Structural insights from vaccine candidates for EV-D68

Fig. 1. Cryo-EM structure of EV-D68 candidate vaccine: Virus-like particle (VLP) from US/2018-23209 B3 subclade. a EV-D68 phylogeny tree shows a prevalence of B3 and A2 subclades (https://nextstrain.org/enterovirus/d68/genome?c=clade_membership). b Surface-rendered representation showing the overall structure of EV-D68 B3 VLP. The VP0, VP1, and VP3 are colored red, forest green, and blue, respectively. c Outlined icosahedral asymmetric unit of EV-D68 B3 VLP, showing the icosahedral symmetry axes (i2, i3, and i5) and quasi-symmetry axis (q3). d B3 VLP density maps are colored by radius from the particle center. The color key shows the radius range from 116 Å to 168 Å, with the particle mean diameter of 284 Å.

Fig. 2. EV-D68 B3 VLP is structurally similar to InVP. a Surface-rendered representation showing the overall structure of EV-D68 B3 InVP. The VP1, VP2, and VP3 are forest green, orange red, and blue, respectively. b Outlined icosahedral asymmetric unit of EV-D68 B3 InVP, showing the icosahedral symmetry axes (i2, i3, and i5) and quasi-symmetry axis (q3). c B3 InVP density maps are colored by radius to particle center. The color key shows the radius range from 100 Å to 155 Å. The particle mean diameter is 255 Å. d Alignment of icosahedral asymmetric unit. B3 VLP is colored in gray. VP4 was present in B3 InVP but not captured in B3 VLP. e Alignment with B3 InVP and VLP VP1 domain. RMSD is calculated based on the C-alpha atom pairs after alignment. f B3 InVP and VLP VP1 alignment at fivefold axis. Black dashed box highlights the DE loop different organization in B3 InVP and VLP. g Alignment with B3 InVP and VLP VP2-VP3 domain. h B3 VLP and InVP VP0 and VP3 alignment at threefold axis. Black dashed box highlights the similar HI loop organization in B3 InVP and VLP.

Fig. 3. Structural differences between EV-D68 B3 InVP and VLP. a Superimposition of EV-D68 B3 InVP and B3 VLP showing the detailed conformational difference. b Summary of EV-D68 B3 VLP structural flexible regions. c Temperature map showing the B3 InVP inner conformational changes between B3 InVP and B3 VLP. B3 InVP is colored by RMSD between two samples. RMSD is calculated based on the C-alpha atom pairs after alignment using VP3 as the reference. Color bar is labeled. Regions highlighted in green represent the flexible parts in B3 VLP. d Similar to c, showing the conformational changes from the B3 InVP surface view.

Fig. 4. EV-D68 A2 VLP and InVP are also structurally similar. a Surface-rendered representation showing the overall structure of EV-D68 A2 VLP. The VP0, VP1, and VP3 are colored magenta, gold, and cyan, respectively. b Outlined icosahedral asymmetric unit of EV-D68 B3 VLP, showing the icosahedral symmetry axes (i2, i3, and i5) and quasi-symmetry axis (q3). c A2 VLP density maps are colored by radius from the particle center. The color key shows the radius range from 116 Å to 168 Å. The A2 VLP particle diameter is 284 Å. d Surface-rendered representation showing the overall structure of EV-D68 A2 InVP. The VP1, VP2, and VP3 are hot pink, gold, and cyan, respectively. e Outlined icosahedral asymmetric unit of EV-D68 A2 InVP, showing the icosahedral symmetry axes (i2, i3, and i5) and quasi-symmetry axis (q3). f A2 InVP density maps are colored by radius to the particle center. The color key shows the radius range from 100 Å to 155 Å. The A2 InVP particle diameter is 255 Å. g Alignment of icosahedral asymmetric unit. A2 InVP is colored in gray. VP4 is present in A2 InVP but not captured in A2 VLP. h Alignment with A2 InVP and VLP VP1 domain. VLP is colored in gray. RMSD is calculated based…

Fig. 5. Subclade comparisons of EV-D68 VLP/procapsid and InVP/mature virion. a–c Zoomed-in view of the icosahedral twofold axis of corresponding maps. Distances are measured by the C-α of VP0 Gln94 and the C-alpha atom of VP3 Pro141. d–f Central sections of the corresponding maps. The inside densities are absent in B1 procapsid (d), B3 VLP (e), and A2 VLP (f). g–i Zoomed-in view of the icosahedral twofold axis of corresponding maps. Distances are measured by the C-alpha atom of VP2 Gln94 and C-α of VP3 Pro141. j–l Central sections of the corresponding maps. The densities of encapsidated genomic RNA are present in B1 mature virion (j), B3 InVP (k), and A2 InVP (l).

Fig. 6. Subclade comparisons of pocket factors in EV-D68 viruses. a Cryo-EM density of pocket factor shown in B1 virus particle and B3 InVP, but absent from A2 InVP. Overlay of the local pocket factor density map in B1 virus particle, B3 InVP, and A2 InVP. Red dashed box highlights the pocket factor density. b VP1 GH loop is shown in both the model and cryo-EM map. Pocket factor density and surrounding amino acids are presented. In B1 virus particle and B3 InVP, the pocket factor densities are indicated by the black arrow. The side chain of I217 was close to the pocket factor. For A2 InVP, I216 occupied similar position as I217 in B1 virus particle and B3 InVP. Pocket factor density was not observed. Red dashed box highlights the overlay of pocket factor density and I216 (A2 InVP) and I217 (B1 virus particle and B3 InVP) in the VP1 GH loop model.

Fig. 7. Sequence variations between EV-D68 circulating B3 and A2 subclades. a EV-D68 A2 substitutions are mapped in B3 InVP, which are highlighted in yellow. b One icosahedral asymmetric unit of EV-D68 B3 InVP. Detailed outward facing A2 substitutions are indicated as yellow dots. c Table for EV-D68 A2 strain substitutions and related structural positions. Substituted residues for VP1, VP2, VP3, and VP4 are numbered by the B3 strain.