Article | Published:

Structure-based energetics of protein interfaces guides foot-and-mouth disease virus vaccine design

Nature Structural & Molecular Biology volume 22, pages 788794 (2015) | Download Citation


Virus capsids are primed for disassembly, yet capsid integrity is key to generating a protective immune response. Foot-and-mouth disease virus (FMDV) capsids comprise identical pentameric protein subunits held together by tenuous noncovalent interactions and are often unstable. Chemically inactivated or recombinant empty capsids, which could form the basis of future vaccines, are even less stable than live virus. Here we devised a computational method to assess the relative stability of protein-protein interfaces and used it to design improved candidate vaccines for two poorly stable, but globally important, serotypes of FMDV: O and SAT2. We used a restrained molecular dynamics strategy to rank mutations predicted to strengthen the pentamer interfaces and applied the results to produce stabilized capsids. Structural analyses and stability assays confirmed the predictions, and vaccinated animals generated improved neutralizing-antibody responses to stabilized particles compared to parental viruses and wild-type capsids.

Access optionsAccess options

Rent or Buy article

Get time limited or full article access on ReadCube.


All prices are NET prices.


Primary accessions

Electron Microscopy Data Bank

Referenced accessions

NCBI Reference Sequence


  1. 1.

    & Foot-and-mouth disease. Clin. Microbiol. Rev. 17, 465–493 (2004).

  2. 2.

    et al. The 2010 foot-and-mouth disease epidemic in Japan. J. Vet. Med. Sci. 74, 399–404 (2012).

  3. 3.

    et al. Characterization of the recent outbreak of foot-and-mouth disease virus serotype SAT2 in Egypt. Arch. Virol. 158, 619–627 (2013).

  4. 4.

    Foot-and-mouth disease. Annu. Rev. Microbiol. 22, 201–244 (1968).

  5. 5.

    FMD vaccines. Virus Res. 91, 81–99 (2003).

  6. 6.

    & Thermal stability of foot-and-mouth disease virus. Arch. Virol. 70, 21–32 (1981).

  7. 7.

    & Comparative immunogenicity of 146S, 75S and 12S particles of foot-and-mouth disease virus. Arch. Virol. 73, 185–191 (1982).

  8. 8.

    , , , & Reconstructing geographical movements and host species transitions of foot-and-mouth disease virus serotype SAT 2. MBio 4, e00591–13 (2013).

  9. 9.

    et al. Role and mechanism of the maturation cleavage of VP0 in poliovirus assembly: structure of the empty capsid assembly intermediate at 2.9 Å resolution. Protein Sci. 3, 1651–1669 (1994).

  10. 10.

    et al. The three-dimensional structure of foot-and-mouth disease virus at 2.9 A resolution. Nature 337, 709–716 (1989).

  11. 11.

    et al. 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 (1996).

  12. 12.

    et al. 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 (1997).

  13. 13.

    et al. The structure and antigenicity of a type C foot-and-mouth disease virus. Structure 2, 123–139 (1994).

  14. 14.

    et al. Sequence-based prediction for vaccine strain selection and identification of antigenic variability in foot-and-mouth disease virus. PLoS Comput. Biol. 6, e1001027 (2010).

  15. 15.

    , , , & 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 (1999).

  16. 16.

    , , & Engineering viable foot-and-mouth disease viruses with increased thermostability as a step in the development of improved vaccines. J. Virol. 82, 12232–12240 (2008).

  17. 17.

    et al. Rational engineering of recombinant picornavirus capsids to produce safe, protective vaccine antigen. PLoS Pathog. 9, e1003255 (2013).

  18. 18.

    et al. The Protein Data Bank. Nucleic Acids Res. 28, 235–242 (2000).

  19. 19.

    et al. A sensor-adaptor mechanism for enterovirus uncoating from structures of EV71. Nat. Struct. Mol. Biol. 19, 424–429 (2012).

  20. 20.

    et al. Picornavirus uncoating intermediate captured in atomic detail. Nat. Commun. 4, 1929 (2013).

  21. 21.

    , & Cryo-electron microscopy reconstruction shows poliovirus 135S particles poised for membrane interaction and RNA release. J. Virol. 88, 1758–1770 (2014).

  22. 22.

    , & Methods used in the structure determination of foot-and-mouth disease virus. Acta Crystallogr. A 49, 45–55 (1993).

  23. 23.

    & Coot: model-building tools for molecular graphics. Acta Crystallogr. D Biol. Crystallogr. 60, 2126–2132 (2004).

  24. 24.

    & Molecular dynamics simulations of biomolecules. Nat. Struct. Biol. 9, 646–652 (2002).

  25. 25.

    , , & Long-timescale molecular dynamics simulations of protein structure and function. Curr. Opin. Struct. Biol. 19, 120–127 (2009).

  26. 26.

    , , , & The epithelial integrin αvβ6 is a receptor for foot-and-mouth disease virus. J. Virol. 74, 4949–4956 (2000).

  27. 27.

    et al. The structure and function of a foot-and-mouth disease virus-oligosaccharide receptor complex. EMBO J. 18, 543–554 (1999).

  28. 28.

    et al. Efficient production of foot-and-mouth disease virus empty capsids in insect cells following down regulation of 3C protease activity. J. Virol. Methods 187, 406–412 (2013).

  29. 29.

    et al. A plate-based high-throughput assay for virus stability and vaccine formulation. J. Virol. Methods 185, 166–170 (2012).

  30. 30.

    , , & Effect of thiomersal on dissociation of intact (146S) foot-and-mouth disease virions into 12S particles as assessed by novel ELISAs specific for either 146S or 12S particles. Vaccine 29, 2682–2690 (2011).

  31. 31.

    et al. In situ macromolecular crystallography using microbeams. Acta Crystallogr. D Biol. Crystallogr. 68, 592–600 (2012).

  32. 32.

    & Deconvolution of fully overlapped reflections from crystals of foot-and-mouth disease virus O1 G67. Acta Crystallogr. D Biol. Crystallogr. 51, 160–167 (1995).

  33. 33.

    , , & Foot-and-mouth disease vaccine potency testing: determination and statistical validation of a model using a serological approach. Vaccine 21, 3240–3248 (2003).

  34. 34.

    , & Comparison between in vitro neutralization titres and in vivo protection against homologous and heterologous challenge induced by vaccines prepared from two serologically distinct variants of foot-and-mouth disease virus, serotype A22. J. Gen. Virol. 73, 727–731 (1992).

  35. 35.

    , , , & A single amino acid substitution in the capsid of foot-and-mouth disease virus can increase acid lability and confer resistance to acid-dependent uncoating inhibition. J. Virol. 84, 2902–2912 (2010).

  36. 36.

    , & Thermostable variants are not generally represented in foot-and-mouth disease virus quasispecies. J. Gen. Virol. 88, 859–864 (2007).

  37. 37.

    , & Variation in the thermal stability of isolates of foot-and-mouth disease type SAT 2 and its significance in the selection of vaccine strains. J. Comp. Pathol. 92, 495–507 (1982).

  38. 38.

    et al. EMAN2: an extensible image processing suite for electron microscopy. J. Struct. Biol. 157, 38–46 (2007).

  39. 39.

    et al. Utility of recombinant integrin αvβ6 as a capture reagent in immunoassays for the diagnosis of foot-and-mouth disease. J. Virol. Methods 127, 69–79 (2005).

  40. 40.

    & Serological response of guinea pigs to inactivated 146S antigens of foot and mouth disease virus after single or repeated inoculations. Rev. Sci. Tech. Off. Int. Epiz 3, 563–574 (1984).

  41. 41.

    et al. Capsid proteins from field strains of foot-and-mouth disease virus confer a pathogenic phenotype in cattle on an attenuated, cell-culture-adapted virus. J. Gen. Virol. 92, 1141–1151 (2011).

  42. 42.

    , , , & Infectious foot-and-mouth disease virus derived from a cloned full-length cDNA. J. Virol. 64, 2467–2473 (1990).

  43. 43.

    , & Studies of genetically defined chimeras of a European type A virus and a South African Territories type 2 virus reveal growth determinants for foot-and-mouth disease virus. J. Gen. Virol. 85, 61–68 (2004).

  44. 44.

    , , , & Highly sensitive fetal goat tongue cell line for detection and isolation of foot-and-mouth disease virus. J. Clin. Microbiol. 47, 3156–3160 (2009).

  45. 45.

    , , & Genetically engineered foot-and-mouth disease viruses with poly(C) tracts of two nucleotides are virulent in mice. J. Virol. 67, 5139–5145 (1993).

  46. 46.

    et al. A versatile ligation-independent cloning method suitable for high-throughput expression screening applications. Nucleic Acids Res. 35, e45 (2007).

  47. 47.

    et al. Viral RNA modulates the acid sensitivity of foot-and-mouth disease virus capsids. J. Virol. 69, 430–438 (1995).

  48. 48.

    et al. 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 (2005).

  49. 49.

    et al. Crystallography & NMR system: a new software suite for macromolecular structure determination. Acta Crystallogr. D Biol. Crystallogr. 54, 905–921 (1998).

  50. 50.

    et al. Electron counting and beam-induced motion correction enable near-atomic-resolution single-particle cryo-EM. Nat. Methods 10, 584–590 (2013).

  51. 51.

    RELION: implementation of a Bayesian approach to cryo-EM structure determination. J. Struct. Biol. 180, 519–530 (2012).

  52. 52.

    et al. UCSF Chimera: a visualization system for exploratory research and analysis. J. Comput. Chem. 25, 1605–1612 (2004).

  53. 53.

    et al. REFMAC5 for the refinement of macromolecular crystal structures. Acta Crystallogr. D Biol. Crystallogr. 67, 355–367 (2011).

  54. 54.

    et al. An atomic model of brome mosaic virus using direct electron detection and real-space optimization. Nat. Commun. 5, 4808 (2014).

Download references


We thank the World Reference Laboratory for VNT determination; beamline staff at the Diamond Light Source for assistance; J. Dong and J. Diprose for computing support; P. Afonine and G. Murshudov for advice on Phenix and Refmac; K. Harlos and T. Walter for help with crystallography; D. Goovaerts and E. Rieder for helpful discussions; and B. Haas (Friedrich-Loeffler-Institut) for providing ZZ_R 127 cells. We are grateful to the Wellcome Trust (WT) for a Translation Award to fund this work (grant no. 089755 to B.C., E.E.F., T.J. and F.F.M.). T.J. and B.C. were funded by the Biotechnology and Biological Sciences Research Council Institute Strategic Programme on Livestock Viral Diseases at The Pirbright Institute. The Oxford Particle Imaging Centre electron microscopy facility was founded by a WT Joint Infrastructure Fund award (060208/Z/00/Z to D.I.S.) and is supported by a WT equipment grant (093305/Z/10/Z to K. Grünewald). The WT, UK Medical Research Council (MRC) and Biotechnology and Biology Research Council also support the National EM facility, which provided the K2 detector. B.C. and D.I.S. are supported as Jenner investigators, J.R. and A.K. are WT supported, and E.E.F. and D.I.S. are supported by the UK MRC (grant no. G100099 to D.I.S.). The work of the WT Centre in Oxford is supported by the WT core award 090532/Z/09/Z.

Author information

Author notes

    • Abhay Kotecha
    •  & Julian Seago

    These authors contributed equally to this work.


  1. Division of Structural Biology, University of Oxford, Oxford, UK.

    • Abhay Kotecha
    • , Jingshan Ren
    • , Claudine Porta
    • , Helen M Ginn
    • , C Alistair Siebert
    • , Juha T Huiskonen
    • , Robert M Esnouf
    • , Elizabeth E Fry
    •  & David I Stuart
  2. Pirbright Institute, Pirbright, UK.

    • Julian Seago
    • , Alison Burman
    • , Claudine Porta
    • , Terry Jackson
    • , Eva Perez-Martin
    •  & Bryan Charleston
  3. Transboundary Animal Disease Programme, Agricultural Research Council-Onderstepoort Veterinary Institute, Onderstepoort, South Africa.

    • Katherine Scott
    •  & Francois F Maree
  4. Animal and Microbial Sciences, University of Reading, Reading, UK.

    • Silvia Loureiro
    •  & Ian M Jones
  5. Merck Sharp & Dohme Animal Health, Cologne, Germany.

    • Guntram Paul
  6. Department of Microbiology and Plant Pathology, University of Pretoria, Pretoria, South Africa.

    • Francois F Maree
  7. Diamond Light Source, Didcot, UK.

    • David I Stuart


  1. Search for Abhay Kotecha in:

  2. Search for Julian Seago in:

  3. Search for Katherine Scott in:

  4. Search for Alison Burman in:

  5. Search for Silvia Loureiro in:

  6. Search for Jingshan Ren in:

  7. Search for Claudine Porta in:

  8. Search for Helen M Ginn in:

  9. Search for Terry Jackson in:

  10. Search for Eva Perez-Martin in:

  11. Search for C Alistair Siebert in:

  12. Search for Guntram Paul in:

  13. Search for Juha T Huiskonen in:

  14. Search for Ian M Jones in:

  15. Search for Robert M Esnouf in:

  16. Search for Elizabeth E Fry in:

  17. Search for Francois F Maree in:

  18. Search for Bryan Charleston in:

  19. Search for David I Stuart in:


A.K., E.E.F., R.M.E. and D.I.S. developed MD-simulation protocols; A.K., J.S., K.S., A.B., S.L. and C.P. prepared samples; J.R., H.M.G., J.T.H., E.P.-M., G.P., C.A.S., F.F.M. and E.E.F. assisted in research; A.K., J.S., F.F.M., E.E.F., T.J., I.M.J., R.M.E., D.I.S. and B.C. designed the study; all authors analyzed data; and A.K., J.S., E.E.F., B.C. and D.I.S. wrote the manuscript.

Competing interests

A number of the stabilizing mutations are patented (patent no. WO 2014154655 A1, patent holders: R.M.E., E.E.F., A.K. and D.I.S.), and work on a VLP-based stabilized vaccine antigen is ongoing at Intervet (MSD Animal Health), in collaboration with the authors (except that F.F.M. and K.S. are not part of this ongoing work). This work was not funded by MSD Animal Health but by a Wellcome Trust Translation Award.

Corresponding authors

Correspondence to Francois F Maree or Bryan Charleston or David I Stuart.

Integrated supplementary information

Supplementary information

PDF files

  1. 1.

    Supplementary Text and Figures

    Supplementary Figures 1–7, Supplementary Tables 1–3 and Supplementary Note

  2. 2.

    Supplementary Data Set 1

    Full length SDS-PAGE gels from truncated panels of Figure 2c

About this article

Publication history