Immunogenicity of a new gorilla adenovirus vaccine candidate for COVID-19

Version published to 10.1016/j.ymthe.2021.04.022

Aug 1, 2021

SciScore for 10.1101/2020.10.22.349951: (What is this?)

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

Table 1: Rigor

Institutional Review Board Statement	IACUC: Animals and in vivo procedures: Animal husbandry and all experimental procedures were approved by the local animal ethics council and were performed in accordance with national and international laws and policies on the protection of animals used for scientific purposes (UE Directive 2010/63/UE; Italian Legislative Decree 26/2014). Consent: SARS-CoV2 microneutralization assay: Human sera derived from post-convalescent COVID-19 patients after signing of an informed consent, in the frame of a project aimed at following up patients.
Randomization	not detected.
Blinding	not detected.
Power Analysis	not detected.
Sex as a biological variable	not detected.
Cell Line Authentication	not detected.

Table 2: Resources

Antibodies
Sentences	Resources
An HA tag, derived from the human influenza hemagglutinin protein, and composed of a 9-amino acid peptide sequence, Tyr-Pro-Tyr-Asp-Val-Pro-Asp-Tyr-Ala, was fused downstream of the last SARS-CoV2 S aa (Thr1273) flanked at its 5’ and 3’ side by a Gly and a Ser, respectively, to facilitate antigen expression detection by the widely commercially available HA antibodies.	human influenza hemagglutinin protein , suggested: None HA suggested: None
Infected cells were blocked at the indicated time points with ice-cold methanol, incubated with Anti-Hexon primary antibody (Abcam) followed by detection with secondary anti-Mouse IgG Peroxidase (Sigma) and Vector NovaRED substrate kit for peroxidase (Vectorlabs).	Anti-Hexon suggested: None anti-Mouse IgG Peroxidase ( Sigma ) suggested: None
50ug of clarified lysates were loaded onto 4-12% acrylamide gel (Thermo Fisher), transferred onto iBlot 2 NC Mini Stacks membranes (Invitrogen) and incubated with the anti-HA (Abcam) or anti-RBD (Sino Biological) primary antibodies.	anti-RBD suggested: None
Detection of the SARS CoV-2 spike protein was achieved by incubation with an anti-rabbit (Sigma) secondary antibody, followed by ECL (Invitrogen) incubation, and images were taken using a Chemidoc (Biorad).	anti-rabbit suggested: None
Cells were then stained with Live/Dead fixable dye (Invitrogen); recombinant human His-tagged ACE-2 protein (RayBiotech) was incubated on cells before adding any antibodies, then detected with anti-His antibody (Sigma).	anti-His suggested: None
Antibody binding was detected with goat anti-mouse IgG mAb conjugated with AlexaFluor 647 fluorochrome (Sigma).	anti-mouse IgG suggested: None
In brief: optimized amount of proteins in 100μl volume were bound to Ni-NTA plates/strips for 1h at 25°C in shaking (1 μg/ml for full length Spike, 2 μg/ml for R121, 0.5 μg/ml for RBD); plates were then incubated with serial dilutions of sera for 2h at 25°C in shaking, then binding was detected with specific alkaline phosphatase-conjugated secondary antibodies (anti-mouse total IgG and anti-monkey total IgG from Sigma and anti-mouse IgG1 and IgG2a from BD Biosciences)	anti-mouse total IgG suggested: None anti-monkey total IgG suggested: None anti-mouse IgG1 suggested: None
ELISpot: For ELISpot assays, MSIP 96 well plates were from Millipore (Multiscreen filter plates) and anti-mouse or anti-monkey IFNγ or IL4 capture and detection antibodies were all from U-CyTech.	anti-mouse suggested: None anti-monkey IFNγ suggested: None IL4 suggested: None
Cells were plated at 1×106 per well in a 96-well round bottom plate and stimulated with either SARS-CoV-2 peptide pool S1 or S2, or with DMSO as negative control, all in presence of anti-CD28 antibody, for 18h (mouse cells) or 5h (monkey cells) in the presence of Brefeldin-A (Sigma).	anti-CD28 suggested: None
Mouse Antibodies: CD3 AlexaFluor647, CD4 BV421, CD8 BUV395, IL-17a PerCP-Cy 5.5, IFNγ BV650, IL2 APC-R700 (from BD Biosciences), IL-4 PE, IL-13 PE (Invitrogen).	CD8 suggested: (BD Biosciences Cat# 563795, RRID:AB_2722501) IL-17a suggested: (BioLegend Cat# 506930, RRID:AB_2686975) PerCP-Cy suggested: None IL-13 PE suggested: None
NPH Antibodies: CD3 AF700, CD4 PerCP-Cy 5.5, CD8 PE (from BD Biosciences)	CD3 suggested: (SouthernBiotech Cat# 8200-27, RRID:AB_2796424) CD4 suggested: None
Experimental Models: Cell Lines
Sentences	Resources
The filtered material was inoculated into monolayers of A549 cultivated in DMEM completed with 10	A549 suggested: None
Briefly, 8×104 HEK293 cells per well were seeded in 96 well plates the day before the assay.	HEK293 suggested: None
HeLa cells infection and S2/ACE2 staining: HeLa cells were plated at 5×105 cells/well in 6-well plates two days prior infection to allow adhesion, then infected with GRAd vectors at multiplicity of infection (MOI) of 150 for 48h.	HeLa suggested: None
ELISA assay on mouse sera were performed with His-tagged SARS-CoV-2 full length Spike protein (produced in baculovirus, SinoBiological), while ELISA with monkey sera were performed with a trimeric his-tagged stabilized SARS-CoV-2 Spike protein produced in-house in Expi293 cells or with a commercial RBD (ACROBiosystems).	Expi293 suggested: RRID:CVCL_D615)
Briefly, 1.5 × 106 HEK293T cells were seeded overnight in a 10 cm diameter Primaria-coated dish (Corning).	HEK293T suggested: None
For infectivity and neutralization assays, either 1.5×104 HuH7 cells or 2×104 VeroE6 cells/well were plated in white 96-well tissue culture plates (Corning) and incubated overnight at 37°C.	HuH7 suggested: None VeroE6 suggested: JCRB Cat# JCRB1819, RRID:CVCL_YQ49)
Experimental Models: Organisms/Strains
Sentences	Resources
Serum from naïve BALB/c animals are routinely tested at 1:100 dilution as negative control in ELISA, and the resulting OD is nearly at the level of secondary Ab alone, therefore for mouse experiments baseline titers cannot be detected, calculated and plotted.	BALB/c suggested: None
Software and Algorithms
Sentences	Resources
Group C adenovirus assignment for GRAd32 was based on a phylogenetic analysis of aligned adenoviral polymerase sequences using a bootstrap-confirmed Maximum Likelihood tree (500 replicates) calculated with MEGA 10.1.7 (21)	MEGA suggested: (Mega BLAST, RRID:SCR_011920)
Polymerase sequences were aligned with MUSCLE (23).	MUSCLE suggested: (MUSCLE, RRID:SCR_011812)
Sample acquisition was performed on a LSR Fortessa-X20 cytofluorimeter (BD biosciences) and sample analysis was performed with FlowJo (TreeStar).	FlowJo suggested: (FlowJo, RRID:SCR_008520)
SARS-CoV2 microneutralization assay: Human sera derived from post-convalescent COVID-19 patients after signing of an informed consent, in the frame of a project aimed at following up patients.	Human suggested: None
: GraphPad Prism version 8 for Windows (GraphPad Software, San Diego, California, USA) was used for graphs and statistical analysis.	GraphPad 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: An explicit section about the limitations of the techniques employed in this study was not found. We encourage authors to address study limitations.

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

Identifier	Status	Title
NCT04528641	Recruiting	GRAd-COV2 Vaccine Against COVID-19

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.

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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Immunogenicity of a new gorilla adenovirus vaccine candidate for COVID-19

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