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

Abstract

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 options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

Figure 1: Dissociation of FMDV capsids into pentameric assemblies and design of the models used for MD simulations.
Figure 2: Growth characteristics and stability of the engineered capsids.
Figure 3: Structural analysis of stable engineered capsids.
Figure 4: Immunogenicity of inactivated wild-type and stabilized viruses.

Accession codes

Primary accessions

Electron Microscopy Data Bank

Protein Data Bank

Referenced accessions

NCBI Reference Sequence

Protein Data Bank

References

  1. 1

    Grubman, M.J. & Baxt, B. Foot-and-mouth disease. Clin. Microbiol. Rev. 17, 465–493 (2004).

    CAS  Article  Google Scholar 

  2. 2

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

    Article  Google Scholar 

  3. 3

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

    CAS  Article  Google Scholar 

  4. 4

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

    CAS  Article  Google Scholar 

  5. 5

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

    CAS  Article  Google Scholar 

  6. 6

    Doel, T.R. & Baccarini, P.J. Thermal stability of foot-and-mouth disease virus. Arch. Virol. 70, 21–32 (1981).

    CAS  Article  Google Scholar 

  7. 7

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

    CAS  Article  Google Scholar 

  8. 8

    Hall, M.D., Knowles, N.J., Wadsworth, J., Rambaut, A. & Woolhouse, M.E.J. Reconstructing geographical movements and host species transitions of foot-and-mouth disease virus serotype SAT 2. MBio 4, e00591–13 (2013).10.1128/mBio.00591-13

  9. 9

    Basavappa, R. 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).

    CAS  Article  Google Scholar 

  10. 10

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

    CAS  Article  Google Scholar 

  11. 11

    Curry, S. 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).

    CAS  Article  Google Scholar 

  12. 12

    Curry, S. 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).

    CAS  PubMed  PubMed Central  Google Scholar 

  13. 13

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

    CAS  Article  Google Scholar 

  14. 14

    Reeve, R. 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).

    Article  Google Scholar 

  15. 15

    Ellard, F.M., Drew, J., Blakemore, W.E., Stuart, D.I. & King, A.M. 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).

    CAS  Article  Google Scholar 

  16. 16

    Mateo, R., Luna, E., Rincón, V. & Mateu, M.G. 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).

    CAS  Article  Google Scholar 

  17. 17

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

    CAS  Article  Google Scholar 

  18. 18

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

    CAS  Article  Google Scholar 

  19. 19

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

    CAS  Article  Google Scholar 

  20. 20

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

    Article  Google Scholar 

  21. 21

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

    Article  Google Scholar 

  22. 22

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

    Article  Google Scholar 

  23. 23

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

    Article  Google Scholar 

  24. 24

    Karplus, M. & McCammon, J.A. Molecular dynamics simulations of biomolecules. Nat. Struct. Biol. 9, 646–652 (2002).

    CAS  Article  Google Scholar 

  25. 25

    Klepeis, J.L., Lindorff-Larsen, K., Dror, R.O. & Shaw, D.E. Long-timescale molecular dynamics simulations of protein structure and function. Curr. Opin. Struct. Biol. 19, 120–127 (2009).

    CAS  Article  Google Scholar 

  26. 26

    Jackson, T., Sheppard, D., Denyer, M., Blakemore, W. & King, A.M. The epithelial integrin αvβ6 is a receptor for foot-and-mouth disease virus. J. Virol. 74, 4949–4956 (2000).

    CAS  Article  Google Scholar 

  27. 27

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

    CAS  Article  Google Scholar 

  28. 28

    Porta, C. 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).

    CAS  Article  Google Scholar 

  29. 29

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

    CAS  Article  Google Scholar 

  30. 30

    Harmsen, M.M., Fijten, H.P., Westra, D.F. & Coco-Martin, J.M. 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).

    CAS  Article  Google Scholar 

  31. 31

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

    CAS  Article  Google Scholar 

  32. 32

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

    CAS  Article  Google Scholar 

  33. 33

    Barnett, P.V., Statham, R.J., Vosloo, W. & Haydon, D.T. Foot-and-mouth disease vaccine potency testing: determination and statistical validation of a model using a serological approach. Vaccine 21, 3240–3248 (2003).

    CAS  Article  Google Scholar 

  34. 34

    Bolwell, C., Parry, N.R. & Rowlands, D.J. 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).

    Article  Google Scholar 

  35. 35

    Martín-Acebes, M.A., Rincon, V., Armas-Portela, R., Mateu, M.G. & Sobrino, F. 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).

    Article  Google Scholar 

  36. 36

    Mateo, R., Luna, E. & Mateu, M.G. Thermostable variants are not generally represented in foot-and-mouth disease virus quasispecies. J. Gen. Virol. 88, 859–864 (2007).

    CAS  Article  Google Scholar 

  37. 37

    Anderson, E.C., Doughty, W.J. & Spooner, P.R. 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).

    CAS  Article  Google Scholar 

  38. 38

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

    CAS  Article  Google Scholar 

  39. 39

    Ferris, N.P. 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).

    CAS  Article  Google Scholar 

  40. 40

    Ferris, N.P. & Donaldson, A.I. 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).

    Article  Google Scholar 

  41. 41

    Bøtner, A. 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).

    Article  Google Scholar 

  42. 42

    Zibert, A., Maass, G., Strebel, K., Falk, M.M. & Beck, E. Infectious foot-and-mouth disease virus derived from a cloned full-length cDNA. J. Virol. 64, 2467–2473 (1990).

    CAS  PubMed  PubMed Central  Google Scholar 

  43. 43

    van Rensburg, H.G., Henry, T.M. & Mason, P.W. 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).

    CAS  Article  Google Scholar 

  44. 44

    Brehm, K.E., Ferris, N.P., Lenk, M., Riebe, R. & Haas, B. Highly sensitive fetal goat tongue cell line for detection and isolation of foot-and-mouth disease virus. J. Clin. Microbiol. 47, 3156–3160 (2009).

    CAS  Article  Google Scholar 

  45. 45

    Rieder, E., Bunch, T., Brown, F. & Mason, P.W. Genetically engineered foot-and-mouth disease viruses with poly(C) tracts of two nucleotides are virulent in mice. J. Virol. 67, 5139–5145 (1993).

    CAS  PubMed  PubMed Central  Google Scholar 

  46. 46

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

    Article  Google Scholar 

  47. 47

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

    CAS  PubMed  PubMed Central  Google Scholar 

  48. 48

    Walter, T.S. 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).

    Article  Google Scholar 

  49. 49

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

    Article  Google Scholar 

  50. 50

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

    CAS  Article  Google Scholar 

  51. 51

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

    CAS  Article  Google Scholar 

  52. 52

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

    CAS  Article  Google Scholar 

  53. 53

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

    CAS  Article  Google Scholar 

  54. 54

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

    CAS  Article  Google Scholar 

Download references

Acknowledgements

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

Affiliations

Authors

Contributions

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.

Corresponding authors

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

Ethics declarations

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.

Integrated supplementary information

Supplementary Figure 1 Sequences and thermostability of cell culture–adapted particles at different pH values.

(a) Sequence alignment of a region of VP2 from representatives of seven FMDV serotypes (VP2 dominates the interactions at the inter-pentamer interface). The position of the α-helix found in all picornaviruses adjacent to the icosahedral 2-fold symmetry axis (residues 87-98) is shown as a red cylinder with position 93 indicated by a star (amino acid sequences are coloured using the Zappo Colour scheme in Jalview, http://www.jalview.org/). (b) The thermostability of infectious O1M wild type viruses with (HS+) and without (HS-) cell adaptation mutations remote from the pentamer interface were measured by fluorescence assay at pH 7.5. Both HS+ and HS- dissociated at 52.5˚C. (c) The engineered mutants are noticeably more thermostable at lower pH. At pH 7.0, the wild-type capsids dissociated at 41.0˚C (compared to 52.0˚C at pH 7.5, Figure 2b), S93Y dissociated at 43.5˚C, S93F at 42.5˚C, S93W at 41.5˚C, S97Q at 42.5˚C and Y98F at 43.4˚C. (d) At pH 6.5, the wild-type capsids dissociated at 30.5˚C, S93W dissociated at 34.0˚C and S93Y and S93F at 38.0˚C, whilst S97Q and Y98F dissociated at 34.0˚C.

Supplementary Figure 2 Effect of storage and heat treatment on inactivated wild-type and engineered O1M virus.

Purified inactivated O1M wild-type and S93Y mutant were analysed for intactness by negative stain EM. (a) ~10% of wild-type capsids are dissociated into pentamers when analysed soon after purification (quantified by taking the mean count of 5 independent areas on the grid), whereas S93Y particles appeared mostly intact. (b) Aliquots of the purified particles were stored at 4˚C and analysed again after ten days. ~80% of the wild-type capsids are dissociated into pentamers, whilst only ~10% dissociation is observed for the S93Y mutant (c) When incubated at 37˚C, wild-type O1M capsids readily dissociated into pentamers. After 20 min only ~5% capsids remained intact (taking the mean count of 5 independent areas on the grid compared to a similar mean count at time 0). (d) In contrast, S93Y mutant capsids were more resistant to heat treatment and ~60% capsids remained intact after 20 min. The scale bar indicates 100nm. (e) To further test the effect of storage, infectious O1M wild-type and mutant S93Y capsids were incubated at 37˚C for up to 72 h and aliquots analysed at 12, 24, 48 and 72 h post incubation by ELISA using single domain llama antibodies that specifically detect intact capsids (146S) or dissociated pentamers. Results are expressed as % of 146S particles relative to the intact antigen (starting antigen). After 72 h, ~50% mutant capsids remained intact whereas for wild-type ~15% capsids remained whole.

Supplementary Figure 3 Indirect sandwich ELISA for detection of FMDV O1Manisa.

The guinea-pig polyclonal antisera to FMDV O1M used in this study was generated by immunization with inactivated, purified 146S (FMD) virus particles in Freund’s Complete as shown previously (Ferris and Donaldson, 1984) and used in an indirect sandwich ELISA for FMDV antigen detection within the FAO World Reference Laboratory for Foot and Mouth Disease (WRL for FMD). Shown is an ELISA (Ferris et al., 2005) using a rabbit anti-FMDV type-O polyclonal serum as a trapping reagent for detection of a dilution series of inactivated, purified FMDV O1Manisa using the guinea-pig polyclonal antisera to O1Manisa as the primary antibody. Similar results were obtained for guinea-pig polyclonal antisera against FMDV A22.

Supplementary Figure 4 Thermostability of engineered O1M recombinant empty capsids.

Recombinant S93F empty capsids were heated to 56˚C for 2 h and sedimented over a 15-45% sucrose gradient. The peak fraction was analysed by EM. (a) The empty capsids were found to be intact, further confirming their improved stability. The scale bar indicates 100nm. (b) High magnification image showing that ~90% capsids are intact. The scale bar indicates 50nm.

Supplementary Figure 5 Thermostability of engineered A22 recombinant empty capsids.

Purified capsids were left untreated or heated to 56˚C for 2 h and sedimented on 15-45% sucrose density gradients. Fractions were taken from the bottom of the gradients and analysed by western blot. Capsid proteins were detected using an anti-FMDV A22 polyclonal antibody. Heated H93F capsids remained intact and migrated to fractions 3-4, as did untreated capsids, whereas wild-type capsids dissociated upon heating, remaining near the top of the gradient.

Supplementary Figure 6 Stability of engineered O1M recombinant empty capsids upon storage at 37 °C.

O1M wild-type and S93F empty capsids were incubated at 37˚C for 24 h or 96 h followed by sedimentation on 15-45% sucrose density gradients. Fractions were taken from the bottom of the gradients and analysed by western blot. Capsid proteins were detected using an anti-FMDV O1M polyclonal antibody. Wild-type capsids readily dissociated and remained near the top of the gradient, whereas mutant S93F capsids remained intact, even after 96 h.

Supplementary Figure 7 Cryo-EM analysis of stabilized FMDV particles.

Purified inactivated O1M and SAT2 S93Y particles were used for data collection by cryoEM. (a) and (b) Representative aligned average (motion corrected) image of O1M VP2 S93Y particles and the corresponding Fourier transform. (c) and (d) Representative aligned average (motion corrected) image of SAT2 VP2 S93Y particles and the corresponding Fourier transform. (e) and (f) FSC curves of the final 3D reconstruction obtained using gold-standard refinement using RELION, marked with the resolution corresponding to a Fourier shell correlation (FSC) of 0.5 and 0.143. The scale bar indicates 100nm.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–7, Supplementary Tables 1–3 and Supplementary Note (PDF 1948 kb)

Supplementary Data Set 1

Full length SDS-PAGE gels from truncated panels of Figure 2c (PDF 3209 kb)

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Kotecha, A., Seago, J., Scott, K. et al. Structure-based energetics of protein interfaces guides foot-and-mouth disease virus vaccine design. Nat Struct Mol Biol 22, 788–794 (2015). https://doi.org/10.1038/nsmb.3096

Download citation

Further reading

Search

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing