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Iterative structure-based improvement of a fusion-glycoprotein vaccine against RSV


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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Figure 1: Design, structure, immunogenicity and informatics of prefusion-stabilized single-chain RSV F glycoproteins from design cycle 1.
Figure 2: Design, properties, structure, immunogenicity and informatics of single-chain RSV F with altered F2-F1 linkers from design cycle 2.
Figure 3: Design, structure, immunogenicity and informatics of single-chain RSV F glycoproteins with interprotomer disulfides (DS2) from design cycle 3.
Figure 4: Design, structure, immunogenicity and informatics from design cycle 4, comprising combinations of interprotomer disulfides (DS2) and other mutations.

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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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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.

Corresponding author

Correspondence to Peter D Kwong.

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Competing interests

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

Integrated supplementary information

Supplementary Figure 1 Properties of single-chain variants.

(a) Size-exclusion chromatography profiles of single-chain variants from design cycle 1 assessed using a Superose 6 Increase 5/150GL (24 ml column volume) gel filtration column. (b) Dynamic light scattering analysis of sc9 DS-Cav1. (c) Engineered single-chain RSV F glycoproteins with interprotomer disulfides shown with the SDS-PAGE shown in uncropped format.

Supplementary Figure 2 Stability of prefusion RSV F glycoprotein immunogens.

Decay curves at 60oC for RSV F glycoprotein immunogens of (a) design cycle 3 and (b) design cycle 4 were assessed by Octet BLI using prefusion-specific antibodies AM14, D25 and MPE8.

Supplementary Figure 3 Structural details of single-chain variants.

(a) Structural alignment of PR-DM and SC-TM RSV F vaccine candidates with sc9-10 A149 Y458C variant. (b) Design cycle 4 comprising combinations of interprotomer disulfides (DS2) and other mutations highlighted here in orange and detailed in Table S1.

Supplementary Figure 4 Immunogenicity of RSV F single-chain variants to RSV CH18537.

Neutralization titers of sera from mice immunized with RSV F variants from strains CH18537 and Long (VR26) tested against RSV CH18537 virus. Titers for each mouse are shown as individual dots, and geometric means are indicated by red horizontal lines. Each group included 10 mice.

Supplementary Figure 5 Immunized mice sera reactivity to D25, AM14, motavizumab (Mota) and MPE8 epitopes.

Sera from mice immunized with single-chain variants of RSV F were assessed for binding to immobilized DS-CAV1 or immobilized DS-Cav1 bound by neutralizing antibodies D25, AM14, Motavizumab, and MPE8 to assess site specific responses. Each group included 10 mice. Responses were measured by Octet Biolayer interferometry (BLI).

Supplementary Figure 6 sc9 DS-Cav1 and sc9-10 DS-Cav1 RSV F variants analyzed in solution through small-angle X-ray scattering.

(a) Normalized SAXS patterns and (b) Kratky plots for sc9 and sc9-10. sc9-10 appears to be a well-folded compact protein, while sc9 appears to be largely unfolded (compact molecules yield Kratky plots that converge to near zero at high q, whereas non-compact or unfolded molecules do not converge to near zero at high q). (c) SAXS-derived structural parameters.

Supplementary information

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Supplementary Figures 1–6 and Supplementary Tables 1–3 (PDF 1232 kb)

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Joyce, M., Zhang, B., Ou, L. et al. Iterative structure-based improvement of a fusion-glycoprotein vaccine against RSV. Nat Struct Mol Biol 23, 811–820 (2016).

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