title: Efficient production of foot-and-mouth disease virus empty capsids in insect cells following down regulation of 3C protease activity.
abstract: Foot-and-mouth disease virus (FMDV) is a significant economically and distributed globally pathogen of Artiodactyla. Current vaccines are chemically inactivated whole virus particles that require large-scale virus growth in strict bio-containment with the associated risks of accidental release or incomplete inactivation. Non-infectious empty capsids are structural mimics of authentic particles with no associated risk and constitute an alternate vaccine candidate. Capsids self-assemble from the processed virus structural proteins, VP0, VP3 and VP1, which are released from the structural protein precursor P1-2A by the action of the virus-encoded 3C protease. To date recombinant empty capsid assembly has been limited by poor expression levels, restricting the development of empty capsids as a viable vaccine. Here expression of the FMDV structural protein precursor P1-2A in insect cells is shown to be efficient but linkage of the cognate 3C protease to the C-terminus reduces expression significantly. Inactivation of the 3C enzyme in a P1-2A-3C cassette allows expression and intermediate levels of 3C activity resulted in efficient processing of the P1-2A precursor into the structural proteins which assembled into empty capsids. Expression was independent of the insect host cell background and leads to capsids that are recognised as authentic by a range of anti-FMDV bovine sera suggesting their feasibility as an alternate vaccine.
authors.affiliation: Institute for Animal Health, Ash Road, Pirbright, Woking GU24 0NF, UK.
authors.forename: Claudine
authors.initials: C
authors.lastname: Porta
authors.forename: Xiaodong
authors.initials: X
authors.lastname: Xu
authors.forename: Silvia
authors.initials: S
authors.lastname: Loureiro
authors.forename: Saravanan
authors.initials: S
authors.lastname: Paramasivam
authors.forename: Junyuan
authors.initials: J
authors.lastname: Ren
authors.forename: Tara
authors.initials: T
authors.lastname: Al-Khalil
authors.forename: Alison
authors.initials: A
authors.lastname: Burman
authors.forename: Terry
authors.initials: T
authors.lastname: Jackson
authors.forename: Graham J
authors.initials: GJ
authors.lastname: Belsham
authors.forename: Stephen
authors.initials: S
authors.lastname: Curry
authors.forename: George P
authors.initials: GP
authors.lastname: Lomonossoff
authors.forename: Satya
authors.initials: S
authors.lastname: Parida
authors.forename: David
authors.initials: D
authors.lastname: Paton
authors.forename: Yanmin
authors.initials: Y
authors.lastname: Li
authors.forename: Ginette
authors.initials: G
authors.lastname: Wilsden
authors.forename: Nigel
authors.initials: N
authors.lastname: Ferris
authors.forename: Ray
authors.initials: R
authors.lastname: Owens
authors.forename: Abhay
authors.initials: A
authors.lastname: Kotecha
authors.forename: Elizabeth
authors.initials: E
authors.lastname: Fry
authors.forename: David I
authors.initials: DI
authors.lastname: Stuart
authors.forename: Bryan
authors.initials: B
authors.lastname: Charleston
authors.forename: Ian M
authors.initials: IM
authors.lastname: Jones
mesh_terms: D000086042:3C Viral Proteases; D000818:Animals; D001709:Biotechnology / Q000379:methods*; D002213:Capsid / Q000276:immunology*; D002460:Cell Line; D003546:Cysteine Endopeptidases / Q000096:biosynthesis* / Q000235:genetics; D015536:Down-Regulation; D029544:Foot-and-Mouth Disease Virus / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification*; D015870:Gene Expression; D007313:Insecta; D013678:Technology, Pharmaceutical / Q000379:methods*; D014764:Viral Proteins / Q000096:biosynthesis* / Q000235:genetics; D014765:Viral Vaccines / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification*
chemical_list: D014764:Viral Proteins; D014765:Viral Vaccines; D003546:Cysteine Endopeptidases; D000086042:3C Viral Proteases