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.
Highlights
Foot-and-mouth disease virus (FMDV) is the prototypic aphthovirus within the family Picornaviridae (reviewed by (Grubman and Baxt, 2004))
A Bombyx baculovirus system encoding a P1-2A-3C sequence of an FMDV Asia 1 isolate was used as a single transcription unit driven by the polyhedrin promoter and the empty capsid material harvested from the haemolymph of the infected silk worms was immunogenic in cattle and led to levels of neutralising antibody associated with protection (Li et al, 2008, 2011)
Previous approaches to recombinant picornavirus particle synthesis in insect cells have included the expression of the complete picornaviruses genomes (Brautigam et al, 1993; Rosen et al, 1993) as well as co-expression of only the structural precursor protein P1 and protease, but the levels of capsid obtained have been variable (Cao et al, 2010; Ko et al, 2005; Li et al, 2008, 2011; Oem et al, 2007; Roosien et al, 1990)
Summary
Foot-and-mouth disease virus (FMDV) is the prototypic aphthovirus within the family Picornaviridae (reviewed by (Grubman and Baxt, 2004)). A Bombyx (silk worm) baculovirus system encoding a P1-2A-3C sequence of an FMDV Asia 1 isolate was used as a single transcription unit driven by the polyhedrin promoter and the empty capsid material harvested from the haemolymph of the infected silk worms was immunogenic in cattle and led to levels of neutralising antibody associated with protection (Li et al, 2008, 2011) Despite these successes, variation associated with both the FMDV serotype and the host cell background used mean that a uniform genetic design capable of producing empty capsids for any serotype has yet to be reported. A new genetic design is described for the expression of empty FMDV capsids in insect cells following infection by recombinant baculoviruses expressing P1-2A-3C with a number of modifications to reduce 3C activity
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