Boosting Neonatal Immunity: Zymosan-Adjuvanted Intranasal Bivalent Vaccine Shields Against Lethal EV-A71 and EV-D68 Infections 2246328

Abstract Introduction Non-polio enteroviruses, particularly EV-D68 and EV-A71, have emerged as major pediatric threats due to their association with acute flaccid myelitis and high mortality. While intranasal vaccines offer a promising route to block viral entry, their effectiveness is limited by poor induction of protective immunity. Zymosan, a yeast cell wall component, has shown its adjuvant potential in multiple vaccine models. This study aims to develop an effective intranasal vaccine incorporating zymosan to protect against EV-D68 and EV-A71 infections. Methods We first prepared an inactivated EV-D68 vaccine to compare immunogenicity across administration routes. Next, we tested monovalent and bivalent intranasal vaccines with zymosan to assess multi-dose and adjuvant effects. Finally, we evaluated vaccine efficacy in a neonatal mouse model. Results Two doses of inactivated EV-D68 with intranasal priming effectively induced mucosal and systemic immunity. A three-dose regimen further strengthened vaccine-specific immunity, with zymosan amplifying responses in both formulations and enhancing serum neutralization. Serum transfer from the zymosan-adjuvanted triple-vaccinated donors afforded significant protection against both viral challenges. Conclusion These findings demonstrated that combining bivalent antigens with zymosan broadened and enhanced the intranasal vaccine efficacy, providing a promising approach for early-life protection against EV-A71 and EV-D68 infections and advancing nasal spray vaccine strategies. Funding Source National Taiwan University Hospital Topic Categories Vaccines and Immunotherapy (VAC)