Efficacy of genotype-matched vaccine against re-emerging genotype V Japanese encephalitis virus

Abstract

Japanese encephalitis (JE), caused by the Japanese encephalitis virus (JEV), is a highly threatening disease with no specific treatment. Fortunately, the development of vaccines has enabled effective defense against JE. However, re-emerging genotype V (GV) JEV poses a challenge as current vaccines are genotype III (GIII)-based and provide suboptimal protection. Given the isolation of GV JEVs from Malaysia, China, and the Republic of Korea, there is a concern about the potential for a broader outbreak. Under the hypothesis that a GV-based vaccine is necessary for effective defense against GV JEV, we developed a pentameric recombinant antigen using cholera toxin B as a scaffold and mucosal adjuvant, which was conjugated with the E protein domain III of GV by genetic fusion. This GV-based vaccine antigen induced a more effective immune response in mice against GV JEV isolates compared to GIII-based antigen and efficiently protected animals from lethal challenges. Furthermore, a bivalent vaccine approach, inoculating simultaneously with GIII- and GV-based antigens, showed protective efficacy against both GIII and GV JEVs. This strategy presents a promising avenue for comprehensive protection in regions facing the threat of diverse JEV genotypes, including both prevalent GIII and GI as well as emerging GV strains.

Keywords: Japanese encephalitis virus; genotype V; neutralizing antibody; recombinant vaccine; bivalent vaccine.

Figure 1.

Flowchart illustrating the process of the design, production, and evaluation of antigens used in this study.

Figure 2.

Efficacy of commercially approved JE-VAX against GIII and GV JEVs. JE-VAX or PBS was administered to mice three times at two-week intervals. Sera were collected two weeks after the final immunization. (A) Specific antibody titres against GIII Nakayama, GV 43279, and GV 43413 (n = 6-10) were determined by ELISA with a serum dilution of 1:240. (B) Neutralizing antibody titres against GIII Nakayama, GV 43279, and GV 43413 (n = 14–19) were determined by PRNT. The red dotted line indicates the detection limit (PRNT₅₀ titre = 10). Seronegative samples were arbitrarily assigned a value of 7. (C) Immunized mice were challenged with GIII Nakayama or GV 43279 (n = 15). **p < 0.01, ***p < 0.001, ****p < 0.0001. Data are pooled from three independent experiments.

Figure 3.

Prediction of 3D structure of vaccine antigens. (A) Sequence alignment of the EDIII amino acid residues from GIII Nakayama, GV 43279, and GV 43413 JEV strains. (B) Predicted 3D structures of antigens with model confidence represented in a colour spectrum. Model confidence was evaluated using the pLDDT score. (C) RMSD-based 3D alignment of predicted EDIII monomers with pentameric vaccine antigen structures. Superimposition is depicted in contrasting colours for clarity. (D) Comparative 3D alignment of predicted structures among vaccine antigens.

Figure 4.

Expression and evaluation of vaccine antigens. (A) Diagram of the expression vector construction. Cha: Chaperna domain; TEV: TEV protease cleavage site; CTB: cholera toxin B subunit; Ag: antigen. (B) Vaccine antigen expression and self-assembly analysed by SDS-PAGE. Non-reducing conditions display pentameric forms, while reducing conditions show monomeric forms. Triangles indicate expected sizes. M: size marker. (C) Pentameric assembly confirmed by GM1 binding assay; serum samples were diluted at 1:240. (D) Vaccine antigen particle sizes determined by Dynamic Light Scattering (DLS). **p < 0.01, ***p < 0.001, ****p < 0.0001.

Figure 5.

Efficacy of monovalent Ag-GIII and Ag-GV. Mice were immunized three times with 20 μg of monovalent vaccine antigens at two-week intervals. Sera and spleens were collected two weeks after the final immunization (n = 8). (A) Specific antibody titres against GIII Nakayama, GV 43279, and GV 43413 were determined by ELISA with a serum dilution of 1:240. (B) Neutralizing antibody titres against GIII Nakayama, GV 43279, and GV 43413 were determined by PRNT. (C) IFN-γ levels in culture supernatants from antigen-restimulated splenocytes isolated from immunized mice, measured after 72 h. (A–C) *p < 0.05, **p < 0.01, ****p < 0.0001. (D) Immunized mice were challenged with GIII Nakayama or GV 43279 (n = 15). * indicates statistical significance versus Ag-neg. # indicates a significant difference between Ag-GIII and Ag-GV: ***p < 0.001, #p < 0.05, ##p < 0.01. Data are pooled from three independent experiments.

Figure 6.

Efficacy of bivalent vaccine antigen. Mice were immunized three times with 20 μg of bivalent vaccine antigens at two-week intervals. Sera and spleens were collected two weeks after the final immunization. (A) Specific antibody titres against GIII Nakayama, GV 43279, and GV 43413 were determined by ELISA with a serum dilution of 1:240 (n = 10). (B) Neutralizing antibody titres against GIII Nakayama, GV 43279, and GV 43413 were determined by PRNT (n = 16–30). (C) IFN-γ levels in culture supernatants from antigen-restimulated splenocytes isolated from immunized mice, measured after 72 h (n = 4). (D) Immunized mice were challenged with either of GIII Nakayama, GV 43279, GV 43413 (n = 15). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. Data are pooled from three independent experiments.