Engineering a Vaccine Platform using Rotavirus A to Express SARS-CoV-2 Spike Epitopes
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Abstract
Human rotavirus (RV) vaccines used worldwide have been developed using live attenuated platforms. The recent development of a reverse genetics system for RVs has delivered the possibility of engineering chimeric viruses expressing heterologous peptides from other virus species to generate polyvalent vaccines. We tested the feasibility of this using two approaches. Firstly, we inserted short SARS-CoV-2 spike peptides into the hypervariable region of the simian SA11 RV strain viral protein (VP) 4. Secondly, we fused the receptor binding domain (RBD) of the SARS-CoV-2 spike protein, or the shorter receptor binding motif (RBM) nested within the RBD, to the C-terminus of non-structural protein (NSP) 3 of the bovine RF strain RV, with or without an intervening T2A peptide. Mutating the hypervariable region of SA11 VP4 impeded viral replication, and for these mutants no cross-reactivity with spike antibodies was detected. To rescue NSP3 mutants, we established a plasmid-based reverse genetics system for the bovine RF strain. Except for the RBD mutant, all NSP3 mutants delivered endpoint titres and replication kinetics comparable to that of the WT virus. In ELISAs, cell lysates of an NSP3 mutant expressing the RBD peptide showed cross reactivity with a SARS-CoV-2 RBD antibody. 3D bovine gut enteroids were susceptible to infection by all NSP3 mutants but only RBM mutant showed cross reactivity with SARS-CoV-2 RBD antibody. The tolerability of large peptide insertions in the NSP3 segment highlights the potential for this approach in the development of vaccine vectors targeting multiple enteric pathogens simultaneously.
IMPORTANCE
We explored the use of rotaviruses (RVs) to express heterologous peptides, using SARS-CoV-2 as an exemplar. Small SARS-CoV-2 peptide insertion (<34 amino acids) into the hypervariable region of the viral protein 4 (VP4) of RV SA11 strain resulted in reduced viral titre and replication, thus limiting its use as a potential vaccine expression platform. To test RF strain for its tolerance for peptide insertions, we constructed a reverse genetics system. NSP3 was C-terminally tagged with SARS-CoV-2 spike peptides of up to 193 amino acids. With a T2A-separated 193 amino acid tag on NSP3, there was little effect on the viral rescue efficiency, titre and replication. Tagged NSP3 elicited cross-reactivity with SARS-CoV-2 spike antibodies in ELISA. This is the first report describing epitope tagging of VP4, and of a reverse genetics system for the RF strain. We highlight the potential for development of RV vaccine vectors targeting multiple enteric pathogens simultaneously.
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SciScore for 10.1101/2022.03.23.485570: (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 not detected. Randomization not detected. Blinding not detected. Power Analysis not detected. Cell Line Authentication not detected. Table 2: Resources
Antibodies Sentences Resources Enteroids were incubated with primary antibodies (Table 4) overnight at 4°C with agitation, then washed three times with PBS and incubated with secondary antibodies (Table 4) and phalloidin (F-actin detection) (1:100) (Invitrogen) for 1 hr at room temperature. F-actin detectionsuggested: NoneExperimental Models: Cell Lines Sentences Resources After 24 hr incubation at 37°C 5% CO2, MA104 cells (1 x 105 cells/well) were added to transfected BSR-T7 cells and co-cultured for 4 days in … SciScore for 10.1101/2022.03.23.485570: (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 not detected. Randomization not detected. Blinding not detected. Power Analysis not detected. Cell Line Authentication not detected. Table 2: Resources
Antibodies Sentences Resources Enteroids were incubated with primary antibodies (Table 4) overnight at 4°C with agitation, then washed three times with PBS and incubated with secondary antibodies (Table 4) and phalloidin (F-actin detection) (1:100) (Invitrogen) for 1 hr at room temperature. F-actin detectionsuggested: NoneExperimental Models: Cell Lines Sentences Resources After 24 hr incubation at 37°C 5% CO2, MA104 cells (1 x 105 cells/well) were added to transfected BSR-T7 cells and co-cultured for 4 days in FBS-free DMEM supplemented with 0.5μg/mL porcine pancreatic trypsin type IX (Sigma-Aldrich) BSR-T7suggested: CCLV Cat# CCLV-RIE 0583, RRID:CVCL_RW96)Recombinant DNA Sentences Resources Plasmid construction: pT7 plasmids used for reverse genetics of SA11 RV were kindly provided by Takeshi Kobayashi [42] through the Addgene plasmid repository against IDs #89162-72. pT7suggested: NoneThe constructs were synthesised by Invitrogen GeneArt on either pMK-RQ (kanamycin resistance), pMA-RQ or pMA-T (ampicillin resistance) vectors. pMK-RQsuggested: NonepMA-RQsuggested: NonepMA-Tsuggested: NoneRF strain NSP3 constructs RBM, T2A-RBM, RBD and T2A-RBD were ordered as gene blocks from Invitrogen GeneArt and cloned into pT7-NSP2SA11 expression plasmid (Addgene #89169) after the NSP2 ORF was removed using SmaI and SalI restriction enzymes. pT7-NSP2SA11suggested: RRID:Addgene_89169)At 70% confluency, monolayers of BSR-T7 cells in 6-well plates were co-transfected with 11 plasmids corresponding to each RV genome segment (2.5μg for plasmids encoding NSP2 and NSP5; 0.8μg for the remaining plasmids) and plasmids encoding two vaccinia virus capping enzyme subunits (pCAG-D1R and pCAG-D12L −0.8μg each) using 16μL Lipofectamine 2000 (Invitrogen) per transfection reaction in a total volume of 200μL of Opti-MEM (Gibco). pCAG-D1Rsuggested: RRID:Addgene_89160)pCAG-D12Lsuggested: RRID:Addgene_89161)Software and Algorithms Sentences Resources The panels of viruses were titred by plaque assays, and the presence of mutations in the target gene segments was confirmed by RT-PCR and Sanger sequencing (GATC Biotech or Genewiz, Germany) as described below. Genewizsuggested: (GENEWIZ, RRID:SCR_003177)Quantification was performed by densitometry of scanned gel images using Image J. Image Jsuggested: (ImageJ, RRID:SCR_003070)The optical density (OD) was measured at 405nm using Cytation™ 3 Cell Imaging Multi-Mode Reader (Agilent) and data was analysed using BioTek Gen5 software (Agilent). Gen5suggested: (Gen5, RRID:SCR_017317)Images were analysed using the Zen Black software and processed using Photoshop v23.1.1. Zen Blacksuggested: (Black Zen software, RRID:SCR_018163)Photoshopsuggested: (Adobe Photoshop, RRID:SCR_014199)Statistical analysis: GraphPad Prism v9 was used for all statistical analyses. GraphPad Prismsuggested: (GraphPad Prism, RRID:SCR_002798)Results from OddPub: Thank you for sharing your data.
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: 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: 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.
Results from scite Reference Check: We found no unreliable references.
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