Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Article
  • Published:

Protective antibodies against Eastern equine encephalitis virus bind to epitopes in domains A and B of the E2 glycoprotein

Nature Microbiology volume 4, pages 187–197 (2019)Cite this article

Subjects

Abstract

Eastern equine encephalitis virus (EEEV) is a mosquito-transmitted alphavirus with a high case mortality rate in humans. EEEV is a biodefence concern because of its potential for aerosol spread and the lack of existing countermeasures. Here, we identify a panel of 18 neutralizing murine monoclonal antibodies (mAbs) against the EEEV E2 glycoprotein, several of which have ‘elite’ activity with 50 and 99% effective inhibitory concentrations (EC50 and EC99) of less than 10 and 100 ng ml−1, respectively. Alanine-scanning mutagenesis and neutralization escape mapping analysis revealed epitopes for these mAbs in domains A or B of the E2 glycoprotein. A majority of the neutralizing mAbs blocked infection at a post-attachment stage, with several inhibiting viral membrane fusion. Administration of one dose of anti-EEEV mAb protected mice from lethal subcutaneous or aerosol challenge. These experiments define the mechanistic basis for neutralization by protective anti-EEEV mAbs and suggest a path forward for treatment and vaccine design.

Access through your institution
Buy or subscribe

This is a preview of subscription content, access via your institution

Access options

Access through your institution

Buy this article

  • Purchase on SpringerLink
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Fig. 1: Characterization of anti-EEEV mAbs.
Fig. 2: Neutralizing activity of anti-EEEV mAbs.
Fig. 3: Neutralizing mAbs map to domain A or B on the E2 glycoprotein.
Fig. 4: Characterization of EEEV mAb escape mutants.
Fig. 5: Anti-EEEV mAbs exhibit in vivo protection.

Similar content being viewed by others

Data availability

The authors declare that all data supporting the findings of this study are available within the paper and its Supplementary Information. The Supplementary Tables provide data on the newly generated mAbs and mutagenesis (alanine and arginine) mapping of the mAb binding sites on EEEV E2 protein.

References

  1. Garlick, J. et al. Locally acquired eastern equine encephalitis virus disease, Arkansas, USA. Emerg. Infect. Dis. 22, 2216–2217 (2016).

    Article  PubMed  PubMed Central  Google Scholar 

  2. Mukerji, S. S., Lam, A. D. & Wilson, M. R. Eastern equine encephalitis treated with intravenous immunoglobulins. Neurohospitalist 6, 29–31 (2016).

    Article  PubMed  PubMed Central  Google Scholar 

  3. Armstrong, P. M. & Andreadis, T. G. Eastern equine encephalitis virus-old enemy, new threat. N. Engl. J. Med. 368, 1670–1673 (2013).

    Article  CAS  PubMed  Google Scholar 

  4. Eastern Equine Encephalitis (Centers for Disease Control and Prevention, 2018); https://www.cdc.gov/easternequineencephalitis/

  5. Lindsey, N. P., Staples, J. E. & Fischer, M. Eastern equine encephalitis virus in the United States, 2003-2016. Am. J. Trop. Med. Hyg. 98, 1472–1477 (2018).

    Article  PubMed  PubMed Central  Google Scholar 

  6. Tyler, K. L. Acute viral encephalitis. N. Engl. J. Med. 379, 557–566 (2018).

    Article  PubMed  Google Scholar 

  7. Tan, Y. et al. Large scale complete genome sequencing and phylodynamic analysis of eastern equine encephalitis virus reveal source-sink transmission dynamics in the United States. J. Virol. https://doi.org/10.1128/JVI.00074-18 (2018).

  8. Hunt, A. R., Frederickson, S., Maruyama, T., Roehrig, J. T. & Blair, C. D. The first human epitope map of the alphaviral E1 and E2 proteins reveals a new E2 epitope with significant virus neutralizing activity. PLoS Negl. Trop. Dis. 4, e739 (2010).

    Article  PubMed  PubMed Central  Google Scholar 

  9. Sherman, M. B. & Weaver, S. C. Structure of the recombinant alphavirus Western equine encephalitis virus revealed by cryoelectron microscopy. J. Virol. 84, 9775–9782 (2010).

    Article  PubMed  PubMed Central  Google Scholar 

  10. Zhang, R. et al. 4.4 Å cryo-EM structure of an enveloped alphavirus Venezuelan equine encephalitis virus. EMBO J. 30, 3854–3863 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Voss, J. E. et al. Glycoprotein organization of Chikungunya virus particles revealed by X-ray crystallography. Nature 468, 709–712 (2010).

    Article  CAS  PubMed  Google Scholar 

  12. Li, L., Jose, J., Xiang, Y., Kuhn, R. J. & Rossmann, M. G. Structural changes of envelope proteins during alphavirus fusion. Nature 468, 705–708 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Smith, T. J. et al. Putative receptor binding sites on alphaviruses as visualized by cryoelectron microscopy. Proc. Natl Acad. Sci. USA 92, 10648–10652 (1995).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Zhao, J. et al. Phage display identifies an Eastern equine encephalitis virus glycoprotein E2-specific B cell epitope. Vet. Immunol. Immunopathol. 148, 364–368 (2012).

    Article  CAS  PubMed  Google Scholar 

  15. EnCheng, S. et al. Analysis of murine B-cell epitopes on Eastern equine encephalitis virus glycoprotein E2. Appl. Microbiol. Biotechnol. 97, 6359–6372 (2013).

    Article  PubMed  Google Scholar 

  16. Roehrig, J. T. et al. Identification of monoclonal antibodies capable of differentiating antigenic varieties of eastern equine encephalitis viruses. Am. J. Trop. Med. Hyg. 42, 394–398 (1990).

    Article  CAS  PubMed  Google Scholar 

  17. Pereboev, A. V., Razumov, I. A., Svyatchenko, V. A. & Loktev, V. B. Glycoproteins E2 of the Venezuelan and eastern equine encephalomyelitis viruses contain multiple cross-reactive epitopes. Arch. Virol. 141, 2191–2205 (1996).

    Article  CAS  PubMed  Google Scholar 

  18. Fox, J. M. et al. Broadly neutralizing alphavirus antibodies bind an epitope on E2 and inhibit entry and egress. Cell 163, 1095–1107 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Smith, S. A. et al. Isolation and characterization of broad and ultrapotent human monoclonal antibodies with therapeutic activity against Chikungunya virus. Cell Host Microbe 18, 86–95 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Hunt, A. R., Frederickson, S., Hinkel, C., Bowdish, K. S. & Roehrig, J. T. A humanized murine monoclonal antibody protects mice either before or after challenge with virulent Venezuelan equine encephalomyelitis virus. J. Gen. Virol. 87, 2467–2476 (2006).

    Article  CAS  PubMed  Google Scholar 

  21. Hülseweh, B. et al. Human-like antibodies neutralizing Western equine encephalitis virus. MAbs 6, 718–727 (2014).

    Article  PubMed  Google Scholar 

  22. Pal, P. et al. Development of a highly protective combination monoclonal antibody therapy against Chikungunya virus. PLoS Pathog. 9, e1003312 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Jin, J. et al. Neutralizing monoclonal antibodies block Chikungunya virus entry and release by targeting an epitope critical to viral pathogenesis. Cell Rep. 13, 2553–2564 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Zhang, R. et al. Mxra8 is a receptor for multiple arthritogenic alphaviruses. Nature 557, 570–574 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Schilte, C. et al. Cutting edge: independent roles for IRF-3 and IRF-7 in hematopoietic and nonhematopoietic cells during host response to Chikungunya infection. J. Immunol. 188, 2967–2971 (2012).

    Article  CAS  PubMed  Google Scholar 

  26. Armstrong, P. M., Prince, N. & Andreadis, T. G. Development of a multi-target TaqMan assay to detect eastern equine encephalitis virus variants in mosquitoes. Vector Borne Zoonotic Dis. 12, 872–876 (2012).

    Article  PubMed  PubMed Central  Google Scholar 

  27. Gardner, C. L., Ebel, G. D., Ryman, K. D. & Klimstra, W. B. Heparan sulfate binding by natural eastern equine encephalitis viruses promotes neurovirulence. Proc. Natl Acad. Sci. USA 108, 16026–16031 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Edwards, J. & Brown, D. T. Sindbis virus-mediated cell fusion from without is a two-step event. J. Gen. Virol. 67, 377–380 (1986).

    Article  PubMed  Google Scholar 

  29. Fong, R. H. et al. Exposure of epitope residues on the outer face of the chikungunya virus envelope trimer determines antibody neutralizing efficacy. J. Virol. 88, 14364–14379 (2014).

    Article  PubMed  PubMed Central  Google Scholar 

  30. Sun, C., Gardner, C. L., Watson, A. M., Ryman, K. D. & Klimstra, W. B. Stable, high-level expression of reporter proteins from improved alphavirus expression vectors to track replication and dissemination during encephalitic and arthritogenic disease. J. Virol. 88, 2035–2046 (2014).

    Article  PubMed  PubMed Central  Google Scholar 

  31. Wust, C. J., Crombie, R. & Brown, A. Passive protection across subgroups of alphaviruses by hyperimmune non-cross-neutralizing anti-Sindbis serum. Proc. Soc. Exp. Biol. Med. 184, 56–63 (1987).

    Article  CAS  PubMed  Google Scholar 

  32. Roehrig, J. T., Hunt, A. R., Kinney, R. M. & Mathews, J. H. In vitro mechanisms of monoclonal antibody neutralization of alphaviruses. Virology 165, 66–73 (1988).

    Article  CAS  PubMed  Google Scholar 

  33. Flynn, D. C., Olmsted, R. A., Mackenzie, J. M.Jr.. & Johnston, R. E. Antibody-mediated activation of Sindbis virus. Virology 166, 82–90 (1988).

    Article  CAS  PubMed  Google Scholar 

  34. Haslwanter, D., Blaas, D., Heinz, F. X. & Stiasny, K. A novel mechanism of antibody-mediated enhancement of flavivirus infection. PLoS Pathog. 13, e1006643 (2017).

    Article  PubMed  PubMed Central  Google Scholar 

  35. Agapov, E. V. et al. Localization of four antigenic sites involved in Venezuelan equine encephalomyelitis virus protection. Arch. Virol. 139, 173–181 (1994).

    Article  CAS  PubMed  Google Scholar 

  36. Pence, D. F., Davis, N. L. & Johnston, R. E. Antigenic and genetic characterization of Sindbis virus monoclonal antibody escape mutants which define a pathogenesis domain on glycoprotein E2. Virology 175, 41–49 (1990).

    Article  CAS  PubMed  Google Scholar 

  37. Vrati, S., Fernon, C. A., Dalgarno, L. & Weir, R. C. Location of a major antigenic site involved in Ross River virus neutralization. Virology 162, 346–353 (1988).

    Article  CAS  PubMed  Google Scholar 

  38. Porta, J. et al. Locking and blocking the viral landscape of an alphavirus with neutralizing antibodies. J. Virol. 88, 9616–9623 (2014).

    Article  PubMed  PubMed Central  Google Scholar 

  39. Watson, A. M. et al. Ribbon scanning confocal for high-speed high-resolution volume imaging of brain. PLoS ONE 12, e0180486 (2017).

    Article  PubMed  PubMed Central  Google Scholar 

  40. Charles, P. C., Walters, E., Margolis, F. & Johnston, R. E. Mechanism of neuroinvasion of Venezuelan equine encephalitis virus in the mouse. Virology 208, 662–671 (1995).

    Article  CAS  PubMed  Google Scholar 

  41. Honnold, S. P. et al. Eastern equine encephalitis virus in mice I: clinical course and outcome are dependent on route of exposure. Virol. J. 12, 152 (2015).

    Article  PubMed  PubMed Central  Google Scholar 

  42. Honnold, S. P. et al. Eastern equine encephalitis virus in mice II: pathogenesis is dependent on route of exposure. Virol. J. 12, 154 (2015).

    Article  PubMed  PubMed Central  Google Scholar 

  43. Pal, P. et al. Chikungunya viruses that escape monoclonal antibody therapy are clinically attenuated, stable, and not purified in mosquitoes. J. Virol. 88, 8213–8226 (2014).

    Article  PubMed  PubMed Central  Google Scholar 

  44. Zhou, T. et al. Structural basis for broad and potent neutralization of HIV-1 by antibody VRC01. Science 329, 811–817 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  45. Law, M. et al. Broadly neutralizing antibodies protect against hepatitis C virus quasispecies challenge. Nat. Med. 14, 25–27 (2008).

    Article  CAS  PubMed  Google Scholar 

  46. Julien, J. P., Lee, P. S. & Wilson, I. A. Structural insights into key sites of vulnerability on HIV-1 Env and influenza HA. Immunol. Rev. 250, 180–198 (2012).

    Article  PubMed  PubMed Central  Google Scholar 

  47. Klimstra, W. B., Ryman, K. D. & Johnston, R. E. Adaptation of Sindbis virus to BHK cells selects for use of heparan sulfate as an attachment receptor. J. Virol. 72, 7357–7366 (1998).

    CAS  PubMed  PubMed Central  Google Scholar 

  48. Aguilar, P. V. et al. Structural and nonstructural protein genome regions of eastern equine encephalitis virus are determinants of interferon sensitivity and murine virulence. J. Virol. 82, 4920–4930 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  49. Gardner, C. L. et al. Antibody preparations from human transchromosomic cows exhibit prophylactic and therapeutic efficacy against Venezuelan equine encephalitis virus. J. Virol. 2, e00226-17 (2017).

Download references

Acknowledgements

This work was supported by the Defense Threat Reduction Agency (grant no. HDTRA1-15-1-0013 to M.S.D. and W.B.K. and grant no. HDTRA1-13-1-0034 to J.E.C) and National Institutes of Health grant no. R01 AI095436 to W.B.K.

Author information

Author notes

  1. Christina L. Gardner

    Present address: United States Army Research Institute for Infectious Diseases, Fort Detrick, MD, USA

Authors and Affiliations

  1. Department of Medicine, Washington University School of Medicine, St. Louis, MO, USA

    Arthur S. Kim, S. Kyle Austin & Michael S. Diamond

  2. Pathology and Immunology, Washington University School of Medicine, St. Louis, MO, USA

    Arthur S. Kim, Adam Zuiani, Katherine Basore, Daved H. Fremont & Michael S. Diamond

  3. Center for Vaccine Research, Department of Immunology, University of Pittsburgh, Pittsburgh, PA, USA

    Christina L. Gardner, Douglas S. Reed, Derek W. Trobaugh, Chengqun Sun & William B. Klimstra

  4. Vanderbilt Vaccine Center, Department of Pediatrics, and Department of Pathology, Microbiology, and Immunology, Vanderbilt University Medical Center, Nashville, TN, USA

    Lauren E. Williamson & James E. Crowe Jr.

  5. Division of Neuroscience, Oregon National Primate Research Center, Oregon Health & Science University, Beaverton, OR, USA

    Mark K. Slifka

  6. Biochemistry and Molecular Biophysics, Washington University School of Medicine, St. Louis, MO, USA

    Daved H. Fremont

  7. Molecular Microbiology, Washington University School of Medicine, St. Louis, MO, USA

    Daved H. Fremont & Michael S. Diamond

  8. The Andrew M. and Jane M. Bursky Center for Human Immunology and Immunotherapy Programs, Washington University School of Medicine, St. Louis, MO, USA

    Michael S. Diamond

Authors
  1. Arthur S. Kim
    View author publications

    Search author on:PubMed Google Scholar

  2. S. Kyle Austin
    View author publications

    Search author on:PubMed Google Scholar

  3. Christina L. Gardner
    View author publications

    Search author on:PubMed Google Scholar

  4. Adam Zuiani
    View author publications

    Search author on:PubMed Google Scholar

  5. Douglas S. Reed
    View author publications

    Search author on:PubMed Google Scholar

  6. Derek W. Trobaugh
    View author publications

    Search author on:PubMed Google Scholar

  7. Chengqun Sun
    View author publications

    Search author on:PubMed Google Scholar

  8. Katherine Basore
    View author publications

    Search author on:PubMed Google Scholar

  9. Lauren E. Williamson
    View author publications

    Search author on:PubMed Google Scholar

  10. James E. Crowe Jr.
    View author publications

    Search author on:PubMed Google Scholar

  11. Mark K. Slifka
    View author publications

    Search author on:PubMed Google Scholar

  12. Daved H. Fremont
    View author publications

    Search author on:PubMed Google Scholar

  13. William B. Klimstra
    View author publications

    Search author on:PubMed Google Scholar

  14. Michael S. Diamond
    View author publications

    Search author on:PubMed Google Scholar

Contributions

A.S.K., S.K.A., A.Z., M.K.S., D.S.R., D.W.T., W.B.K. and M.S.D. designed the experiments. A.S.K., S.K.A., C.L.G., A.Z., D.W.T., C.S. and K.B. performed the experiments. A.S.K., S.K.A, C.L.G., D.H.F., A.Z., D.W.T. and K.B. performed the data analysis. L.E.W., J.E.C. and D.H.F. contributed key reagents. D.S.R. and D.H.F. contributed the methodology. A.S.K. and M.S.D. wrote the initial draft of the manuscript, with the other authors providing comments and edits to the final version.

Corresponding author

Correspondence to Michael S. Diamond.

Ethics declarations

Competing interests

M.S.D. is a consultant for InBios International and is on the Scientific Advisory Board of Moderna. J.E.C. has served as a consultant for Takeda Vaccines, Sanofi Pasteur, Pfizer and Novavax, is on the Scientific Advisory Boards of CompuVax, GigaGen, Meissa Vaccines, PaxVax, and is the Founder of IDBiologics.

Additional information

Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary information

Supplementary Information

Supplementary Figures 1–6.

Reporting Summary

Supplementary Table 1

Characteristics of anti-EEEV mAbs.

Supplementary Table 2

Relative binding of anti-EEEV mAbs to E2 alanine mutants.

Supplementary Table 3

Relative binding of anti-EEEV mAbs to E2 select arginine mutants.

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Kim, A.S., Austin, S.K., Gardner, C.L. et al. Protective antibodies against Eastern equine encephalitis virus bind to epitopes in domains A and B of the E2 glycoprotein. Nat Microbiol 4, 187–197 (2019). https://doi.org/10.1038/s41564-018-0286-4

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue date:

  • DOI: https://doi.org/10.1038/s41564-018-0286-4

This article is cited by

Search

Advanced search

Quick links

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