Abstract

INTRODUCTION Effective pulmonary vaccination remains limited by the pulmonary surfactant (PS) barrier and inefficient intracellular delivery of vaccine cargo to alveolar antigen-presenting cells, particularly alveolar macrophages. Inspired by the natural compatibility of alveolar macrophage-derived vesicles with the alveolar environment, we developed biomimetic alveolar macrophage membrane vesicles (AMVs) as a mucosal nanocarrier platform. OBJECTIVES This study aimed to develop a nanovaccine platform that addresses key extracellular and intracellular barriers in the lung and to evaluate its immunogenicity and protective efficacy in multiple respiratory pathogen models. METHODS AMVs were engineered to improve performance in the PS environment and to achieve preferential uptake by alveolar macrophages. An Antigen Capture and Cytosolic Delivery System (ACCDS) was incorporated, comprising: (1) an engineered surfactant protein A domain for broad pathogen binding; (2) a pH-responsive listeriolysin O module designed to facilitate endo/lysosomal escape and enhance cytosolic access of cargo; and (3) encapsulated Poly(I:C) to activate TLR3 and support RIG-I/MDA5-associated signaling. RESULTS AMV-ACCDS-Poly(I:C) showed improved delivery performance in the PS-associated environment and preferential uptake by alveolar macrophages compared with synthetic nanoparticles and a commercial transfection reagent. Dual innate activation was associated with stronger IFN-β and IL-18 responses than those induced by the tested control formulations. The platform promoted the local establishment and/or differentiation of lung-resident memory-like CD8+ T cells and was accompanied by IL-18-associated metabolic remodeling. In prime-boost studies, it conferred complete protection in lethal influenza and pseudorabies virus challenge models and reduced pulmonary burden in a Mycoplasma infection model. CONCLUSION