A Single Dose of a Hybrid hAdV5-Based Anti-COVID-19 Vaccine Induces a Long-Lasting Immune Response and Broad Coverage against VOC

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1. Version published to 10.3390/vaccines9101106

   Sep 29, 2021
2. 

   ScreenIT

   Aug 19, 2021

   SciScore for 10.1101/2021.08.11.455942: (What is this?)

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

   Table 1: Rigor

   Ethics	IACUC: Mice were maintained under specific pathogen-free conditions at the Institute Leloir animal facility and all experiments were conducted in accordance with animal use guidelines and protocols approved by the Institutional Animal Care and Use Committee (IACUC protocol 69).
   Sex as a biological variable	Mice immunization: Six- to eight-week-old male BALB/c mice (obtained from the animal facility of the Veterinary School, University of La Plata, Argentina) were immunized with 109 or 1010 viral particles (vp) of Ad.C (empty vector), CoroVaxG.5 or CoroVaxG.
   Randomization	not detected.
   Blinding	not detected.
   Power Analysis	not detected.
   Cell Line Authentication	not detected.

   Table 2: Resources

   Antibodies
   S…

   SciScore for 10.1101/2021.08.11.455942: (What is this?)

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

   Table 1: Rigor

   Ethics	IACUC: Mice were maintained under specific pathogen-free conditions at the Institute Leloir animal facility and all experiments were conducted in accordance with animal use guidelines and protocols approved by the Institutional Animal Care and Use Committee (IACUC protocol 69).
   Sex as a biological variable	Mice immunization: Six- to eight-week-old male BALB/c mice (obtained from the animal facility of the Veterinary School, University of La Plata, Argentina) were immunized with 109 or 1010 viral particles (vp) of Ad.C (empty vector), CoroVaxG.5 or CoroVaxG.
   Randomization	not detected.
   Blinding	not detected.
   Power Analysis	not detected.
   Cell Line Authentication	not detected.

   Table 2: Resources

   Antibodies
   Sentences	Resources
   ELISA: Sera from all mice were collected at different time points after immunization and evaluated for SARS-CoV-2-S-specific IgG antibodies using ELISA.	SARS-CoV-2-S-specific IgG suggested: None
   Sera collected at week 4 after vaccination were also tested for SARS-CoV-2-S-specific IgG1 and IgG2a antibodies using ELISA.	SARS-CoV-2-S-specific IgG1 suggested: None IgG2a suggested: None
   Plates were washed and bound specific IgG was detected with a HRP-conjugated goat anti-mouse IgG H&L antibody (ab6789, Abcam) diluted 1: 10,000 in blocking buffer.	anti-mouse IgG suggested: (Abcam Cat# ab6789, RRID:AB_955439)
   Neutralization of authentic SARS-CoV-2 virus: Neutralizing antibody (nAb) titers against SARS-CoV2 were defined according to the following protocol.	SARS-CoV2 suggested: None
   Experimental Models: Cell Lines
   Sentences	Resources
   CRL-3216), Vero cells (CCL-81), Hs 729T (HTB-153) and THP-1 (TIB-202) cells were obtained from the ATCC (Manassas, VA, USA)	Vero suggested: None THP-1 suggested: None
   HEK293 cells were purchased from Microbix Biosystems Inc (Mississauga, Canada) and 911 cells were already described63.	HEK293 suggested: None
   HEK293T-hACE2 cells were already described64.	HEK293T-hACE2 suggested: RRID:CVCL_A7UK)
   For the selection of the most appropriate promoter, HEK293T cells grown in 24-well plates were co-transfected with 1 μg of the different plasmids and 100 ng of pS-CMV-Renilla, using Lipofectamine 2000 (Thermo Fisher Scientific, CA, USA).	HEK293T suggested: None
   The viruses were propagated in HEK-293 cells in CellSTACK® cell culture chambers (Corning, Arizona, USA), purified by double CsCl density gradient centrifugation and stored in 10% glycerol in single-use aliquots at –80°C.	HEK-293 suggested: None
   HEK-293T cells growing in Optimem media (Gibco, MD, USA) with 2% of FBS were transduced with G*ΔG-VSV at a multiplicity of infection of four.	HEK-293T suggested: CCLV Cat# CCLV-RIE 1018, RRID:CVCL_0063)
   Next, 100 μl of 5×105/mL HEK293T-ACE2 cells were added to the pseudovirus-serum mixture and incubated at 37 °C, 5% CO2 for 20-24 h.	HEK293T-ACE2 suggested: None
   Experimental Models: Organisms/Strains
   Sentences	Resources
   For RBD inoculations eight-week-old male BALB/c mice were immunized with 7.5 μg of the receptor binding domain of Spike protein (kindly gifted by Andrea Gamarnik, Argentina) in 75 μL Complete Freund’s Adjuvant (CFA, Sigma, St. Louis, MO) via subcutaneous injection and boosted 2 weeks later with 5 μg of RBD in 100 μL Incomplete Freund’s Adjuvant (IFA, Sigma).	BALB/c suggested: None
   Recombinant DNA
   Sentences	Resources
   Promoter and fiber selection: Promoters were synthesized by Genscript (NJ, USA) with NotI / (XhoI-StuI) flanking restriction sites and cloned in the NotI / StuI sites of the vector pShuttle-I-XP-Luc63 to obtain pShuttle-Pr1-Luc and pShuttle-Pr2-Luc.	pShuttle-I-XP-Luc63 suggested: None pShuttle-Pr1-Luc suggested: None
   The vectors pAd-SV40-Luc, and pS-CMV-Renilla were previously described63.	pAd-SV40-Luc suggested: None pS-CMV-Renilla suggested: None
   The synthesized 4,650 bp fragment was cloned into the pShuttle-Pr2-Luc vector digested with StuI / SalI to exchange the luciferase ORF by the designed Spike cassette, downstream of Pr2.	pShuttle-Pr2-Luc suggested: None
   The sequence of the resulting plasmid pS-Spike(D614G)-PP was confirmed by sequencing (Macrogen, Seul, Korea).	pS-Spike(D614G)-PP suggested: None
   To construct the non-replicating adenoviruses, the plasmid pS-Spike(D614G)-PP was linearized with PmeI and co-transformed with E1/E3 (pCoroVaxG.5) or E1 (pCoroVaxG.3) deleted adenoviral backbone vectors in electrocompetent BJ5183 bacteria.	pCoroVaxG.5 suggested: None pCoroVaxG.3 suggested: None
   Briefly, the full length cDNA of Spike-D614G, and the Spike variants B.1.1.7 (alpha, first identified in the UK) and P.1 (gamma, first identified in Manaos, Brazil), were cloned into the eukaryotic expression vector pcDNA3.1, using the EcoRV restriction site and blunt-end ligation strategy, to generate the recombinant plasmids pcDNA-3.1-Spike-D614G, pcDNA-3.1-Spike-B,1,1,7 and pcDNA-3.1-Spike-P.1..	pcDNA3.1 suggested: RRID:Addgene_79663) pcDNA-3.1-Spike-D614G suggested: None pcDNA-3.1-Spike-B,1,1,7 suggested: None pcDNA-3.1-Spike-P.1 suggested: None
   Software and Algorithms
   Sentences	Resources
   Protein extracts were separated by SDS-PAGE with a 10% gel and transferred to nitrocellulose membranes (Bio-Rad Laboratories).	Bio-Rad Laboratories suggested: (Bio-Rad Laboratories, RRID:SCR_008426)
   Semi-quantifications of WB assays were performed by densitometry using the ImageJ software 1.53 (Wayne Rasband, NIH, USA), normalizing by β-actin expression.	ImageJ suggested: (ImageJ, RRID:SCR_003070)
   For each IgG subclass reference, a standard curve was plotted using GraphPad Prism 8.0 generating a four-parameter logistical (4PL) fit of the OD 450 nm at each serial antibody dilution.	GraphPad Prism suggested: (GraphPad Prism, RRID:SCR_002798)
   Cells were acquired on a FACSAria Fussion cytometer and analysis was performed using Flow Jo version 10.7.1.	Flow Jo 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 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.
   • No funding statement was detected.
   • 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 10.1101/2021.08.11.455942v1 on bioRxiv

   Aug 11, 2021