Self-assembled EDIII/NS1 nanoparticle vaccines elicit protective immune responses against Japanese encephalitis virus

Japanese encephalitis virus (JEV) is a mosquito-borne orthoflavivirus that can cause severe neurological disease in humans and reproductive disorders in swine, posing a persistent threat to public health and the swine industry. Despite being vaccine-preventable, the virus remains endemic in 24 countries and territories throughout Asia and Oceania, underscoring the urgent need for safer and more effective vaccines to counter this ongoing threat. In this study, we developed the nanoparticle vaccines by covalently conjugating 60 copies of EDIII or NS1 protein subunits to the self-assembling mi3 protein via the SpyTag/SpyCatcher system. Transmission electron microscopy (TEM) and dynamic light scattering (DLS) analyses revealed average particle sizes of approximately 27.8 nm for mi3-EDIII and 36.0 nm for mi3-NS1 nanoparticles. Compared to the EDIII subunit vaccine, the mi3-EDIII nanoparticle vaccine induced stronger neutralizing antibodies, but not significantly enhanced cellular immunity. Notably, combining mi3-EDIII with mi3-NS1 further increased neutralizing antibody titers and stimulated CD4⁺ T cell mediated immune responses, leading to effective protection against JEV challenge. These results provide a foundation for developing improved JEV vaccines and offer valuable insights for next-generation vaccine design.