title: A Cysteine Zipper Stabilizes a Pre-Fusion F Glycoprotein Vaccine for Respiratory Syncytial Virus.
abstract: Recombinant subunit vaccines should contain minimal non-pathogen motifs to reduce potential off-target reactivity. We recently developed a vaccine antigen against respiratory syncytial virus (RSV), which comprised the fusion (F) glycoprotein stabilized in its pre-fusion trimeric conformation by "DS-Cav1" mutations and by an appended C-terminal trimerization motif or "foldon" from T4-bacteriophage fibritin. Here we investigate the creation of a cysteine zipper to allow for the removal of the phage foldon, while maintaining the immunogenicity of the parent DS-Cav1+foldon antigen. Constructs without foldon yielded RSV F monomers, and enzymatic removal of the phage foldon from pre-fusion F trimers resulted in their dissociation into monomers. Because the native C terminus of the pre-fusion RSV F ectodomain encompasses a viral trimeric coiled-coil, we explored whether introduction of cysteine residues capable of forming inter-protomer disulfides might allow for stable trimers. Structural modeling indicated the introduced cysteines to form disulfide "rings", with each ring comprising a different set of inward facing residues of the coiled-coil. Three sets of rings could be placed within the native RSV F coiled-coil, and additional rings could be added by duplicating portions of the coiled-coil. High levels of neutralizing activity in mice, equivalent to that of the parent DS-Cav1+foldon antigen, were elicited by a 4-ring stabilized RSV F trimer with no foldon. Structure-based alteration of a viral coiled-coil to create a cysteine zipper thus allows a phage trimerization motif to be removed from a candidate vaccine antigen.
authors.affiliation: Vaccine Research Center, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland, United States of America.
authors.forename: Guillaume B E
authors.initials: GB
authors.lastname: Stewart-Jones
authors.affiliation: Vaccine Research Center, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland, United States of America.
authors.forename: Paul V
authors.initials: PV
authors.lastname: Thomas
authors.affiliation: Vaccine Research Center, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland, United States of America.
authors.forename: Man
authors.initials: M
authors.lastname: Chen
authors.affiliation: Vaccine Research Center, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland, United States of America.
authors.forename: Aliaksandr
authors.initials: A
authors.lastname: Druz
authors.affiliation: Vaccine Research Center, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland, United States of America.
authors.forename: M Gordon
authors.initials: MG
authors.lastname: Joyce
authors.affiliation: Vaccine Research Center, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland, United States of America.
authors.forename: Wing-Pui
authors.initials: WP
authors.lastname: Kong
authors.affiliation: Vaccine Research Center, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland, United States of America.
authors.forename: Mallika
authors.initials: M
authors.lastname: Sastry
authors.affiliation: Vaccine Research Center, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland, United States of America.
authors.forename: Cinque
authors.initials: C
authors.lastname: Soto
authors.affiliation: Vaccine Research Center, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland, United States of America.
authors.forename: Yongping
authors.initials: Y
authors.lastname: Yang
authors.affiliation: Vaccine Research Center, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland, United States of America.
authors.forename: Baoshan
authors.initials: B
authors.lastname: Zhang
authors.affiliation: Vaccine Research Center, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland, United States of America.
authors.forename: Lei
authors.initials: L
authors.lastname: Chen
authors.affiliation: Vaccine Research Center, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland, United States of America.
authors.forename: Gwo-Yu
authors.initials: GY
authors.lastname: Chuang
authors.affiliation: Vaccine Research Center, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland, United States of America.
authors.forename: Ivelin S
authors.initials: IS
authors.lastname: Georgiev
authors.affiliation: Vaccine Research Center, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland, United States of America; Department of Biochemistry, Geisel School of Medicine at Dartmouth, Hanover, New Hampshire, United States of America.
authors.forename: Jason S
authors.initials: JS
authors.lastname: McLellan
authors.affiliation: Vaccine Research Center, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland, United States of America.
authors.forename: Sanjay
authors.initials: S
authors.lastname: Srivatsan
authors.affiliation: Vaccine Research Center, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland, United States of America.
authors.forename: Tongqing
authors.initials: T
authors.lastname: Zhou
authors.affiliation: Electron Microscopy Laboratory, Cancer Research Technology Program, Leidos Biomedical Research, Inc., Frederick National Laboratory for Cancer Research, Frederick, Maryland, United States of America.
authors.forename: Ulrich
authors.initials: U
authors.lastname: Baxa
authors.affiliation: Vaccine Research Center, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland, United States of America.
authors.forename: John R
authors.initials: JR
authors.lastname: Mascola
authors.affiliation: Vaccine Research Center, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland, United States of America.
authors.forename: Barney S
authors.initials: BS
authors.lastname: Graham
authors.affiliation: Vaccine Research Center, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland, United States of America.
authors.forename: Peter D
authors.initials: PD
authors.lastname: Kwong
mesh_terms: D000818:Animals; D057134:Antibodies, Neutralizing / Q000276:immunology; D000956:Antigens, Viral / Q000737:chemistry / Q000276:immunology*; D017122:Bacteriophage T4 / Q000276:immunology; D051242:Caveolin 1 / Q000737:chemistry / Q000235:genetics / Q000276:immunology; D002460:Cell Line; D003545:Cysteine / Q000737:chemistry*; D007114:Immunization; D051379:Mice; D008807:Mice, Inbred BALB C; D008810:Mice, Inbred C57BL; D022261:Respiratory Syncytial Virus Vaccines / Q000276:immunology*; D012136:Respiratory Syncytial Viruses / Q000235:genetics / Q000276:immunology*; D022223:Vaccines, Subunit / Q000276:immunology; D014760:Viral Fusion Proteins / Q000276:immunology*; D014764:Viral Proteins / Q000276:immunology
chemical_list: D057134:Antibodies, Neutralizing; D000956:Antigens, Viral; C495541:Cav1 protein, mouse; D051242:Caveolin 1; D022261:Respiratory Syncytial Virus Vaccines; D022223:Vaccines, Subunit; D014760:Viral Fusion Proteins; D014764:Viral Proteins; C057839:fibritin protein, Enterobacteria phage T4; D003545:Cysteine