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Simplified Reverse Genetics Method to Recover Recombinant Rotaviruses Expressing Reporter Proteins
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Using Species a Rotavirus Reverse Genetics to Engineer Chimeric Viruses Expressing SARS-CoV-2 Spike Epitopes
Ola Diebold1, Victoria Gonzalez1, Luca Venditti2
1Infection and Immunity Division, Roslin Institute, University of Edinburghgrid.4305.2, Easter Bush Campus, Midlothian, United Kingdom.
Journal of Virology
|June 27, 2022
Summary
Rotaviruses (RVs) can be engineered to express foreign peptides, like those from SARS-CoV-2, for potential polyvalent vaccines. However, insertions into VP4 reduced replication, while NSP3 insertions showed greater tolerance and elicited antibody responses.
Area of Science:
- Virology
- Vaccinology
- Molecular Biology
Background:
- Live attenuated rotavirus (RVA) vaccines are widely used globally.
- Reverse genetics systems for rotaviruses (RVs) enable engineering of chimeric viruses.
- This technology allows for the expression of heterologous peptides for polyvalent vaccine development.
Purpose of the Study:
- To assess the feasibility of engineering rotaviruses to express heterologous peptides from SARS-CoV-2.
- To develop potential vaccine vectors targeting multiple enteric pathogens simultaneously.
Main Methods:
- Insertion of SARS-CoV-2 spike peptides into the hypervariable region of simian RV SA11 strain's viral protein (VP) 4.
- Fusion of SARS-CoV-2 spike receptor binding domain (RBD) or receptor binding motif (RBM) to the C terminus of bovine RV RF strain's nonstructural protein (NSP) 3, with or without a T2A peptide.
- Establishment of a plasmid-based reverse genetics system for the bovine RV RF strain to rescue NSP3 mutants.
- Analysis of viral replication, infectivity titers, and cross-reactivity with SARS-CoV-2 antibodies using ELISAs and bovine gut enteroids.
Main Results:
- Mutations in SA11 VP4's hypervariable region impeded viral replication and showed no cross-reactivity with spike antibodies.
- Most bovine RV RF NSP3 mutants, except for one RBD mutant with a rescue defect, showed comparable replication kinetics and infectivity titers to wild-type virus.
- Cell lysates of an NSP3 mutant expressing the RBD peptide demonstrated cross-reactivity with a SARS-CoV-2 RBD antibody in ELISAs.
- Bovine gut enteroids were susceptible to infection by NSP3 mutants, with cross-reactivity to SARS-CoV-2 RBD antibody observed only for the RBM mutant.
Conclusions:
- Insertion of small SARS-CoV-2 peptides into RV VP4 limits viral replication, indicating low tolerance at this site.
- Large SARS-CoV-2 peptide insertions at the C terminus of RV NSP3 are tolerated, especially with a T2A element, without significantly affecting viral properties.
- Engineered rotaviruses expressing SARS-CoV-2 peptides show potential as vaccine vectors for targeting multiple enteric pathogens.
