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Recognition determinants of broadly neutralizing human antibodies against dengue viruses

Abstract

Dengue disease is caused by four different flavivirus1 serotypes, which infect 390 million people yearly with 25% symptomatic cases2 and for which no licensed vaccine is available. Recent phase III vaccine trials showed partial protection, and in particular no protection for dengue virus serotype 2 (refs 3, 4). Structural studies so far have characterized only epitopes recognized by serotype-specific human antibodies5,6. We recently isolated human antibodies potently neutralizing all four dengue virus serotypes7. Here we describe the X-ray structures of four of these broadly neutralizing antibodies in complex with the envelope glycoprotein E from dengue virus serotype 2, revealing that the recognition determinants are at a serotype-invariant site at the E-dimer interface, including the exposed main chain of the E fusion loop8 and the two conserved glycan chains. This ‘E-dimer-dependent epitope’ is also the binding site for the viral glycoprotein prM during virus maturation in the secretory pathway of the infected cell9, explaining its conservation across serotypes and highlighting an Achilles’ heel of the virus with respect to antibody neutralization. These findings will be instrumental for devising novel immunogens to protect simultaneously against all four serotypes of dengue virus.

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Figure 1: DENV-2 sE in complex with four EDE bnAbs.
Figure 2: Comparison of paratopes.
Figure 3: Exposed main-chain atoms in the epitope.

Accession codes

Primary accessions

GenBank/EMBL/DDBJ

Protein Data Bank

Data deposits

Coordinates and structure factor amplitudes have been deposited in the Protein Data Bank under accession numbers 4UTC, 4UTA, 4UT9, 4UTB and 4UT6 respectively for the structures of DENV-2 sE unliganded and in complex with EDE1 C8, EDE1 C10, EDE2 A11 and EDE2 B7, and 4UT7 for the structure of the unliganded scFv of EDE2 A11. The sequence of prM/sE fragment from Den2_FGA-02 has been deposited in GenBank under accession number KM087965. Patent application (UK 1413086.8) was deposited.

References

  1. Lindenbach, B., Thiel, H. & Rice, C. Flaviviridae: the Viruses and their Replication 5th edn, Vol. 1, 1101–1152 (Lippincott Williams & Wilkins, 2007)

    Google Scholar 

  2. Bhatt, S. et al. The global distribution and burden of dengue. Nature 496, 504–507 (2013)

    ADS  CAS  Article  PubMed  PubMed Central  Google Scholar 

  3. Capeding, M. R. et al. Clinical efficacy and safety of a novel tetravalent dengue vaccine in healthy children in Asia: a phase 3, randomised, observer-masked, placebo-controlled trial. Lancet 384, 1358–1365 (2014)

    CAS  PubMed  Google Scholar 

  4. Normile, D. Tropical diseases. Dengue vaccine trial poses public health quandary. Science 345, 367–368 (2014)

    ADS  CAS  PubMed  Google Scholar 

  5. Fibriansah, G. et al. A potent anti-dengue human antibody preferentially recognizes the conformation of E protein monomers assembled on the virus surface. EMBO Mol. Med. 6, 358–371 (2014)

    CAS  PubMed  PubMed Central  Google Scholar 

  6. Teoh, E. P. et al. The structural basis for serotype-specific neutralization of dengue virus by a human antibody. Sci. Translat. Med. 4, 139–183 (2012)

    Google Scholar 

  7. Dejnirattisai, W. et al. A new class of highly potent broadly neutralizing antibodies isolated from dengue viremic patients. Nature Immunol. http://dx.doi.org/10.1038/ni.3058 (15, December 2014)

  8. Modis, Y., Ogata, S., Clements, D. & Harrison, S. C. A ligand-binding pocket in the dengue virus envelope glycoprotein. Proc. Natl Acad. Sci. USA 100, 6986–6991 (2003)

    ADS  CAS  PubMed  PubMed Central  Google Scholar 

  9. Yu, I. M. et al. Structure of the immature dengue virus at low pH primes proteolytic maturation. Science 319, 1834–1837 (2008)

    ADS  CAS  PubMed  Google Scholar 

  10. Kuhn, R. J. et al. Structure of dengue virus: implications for flavivirus organization, maturation, and fusion. Cell 108, 717–725 (2002)

    CAS  PubMed  PubMed Central  Google Scholar 

  11. Zhang, X. et al. Cryo-EM structure of the mature dengue virus at 3.5-A resolution. Nature Struct. Mol. Biol. 20, 105–110 (2013)

    Google Scholar 

  12. Zhang, Y. et al. Conformational changes of the flavivirus E glycoprotein. Structure 12, 1607–1618 (2004)

    CAS  PubMed  PubMed Central  Google Scholar 

  13. Cockburn, J. J. et al. Structural insights into the neutralization mechanism of a higher primate antibody against dengue virus. EMBO J. 31, 767–779 (2012)

    CAS  PubMed  Google Scholar 

  14. Rey, F. A., Heinz, F. X., Mandl, C., Kunz, C. & Harrison, S. C. The envelope glycoprotein from tick-borne encephalitis virus at 2 Å resolution. Nature 375, 291–298 (1995)

    ADS  CAS  PubMed  Google Scholar 

  15. Lawrence, M. C. & Colman, P. M. Shape complementarity at protein/protein interfaces. J. Mol. Biol. 234, 946–950 (1993)

    CAS  PubMed  Google Scholar 

  16. Cockburn, J. J. et al. Mechanism of dengue virus broad cross-neutralization by a monoclonal antibody. Structure 20, 303–314 (2012)

    CAS  PubMed  Google Scholar 

  17. Lok, S. M. et al. Binding of a neutralizing antibody to dengue virus alters the arrangement of surface glycoproteins. Nature Struct. Mol. Biol. 15, 312–317 (2008)

    CAS  Google Scholar 

  18. Rodenhuis-Zybert, I. A. et al. A fusion-loop antibody enhances the infectious properties of immature flavivirus particles. J. Virol. 85, 11800–11808 (2011)

    CAS  PubMed  PubMed Central  Google Scholar 

  19. Zhang, Y. et al. Structures of immature flavivirus particles. EMBO J. 22, 2604–2613 (2003)

    CAS  PubMed  PubMed Central  Google Scholar 

  20. Kostyuchenko, V. A., Zhang, Q., Tan, J. L., Ng, T. S. & Lok, S. M. Immature and mature dengue serotype 1 virus structures provide insight into the maturation process. J. Virol. 87, 7700–7707 (2013)

    CAS  PubMed  PubMed Central  Google Scholar 

  21. Li, L. et al. The flavivirus precursor membrane-envelope protein complex: structure and maturation. Science 319, 1830–1834 (2008)

    ADS  CAS  PubMed  Google Scholar 

  22. Plevka, P. et al. Maturation of flaviviruses starts from one or more icosahedrally independent nucleation centres. EMBO Rep. 12, 602–606 (2011)

    CAS  PubMed  PubMed Central  Google Scholar 

  23. Plevka, P., Battisti, A. J., Sheng, J. & Rossmann, M. G. Mechanism for maturation-related reorganization of flavivirus glycoproteins. J. Struct. Biol. 185, 27–31 (2014)

    CAS  PubMed  Google Scholar 

  24. Changeux, J. P. & Edelstein, S. Conformational selection or induced fit? 50 years of debate resolved. F1000 Biol. Rep. 3, 19 (2011)

    PubMed  PubMed Central  Google Scholar 

  25. de Alwis, R. et al. Identification of human neutralizing antibodies that bind to complex epitopes on dengue virions. Proc. Natl Acad. Sci. USA 109, 7439–7444 (2012)

    ADS  CAS  PubMed  PubMed Central  Google Scholar 

  26. Kaufmann, B. et al. Neutralization of West Nile virus by cross-linking of its surface proteins with Fab fragments of the human monoclonal antibody CR4354. Proc. Natl Acad. Sci. USA 107, 18950–18955 (2010)

    ADS  CAS  PubMed  PubMed Central  Google Scholar 

  27. Fibriansah, G. et al. Structural changes of dengue virus when exposed to 37°C. J. Virol. 87, 7585–7592 (2013)

    CAS  PubMed  PubMed Central  Google Scholar 

  28. Zhang, X. et al. Dengue structure differs at the temperatures of its human and mosquito hosts. Proc. Natl Acad. Sci. USA 110, 6795–6799 (2013)

    ADS  CAS  PubMed  PubMed Central  Google Scholar 

  29. Jarmer, J. et al. Variation of the specificity of the human antibody responses after tick-borne encephalitis virus infection and vaccination. J. Virol. 88, 13845–13857 (2014)

    PubMed  PubMed Central  Google Scholar 

  30. McLellan, J. S. et al. Structure-based design of a fusion glycoprotein vaccine for respiratory syncytial virus. Science 342, 592–598 (2013)

    ADS  CAS  PubMed  PubMed Central  Google Scholar 

  31. Gilmartin, A. A. et al. High-level secretion of recombinant monomeric murine and human single-chain Fv antibodies from Drosophila S2 cells. Protein Eng. Des. Sel. 25, 59–66 (2012)

    CAS  PubMed  Google Scholar 

  32. Backovic, M. et al. Efficient method for production of high yields of Fab fragments in Drosophila S2 cells. Protein Eng. Des. Sel. 23, 169–174 (2010)

    CAS  PubMed  Google Scholar 

  33. Kabsch, W. XDS. Acta Crystallogr. D 66, 125–132 (2010)

    CAS  PubMed  PubMed Central  Google Scholar 

  34. Evans, P. R. & Murshudov, G. N. How good are my data and what is the resolution? Acta Crystallogr. D 69, 1204–1214 (2013)

    CAS  PubMed  PubMed Central  Google Scholar 

  35. Winn, M. D. et al. Overview of the CCP4 suite and current developments. Acta Crystallogr. D 67, 235–242 (2011)

    CAS  PubMed  PubMed Central  Google Scholar 

  36. McCoy, A. J. et al. Phaser crystallographic software. J. Appl. Cryst. 40, 658–674 (2007)

    CAS  Google Scholar 

  37. Navaza, J. Implementation of molecular replacement in AMoRe. Acta Crystallogr. D 57, 1367–1372 (2001)

    CAS  PubMed  Google Scholar 

  38. Emsley, P., Lohkamp, B., Scott, W. G. & Cowtan, K. Features and development of Coot. Acta Crystallogr. D 66, 486–501 (2010)

    CAS  PubMed  PubMed Central  Google Scholar 

  39. Blanc, E. et al. Refinement of severely incomplete structures with maximum likelihood in BUSTER-TNT. Acta Crystallogr. D 60, 2210–2221 (2004)

    CAS  PubMed  Google Scholar 

  40. Winn, M. D., Murshudov, G. N. & Papiz, M. Z. Macromolecular TLS refinement in REFMAC at moderate resolutions. Methods Enzymol. 374, 300–321 (2003)

    CAS  PubMed  Google Scholar 

  41. Afonine, P. V. et al. Towards automated crystallographic structure refinement with phenix. refine. Acta Crystallogr. D 68, 352–367 (2012)

    CAS  PubMed  PubMed Central  Google Scholar 

  42. Lefranc, M. P. et al. IMGT, the international ImMunoGeneTics information system. Nucleic Acids Res. 37, D1006–D1012 (2009)

    CAS  PubMed  Google Scholar 

  43. Wu, T. T. & Kabat, E. A. An analysis of the sequences of the variable regions of Bence Jones proteins and myeloma light chains and their implications for antibody complementarity. J. Exp. Med. 132, 211–250 (1970)

    CAS  PubMed  PubMed Central  Google Scholar 

  44. Larkin, M. A. et al. Clustal W and Clustal X version 2.0. Bioinformatics 23, 2947–2948 (2007)

    CAS  PubMed  Google Scholar 

  45. Goujon, M. et al. A new bioinformatics analysis tools framework at EMBL-EBI. Nucleic Acids Res. 38, W695–699 (2010)

    ADS  CAS  PubMed  PubMed Central  Google Scholar 

  46. Gouet, P., Courcelle, E., Stuart, D. I. & Metoz, F. ESPript: analysis of multiple sequence alignments in PostScript. Bioinformatics 15, 305–308 (1999)

    CAS  PubMed  Google Scholar 

  47. Baker, N. A., Sept, D., Joseph, S., Holst, M. J. & McCammon, J. A. Electrostatics of nanosystems: application to microtubules and the ribosome. Proc. Natl Acad. Sci. USA 98, 10037–10041 (2001)

    ADS  CAS  PubMed  PubMed Central  Google Scholar 

  48. Dolinsky, T. J., Nielsen, J. E., McCammon, J. A. & Baker, N. A. PDB2PQR: an automated pipeline for the setup of Poisson-Boltzmann electrostatics calculations. Nucleic Acids Res. 32, W665–W667 (2004)

    CAS  PubMed  PubMed Central  Google Scholar 

  49. Chen, V. B. et al. MolProbity: all-atom structure validation for macromolecular crystallography. Acta Crystallogr. D 66, 12–21 (2010)

    CAS  PubMed  Google Scholar 

Download references

Acknowledgements

This work was made possible by a Pediatrics Dengue Vaccine Initiative grant to F.A.R., allowing the set up of a production facility of recombinant DENV sE. The co-crystallization with the bnAbs was done with European Union funding (DenFree consortium) to F.A.R. and G.R.S./J.M. F.A.R. acknowledges support from Insitut Pasteur, from the French Government’s ‘Investissements d’Avenir’ program: Laboratoire d’Excellence ‘Integrative Biology of Emerging Infectious Diseases’ (grant number ANR-10-LABX-62-IBEID) and the CNRS. G.S.R. and J.M. were supported by the Medical Research Council, UK, the Wellcome Trust, UK, the National Institute for Health Research Biomedical Research Centre, Funding Scheme. G.R.S. is a Wellcome Trust Senior investigator. We thank staffs at beam lines PROXIMA-1 and PROXIMA-2 at the SOLEIL synchrotron (St Aubin, France) and ID23-2 and ID29 at the European Synchrotron Radiation Facility (Grenoble, France). We thank A. Sakuntabhai for coordination of the DenFree grant, G. Bricogne for advice on diffraction data collection strategies, J. Cockburn and P.-Y. Lozach for help with the initial sE constructs, and S. Halstead and S. Kliks for support through the Pediatrics Dengue Vaccine Initiative.

Author information

Authors and Affiliations

Authors

Contributions

J.M., G.R.S. and F.A.R. conceived the experiments. W.W. and W.D. made the constructs for production of antibody fragments in S2 cells. M.E.N.S. and C.M.K. made the constructs for production of recombinant sE, and crystallized the unliganded form of DENV-2 FGA02 sE. C.G.B. and S.P. produced large amounts of sE protein for crystallization. A.R. and G.B.S. prepared the recombinant bnAb fragments and sE for crystallization. A.R. and A.H. optimized the crystals of the complexes. P.G.C., W.E.S., S.D., M.C.V. and A.R. collected and processed the diffraction data. P.G.C., M.C.V. and S.D. determined the structures and refined the atomic models. P.De., F.A.S. and F.A.R. conceived the protocols for production of recombinant sE. P.Du. provided a plasmid containing the envelope protein of DENV-2 FGA02 strain circulating in French Guiana in 2002. F.A.R. wrote the paper with the help of A.R., P.G.C., G.B.S., M.C.V. and S.D.

Corresponding authors

Correspondence to Juthathip Mongkolsapaya, Gavin R. Screaton or Félix A. Rey.

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Competing interests

The authors declare no competing financial interests.

Extended data figures and tables

Extended Data Figure 1 Overall complexes and footprints of the bnAbs on the sE dimer.

ad, Each row corresponds to a different sE/bnAb complex (except for the first one, which shows the unliganded sE dimer) and each column displays the same orientation, as labelled. In the first two columns the sE dimer is depicted as ribbons and the bnAb variable domains as surface coloured as in Fig. 1. In the side view (left column) the viral membrane would be underneath, whereas the bottom view (middle column) corresponds to the sE dimer seen from the viral membrane with the antibodies visible across the sE ribbons. The top view (right column) shows the sE surface as presented to the immune system on the viral particle, showing the footprint of the antibodies (green) with a white depth-cuing fog. For clarity, a white outline delimits the green footprint on the blue surface of domain III. As a guide, in the top-left panel the glycan chains of foreground and background subunits are labelled in red and black respectively. In the middle and right columns, the two-fold axis of the sE dimer is marked by a black ellipse at the centre. The fusion loop and the ij loop are labelled on the top-middle panel, and can be seen in the other rows in contact with the bnAbs. A red star in the left panels of rows c and d marks the location of the 150 loop, which is disordered in the complexes with the EDE1 bnAbs. This loop bears the N153 glycan recognized by the EDE2 bnAbs, as seen in row b, left panel (glycan shown as sticks with carbon atoms coloured red). In contrast, all the bnAbs are seen contacting the N67 glycan, with C8 displaying the most contacts (row c, left panel, N67 glycan as sticks with carbon atoms yellow). A blue star in row c shows a disordered loop in domain III. Note that EDE1 C10 (row d) inserts deeper into the sE dimer than the others bnAbs.

Extended Data Figure 2 Electrostatic potential of paratopes and epitope.

‘Open book’ representation of the complexes, with negative and positive potential displayed and coloured according to the bar underneath. Because certain regions are disordered in the complexes, the unliganded DENV-2 sE dimer model, generated as described in the Methods section, was used to calculate the surface electrostatic potential of the sE dimer. Corresponding areas in contact are indicated by ovals as in Fig. 2.

Extended Data Figure 3 Unliganded bnAb A11 and EDE2 bnAbs in interactions with DENV-2 sE.

a, The structure of the unliganded EDE2 A11 scFv (red, 1.7 Å resolution) superposed to the variable domain of Fab A11 in complex with DENV-2 sE (yellow, 3.8 Å resolution), to show that the same conformation is retained in the sE/Fab fragment complex. b, Stereo view showing the superposed B7 (green) and A11 (yellow) variable domains, together with the 150 loop extracted from the structures of the corresponding Fab/DENV-2 sE complexes. Note that the main chain of the 150 loop adopts different conformations in the two complexes, mainly because of the hydroxyl group the Y99 side chain in the CDR H3 of B7 makes a hydrogen bond with sE T155. A11 has a phenylalanine at this position, and so lacks the hydroxyl group. The sE protein in the complex with A11 displays the same conformation as the unliganded sE (not shown). c, Histograms of the atomic contacts of B7 (above the sE sequence) and A11 (below the sequence) according to the key at the bottom (see also detailed data and explanations in Supplementary Information). The secondary structure elements of the DENV-2 sE protein are indicated above the sequence, as a guide.

Extended Data Figure 4 Residues involved in bnAb/antigen interactions.

a, Amino-acid sequence alignment of sE from the four DENV serotypes, with residues in black or light blue background highlighting identity and similarity, respectively, across serotypes. Secondary structure elements are indicated underneath, with tertiary organization given by colours as in Fig. 1. DENV-2 sE residues contacted by the bnAbs are marked above, according to the code of the key (bottom-right insert). Full and empty symbols correspond to contacts on the reference subunit (defined as the one contributing the fusion loop to the epitope) and opposite subunit, respectively. Coloured boxes highlight the five distinct regions of sE making up the epitopes, matching Fig. 1c. The histogram displaying the number of atomic contacts per sE residue by each bnAb is provided as Supplementary Information. Because the EDE2 B7 and A11 contacts are very similar, only the B7 contacts are shown here. The question mark on the 150 loop indicates residues likely to contact the EDE1 bnAbs, but which are not visible in the structure because the loop is disordered. b, Sequence alignment of the four bnAbs crystallized and with the framework and CDR regions in grey and white background (in Kabat numbering43), respectively. Blue lines over the sequence mark the CDRs in the IMGT convention42. Somatic mutations are in red with germline residues in smaller font underneath. Residues arising from the recombination process are in green. A symbol above the sequence indicates the sE segment contacted, according to the key of the bottom-right inset. The secondary structure elements of the EDE1 C8 Ig β-barrels are indicated above the sequence, as guide.

Extended Data Figure 5 Epitopes and paratopes.

The epitope area of DENV-2 sE from three different complexes is highlighted in green and dark grey, with relevant side chains as sticks, corresponding to residues interacting with the heavy and light chain, respectively (left panels). The variable domain of the corresponding bnAb is shown in side view, with interacting side chains labelled. Heavy and light chains are in dark and light grey, respectively, with somatic mutations in red and residues that arose through the recombination process (third CDR in each chain) in green. a, EDE2 B7 complex; b, EDE1 C8 complex; c, EDE1 C10 complex.

Extended Data Figure 6 Key interactions of the bnAbs with sE.

a, The right panel shows the sE dimer in ribbons, with the framed area enlarged in the left panel to show the epitope, with main features labelled. b, sE dimer in complex with bnAb EDE2 B7, c with EDE1 C8 and d with EDE1 C10. The sE dimer surface is shown in a semi-transparent representation with the ribbons visible through. The glycan residues were not included in the surface, and are displayed as sticks. The relevant CDR loops of the bnAbs are shown as ribbons with side chains as sticks on top of the sE protein, coloured as in Fig. 1. The orientation of the left panel in rows b–d corresponds to the enlargement of row a, and the right panel is a view along the arrow in Fig. 1b (main text). Hydrogen bonds are displayed as dotted lines. The circle in the left panel of d highlights a deep contact of EDE1 C10 CDR-H3 into the DENV-2 sE dimer (see also Extended Data Fig. 1d, left panel).

Extended Data Figure 7 Interactions with the glycan chains.

Ribbon representation of a the EDE2 A11 Fab and b the EDE1 C8 Fab in complex with DENV-2 sE, coloured as in Fig. 1. The simulated annealing omit maps contoured at 1σ (cyan) or 0.6σ (gold) show clear density for the N153 (in a) and N67 glycans (in a and b) (black arrows). To create an unbiased map, all glycan atoms were removed from the structures, all B factors were reset to 20 Å and the structures were re-refined using torsion dynamics simulated annealing. Note that the antibody spans the two glycans across the dimer interface (as also shown in Fig. 1). c, Views down the black arrow in a (left panel) and the arrow in b (right panel), through the glycan chain. The key to the sugar connectivity and nomenclature is framed at the centre. d, Contacts of the sugar residues with the antibodies, coded according to the key.

Extended Data Table 1 Crystallization conditions, data collection and refinement statistics
Extended Data Table 2 Buried surface areas and surface complementarity in the various DENV sE–EDE complexes

Supplementary information

Supplementary Information

This file contains Supplementary Text and Data and Supplementary Table 1. (PDF 7222 kb)

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Rouvinski, A., Guardado-Calvo, P., Barba-Spaeth, G. et al. Recognition determinants of broadly neutralizing human antibodies against dengue viruses. Nature 520, 109–113 (2015). https://doi.org/10.1038/nature14130

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