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A Cysteine Zipper Stabilizes a Pre-Fusion F Glycoprotein Vaccine for Respiratory Syncytial Virus.

Stewart-Jones GB, Thomas PV, Chen M, Druz A, Joyce MG, Kong WP, Sastry M, Soto C, Yang Y, Zhang B, Chen L, Chuang GY, Georgiev IS, McLellan JS, Srivatsan S, Zhou T, Baxa U, Mascola JR, Graham BS, Kwong PD - PLoS ONE (2015)

Bottom Line: Recombinant subunit vaccines should contain minimal non-pathogen motifs to reduce potential off-target reactivity.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.

View Article: PubMed Central - PubMed

Affiliation: Vaccine Research Center, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland, United States of America.

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.

No MeSH data available.


Related in: MedlinePlus

Design of pre-fusion RSV F with inter-protomer disulfide rings within the viral α10 coiled-coil.(A) Pre-fusion DS-Cav1 RSV F trimer structure with T4 fibritin foldon trimerization domain colored in blue showing residues L512 and L513 in green. (B) Designed pre-fusion RSV F with inter-protomer disulfide rings A-E within the α10 coiled-coil. Pre-fusion DS-Cav1 RSV F trimer structure with extended α10 helix modelled and depiction of cysteines introduced into the coiled-coil core to covalently link the three protomers. (C) 2-dimensional complementation grid exemplifying 2 rings where combinations of covalent closure of the coiled-coil by a single pair of cysteines are shown in red (cis-circularization) and multiple pairs of cysteines shown in orange (trans-circularization) and incomplete covalent circularization of the protomers shown in white. (D) Wheel diagram representations of the α10 coiled-coil wild-type (left) and 5-ring engineered coiled-coil (right) as generated by DrawCoil 1.0: http://www.grigoryanlab.org/drawcoil/ [32].
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pone.0128779.g002: Design of pre-fusion RSV F with inter-protomer disulfide rings within the viral α10 coiled-coil.(A) Pre-fusion DS-Cav1 RSV F trimer structure with T4 fibritin foldon trimerization domain colored in blue showing residues L512 and L513 in green. (B) Designed pre-fusion RSV F with inter-protomer disulfide rings A-E within the α10 coiled-coil. Pre-fusion DS-Cav1 RSV F trimer structure with extended α10 helix modelled and depiction of cysteines introduced into the coiled-coil core to covalently link the three protomers. (C) 2-dimensional complementation grid exemplifying 2 rings where combinations of covalent closure of the coiled-coil by a single pair of cysteines are shown in red (cis-circularization) and multiple pairs of cysteines shown in orange (trans-circularization) and incomplete covalent circularization of the protomers shown in white. (D) Wheel diagram representations of the α10 coiled-coil wild-type (left) and 5-ring engineered coiled-coil (right) as generated by DrawCoil 1.0: http://www.grigoryanlab.org/drawcoil/ [32].

Mentions: Since we could not use D25 selection to identify stable trimers, we chose to focus on interprotomer disulfides, the formation of which could be assessed by non-reducing SDS-PAGE. The trimeric structure of pre-fusion RSV F [10] contains a membrane-proximal helix (α10), which forms a left-handed coiled-coil with C3 symmetry about the trimer axis with a tilt angle of about 30 degrees (Fig 2). We attempted to stabilize the RSV F trimer by introducing interprotomer disulfide bonds in the RSV F C-terminal coiled-coil that would allow for stable trimers. Because the foldon is attached to the C-terminus of this helix, these interprotomer disulfides would stabilize the trimer at a position similar to that of the foldon in the parent RSV F DS-Cav1+foldon.


A Cysteine Zipper Stabilizes a Pre-Fusion F Glycoprotein Vaccine for Respiratory Syncytial Virus.

Stewart-Jones GB, Thomas PV, Chen M, Druz A, Joyce MG, Kong WP, Sastry M, Soto C, Yang Y, Zhang B, Chen L, Chuang GY, Georgiev IS, McLellan JS, Srivatsan S, Zhou T, Baxa U, Mascola JR, Graham BS, Kwong PD - PLoS ONE (2015)

Design of pre-fusion RSV F with inter-protomer disulfide rings within the viral α10 coiled-coil.(A) Pre-fusion DS-Cav1 RSV F trimer structure with T4 fibritin foldon trimerization domain colored in blue showing residues L512 and L513 in green. (B) Designed pre-fusion RSV F with inter-protomer disulfide rings A-E within the α10 coiled-coil. Pre-fusion DS-Cav1 RSV F trimer structure with extended α10 helix modelled and depiction of cysteines introduced into the coiled-coil core to covalently link the three protomers. (C) 2-dimensional complementation grid exemplifying 2 rings where combinations of covalent closure of the coiled-coil by a single pair of cysteines are shown in red (cis-circularization) and multiple pairs of cysteines shown in orange (trans-circularization) and incomplete covalent circularization of the protomers shown in white. (D) Wheel diagram representations of the α10 coiled-coil wild-type (left) and 5-ring engineered coiled-coil (right) as generated by DrawCoil 1.0: http://www.grigoryanlab.org/drawcoil/ [32].
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Related In: Results  -  Collection

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pone.0128779.g002: Design of pre-fusion RSV F with inter-protomer disulfide rings within the viral α10 coiled-coil.(A) Pre-fusion DS-Cav1 RSV F trimer structure with T4 fibritin foldon trimerization domain colored in blue showing residues L512 and L513 in green. (B) Designed pre-fusion RSV F with inter-protomer disulfide rings A-E within the α10 coiled-coil. Pre-fusion DS-Cav1 RSV F trimer structure with extended α10 helix modelled and depiction of cysteines introduced into the coiled-coil core to covalently link the three protomers. (C) 2-dimensional complementation grid exemplifying 2 rings where combinations of covalent closure of the coiled-coil by a single pair of cysteines are shown in red (cis-circularization) and multiple pairs of cysteines shown in orange (trans-circularization) and incomplete covalent circularization of the protomers shown in white. (D) Wheel diagram representations of the α10 coiled-coil wild-type (left) and 5-ring engineered coiled-coil (right) as generated by DrawCoil 1.0: http://www.grigoryanlab.org/drawcoil/ [32].
Mentions: Since we could not use D25 selection to identify stable trimers, we chose to focus on interprotomer disulfides, the formation of which could be assessed by non-reducing SDS-PAGE. The trimeric structure of pre-fusion RSV F [10] contains a membrane-proximal helix (α10), which forms a left-handed coiled-coil with C3 symmetry about the trimer axis with a tilt angle of about 30 degrees (Fig 2). We attempted to stabilize the RSV F trimer by introducing interprotomer disulfide bonds in the RSV F C-terminal coiled-coil that would allow for stable trimers. Because the foldon is attached to the C-terminus of this helix, these interprotomer disulfides would stabilize the trimer at a position similar to that of the foldon in the parent RSV F DS-Cav1+foldon.

Bottom Line: Recombinant subunit vaccines should contain minimal non-pathogen motifs to reduce potential off-target reactivity.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.

View Article: PubMed Central - PubMed

Affiliation: Vaccine Research Center, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland, United States of America.

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.

No MeSH data available.


Related in: MedlinePlus