Adenovirus-vectored SARS-CoV-2 vaccine expressing S1-N fusion protein

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1. Version published to 10.1093/abt/tbac015

   Jul 1, 2022
2. 

   ScreenIT

   May 14, 2022

   SciScore for 10.1101/2022.05.09.491179: (What is this?)

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

   Table 1: Rigor

   Ethics	Field Sample Permit: Mice were maintained under specific pathogen-free conditions at the University of Pittsburgh, and all experiments were conducted in accordance with animal use guidelines and protocols approved by the University of Pittsburgh’s Institutional Animal Care and Use (IACUC) Committee. IACUC: Mice were maintained under specific pathogen-free conditions at the University of Pittsburgh, and all experiments were conducted in accordance with animal use guidelines and protocols approved by the University of Pittsburgh’s Institutional Animal Care and Use (IACUC) Committee.
   Sex as a biological variable	not detected.
   Randomization	not detected.
   Blinding	not detected.
   Power Analysis	not detected.

   SciScore for 10.1101/2022.05.09.491179: (What is this?)

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

   Table 1: Rigor

   Ethics	Field Sample Permit: Mice were maintained under specific pathogen-free conditions at the University of Pittsburgh, and all experiments were conducted in accordance with animal use guidelines and protocols approved by the University of Pittsburgh’s Institutional Animal Care and Use (IACUC) Committee. IACUC: Mice were maintained under specific pathogen-free conditions at the University of Pittsburgh, and all experiments were conducted in accordance with animal use guidelines and protocols approved by the University of Pittsburgh’s Institutional Animal Care and Use (IACUC) Committee.
   Sex as a biological variable	not detected.
   Randomization	not detected.
   Blinding	not detected.
   Power Analysis	not detected.
   Cell Line Authentication	not detected.

   Table 2: Resources

   Antibodies
   Sentences	Resources
   After blocking for 1 hour at room temperature (RT) with 5% non-fat milk in TBS-T, rabbit anti-SARS-CoV spike polyclonal antibody (1:3000) (Sino Biological), or rabbit anti-SARS-CoV nucleoprotein (1:3,000	anti-SARS-CoV suggested: None anti-SARS-CoV nucleoprotein suggested: None
   ) (Sino Biological) was added and incubated overnight at 4°C as primary antibody, and horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG (1:10000) (Jackson immunoresearch) was added and incubated at RT for 1 hours as secondary antibody.	anti-rabbit IgG suggested: None
   ELISA: Sera from all mice were collected prior to immunization (week 0) and at weeks indicated after immunization and evaluated for SARS-CoV-2-S1-specific IgG, IgG1, and IgG2a antibodies using ELISA [34].	SARS-CoV-2-S1-specific IgG suggested: None IgG1 suggested: None IgG2a suggested: None
   SARS-CoV-2 microneutralization assay: Neutralizing antibody (NT-Ab) titers against SARS-CoV2 were defined according to the following protocol [78, 79].	SARS-CoV2 suggested: None
   Spots were counted using a binocular on a dissecting microscope and the detected numbers of IgG anti-SARS-CoV-2-S1 and anti- SARS-CoV-2-N antibody forming cells were calculated per million bone marrow cells.	anti-SARS-CoV-2-S1 suggested: None anti- SARS-CoV-2-N suggested: None
   Experimental Models: Cell Lines
   Sentences	Resources
   For Expi293 cell transfection, we used ExpiFectamieTM 293 Transfection Kit (ThermoFisher) and followed the manufacturer’s instructions.	Expi293 suggested: RRID:CVCL_D615)
   The supernatants of A549 cells transduced with Ad5.SARS-CoV-2-S1N, Ad5.SARS-CoV-2-S1, and AdΨ5 were subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot.	A549 suggested: None
   After 1 hour incubation at 33°C 5%CO2, 3×104 VERO E6 cells [VERO C1008 (Vero 76, clone E6, Vero E6); ATCC® CRL-1586TM] were added to each well.	VERO E6 suggested: None
   Experimental Models: Organisms/Strains
   Sentences	Resources
   Animals and immunization: For single immunization experiment, BALB/cJ mice (n = 5 animals per group in each independent experiment) were vaccinated by either I.N. delivery or S.C. injection of 5×1010 viral particles (v.p.) of AdΨ5 (a null Ad5 vector negative control), Ad5.SARS-CoV-2-S1, or by Ad5.SARS-CoV-2-S1N.	BALB/cJ suggested: None
   Recombinant DNA
   Sentences	Resources
   The coding sequence of N (GenBank NC_045512) having Sac I & Sal I in 5’ end and Not I & Apa I in 3’ end was synthesized and cloned in Sac I/ApaI sites in pCMV-3Tag-4A generated in pCMV3/SARS-CoV- 2-N (GenScript).	pCMV-3Tag-4A suggested: None pCMV3/SARS-CoV- 2-N suggested: None
   For the construction of pAd/SARS-CoV-2-S1N, BamH I-6H-Not I of pAd/SARS-CoV-2-S1 was replace with Nucleoprotein gene digested with BamH I & Not I after amplified with NP-S (5’-GACGGATCCATGTCTGATAATGGACCCC-3’) & T7 (5’-TAATACGACTCACTATAGGG-3’) primers from pCMV3/SARS-CoV-2-NP.	pAd/SARS-CoV-2-S1N suggested: None pAd/SARS-CoV-2-S1 suggested: None pCMV3/SARS-CoV-2-NP suggested: None
   For B.1.351 variant, SARS-CoV-2-S1 mutated (Del144; K417N; E484K; N501Y; A570D; D614G) was synthesized based on above codon-optimized SARS-CoV-2-S1 from Wuhan. pAd/SARS- CoV-2-S1WU and pAd/SARS-CoV-2-SAS1SA were then created by subcloning the codon- optimized SARS-CoV-2-S1 inserts into the shuttle vector, pAdlox (GenBank U62024), at SalI/NotI sites.	pAd/SARS-CoV-2-SAS1SA suggested: None
   Software and Algorithms
   Sentences	Resources
   Samples were run on an Aurora (Cytek) flow cytometer and analyzed with FlowJo v10 software (BD Biosciences)	FlowJo suggested: (FlowJo, RRID:SCR_008520)
   Statistical analysis: Statistical analyses were performed using GraphPad Prism v9 (San Diego, CA)	GraphPad Prism 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:

   An important limitation regarding our findings concerning intranasal vaccination of Ad5.SARS- CoV-2-S1N is that we did not investigate key mucosal immunity aspects, such as IgA production. Our future studies will mechanistically investigate the kinetics of intranasal immunization through isolation of tissues in closer proximity to the nasal cavity. Particularly, the lack of induction of CD4+ and CD8+ T cell response to intranasal vaccination could be attributed to isolating splenocytes, rather than mucosa-associated lymph node tissue (MALT) or conducting bronchioalveolar lavage (BAL). In our future studies, we will isolate MALT and lung tissue post intranasal vaccination to investigate the localized cellular immune response post intranasal immunization. Our future studies will also include conducting BAL on intranasally immunized mice to better investigate local immunity. We believe that the increasing CD8+ T cell response through including the N protein in vaccine formulation will not only help by introducing more conserved regions of SARS-CoV-2 to the immune system, potentially allowing for resistance against emerging variants, but will also assist in viral clearance. This is particularly important in the context of long covid and in populations that are at high risk of SARS-CoV-2 morbidity and mortality where the T cell response has been shown to play an important role [67–71]. While previous clinical translation of Ad vaccines has been hampered by pre-existing immunity ag...

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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.
   • 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 10.1101/2022.05.09.491179v1 on bioRxiv

   May 10, 2022
4. 

   Version published to 10.1101/2022.05.09.491179v2 on bioRxiv

   May 10, 2022