Structure-based design of vaccines, particularly the iterative optimization used so successfully in the structure-based design of drugs, has been a long-sought goal. We previously developed a first-generation vaccine antigen called DS-Cav1, comprising a prefusion-stabilized form of the fusion (F) glycoprotein, which elicits high-titer protective responses against respiratory syncytial virus (RSV) in mice and macaques. Here we report the improvement of DS-Cav1 through iterative cycles of structure-based design that significantly increased the titer of RSV-protective responses. The resultant second-generation 'DS2'-stabilized immunogens have their F subunits genetically linked, their fusion peptides deleted and their interprotomer movements stabilized by an additional disulfide bond. These DS2 immunogens are promising vaccine candidates with superior attributes, such as their lack of a requirement for furin cleavage and their increased antigenic stability against heat inactivation. The iterative structure-based improvement described here may have utility in the optimization of other vaccine antigens.

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We thank M. Moore and A. Hotard (Department of Pediatrics, Emory University, and Children's Healthcare of Atlanta) for engineered RSVs used in neutralization assays and protocols. We thank J. Stuckey for assistance with figures, and L. Shapiro and members of the Structural Biology Section, the Structural Bioinformatics Core Section, the Virology Core Section of the Virology Laboratory and the Viral Pathogenesis Laboratory, Vaccine Research Center, NIAID, NIH for discussions and comments on the manuscript. Funding was provided by the Intramural Research Program of the Vaccine Research Center, National Institute of Allergy and Infectious Diseases, National Institutes of Health for M.G.J., B.Z., L.O., M.C., G.-Y.C., A.D., W.-P.K., Y.-T.L., E.J.R., Y.T., Y.Y., I.S.G., C.R.L., M.P., M.S., C.S., G.B.E.S.-J., P.V.T., J.G.V.G., J.R.M., B.S.G. and P.D.K., and by the Gates Foundation Global Health Vaccine Accelerator Platform (GH-VAP), no. OPP1126258, to M.G. and K.K.L. This project was funded in part by Federal funds from the Frederick National Laboratory for Cancer Research, National Institutes of Health, under contract HHSN261200800001E. Leidos Biomedical Research, Inc. provided support in the form of salaries for Y.T. and U.B. Use of the Stanford Synchrotron Radiation Lightsource, SLAC National Accelerator Laboratory by M.G. and K.K.L. was supported by the US Department of Energy, Office of Science, Office of Basic Energy Sciences, under contract no. DE-AC02-76SF00515. Use of sector 22 (Southeast Region Collaborative Access team) at the Advanced Photon Source by M.G.J. and P.D.K. was supported by the US Department of Energy, Basic Energy Sciences, Office of Science, under contract no. W-31-109-Eng-38.

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Author notes

    • M Gordon Joyce
    •  & Baoshan Zhang

    These authors contributed equally to this work.


  1. Vaccine Research Center, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland, USA.

    • M Gordon Joyce
    • , Baoshan Zhang
    • , Li Ou
    • , Man Chen
    • , Gwo-Yu Chuang
    • , Aliaksandr Druz
    • , Wing-Pui Kong
    • , Yen-Ting Lai
    • , Emily J Rundlet
    • , Yongping Yang
    • , Ivelin S Georgiev
    • , Christopher R Lees
    • , Marie Pancera
    • , Mallika Sastry
    • , Cinque Soto
    • , Guillaume B E Stewart-Jones
    • , Paul V Thomas
    • , Joseph G Van Galen
    • , John R Mascola
    • , Barney S Graham
    •  & Peter D Kwong
  2. Electron Microscopy Laboratory, Cancer Research Technology Program, Leidos Biomedical Research, Inc., Frederick National Laboratory for Cancer Research, Frederick, Maryland, USA.

    • Yaroslav Tsybovsky
    •  & Ulrich Baxa
  3. Department of Medicinal Chemistry, University of Washington, Seattle, Washington, USA.

    • Miklos Guttman
    •  & Kelly K Lee


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M.G.J., B.Z., B.S.G. and P.D.K. conceived, designed and coordinated the study; M.G.J., B.Z., L.O., G.-Y.C. and P.D.K. wrote and revised the manuscript and generated figures; M.G.J., B.Z., L.O., M.C., A.D., W.-P.K., Y.-T.L., E.J.R., Y.T., Y.Y., M.G., C.R.L., M.S., G.B.E.S.-J., P.V.T., J.G.V.G. and U.B. performed experiments; M.G.J., B.Z., I.S.G., M.P., C.S., B.S.G. and P.D.K. designed stabilized prefusion F immunogens. G.-Y.C. and P.D.K. carried out bioinformatics analyses. M.G.J., B.Z., L.O., M.C., G.-Y.C., M.G., K.K.L., J.R.M., B.S.G. and P.D.K. analyzed data. L.O., M.C., G.-Y.C., A.D., W.-P.K., Y.-T.L., E.J.R., Y.T. and Y.Y. contributed equally to this study. All authors read and approved the manuscript.

Competing interests

The NIH has filed patents USPTO 20160046675 and USPTO 20140271699 on the use of prefusion-stabilized RSV F glycoproteins.

Corresponding author

Correspondence to Peter D Kwong.

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