A chimeric trivalent Echovirus vaccine designed by loop substitution elicits cross-neutralizing immunity against serotypes 11, 18, and 30

Figures

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  Fig 1. Phylogenetic construction of Echovirus. E11, E18, and E30 were located on different branches in phylogenetic analysis based on VP1 sequences.

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  Fig 2. Mouse anti-E11/18/30-VP1 serum was collected and used to screen B-cell epitopes. (A) SDS-PAGE of E11-VP1, E18-VP1, and E30-VP1. (B) Immune schedule of mice. (C) Changes of antibody titers during the immunization induced by E11/18/30-VP1 were detected by ELISA. The dashed line represents the lower limit of detection (sera were collected before immunization). Binding of the epitopes with E11-VP1 (D), E18-VP1 (E), and E30-VP1 (F) antiserum detected by ELISA. Screened epitopes in the structure of E11-VP1 (G), E18-VP1 (H), and E30-VP1 (I). The receptor-binding regions are highlighted and enclosed by black boxes. E11-VP1, E18-VP1, and E30-VP1 were colored green, epitopes were colored red. **P < 0.01; ***P < 0.001; ****P < 0.0001.

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  Fig 3. E11-Peptide 2, E18-Peptide 3, and E30-Peptide 4 were immunized and showed overlapping binding ability. (A) Sequence logo and sequence alignments of E11-Peptide 2, E18-Peptide 3, and E30-Peptide 4 were obtained from WebLogo 3 and MEGA 11. (B) Immune schedule of mice. (C) Changes of antibody titers during the immunization induced by E11-Peptide 2, E18-Peptide 3, and E30-Peptide 4 were detected by ELISA. The dashed line represents the lower limit of detection (sera were collected before immunization). Binding of the E11-Peptide 2, E18-Peptide 3, E30-Peptide 4, or VP1s antiserum with E11-VP1 (D), E18-VP1 (E), and E30-VP1 (F) detected by ELISA. *P < 0.05; ***P < 0.001; ****P < 0.0001.

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  Fig 4. A trivalent chimeric vaccine (VP1-III) was designed by inserting E30-Peptide 1, E18-Peptide 2, and E30-Peptide 6 into E11-VP1, and the structure was predicted. (A) Design of the vaccine, epitopes were shown as yellow modules. (B) Prediction model of VP1-III structure, in which inserted epitopes were compared among E11/18/30 peptides. The region spanning amino acids 82–97 of E30-Peptides 1, 121–142 of E18-Peptides 2, 143–175 of E11-Peptide 2, and 252–273 of E30-Peptides 6 was selected for substitution in the chimeric VP1 construct. VP1-III, E11-VP1, E18-VP1, and E30-VP1 were colored green, blue, orange, and red, respectively.

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  Fig 5. VP1-III was expressed, and the antiserum titer was obtained to study humoral immunity. (A) SDS-PAGE of VP1-III. (B) Immunization schedule of mice. (C and D) Changes in antibody titers during the immunization induced by E11&18&30-VP1 and VP1-III were detected by ELISA, respectively. (E) Binding of E11&18&30-VP1 and VP1-III antiserum with E11-VP1, E18-VP1, and E30-VP1 detected by ELISA, respectively. (F) Area under the curve (AUC) analysis of three experimental groups represented in panel E. Arb. Units, arbitrary units. ****P < 0.0001.

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  Fig 6. VP1-III-mRNA was designed and produced, and the antiserum titer was obtained to study humoral immunity. (A) Schematic diagram of plasmids pUC19-VP1-III. (B) Schematic diagram of mRNA structure. (C) Schematic illustration of the mRNA-LNP formulation process. (D) Particle size graph and zeta potential of mRNA-LNP. (E) TEM of mRNA-LNP. (F) Changes in antibody titers during the immunization induced by 1 and 10 µg VP1-III-mRNA detected by ELISA. (G) Binding of VP1-III-mRNA antiserum with E11-VP1, E18-VP1, and E30-VP1 detected by ELISA. (H) AUC of three experimental groups represented in panel G. ****P < 0.0001.

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  Fig 7. Immunogenicity of VP1-III and VP1-III-mRNA. Neutralization of E11 (A), E18 (B), and E30 (C) viruses by E11&18&30-VP1, VP1-III, and VP1-III-mRNA antisera was detected by relative plaque formation and plaque reduction neutralization test 50 (PRNT50). (D) Examples of plaque counting. Antiserums of E11&18&30-VP1, VP1-III, and VP1-III-mRNA were diluted at a ratio of 1:32. Neutralization of E11 (E), E18 (F), and E30 (G) viruses by antisera, as determined by viral RNA quantification. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.

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  Fig 8. VP1-III and VP1-III-mRNA induced potent cell immunity. Splenocytes were isolated from immunized mice at day 35 post initial immunization and stimulated with the corresponding epitope peptides. Antigen-specific cellular immune responses were assessed by measuring (A) splenocyte proliferation, (B) IFN-γ secretion, (C) IL-4 secretion, and the (D) IFN-γ/IL-4 ratio in mice immunized with E11&18&30-VP1, VP1-III, or VP1-III-mRNA, respectively. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.