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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

Characteristics of RSV F DS-Cav1 with and without C-terminal phage foldon(A) S200 size-exclusion chromatography profile for RSV F DS-Cav1 with and without foldon in the expression construct illustrating the relative positions of purified trimer (T) and monomer (M) and DS-Cav1xFd (DS-Cav1 with a thrombin cleavage site between the α10 helix and the foldon) before and after treatment with thrombin, showing the conversion of trimer to monomer when the foldon is removed. (B) SDS-PAGE analysis of (1) DS-Cav1 monomer expressed without foldon, (2) DS-Cav1-foldon (Fd) with C-terminal purification his6 and Strep-tag II tags removed, (3) DS-Cav1-with thrombin cleavable foldon (xFd) and C-terminal purification his6 and Strep-tag II tags (4) DS-Cav1-thrombin cleavable foldon (xFd) after treatment with thrombin.
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pone.0128779.g001: Characteristics of RSV F DS-Cav1 with and without C-terminal phage foldon(A) S200 size-exclusion chromatography profile for RSV F DS-Cav1 with and without foldon in the expression construct illustrating the relative positions of purified trimer (T) and monomer (M) and DS-Cav1xFd (DS-Cav1 with a thrombin cleavage site between the α10 helix and the foldon) before and after treatment with thrombin, showing the conversion of trimer to monomer when the foldon is removed. (B) SDS-PAGE analysis of (1) DS-Cav1 monomer expressed without foldon, (2) DS-Cav1-foldon (Fd) with C-terminal purification his6 and Strep-tag II tags removed, (3) DS-Cav1-with thrombin cleavable foldon (xFd) and C-terminal purification his6 and Strep-tag II tags (4) DS-Cav1-thrombin cleavable foldon (xFd) after treatment with thrombin.

Mentions: Each protomer of the trimeric DS-Cav1 spirals around a central 3-fold axis, with a RSV-only (no foldon) interface of over 2000 Å2. As this large interface might form a stable trimer, we first tested a DS-Cav1 without foldon, created by introducing a stop codon prior to the C-terminal foldon motif. Expression of this DS-Cav1 without foldon resulted in a substantially lower ratio of D25 to motavizumab ELISA compared to DS-Cav1 with C-terminal foldon (S1 Table); when purified by tandem affinity chromatography and size-exclusion chromatography, DS-Cav1 without foldon eluted at a position corresponding to 55 kDa, substantially after that of DS-Cav1 with foldon, which elutes at a position corresponding to 165 kDa. We also created a DS-Cav1 construct with a thrombin cleavage site inserted between RSV F and foldon motif (DS-Cav1xFd). This construct initially eluted as a trimer of 165 kDa; however, after enzymatic cleavage to remove the foldon, it dissociated into 55 kDa monomers (Fig 1A and 1B).


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)

Characteristics of RSV F DS-Cav1 with and without C-terminal phage foldon(A) S200 size-exclusion chromatography profile for RSV F DS-Cav1 with and without foldon in the expression construct illustrating the relative positions of purified trimer (T) and monomer (M) and DS-Cav1xFd (DS-Cav1 with a thrombin cleavage site between the α10 helix and the foldon) before and after treatment with thrombin, showing the conversion of trimer to monomer when the foldon is removed. (B) SDS-PAGE analysis of (1) DS-Cav1 monomer expressed without foldon, (2) DS-Cav1-foldon (Fd) with C-terminal purification his6 and Strep-tag II tags removed, (3) DS-Cav1-with thrombin cleavable foldon (xFd) and C-terminal purification his6 and Strep-tag II tags (4) DS-Cav1-thrombin cleavable foldon (xFd) after treatment with thrombin.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0128779.g001: Characteristics of RSV F DS-Cav1 with and without C-terminal phage foldon(A) S200 size-exclusion chromatography profile for RSV F DS-Cav1 with and without foldon in the expression construct illustrating the relative positions of purified trimer (T) and monomer (M) and DS-Cav1xFd (DS-Cav1 with a thrombin cleavage site between the α10 helix and the foldon) before and after treatment with thrombin, showing the conversion of trimer to monomer when the foldon is removed. (B) SDS-PAGE analysis of (1) DS-Cav1 monomer expressed without foldon, (2) DS-Cav1-foldon (Fd) with C-terminal purification his6 and Strep-tag II tags removed, (3) DS-Cav1-with thrombin cleavable foldon (xFd) and C-terminal purification his6 and Strep-tag II tags (4) DS-Cav1-thrombin cleavable foldon (xFd) after treatment with thrombin.
Mentions: Each protomer of the trimeric DS-Cav1 spirals around a central 3-fold axis, with a RSV-only (no foldon) interface of over 2000 Å2. As this large interface might form a stable trimer, we first tested a DS-Cav1 without foldon, created by introducing a stop codon prior to the C-terminal foldon motif. Expression of this DS-Cav1 without foldon resulted in a substantially lower ratio of D25 to motavizumab ELISA compared to DS-Cav1 with C-terminal foldon (S1 Table); when purified by tandem affinity chromatography and size-exclusion chromatography, DS-Cav1 without foldon eluted at a position corresponding to 55 kDa, substantially after that of DS-Cav1 with foldon, which elutes at a position corresponding to 165 kDa. We also created a DS-Cav1 construct with a thrombin cleavage site inserted between RSV F and foldon motif (DS-Cav1xFd). This construct initially eluted as a trimer of 165 kDa; however, after enzymatic cleavage to remove the foldon, it dissociated into 55 kDa monomers (Fig 1A and 1B).

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