Fig 1.
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.
Fig 2.
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].
Fig 3.
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.
Table 1.
Antigenic and physical characteristics of interprotomer-disulfide stabilized RSV F glycoprotein trimers.1
Fig 4.
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.
Fig 5.
Immunogenicity of inter-protomer disulfide stabilized RSV F variants.
Neutralization titers of sera from mice immunized with 10 μg of RSV F variants in the presence of 50 μg of poly I:C adjuvant are shown (10 mice/group). Titers from individual mice are shown as individual colored dots, and the geometric mean of each group is indicated by a red horizontal line. Protective threshold [18] at a neutralization titer of 100 is indicated by a dotted line, and statistical significance as assessed by a Mann-Whitney Unpaired non-parametric two-tailed test followed by false discovery rate correction are shown for post-fusion, ring A, ring ABCD, and DS-Cav1 RSV F immunized groups.