Comparative immunogenic and structural characterization of virus-like particle and inactivated whole-virion vaccines against enterovirus D68

Figure 1. Preparation and characterization of IWV and VLP (A) Schematic representation of the construction of plasmids encoding EV-D68 P1 and 3CD. (B) Workflow for the expression and purification of VLP. (C–F) SDS-PAGE analysis of purified IWV and VLP. (C and D) VLP expressed in (C) Expi293F cells and (D) ExpiCHO-S cells purified with sucrose. The Hsp and Hsc identified by mass spectrometry are labeled in red lines. (E) VLP was expressed in ExpiCHO-S cells and further purified using both sucrose and iodixanol (OptiPrep) gradients. (F) IWV was purified by sucrose gradient ultracentrifugation. (G) IWV and VLP particle size distribution was measured by dynamic light scattering. (H) Representative negative-stain TEM images of IWV and VLP. Scale bars, 100 nm. (I) The thermal stability of the non-inactivated virus, IWV, and VLP was assessed using differential scanning fluorimetry (DSF). Data represent the mean of four independent measurements (n = 4) for each sample.

Figure 2. IWV and VLP immunogenicity (A–D) Female BALB/c mice were immunized subcutaneously with 0.1 μg of either IWV or VLP. (A and B) Plasma IgG levels specific to whole virus, IWV, or VLP following (A) prime and (B) boost immunization. (C) Neutralizing antibody titers against the MO strain were measured after boost immunization. (D) Viral loads in nasal turbinates and lungs following intranasal challenge with the MO strain. (E and F) Passive transfer protection assay in neonatal mice. Briefly, 1-day-old naive BALB/c mice were intraperitoneally injected with pooled sera collected from immunized mice 7 days after boost immunization, followed by intraperitoneal challenge with the MO strain 1 day later. (E) Limb paralysis and (F) survival rates were monitored every day for 14 days after infection. (G and H) Dose-dependent responses to IWV or VLP vaccines (ranging from 0.004 to 2.5 μg) were immunized subcutaneously. (G) Virus-specific IgG levels in plasma after boost immunization. (H) Neutralizing antibody titers post-boost. (A–D, G, and H) Each group consisted of n = 5 mice. (E and F) Each group comprised 13 or 14 mice. (A–D, G, and H) Data are presented as mean ± SD. (C, D, and H) Dotted…

Figure 3. Dispensable role of TLR4 signaling in the vaccine-induced immune response Female C3H/HeN (wild-type) and C3H/HeJ (TLR4-deficient) mice were immunized subcutaneously with 0.1 μg of IWV or VLP. (A) Virus-specific IgG levels in plasma following prime (left) and boost (right) immunizations. (B) Neutralizing antibody titers measured after boost immunization. n = 5 per group. (A, B) Data are presented as mean ± SD. (B) Dotted line represents the limit of detection. Statistical analysis was performed using 200-fold-diluted (Prime) or 5000-fold-diluted (Boost) plasma samples. “ns” denotes not significant. ∗∗∗∗p < 0.0001, as determined by Tukey’s test.

Figure 4. Epitope specificities of IgG induced by IWV and VLP vaccines (A) Localization of neutralizing antigenic sites I–IV and corresponding epitope peptide sequences. Neutralizing antigenic sites I–IV were mapped onto a single icosahedral asymmetric unit of the EV-D68 MO strain capsid, based on the previously reported cryo-EM structure (PDB: 6CSG). VP1, VP2, and VP3 are shown in gray, pink, and cyan, respectively. Neutralizing antigenic sites I, II, III, and IV are highlighted in yellow, green, blue, and magenta, respectively. The accompanying table lists the sequences of epitope peptides spanning the antigenic sites; residues constituting the neutralizing antigenic sites are indicated in red. Molecular graphics were generated using UCSF ChimeraX v1.9. (B) Plasma IgG levels specific to epitope peptides following boost immunization with either IWV or VLP. (C) Plasma IgG reactivity to mutant VLPs following boost immunization with IWV or wild-type VLP. Then, 1,000-fold diluted plasma samples were used. Details of the mutations in each mutant are summarized in the table on the right. (B–C) n = 5 per group. Data are presented as mean ± SD. (B) Statistical comparisons were performed using…

Figure 5. Cryo-EM structure of EV-D68 MO strain VLP (A) Cryo-EM density maps showing the overall structures of the mature virion (PDB: 6CSG),31 empty particle (PDB: 6CRU),31 and VLP of the EV-D68 MO strain. A schematic illustration of the viral particle is shown in the left-most. The 5-, 3-, and 2-fold symmetry axes are indicated by a pentagon, triangle, and circle, respectively. (B) Ribbon representations of the mature virion (PDB: 6CSG), empty particle (PDB: 6CRU), and VLP structures around the 2-fold axis. The 3-fold and 2-fold axes are denoted by a triangle and circle, respectively. (C) Structural comparison of the icosahedral asymmetric units of the VLP with those of the mature virion (left, PDB: 6CSG) and the empty particle (middle, PDB: 6CRU). VP1, VP0, and VP3 of the VLP were superimposed onto the corresponding subunits of the mature virion and the empty particle. Representative structures are shown based on superposition via VP1. All structures are depicted as ribbon models. The table on the right summarizes the number of pruned atom pairs and root-mean-square deviation (RMSD) values for each superposition.