title: Chimaeric virus-like particles derived from consensus genome sequences of human rotavirus strains co-circulating in Africa.
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.
authors.affiliation: Biochemistry, Centre of Human Metabonomics, North-West University, Potchefstroom, South Africa; Institute of Infection and Global Health, University of Liverpool, Liverpool, United Kingdom.
authors.forename: Khuzwayo C
authors.initials: KC
authors.lastname: Jere
authors.affiliation: Biochemistry, Centre of Human Metabonomics, North-West University, Potchefstroom, South Africa; Department of Microbiology, Biochemistry and Food Biotechnology, University of the Free State, Bloemfontein, South Africa.
authors.forename: Hester G
authors.initials: HG
authors.lastname: O'Neill
authors.affiliation: Biochemistry, Centre of Human Metabonomics, North-West University, Potchefstroom, South Africa; Deltamune (Pty.) Ltd., Lyttelton, Centurion, South Africa.
authors.forename: A Christiaan
authors.initials: AC
authors.lastname: Potgieter
authors.affiliation: Biochemistry, Centre of Human Metabonomics, North-West University, Potchefstroom, South Africa.
authors.forename: Alberdina A
authors.initials: AA
authors.lastname: van Dijk
mesh_terms: D000349:Africa; D000818:Animals; D036022:Capsid Proteins / Q000096:biosynthesis / Q000235:genetics; D016384:Consensus Sequence; D016679:Genome, Viral; D006801:Humans; D016366:Open Reading Frames; D012401:Rotavirus / Q000276:immunology*; D012400:Rotavirus Infections / Q000517:prevention & control* / Q000821:virology; D061987:Sf9 Cells; D018411:Spodoptera; D014611:Vaccination; D058425:Vaccines, Virus-Like Particle / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification; D014765:Viral Vaccines / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification
chemical_list: D036022:Capsid Proteins; D058425:Vaccines, Virus-Like Particle; D014765:Viral Vaccines