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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

Negative stain-electron microscopy of RSV F glycoproteins stabilized by different C-terminal motifs.(A-J) shows typical 2D averaged classes of particles of negatively stained specimens for DS-Cav1 with various disulfide coiled-coil motifs. (A) DS-Cav control; (B) ring A with foldon; (C) ring A without foldon; (D) rings AB with foldon; (E) rings AB without foldon; (F) post-fusion F; (G) rings BCD without foldon; (H) rings ABCD without foldon; (I) rings BCDE without foldon; (J) rings ABCDE without foldon. The scale bar is 10 nm.
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pone.0128779.g004: Negative stain-electron microscopy of RSV F glycoproteins stabilized by different C-terminal motifs.(A-J) shows typical 2D averaged classes of particles of negatively stained specimens for DS-Cav1 with various disulfide coiled-coil motifs. (A) DS-Cav control; (B) ring A with foldon; (C) ring A without foldon; (D) rings AB with foldon; (E) rings AB without foldon; (F) post-fusion F; (G) rings BCD without foldon; (H) rings ABCD without foldon; (I) rings BCDE without foldon; (J) rings ABCDE without foldon. The scale bar is 10 nm.

Mentions: Analysis of the antigenic and physical characteristics of the disulfide linked DS-Cav1 variants after foldon removal and purification of the trimeric species showed broad similarity to the parental DS-Cav1 molecule (Table 1). 1-ring disulfide-linked and 2-ring disulfide linked RSV F trimers were analyzed by negative-stain electron microscopy, to quantify the presence of pre-fusion RSV F trimers (Figs 4A–4J and S1A–S1J). 10μg of each protein were adjuvanted with 50μg poly IC and used to immunize groups of 10 CB6F1/J mice twice with a 3 week interval. Week 5 sera for the 1-ring disulfide linked trimers showed no significant improvement in RSV subtype A neutralization titers compared to post-fusion with reciprocal geometric mean titer (GMT) EC50s ranging from 3.5 to 332; however two mice in the “Rings AB” DS-Cav1 stabilized group showed neutralization titers similar to DS-Cav1 (Fig 5) and the 2-ring variant group showed a higher geometric mean titer (GMT) (EC50 = 745) than all other 1-ring variants and all mice in this group had sera above the protective threshold (7) (EC50 = 100) at week 5.


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)

Negative stain-electron microscopy of RSV F glycoproteins stabilized by different C-terminal motifs.(A-J) shows typical 2D averaged classes of particles of negatively stained specimens for DS-Cav1 with various disulfide coiled-coil motifs. (A) DS-Cav control; (B) ring A with foldon; (C) ring A without foldon; (D) rings AB with foldon; (E) rings AB without foldon; (F) post-fusion F; (G) rings BCD without foldon; (H) rings ABCD without foldon; (I) rings BCDE without foldon; (J) rings ABCDE without foldon. The scale bar is 10 nm.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4476739&req=5

pone.0128779.g004: Negative stain-electron microscopy of RSV F glycoproteins stabilized by different C-terminal motifs.(A-J) shows typical 2D averaged classes of particles of negatively stained specimens for DS-Cav1 with various disulfide coiled-coil motifs. (A) DS-Cav control; (B) ring A with foldon; (C) ring A without foldon; (D) rings AB with foldon; (E) rings AB without foldon; (F) post-fusion F; (G) rings BCD without foldon; (H) rings ABCD without foldon; (I) rings BCDE without foldon; (J) rings ABCDE without foldon. The scale bar is 10 nm.
Mentions: Analysis of the antigenic and physical characteristics of the disulfide linked DS-Cav1 variants after foldon removal and purification of the trimeric species showed broad similarity to the parental DS-Cav1 molecule (Table 1). 1-ring disulfide-linked and 2-ring disulfide linked RSV F trimers were analyzed by negative-stain electron microscopy, to quantify the presence of pre-fusion RSV F trimers (Figs 4A–4J and S1A–S1J). 10μg of each protein were adjuvanted with 50μg poly IC and used to immunize groups of 10 CB6F1/J mice twice with a 3 week interval. Week 5 sera for the 1-ring disulfide linked trimers showed no significant improvement in RSV subtype A neutralization titers compared to post-fusion with reciprocal geometric mean titer (GMT) EC50s ranging from 3.5 to 332; however two mice in the “Rings AB” DS-Cav1 stabilized group showed neutralization titers similar to DS-Cav1 (Fig 5) and the 2-ring variant group showed a higher geometric mean titer (GMT) (EC50 = 745) than all other 1-ring variants and all mice in this group had sera above the protective threshold (7) (EC50 = 100) at week 5.

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