is bound by the immobilized antibody.	HIV-1 Gag suggested: None
   The purity was monitored by using SDS-PAGE and the proteins were confirmed using Western blot with anti-(his)6 antibodies (Qiagen) or specific anti-SARS-CoV-2 S protein antibody and horseradish peroxidase-conjugated secondary antibody (PerkinElmer).	anti- suggested: None his)6 suggested: None anti-SARS-CoV-2 S protein suggested: None
   After washes with PBST, the plates were incubated with anti-SARS-CoV-2 S antibody (1:2000) at 37 °C for 1 h.	anti-SARS-CoV-2 S suggested: None
   The tissue sectioned were blocked with 5% normal goat serum and 1 % BSA in 1x PBST for 1 h, followed by incubation with rabbit anti-N primary antibody at 1:50 dilution (Anti-SARS-CoV-2 polyclonal antibody) overnight at 4°C.	anti-N suggested: None Anti-SARS-CoV-2 suggested: None
   Then the tissue was incubated with goat anti-rabbit HRP secondary antibody at 1:500 dilutions for 1 h and visualized by incubation with 3,3-diaminobenzidine (DAB) substrate and counterstained with hematoxylin.	anti-rabbit HRP suggested: None
   Sera antibody titer evaluation: Anti-S protein ELISA were used to determine sera IgG titer.	Anti-S protein ELISA suggested: None
   Mouse polyclonal anti-S protein primary antibody and HRP-conjugated secondary antibody were sequentially added.	anti-S protein suggested: None
   FACS analysis and sorting of S protein-specific B cells: Splenocytes isolated from Smg immunized mice were incubated with 2 μg/ml S protein at 4 °C for 1 h, followed by washing and incubation with an antibody cocktail containing the following antibodies: PE-Cy7-conjugated anti-CD19 (Biolegend, 6D5)	anti-CD19 suggested: None
   Experimental Models: Cell Lines
   Sentences	Resources
   Cell culture: HEK 293T (Homo sapiens, embryonic kidney), HEK293T-ACE2 (293T cells stably expressed hACE2), Vero E6 (Cercopithecus aethiops, kidney), Vero E6-ACE2 (Vero E6 cells stably expressed hACE2), A549 (Homo sapiens, lung) and A549-ACE2 cells (A549 cells stably expressed hACE2) were cultured in Dulbecco’s modified Eagle medium (DMEM, high glucose; GIBCO, Cat#11995065).	HEK293T-ACE2 suggested: None A549 suggested: None A549-ACE2 suggested: None
   Calu1 (Homo sapiens, lung) and Calu1-ACE2 cells (Calu1 cells stably expressed hACE2) were cultured in DMEM/F-12 (GIBCO, Cat#11330032).	Calu1-ACE2 suggested: None Calu1 suggested: None
   Calu3 (Homo sapiens, lung) and Calu3-ACE2 cells (Calu3 cells stably expressed hACE2) were cultured in Minimum Essential Medium (MEM; GIBCO, Cat#11095080).	Calu3-ACE2 suggested: None Calu3 suggested: None
   HEK293EBNA (ATCC number CRL-10852) and HEK293S GnTI− (ATCC® CRL-3022™) cells were cultured in Freestyle 293 expression medium (Invitrogen) supplemented with 0.5% bovine calf serum.	HEK293EBNA suggested: ATCC Cat# CRL-10852, RRID:CVCL_6974) HEK293S suggested: ATCC Cat# CRL-3022, RRID:CVCL_A785)
   For production of pseudotyped viruses and for the assay of virus entry, the day before transfection, HEK293T cells should be plated 18 to 24 h prior to transfection so that the cell density should be 50-80% confluent at the time of transfection.	HEK293T suggested: None
   Plasmid constructions, expression and purification of SARS-CoV-2 S protein in HEK293T and BEAS-2B cells: SARS-CoV-2 S protein stable trimer with a HisTag is the ectodomain of SARS-CoV-2 S protein which contains residues 1-1208.	BEAS-2B suggested: None
   This expression vector was used to transiently transfect human epithelial kidney (HEK) 293T cells or BEAS-2B (Homo sapiens, lung) using Mirus TransIT®-LT1	HEK suggested: None
   The mixture was then inoculated with 10,000 HEK-293T cells stably expressing human ACE2 gene in 96-well plate.	HEK-293T suggested: None
   CPE-based neutralization assay: For CPE-based neutralization assay, Vero E6 cells were plated onto a 6-well plate at 2×105 cells/well overnight for 90% confluence.	Vero E6 suggested: RRID:CVCL_A7UJ)
   Binding of antibody with S protein expressing 293T surface: 293T cells were transfected with pcDNA6/Spike-P2A-eGFP.	293T suggested: None
   Experimental Models: Organisms/Strains
   Sentences	Resources
   BEAS-2B (Homo sapiens, lung) were cultured in RPMI 1640 (GIBCO, Cat#22400089).	BEAS-2B suggested: None
   Glycans categorization follows previous studies according to the composition detected and visualized by Graphpad Prism 9.0.0.13 Animal vaccination and virus challenge: For mice vaccination study, female 6- to 8-week-old BALB/c mice (n= 5) were immunized intramuscularly with 20ug purified Sfg or Smg proteins mixed with aluminum hydroxide (20ug) at day 0, day 14 and day 56.	BALB/c suggested: None
   Recombinant DNA
   Sentences	Resources
   Pseudotyped viruses incorporated with spike protein from either full-length, variants or glycosylation mutants were constructed and cloned into expression plasmid pVax to generate the envelope recombinant plasmids.	pVax suggested: None
   Luciferase-expressing HIV-1 genome plasmid (pNL4-3.luc.RE) and a plasmid expressing SARS-CoV-2 spike, other representative spike constructs in different variants, or glycosylation mutants were co-transfected according to the manufacturer’s instructions.	pNL4-3.luc.RE suggested: None
   , thrombin cleavage site and 6xHisTag at the C-terminus of the SARS-CoV-2 S protein was created and subsequently cloned into the mammalian expression vector pcDNA.	pcDNA suggested: RRID:Addgene_66792)
   The modified S protein sequence was cloned into pTT vector for protein expression and purification.20 Expression and purification methods are modified from the previously published.	pTT suggested: RRID:Addgene_44006)
   Pseudovirus neutralization assay for serum and antibody study: For production and purification of SARS-CoV-2 pseudotyped lentivirus, the pseudotyped lentivirus carrying SARS-CoV-2 S protein was generated by transiently transfecting HEK-293T cells with pCMV-ΔR8.91, pLAS2w.Fluc.	pCMV-ΔR8.91 suggested: None pLAS2w suggested: None
   Ppuro and pcDNA3.1-nCoV-SΔ18 (or pcDNA3.1-nCoV-SΔ18 D614G).	pcDNA3.1-nCoV-SΔ18 suggested: None pcDNA3.1-nCoV-SΔ18 D614G suggested: None
   Binding of antibody with S protein expressing 293T surface: 293T cells were transfected with pcDNA6/Spike-P2A-eGFP.	pcDNA6/Spike-P2A-eGFP suggested: None
   Software and Algorithms
   Sentences	Resources
   The default scripts, parameters, and pre-optimized model generated by CHARMM-GUI were used as the input for the OpenMM program.	OpenMM suggested: (OpenMM, RRID:SCR_000436)
   The protein 3D visualization in Figure 1 and Figure S1(A) were drawn by the PyMOL program53.	PyMOL suggested: (PyMOL, RRID:SCR_000305)
   Glycans categorization follows previous studies according to the composition detected and visualized by Graphpad Prism 9.0.0.13 Animal vaccination and virus challenge: For mice vaccination study, female 6- to 8-week-old BALB/c mice (n= 5) were immunized intramuscularly with 20ug purified Sfg or Smg proteins mixed with aluminum hydroxide (20ug) at day 0, day 14 and day 56.	Graphpad Prism suggested: (GraphPad Prism, RRID:SCR_002798)
   The percentage of positive cells was quantified using FACS Canto II and the data were analyzed with FlowJo.	FlowJo suggested: (FlowJo, RRID:SCR_008520)

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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 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 page 10. 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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   Read the original source
3. 

   Version published to 10.1101/2021.05.25.445523v1 on bioRxiv

   May 25, 2021