Inverted HA-EV immunization elicits stalk-specific influenza immunity and cross-protection in mice

Enhancing protective immunity in the respiratory tract is crucial to combat influenza infection and transmission. Developing mucosal universal influenza vaccines requires effective delivery platforms to overcome the respiratory mucosal barrier and stimulate appropriate innate immune reactions, thereby bridging adaptive immune responses with minimal necessary inflammation. Meanwhile, the vaccine platforms must be biocompatible. This study employed cell-derived extracellular vesicles (EVs) as a mucosal universal influenza vaccine platform. By conjugating influenza hemagglutinin (HA) onto EV surfaces through HA-receptor interaction, we achieved an upside-down (inverted) influenza HA configuration that exposed the conserved HA stalk region while partially hiding the globular head domain. Intranasal immunization with the resulting EVs induced robust HA stalk- and virus-specific serum antibody and mucosal immune responses in mice, protecting against heterologous virus infection. Notably, EVs derived from the lung epithelial cell line A549 induced superior cross-reactive antibodies and enhanced protection upon intranasal immunization. EVs conjugating multivalent HA elicited broadly cross-reactive antibody and cellular responses against different influenza strains. Our results demonstrated that EVs conjugating multiple inverted HAs represented an effective strategy for developing a mucosal universal influenza vaccine.