Modular capsid decoration boosts adenovirus vaccine-induced humoral immunity against SARS-CoV-2

SciScore for 10.1101/2022.02.20.480711: (What is this?)

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

Table 1: Rigor

Ethics	not detected.
Sex as a biological variable	Female BALB/c mice (age 6-8 weeks, Envigo), housed in specific-pathogen free environments, were immunized intramuscularly by injection of 50 μL of vaccine formulated in endotoxin-free PBS (Gibco) into both hind limbs of each animal (100 μL total).
Randomization	not detected.
Blinding	not detected.
Power Analysis	not detected.
Cell Line Authentication	not detected.

Table 2: Resources

Antibodies
Sentences	Resources
Mouse monoclonal anti-hexon antibody (B025/AD51, Thermo-Fisher) was added at 1:1000 dilution in 1% w/v milk in PBS and incubated for 1 h at 25°C.	anti-hexon suggested: (DSHB Cat# TC31-27F11.C2, RRID:AB_1553403)
Cells were washed with 1% w/v milk in PBS prior …

SciScore for 10.1101/2022.02.20.480711: (What is this?)

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

Table 1: Rigor

Ethics	not detected.
Sex as a biological variable	Female BALB/c mice (age 6-8 weeks, Envigo), housed in specific-pathogen free environments, were immunized intramuscularly by injection of 50 μL of vaccine formulated in endotoxin-free PBS (Gibco) into both hind limbs of each animal (100 μL total).
Randomization	not detected.
Blinding	not detected.
Power Analysis	not detected.
Cell Line Authentication	not detected.

Table 2: Resources

Antibodies
Sentences	Resources
Mouse monoclonal anti-hexon antibody (B025/AD51, Thermo-Fisher) was added at 1:1000 dilution in 1% w/v milk in PBS and incubated for 1 h at 25°C.	anti-hexon suggested: (DSHB Cat# TC31-27F11.C2, RRID:AB_1553403)
Cells were washed with 1% w/v milk in PBS prior to addition of a secondary goat anti-mouse alkaline phosphatase (ALP) conjugated antibody (STAR117A, BioRad) at 1:1000 dilution in 1% w/v milk in PBS.	anti-mouse alkaline phosphatase (ALP suggested: (CEDARLANE Cat# CLF004ALP, RRID:AB_10060171)
Coupling efficiency was assessed by comparing band intensities of unconjugated hexon-Tag in ligand decorated samples to undecorated (control) samples using Image J: Anti-vector antibody neutralization assay: For assessment of vector neutralization by potent neutralising mouse monoclonal antibody (mAb) 9C12 (24) (Developmental Studies Hybridoma Bank, University of Iowa), Ad5 vectors expressing GFP were incubated with serially diluted mAb 9C12 antibody at a 1:1 ratio in complete media for 1 h at 37°C.	Anti-vector suggested: None
For assessment of vector neutralization by serum containing anti-adenovirus antibodies, serum samples were obtained by immunizing C57BL/6 mice with 1E+8 ifu of an Ad5 vector expressing ovalbumin (vector had an unmodified hexon).	anti-adenovirus suggested: None
Experimental Models: Cell Lines
Sentences	Resources
Linearized DNA was transfected (Lipofectamine 2000, Invitrogen) into E1-complementing Human Embryonic Kidney (HEK) 293 cell lines; either 293A cells (Invitrogen) for Ad vectors expressing GFP and DogCatcher-NANP18, or 293TREX cells (Invitrogen) for Ad vectors expressing SARS-CoV-2 spike.	HEK suggested: None 293 suggested: None 293A suggested: None 293TREX suggested: None
Titration of recombinant adenoviruses: Infectious titer of vector preparations was assessed by single cell infectivity assay on HEK293A cells or 293TREX cells.	HEK293A suggested: None
Diluted serum was incubated with Ad5(GFP) vectors, the mix incubated on HEK293 cells, and bulk GFP fluorescence read 24 h later as described above.	HEK293 suggested: None
Assessment of coagulation Factor X-mediated vector transduction of SKOV3 cells: SKOV3 cells (human ovary adenocarcinoma) were obtained from Public Health England and cultured in McCoy’s 5a media with 2 mM Glutamine and 15% v/v fetal bovine serum (complete McCoy’s).	SKOV3 suggested: RRID:CVCL_5J11)
SARS-CoV-2 pseudovirus neutralization (pVNT) assay: Pseudotyped HIV-1 viruses incorporating the SARS-CoV-2 full-length spike (Wuhan strain or B.1.1.7, B.1.617.2 or B.1.351 variants of concern) were generated and SARS-CoV-2 pVNT assays performed as previously described using Hela cells stably expressing ACE2 as target cells (28).	Hela suggested: CLS Cat# 300194/p772_HeLa, RRID:CVCL_0030)
Experimental Models: Organisms/Strains
Sentences	Resources
For assessment of vector neutralization by serum containing anti-adenovirus antibodies, serum samples were obtained by immunizing C57BL/6 mice with 1E+8 ifu of an Ad5 vector expressing ovalbumin (vector had an unmodified hexon).	C57BL/6 suggested: None
Female BALB/c mice (age 6-8 weeks, Envigo), housed in specific-pathogen free environments, were immunized intramuscularly by injection of 50 μL of vaccine formulated in endotoxin-free PBS (Gibco) into both hind limbs of each animal (100 μL total).	BALB/c suggested: None
Recombinant DNA
Sentences	Resources
Bacterial artificial chromosome (BAC) sequences from pBELOBAC11 (NEB) were amplified using forward (5’-TTAATTAAcgtcgaccaattctcatg) and reverse (5’-TTAATTAAgtcgacagcgacacacttg) primers to introduce PacI sites at either end of the BAC cassette.	pBELOBAC11 suggested: RRID:Addgene_60342)
The entire Ad5(GFP) genome was subsequently cloned into the BAC with PacI, to generate pBAC-Ad5(GFP).	pBAC-Ad5 suggested: None
Recombinant vectors expressing DogCatcher-NANP18 and SARS-CoV-2 spike (residues 1-1208 Wuhan strain, codon-optimized for mammalian expression and including stabilizing mutations K986P and V987P and mutation of the furin cleavage site 682-GSAS-685 (60)) were generated through cloning of gene constructs into pENTR4.	pENTR4 suggested: RRID:Addgene_26366)
Protein production and purification: DNA sequences for expression of DogCatcher, DogCatcher-NANP9, DogCatcher-NANP18 and SpyCatcher were cloned into expression plasmid pET45(+) (EMD Millipore) for protein production in BL21(DE3) E. coli (NEB).	pET45 suggested: None
DNA sequences for expression of DogCatcher fused to SARS-CoV-2 spike receptor binding domain (Wuhan strain, residues 319-532) (DogCatcher-RBD), were cloned into mammalian protein expression plasmid pcDNA3.4.	pcDNA3.4 suggested: RRID:Addgene_131198)
Software and Algorithms
Sentences	Resources
Coupling efficiency was assessed by comparing band intensities of unconjugated hexon-Tag in ligand decorated samples to undecorated (control) samples using Image J: Anti-vector antibody neutralization assay: For assessment of vector neutralization by potent neutralising mouse monoclonal antibody (mAb) 9C12 (24) (Developmental Studies Hybridoma Bank, University of Iowa), Ad5 vectors expressing GFP were incubated with serially diluted mAb 9C12 antibody at a 1:1 ratio in complete media for 1 h at 37°C.	Image J suggested: (ImageJ, RRID:SCR_003070)
Automated data-collection was performed with Leginon software (65) at a nominal magnification of 28,000×, corresponding to a pixel size of 5.19 Å.	Leginon suggested: (Leginon, RRID:SCR_016731)
Statistics: Statistical analyses were performed in GraphPad Prism 9.	GraphPad Prism suggested: (GraphPad Prism, RRID:SCR_002798)

Results from OddPub: Thank you for sharing your data.

Results from LimitationRecognizer: We detected the following sentences addressing limitations in the study:

Further investigation will be required to comprehensively determine limitations for capsid display in terms of size and structure of ligands, but similar sized receptor-binding domains of other viral proteins, including influenza hemagglutinin (∼25 kDa), have been expressed independently as recombinant proteins and displayed on VLPs (56) implying that this could be a generalizable concept. Capsid decoration using our protein superglue technology is simple, requiring only co-incubation of spontaneously and irreversibly reacting components with no chemical modification required. A similar conjugation process has already been scaled under good manufacturing practice (GMP) during development of a VLP-based SARS-CoV-2 vaccine currently in Phase I/II clinical trials (19). The ability of our adenovirus-based platform to induce both robust cellular and humoral immunity and to enhance efficacy of multi-shot regimens could be advantageous for applications beyond prophylactic vaccines, including therapeutic vaccines against chronic viral pathogens and cancer. Methods of rapid and customizable covalent decoration of Ad capsids could also be utilized for development of personalized therapies. In prophylactic settings, adenovirus capsid decoration could be utilized in the design of pan-coronavirus and pan-influenza vaccines; combining broad and conserved T cell immunity from encoded antigens with exchangeable capsid ligands delivering potent neutralizing humoral immunity.

Results from TrialIdentifier: No clinical trial numbers were referenced.

Results from Barzooka: We did not find any issues relating to the usage of bar graphs.

Results from JetFighter: Please consider improving the rainbow (“jet”) colormap(s) used on page 27. At least one figure is not accessible to readers with colorblindness and/or is not true to the data, i.e. not perceptually uniform.

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.

Results from scite Reference Check: We found no unreliable references.

Read the original source

Modular capsid decoration boosts adenovirus vaccine-induced humoral immunity against SARS-CoV-2

This article has been Reviewed by the following groups

Listed in

Abstract

Article activity feed