mRNA vaccine expressing enterovirus D68 virus-like particles induces potent neutralizing antibodies and protects against infection

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  Figure 1. mRNA vaccine for VP1 induced anti-EV-D68 IgG but not neutralizing antibodies against EV-D68 (A) Schematic representation of the evaluated mRNAs. VP1 (red) is derived from a part of the structural protein of the EV-D68 MO strain. SPIL-2-VP1 or SPIgGκ-VP1 has secretory signal peptide derived from IL-2 (green) or IgGκ (blue) on the N terminus of VP1, respectively. (B) In vitro expression analysis of VP1. VP1 mRNA was transfected into HEK293 cells, and the culture supernatant or cell lysate was analyzed by western blotting with an anti-EV-D68 VP1 polyclonal antibody. The lane of EV-D68 means virus as a positive control. The viral VP1 is indicated by a red arrowhead, and the non-specific band is indicated by a gray arrowhead. (C) Vaccination and experiment schema of (D and E). Mice were vaccinated intramuscularly on days 0 and 21, and the plasma was collected on day 28. All the doses contained 2 μg mRNA. (D) Anti-EV-D68 IgG in plasma evaluated with ELISA. (E) Neutralizing antibody titers against EV-D68 measured on day 28. (D and E) Points are presented as individual data, and lines are presented as the median. Dotted line indicates the detection limit. ∗∗∗∗p < 0.0001; N.D., not…

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  Figure 2. mRNA vaccine expressing VLP induced anti-EV-D68 IgG and neutralizing antibodies in blood (A) Schematic representation of the mRNA vaccine expressing VLP. By co-administration of P1 mRNA-LNP and 3CD mRNA-LNP, P1 and 3CD were co-expressed in the cell. P1 was cleaved into VP0/VP3/VP1 by 3CD, and the VP0/VP3/VP1 were assembled into VLPs. (B) In vitro expression analysis of EV-D68 VLP. The culture supernatants and cell lysates from HEK293 transfected with P1 and 3CD mRNA (1:1 on weight) were analyzed by western blotting using anti-EV-D68 VP1 polyclonal antibody. (C) Confirmation of VLP formation by density gradient. The culture supernatants from HEK293 transfected with P1 and 3CD mRNA (1:1 on weight) were subjected to 10%–40% sucrose gradient, following by western blotting using anti-EV-D68 VP1 polyclonal antibody. As control, culture supernatant of EV-D68-inoculated cells was tested in parallel. (D) In vivo expression analysis of EV-D68 VLP. Mixture of P1 mRNA-LNP and 3CD mRNA-LNP (1:1 on weight, total 2 μg of mRNA) was injected into muscle, and the expression of EV-D68 VLP was analyzed with western blotting using the anti-EV-D68 VP1 polyclonal antibody. (B and D) The lane of EV-D68…

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  Figure 3. mRNA vaccine expressing VLP had the potential to protect from EV-D68 infection and paralysis (A) Vaccination and experiment schema of (B). (B) The protection effect of the mRNA vaccine expressing VLP (P1:3CD = 1:1 on weight, total 2 μg of mRNA). Mice were intranasally challenged with the MO strain after vaccination, and viral titers in the nasal turbinate and lungs were determined. (C) Experiment schema to evaluate protective effect against infection in spinal cord and EV-D68-related paralysis. (D and E) The protecting effect of the antisera from vaccinated mice. Limb paralysis (D) and rate of survival (E) in the challenged mice were monitored every day for 14 days after the serum transfer. n = 14 in the control serum group, and n = 15 in the vaccinated serum group. (F) The viral titers in the spinal cord were evaluated 4 days after challenge. (B and F) Dotted lines indicate detection limits. ∗∗∗∗p < 0.0001 as indicated by Student’s t test.

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  Figure 4. Anti-EV-D68 IgG and neutralizing antibodies were induced more potently by the mRNA vaccine expressing VLP than IWV (A–D) EV-D68 specific (A) IgG, (B) IgG1, (C) IgG2a, and (D) IgG2b titer after boost vaccination of the mRNA vaccine and IWV. The mRNA vaccine (2, 0.4, 0.08, 0.016, or 0.0032 μg of mRNA) or IWV (10, 2, 0.4, 0.08, or 0.016 μg of protein) immunizations were done twice, and anti-EV-D68 IgG in the plasma was evaluated with ELISA. (E) Neutralizing antibody titers against the homologous MO strain of EV-D68 after boost immunization with mRNA vaccine or IWV. (F) The persistence of anti-EV-D68 MO IgG antibodies in plasma. Data are shown as means ± SD. (G, H) The IgG titers against the heterologous (G) IL strain and (H) KY strain. (I–L) Neutralizing antibody titers against the (I) IL, (J) KY, (K) 1520-Yamagata-2015, and (L) EVD68/Yamagata.JPN/2023-89 strains. (M, N) Booster effect of the third dose. The neutralizing antibody titer against (M) MO strain and (N) KY strain in the plasma was quantified after the third vaccination. (E and I–N) N.D., not detectable. (A–E and G–N) Points are presented as individual data, and lines are presented as the median. (A–N) Dotted lines…

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  Figure 5. mRNA vaccine expressing VLP induced mucosal antibodies against EV-D68 (A–F) The mRNA vaccine (P1:3CD = 1:1 on weight, total 2 μg as mRNA) and IWV (2 μg as protein) were immunized twice, and the nasal wash, BALF, and immune cells in nasal tissue were collected. (A) Anti-EV-D68 IgG and IgA in the nasal wash. (B) Anti-EV-D68 IgG and IgA in the BALF. (C and D) Neutralizing antibody titers against EV-D68 in (C) nasal wash and (D) BALF. (E and F) The number of ASCs in the nasal tissue quantified by ELISpot assay. The number of (E) anti-EV-D68 IgG-ASC and (F) IgA-ASC were represented as spots per tissue. (G) The tissue distribution and expression of mRNA-LNP administrated intramuscularly or intranasally. The mRNA-LNP encoding firefly luciferase was administered to mice intramuscularly (i.m.) or intranasally (i.n.). The luciferase activity in the homogenate of nasal turbinate was quantified. The luminescence was standardized by tissue weight. (C–G) N.D., not detectable. (A–G) Points are presented as individual data, and lines are presented as the median. Dotted lines indicate detection limits. ∗p < 0.05; ∗∗∗p < 0.001; ∗∗∗∗p < 0.0001; ns, not significant as indicated by Tukey’s test.

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  Figure 6. mRNA vaccine expressing VLP induced cellular immunity, contributing to CD8 T+ cell-dependent cross-protection against CVB3 (A) IFN-γ response in splenocytes. The mRNA vaccine (P1:3CD = 1:1 on weight, total 2 μg of mRNA) and IWV (2 μg of protein) immunizations were performed twice before the splenocytes were collected. The splenocytes were cultured in the absence (non-treat) or presence of a mixture of recombinant 3C and 3D, 3C, or 3D protein of EV-D68, and the IFN-γ in the culture supernatants were quantified by ELISA. Points are presented as individual data, and lines are presented as the median. Dotted lines indicate detection limits. (B) Experiment schema for (C–E). The P1 or 3CD mRNA vaccine (1 μg of mRNA per dose) immunizations were performed twice, and CVB3 challenge was started on day 30. For CD4+ or CD8+ T cell depletion, mice vaccinated with 3CD mRNA were administered anti-CD4 (αCD4), anti-CD8 (αCD8), or their isotype control antibodies 1 day before the challenge. (C) The weight change after CVB3 challenge. (D, E) Influence of CD4+ or CD8+ T cell depletion on the efficacy of EV-D68 3CD against CVB3 challenge. (C–E) Data are shown as means ± SD. Dotted line represents the…