Foot-and-mouth disease remains a major plague of livestock and outbreaks are often economically catastrophic. Current inactivated virus vaccines require expensive high containment facilities for their production and maintenance of a cold-chain for their activity. We have addressed both of these major drawbacks. Firstly we have developed methods to efficiently express recombinant empty capsids. Expression constructs aimed at lowering the levels and activity of the viral protease required for the cleavage of the capsid protein precursor were used; this enabled the synthesis of empty A-serotype capsids in eukaryotic cells at levels potentially attractive to industry using both vaccinia virus and baculovirus driven expression. Secondly we have enhanced capsid stability by incorporating a rationally designed mutation, and shown by X-ray crystallography that stabilised and wild-type empty capsids have essentially the same structure as intact virus. Cattle vaccinated with recombinant capsids showed sustained virus neutralisation titres and protection from challenge 34 weeks after immunization. This approach to vaccine antigen production has several potential advantages over current technologies by reducing production costs, eliminating the risk of infectivity and enhancing the temperature stability of the product. Similar strategies that will optimize host cell viability during expression of a foreign toxic gene and/or improve capsid stability could allow the production of safe vaccines for other pathogenic picornaviruses of humans and animals.
References
[1]
Basavappa R, Syed R, Flore O, Icenogle JP, Filman DJ, et al. (1994) Role and mechanism of the maturation cleavage of VP0 in poliovirus assembly: structure of the empty capsid assembly intermediate at 2.9 A resolution. Protein Sci 3: 1651–1669. doi: 10.1002/pro.5560031005
[2]
Curry S, Fry E, Blakemore W, Abu-Ghazaleh R, Jackson T, et al. (1997) Dissecting the roles of VP0 cleavage and RNA packaging in picornavirus capsid stabilization: the structure of empty capsids of foot-and-mouth disease virus. J Virol 71: 9743–9752.
[3]
Acharya R, Fry E, Stuart D, Fox G, Rowlands D, et al. (1989) The three-dimensional structure of foot-and-mouth disease virus at 2.9 ？ resolution. Nature 337: 709–716. doi: 10.1038/337709a0
[4]
Ellard FM, Drew J, Blakemore WE, Stuart DI, King AM (1999) Evidence for the role of His-142 of protein 1C in the acid-induced disassembly of foot-and-mouth disease virus capsids. J Gen Virol 80: 1911–1918.
[5]
Hogle JM (2002) Poliovirus cell entry: common structural themes in viral cell entry pathways. Annu Rev Microbiol 56: 677–702. doi: 10.1146/annurev.micro.56.012302.160757
[6]
Wang X, Peng W, Ren J, Hu Z, Xu J, et al. (2012) A sensor-adaptor mechanism for enterovirus uncoating from structures of EV71. Nat Struct Mol Biol 19: 424–429. doi: 10.1038/nsmb.2255
Ansardi DC, Porter DC, Morrow CD (1991) Coinfection with recombinant vaccinia viruses expressing poliovirus P1 and P3 proteins results in polyprotein processing and formation of empty capsid structures. J Virol 65: 2088–2092.
[9]
Abrams CC, King AM, Belsham GJ (1995) Assembly of foot-and-mouth disease virus empty capsids synthesized by a vaccinia virus expression system. J Gen Virol 76: 3089–3098. doi: 10.1099/0022-1317-76-12-3089
[10]
Roosien J, Belsham GJ, Ryan MD, King AM, Vlak JM (1990) Synthesis of foot-and-mouth disease virus capsid proteins in insect cells using baculovirus expression vectors. J Gen Virol 71: 1703–1711. doi: 10.1099/0022-1317-71-8-1703
[11]
Fry EE, Newman JW, Curry S, Najjam S, Jackson T, et al. (2005) Structure of toot-and-mouth disease virus serotype A10 61 alone and complexed with oligosaccharide receptor: receptor conservation in the face of antigenic variation. J Gen Virol 86: 1909–1920. doi: 10.1099/vir.0.80730-0
[12]
Lea S, Hernandez J, Blakemore W, Brocchi E, Curry S, et al. (1994) The structure and antigenicity of a type C foot-and-mouth disease virus. Structure 2: 123–139. doi: 10.1016/s0969-2126(00)00014-9
[13]
Mateo R, Luna E, Rincon V, Mateu MG (2008) Engineering viable foot-and-mouth disease viruses with increased thermostability as a step in the development of improved vaccines. J Virol 82: 12232–12240. doi: 10.1128/jvi.01553-08
[14]
King AM, Burman, A, Audonnet, JC Antonie, MF (2009) Vaccine against foot-and-mouth disease. US Patent No7,331,182 B2.
[15]
Lopez-Lastra M, Rivas A, Barria MI (2005) Protein synthesis in eukaryotes: the growing biological relevance of cap-independent translation initiation. Biol Res 38: 121–146. doi: 10.4067/s0716-97602005000200003
[16]
Fuerst TR, Niles EG, Studier FW, Moss B (1986) Eukaryotic transient-expression system based on recombinant vaccinia virus that synthesizes bacteriophage T7 RNA polymerase. Proc Natl Acad Sci USA 83: 8122–8126. doi: 10.1073/pnas.83.21.8122
[17]
Drugmand JC, Schneider YJ, Agathos SN (2012) Insect cells as factories for biomanufacturing. Biotechnol Adv 30: 1140–1157. doi: 10.1016/j.biotechadv.2011.09.014
[18]
Berrow NS, Alderton D, Sainsbury S, Nettleship J, Assenberg R, et al. (2007) A versatile ligation-independent cloning method suitable for high-throughput expression screening applications. Nucleic Acids Res 35: e45. doi: 10.1093/nar/gkm047
[19]
Porta C, Xu X, Loureiro S, Paramasivam S, Ren J, et al. (2013) Efficient production of foot-and-mouth disease virus empty capsids in insect cells following downregulation of 3C protease activity. J Virol Methods 187: 406–412. doi: 10.1016/j.jviromet.2012.11.011
[20]
Axford D, Owen RL, Aishima J, Foadi J, Morgan AW, et al. (2012) In situ macromolecular crystallography using microbeams. Acta Crystallogr D Biol Crystallogr 68: 592–600. doi: 10.1107/s0907444912006749
[21]
Curry S, Fry E, Blakemore W, Abu-Ghazaleh R, Jackson T, et al. (1996) Perturbations in the surface structure of A22 Iraq foot-and-mouth disease virus accompanying coupled changes in host cell specificity and antigenicity. Structure 4: 135–145. doi: 10.1016/s0969-2126(96)00017-2
[22]
Cox SJ, Carr BV, Parida S, Hamblin PA, Prentice H, et al. (2010) Longevity of protection in cattle following immunisation with emergency FMD A22 serotype vaccine from the UK strategic reserve. Vaccine 28: 2318–2322. doi: 10.1016/j.vaccine.2009.12.065
[23]
Wikoff WR, Liljas L, Duda RL, Tsuruta H, Hendrix RW, et al. (2000) Topologically linked protein rings in the bacteriophage HK97 capsid. Science 289: 2129–2133. doi: 10.1126/science.289.5487.2129
[24]
Delamarre L, Couture R, Mellman I, Trombetta ES (2006) Enhancing immunogenicity by limiting susceptibility to lysosomal proteolysis. J Exp Med 203: 2049–2055. doi: 10.1084/jem.20052442
[25]
Ohkuri T, Nagatomo S, Oda K, So T, Imoto T, et al. (2010) A protein's conformational stability is an immunologically dominant factor: evidence that free-energy barriers for protein unfolding limit the immunogenicity of foreign proteins. J Immunol 185: 4199–4205. doi: 10.4049/jimmunol.0902249
Emsley P, Cowtan K (2004) Coot: model-building tools for molecular graphics. Acta Crystallogr D Biol Crystallogr 60: 2126–2132. doi: 10.1107/s0907444904019158
[28]
Walter TS, Diprose JM, Mayo CJ, Siebold C, Pickford MG, et al. (2005) A procedure for setting up high-throughput nanolitre crystallization experiments. Crystallization workflow for initial screening, automated storage, imaging and optimization. Acta Crystallogr D Biol Crystallogr 61: 651–657. doi: 10.1107/s0907444905007808
[29]
Brunger AT, Adams PD, Clore GM, DeLano WL, Gros P, et al. (1998) Crystallography & NMR system: A new software suite for macromolecular structure determination. Acta Crystallogr D Biol Crystallogr 54: 905–921. doi: 10.1107/s0907444998003254
[30]
Reid SM, Ebert K, Bachanek-Bankowska K, Batten C, Sanders A, et al. (2009) Performance of real-time reverse transcription polymerase chain reaction for the detection of foot-and-mouth disease virus during field outbreaks in the United Kingdom in 2007. J Vet Diagn Invest 21: 321–330. doi: 10.1177/104063870902100303
