Abstract
Rotavirus virus-like particles (RV-VLPs) are potential alternative non-live vaccine candidates due to their high immunogenicity. They mimic the natural conformation of native viral proteins but cannot replicate because they do not contain genomic material which makes them safe. To date, most RV-VLPs have been derived from cell culture adapted strains or common G1 and G3 rotaviruses that have been circulating in communities for some time. In this study, chimaeric RV-VLPs were generated from the consensus sequences of African rotaviruses (G2, G8, G9 or G12 strains associated with either P[4], P[6] or P[8] genotypes) characterised directly from human stool samples without prior adaptation of the wild type strains to cell culture. Codon-optimised sequences for insect cell expression of genome segments 2 (VP2), 4 (VP4), 6 (VP6) and 9 (VP7) were cloned into a modified pFASTBAC vector, which allowed simultaneous expression of up to four genes using the Bac-to-Bac Baculovirus Expression System (BEVS; Invitrogen). Several combinations of the genome segments originating from different field strains were cloned to produce double-layered RV-VLPs (dRV-VLP; VP2/6), triple-layered RV-VLPs (tRV-VLP; VP2/6/7 or VP2/6/7/4) and chimaeric tRV-VLPs. The RV-VLPs were produced by infecting Spodoptera frugiperda 9 and Trichoplusia ni cells with recombinant baculoviruses using multi-cistronic, dual co-infection and stepwise-infection expression strategies. The size and morphology of the RV-VLPs, as determined by transmission electron microscopy, revealed successful production of RV-VLPs. The novel approach of producing tRV-VLPs, by using the consensus insect cell codon-optimised nucleotide sequence derived from dsRNA extracted directly from clinical specimens, should speed-up vaccine research and development by by-passing the need to adapt rotaviruses to cell culture. Other problems associated with cell culture adaptation, such as possible changes in epitopes, can also be circumvented. Thus, it is now possible to generate tRV-VLPs for evaluation as non-live vaccine candidates for any human or animal field rotavirus strain.
Highlights
Human rotaviruses are the main cause of severe infant gastroenteritis
Since the baculovirus polh is a stronger promoter than p10 [19,23,55], the cloning strategy of the open reading frames (ORFs) coding structural rotavirus proteins was based on the number of copies required for each protein to form the viral capsid
Evaluation of the assembly of baculovirus-expressed rotavirus proteins into chimaeric rotavirus virus-like particles To generate chimaeric Rotavirus virus-like particles (RV-VLPs), the VP2/VP6 proteins were co-expressed with outer capsid proteins that comprised of various combinations of VP4 and VP7 consisting of G2, G8, G9 or G12 associated with either P[4], P[6] or P[8] genotypes
Summary
Human rotaviruses are the main cause of severe infant gastroenteritis. Rotavirus disease is associated with approximately 453 000 annual childhood deaths of which most occur in developing countries [1]. RotaTeq and Rotarix vaccines were recommended by the WHO for routine use in children across the globe [2,3]. These vaccines have been shown to be effective in preventing severe rotavirus disease [4,5], their use has revealed some shortcomings. Their high cost is beyond reach of most developing nations. Rotarix was adopted under the assumption that it would render cross-reactive antibody protection whereas the VP4 and VP7 of RotaTeq represented the most prevalent serotypes of the strains that were circulating at that time. There is a need for further development of alternative rotavirus vaccine candidates and strategies
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Disclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.