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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Hall, C.B. et al. The burden of respiratory syncytial virus infection in young children. N. Engl. J. Med. 360, 588–598 (2009).
Shay, D.K. et al. Bronchiolitis-associated hospitalizations among US children, 1980–1996. J. Am. Med. Assoc. 282, 1440–1446 (1999).
Thompson, W.W. et al. Mortality associated with influenza and respiratory syncytial virus in the United States. J. Am. Med. Assoc. 289, 179–186 (2003).
Nair, H. et al. Global burden of acute lower respiratory infections due to respiratory syncytial virus in young children: a systematic review and meta-analysis. Lancet 375, 1545–1555 (2010).
van den Berg, J.P., Westerbeek, E.A., van der Klis, F.R., Berbers, G.A. & van Elburg, R.M. Transplacental transport of IgG antibodies to preterm infants: a review of the literature. Early Hum. Dev. 87, 67–72 (2011).
Hobbs, J.R. & Davis, J.A. Serum gamma-G-globulin levels and gestational age in premature babies. Lancet 1, 757–759 (1967).
Leuridan, E. et al. Early waning of maternal measles antibodies in era of measles elimination: longitudinal study. Br. Med. J. 340, c1626 (2010).
Sarvas, H., Seppälä, I., Kurikka, S., Siegberg, R. & Mäkelä, O. Half-life of the maternal IgG1 allotype in infants. J. Clin. Immunol. 13, 145–151 (1993).
Lee, M.S. et al. Half-life of human parainfluenza virus type 3 (hPIV3) maternal antibody and cumulative proportion of hPIV3 infection in young infants. J. Infect. Dis. 183, 1281–1284 (2001).
Hall, C.B. et al. Respiratory syncytial virus-associated hospitalizations among children less than 24 months of age. Pediatrics 132, e341–e348 (2013).
Palivizumab: new indication. Moderate reduction in hospitalisation rate. Prescrire Int. 13, 213–216 (2004).
The IMpact-RSV Study Group. Palivizumab, a humanized respiratory syncytial virus monoclonal antibody, reduces hospitalization from respiratory syncytial virus infection in high-risk infants. Pediatrics 102, 531–537 (1998).
Glenn, G.M. et al. A randomized, blinded, controlled, dose-ranging study of a respiratory syncytial virus recombinant fusion (F) nanoparticle vaccine in healthy women of childbearing age. J. Infect. Dis. 213, 411–422 (2016).
Swanson, K.A. et al. Structural basis for immunization with postfusion respiratory syncytial virus fusion F glycoprotein (RSV F) to elicit high neutralizing antibody titers. Proc. Natl. Acad. Sci. USA 108, 9619–9624 (2011).
Modjarrad, K. et al. WHO consultation on respiratory syncytial virus vaccine development report from a World Health Organization Meeting held on 23-24 March 2015. Vaccine 34, 190–197 (2016).
Karron, R.A., Buchholz, U.J. & Collins, P.L. Live-attenuated respiratory syncytial virus vaccines. Curr. Top. Microbiol. Immunol. 372, 259–284 (2013).
Graham, B.S., Modjarrad, K. & McLellan, J.S. Novel antigens for RSV vaccines. Curr. Opin. Immunol. 35, 30–38 (2015).
Pollack, P. & Groothuis, J.R. Development and use of palivizumab (Synagis): a passive immunoprophylactic agent for RSV. J. Infect. Chemother. 8, 201–206 (2002).
Young, J. Development of a potent respiratory syncytial virus-specific monoclonal antibody for the prevention of serious lower respiratory tract disease in infants. Respir. Med. 96 (Suppl. B), S31–S35 (2002).
Johnson, S. et al. Development of a humanized monoclonal antibody (MEDI-493) with potent in vitro and in vivo activity against respiratory syncytial virus. J. Infect. Dis. 176, 1215–1224 (1997).
Colman, P.M. & Lawrence, M.C. The structural biology of type I viral membrane fusion. Nat. Rev. Mol. Cell Biol. 4, 309–319 (2003).
Bolt, G., Pedersen, L.Ø. & Birkeslund, H.H. Cleavage of the respiratory syncytial virus fusion protein is required for its surface expression: role of furin. Virus Res. 68, 25–33 (2000).
Liljeroos, L., Krzyzaniak, M.A., Helenius, A. & Butcher, S.J. Architecture of respiratory syncytial virus revealed by electron cryotomography. Proc. Natl. Acad. Sci. USA 110, 11133–11138 (2013).
McLellan, J.S., Yang, Y., Graham, B.S. & Kwong, P.D. Structure of respiratory syncytial virus fusion glycoprotein in the postfusion conformation reveals preservation of neutralizing epitopes. J. Virol. 85, 7788–7796 (2011).
Magro, M. et al. Neutralizing antibodies against the preactive form of respiratory syncytial virus fusion protein offer unique possibilities for clinical intervention. Proc. Natl. Acad. Sci. USA 109, 3089–3094 (2012).
Corti, D. et al. Cross-neutralization of four paramyxoviruses by a human monoclonal antibody. Nature 501, 439–443 (2013).
Gilman, M.S. et al. Characterization of a prefusion-specific antibody that recognizes a quaternary, cleavage-dependent epitope on the RSV fusion glycoprotein. PLoS Pathog. 11, e1005035 (2015).
Kwakkenbos, M.J. et al. Generation of stable monoclonal antibody–producing B cell receptor–positive human memory B cells by genetic programming. Nat. Med. 16, 123–128 (2010).
McLellan, J.S. et al. Structure of RSV fusion glycoprotein trimer bound to a prefusion-specific neutralizing antibody. Science 340, 1113–1117 (2013).
Ngwuta, J.O. et al. Prefusion F-specific antibodies determine the magnitude of RSV neutralizing activity in human sera. Sci. Transl. Med. 7, 309ra162 (2015).
McLellan, J.S. et al. Structure-based design of a fusion glycoprotein vaccine for respiratory syncytial virus. Science 342, 592–598 (2013).
Krarup, A. et al. A highly stable prefusion RSV F vaccine derived from structural analysis of the fusion mechanism. Nat. Commun. 6, 8143 (2015).
Johnson, J.E., Gonzales, R.A., Olson, S.J., Wright, P.F. & Graham, B.S. The histopathology of fatal untreated human respiratory syncytial virus infection. Mod. Pathol. 20, 108–119 (2007).
Langley, J.M. et al. A dose-ranging study of a subunit respiratory syncytial virus subtype A vaccine with and without aluminum phosphate adjuvantation in adults 65 years of age. Vaccine 27, 5913–5919 (2009).
Paradiso, P.R. et al. Safety and immunogenicity of a subunit respiratory syncytial virus vaccine in children 24 to 48 months old. Pediatr. Infect. Dis. J. 13, 792–798 (1994).
Tristram, D.A., Welliver, R.C., Hogerman, D.A., Hildreth, S.W. & Paradiso, P. Second-year surveillance of recipients of a respiratory syncytial virus (RSV) F protein subunit vaccine, PFP-1: evaluation of antibody persistence and possible disease enhancement. Vaccine 12, 551–556 (1994).
Cohen, J. 2013 runners-up: in vaccine design, looks do matter. Science 342, 1442–1443 (2013).
Georgiev, I.S. et al. Single-chain soluble BG505.SOSIP gp140 trimers as structural and antigenic mimics of mature closed HIV-1 Env. J. Virol. 89, 5318–5329 (2015).
Sharma, S.K. et al. Cleavage-independent HIV-1 Env trimers engineered as soluble native spike mimetics for vaccine design. Cell Rep. 11, 539–550 (2015).
Begoña Ruiz-Argüello, M. et al. Effect of proteolytic processing at two distinct sites on shape and aggregation of an anchorless fusion protein of human respiratory syncytial virus and fate of the intervening segment. Virology 298, 317–326 (2002).
Coates, H.V., Kendrick, L. & Chanock, R.M. Antigenic differences between two strains of respiratory syncytial virus. Proc. Soc. Exp. Biol. Med. 112, 958–964 (1963).
Putnam, C.D., Hammel, M., Hura, G.L. & Tainer, J.A. X-ray solution scattering (SAXS) combined with crystallography and computation: defining accurate macromolecular structures, conformations and assemblies in solution. Q. Rev. Biophys. 40, 191–285 (2007).
Rambo, R.P. & Tainer, J.A. Characterizing flexible and intrinsically unstructured biological macromolecules by SAS using the Porod-Debye law. Biopolymers 95, 559–571 (2011).
Liang, B. et al. Enhanced neutralizing antibody response induced by respiratory syncytial virus prefusion F protein expressed by a vaccine candidate. J. Virol. 89, 9499–9510 (2015).
Godley, L. et al. Introduction of intersubunit disulfide bonds in the membrane-distal region of the influenza hemagglutinin abolishes membrane fusion activity. Cell 68, 635–645 (1992).
Kwon, Y.D. et al. Crystal structure, conformational fixation and entry-related interactions of mature ligand-free HIV-1 Env. Nat. Struct. Mol. Biol. 22, 522–531 (2015).
Wong, J.J., Paterson, R.G., Lamb, R.A. & Jardetzky, T.S. Structure and stabilization of the Hendra virus F glycoprotein in its prefusion form. Proc. Natl. Acad. Sci. USA 113, 1056–1061 (2016).
Wen, X. et al. Structure of the human metapneumovirus fusion protein with neutralizing antibody identifies a pneumovirus antigenic site. Nat. Struct. Mol. Biol. 19, 461–463 (2012).
Welch, B.D. et al. Structure of the cleavage-activated prefusion form of the parainfluenza virus 5 fusion protein. Proc. Natl. Acad. Sci. USA 109, 16672–16677 (2012).
Nabel, G.J. Philosophy of science: the coordinates of truth. Science 326, 53–54 (2009).
Klebe, G. Recent developments in structure-based drug design. J. Mol. Med. (Berl.) 78, 269–281 (2000).
Pancera, M. et al. N332-directed broadly neutralizing antibodies use diverse modes of HIV-1 recognition: inferences from heavy-light chain complementation of function. PLoS One 8, e55701 (2013).
McLellan, J.S. et al. Structural basis of respiratory syncytial virus neutralization by motavizumab. Nat. Struct. Mol. Biol. 17, 248–250 (2010).
Heymann, J.B. & Belnap, D.M. Bsoft: image processing and molecular modeling for electron microscopy. J. Struct. Biol. 157, 3–18 (2007).
Ludtke, S.J., Baldwin, P.R. & Chiu, W. EMAN: semiautomated software for high-resolution single-particle reconstructions. J. Struct. Biol. 128, 82–97 (1999).
Stewart-Jones, G.B. et al. A cysteine zipper stabilizes a pre-fusion F glycoprotein vaccine for respiratory syncytial virus. PLoS One 10, e0128779 (2015).
Graham, B.S., Perkins, M.D., Wright, P.F. & Karzon, D.T. Primary respiratory syncytial virus infection in mice. J. Med. Virol. 26, 153–162 (1988).
Hotard, A.L. et al. A stabilized respiratory syncytial virus reverse genetics system amenable to recombination-mediated mutagenesis. Virology 434, 129–136 (2012).
Majeed, S. et al. Enhancing protein crystallization through precipitant synergy. Structure 11, 1061–1070 (2003).
Otwinowski, Z. & Minor, W. Processing of X-ray diffraction data collected in oscillation mode. Methods Enzymol. 276, 307–326 (1997).
McCoy, A.J. et al. Phaser crystallographic software. J. Appl. Crystallogr. 40, 658–674 (2007).
Emsley, P., Lohkamp, B., Scott, W.G. & Cowtan, K. Features and development of Coot. Acta Crystallogr. D Biol. Crystallogr. 66, 486–501 (2010).
Adams, P.D. et al. PHENIX: a comprehensive Python-based system for macromolecular structure solution. Acta Crystallogr. D Biol. Crystallogr. 66, 213–221 (2010).
Guttman, M., Weinkam, P., Sali, A. & Lee, K.K. All-atom ensemble modeling to analyze small-angle x-ray scattering of glycosylated proteins. Structure 21, 321–331 (2013).
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.
The NIH has filed patents USPTO 20160046675 and USPTO 20140271699 on the use of prefusion-stabilized RSV F glycoproteins.
Integrated supplementary information
(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.
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.
(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.
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.
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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). https://doi.org/10.1038/nsmb.3267
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