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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

α10 helix cysteines form inter-protomer disulfides that efficiently cross-link RSV F trimers.(A) Non-reducing and (B) reducing SDS-PAGE of engineered RSV F glycoproteins with a C-terminal, thrombin-cleavable foldon after tandem purification by his6 and Strep-tag II tags and visualized by Coomassie stain. Bands corresponding to covalently linked RSV F trimer (T), dimer (D), and RSV F monomer (M) are indicated on panel A. Bands corresponding to RSV polypeptides F1 and F2 are indicated on panel B. Lanes 1, DS-Cav1-Fd; 2, 1-A; 3, 1-A-F505W+SM; 4, 1-A-long; 5, 1-B; 6, 1-C; 7, 2-AB; 8, 3-ABC; 9, 3-BCD; 10, 4-ABCD-1; 11, 4-ABCD-2; 12, 4-BCDE; 13, 5-ABCDE. (C) Size exclusion chromatography of representative engineered RSV F glycoproteins after cleavage of the foldon compared to DS-Cav1-Fd. Peaks corresponding to covalently linked RSV F trimer (T), monomeric thrombin-cleaved RSV F (M), thrombin-cleaved foldon (Fd) are indicated on the chromatogram. (D) Calculated relative proportions of triple-linked, double-linked and unlinked monomeric RSV F protomers with one to five disulfide rings in the α10 helices. (E) Calculated relative proportions of cis- and trans-circularized crosslinking based on the assumption of an equal probability of neighboring cysteines in the α10 helices forming a covalent bond between helices of the coiled-coil to create a covalently ‘circularized’ ring within the coiled-coil.
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pone.0128779.g003: α10 helix cysteines form inter-protomer disulfides that efficiently cross-link RSV F trimers.(A) Non-reducing and (B) reducing SDS-PAGE of engineered RSV F glycoproteins with a C-terminal, thrombin-cleavable foldon after tandem purification by his6 and Strep-tag II tags and visualized by Coomassie stain. Bands corresponding to covalently linked RSV F trimer (T), dimer (D), and RSV F monomer (M) are indicated on panel A. Bands corresponding to RSV polypeptides F1 and F2 are indicated on panel B. Lanes 1, DS-Cav1-Fd; 2, 1-A; 3, 1-A-F505W+SM; 4, 1-A-long; 5, 1-B; 6, 1-C; 7, 2-AB; 8, 3-ABC; 9, 3-BCD; 10, 4-ABCD-1; 11, 4-ABCD-2; 12, 4-BCDE; 13, 5-ABCDE. (C) Size exclusion chromatography of representative engineered RSV F glycoproteins after cleavage of the foldon compared to DS-Cav1-Fd. Peaks corresponding to covalently linked RSV F trimer (T), monomeric thrombin-cleaved RSV F (M), thrombin-cleaved foldon (Fd) are indicated on the chromatogram. (D) Calculated relative proportions of triple-linked, double-linked and unlinked monomeric RSV F protomers with one to five disulfide rings in the α10 helices. (E) Calculated relative proportions of cis- and trans-circularized crosslinking based on the assumption of an equal probability of neighboring cysteines in the α10 helices forming a covalent bond between helices of the coiled-coil to create a covalently ‘circularized’ ring within the coiled-coil.

Mentions: One design (DS-Cav1 1-A-F505W+SM) contained Ring A and additional mutations that were intended to enhance the stability of the α10 coiled-coil by a cavity-filling substitution in the coiled-coil core (F505W) and by mutations at solvent-exposed regions that might stabilize the pre-fusion conformation. Purification of the DS-Cav1 1-A-F505W+SM protein revealed this design to form inter-protomer disulfide bonds as shown by reducing and non-reducing SDS-PAGE (Fig 3A and 3B) and by elution on size-exclusion chromatography at a position corresponding to the predicted trimer molecular weight (165 kDa) (Fig 3C) (Table 1). However, digestion with thrombin and size-exclusion purification revealed a significant fraction (>90%) to separate into RSV F dimers and monomers. Through successive rounds of size-exclusion chromatography, a pure trimeric fraction was isolated and used for antigenic and physical analyses and immunization studies.


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)

α10 helix cysteines form inter-protomer disulfides that efficiently cross-link RSV F trimers.(A) Non-reducing and (B) reducing SDS-PAGE of engineered RSV F glycoproteins with a C-terminal, thrombin-cleavable foldon after tandem purification by his6 and Strep-tag II tags and visualized by Coomassie stain. Bands corresponding to covalently linked RSV F trimer (T), dimer (D), and RSV F monomer (M) are indicated on panel A. Bands corresponding to RSV polypeptides F1 and F2 are indicated on panel B. Lanes 1, DS-Cav1-Fd; 2, 1-A; 3, 1-A-F505W+SM; 4, 1-A-long; 5, 1-B; 6, 1-C; 7, 2-AB; 8, 3-ABC; 9, 3-BCD; 10, 4-ABCD-1; 11, 4-ABCD-2; 12, 4-BCDE; 13, 5-ABCDE. (C) Size exclusion chromatography of representative engineered RSV F glycoproteins after cleavage of the foldon compared to DS-Cav1-Fd. Peaks corresponding to covalently linked RSV F trimer (T), monomeric thrombin-cleaved RSV F (M), thrombin-cleaved foldon (Fd) are indicated on the chromatogram. (D) Calculated relative proportions of triple-linked, double-linked and unlinked monomeric RSV F protomers with one to five disulfide rings in the α10 helices. (E) Calculated relative proportions of cis- and trans-circularized crosslinking based on the assumption of an equal probability of neighboring cysteines in the α10 helices forming a covalent bond between helices of the coiled-coil to create a covalently ‘circularized’ ring within the coiled-coil.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4476739&req=5

pone.0128779.g003: α10 helix cysteines form inter-protomer disulfides that efficiently cross-link RSV F trimers.(A) Non-reducing and (B) reducing SDS-PAGE of engineered RSV F glycoproteins with a C-terminal, thrombin-cleavable foldon after tandem purification by his6 and Strep-tag II tags and visualized by Coomassie stain. Bands corresponding to covalently linked RSV F trimer (T), dimer (D), and RSV F monomer (M) are indicated on panel A. Bands corresponding to RSV polypeptides F1 and F2 are indicated on panel B. Lanes 1, DS-Cav1-Fd; 2, 1-A; 3, 1-A-F505W+SM; 4, 1-A-long; 5, 1-B; 6, 1-C; 7, 2-AB; 8, 3-ABC; 9, 3-BCD; 10, 4-ABCD-1; 11, 4-ABCD-2; 12, 4-BCDE; 13, 5-ABCDE. (C) Size exclusion chromatography of representative engineered RSV F glycoproteins after cleavage of the foldon compared to DS-Cav1-Fd. Peaks corresponding to covalently linked RSV F trimer (T), monomeric thrombin-cleaved RSV F (M), thrombin-cleaved foldon (Fd) are indicated on the chromatogram. (D) Calculated relative proportions of triple-linked, double-linked and unlinked monomeric RSV F protomers with one to five disulfide rings in the α10 helices. (E) Calculated relative proportions of cis- and trans-circularized crosslinking based on the assumption of an equal probability of neighboring cysteines in the α10 helices forming a covalent bond between helices of the coiled-coil to create a covalently ‘circularized’ ring within the coiled-coil.
Mentions: One design (DS-Cav1 1-A-F505W+SM) contained Ring A and additional mutations that were intended to enhance the stability of the α10 coiled-coil by a cavity-filling substitution in the coiled-coil core (F505W) and by mutations at solvent-exposed regions that might stabilize the pre-fusion conformation. Purification of the DS-Cav1 1-A-F505W+SM protein revealed this design to form inter-protomer disulfide bonds as shown by reducing and non-reducing SDS-PAGE (Fig 3A and 3B) and by elution on size-exclusion chromatography at a position corresponding to the predicted trimer molecular weight (165 kDa) (Fig 3C) (Table 1). However, digestion with thrombin and size-exclusion purification revealed a significant fraction (>90%) to separate into RSV F dimers and monomers. Through successive rounds of size-exclusion chromatography, a pure trimeric fraction was isolated and used for antigenic and physical analyses and immunization studies.

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