Tracking spatio-temporal dynamics of early immune responses to an intranasal OMV-based pneumococcal vaccine in mice

Outer membrane vesicle (OMV)-based vaccines elicit strong immune responses and have emerged as a versatile platform for targeting multiple pathogens, yet the mechanisms underlying their efficacy remain incompletely understood. Here, we investigated the early immune response following intranasal administration of an OMV-based pneumococcal vaccine in mice. Using in vivo bioluminescence imaging, spectral flow cytometry, and high-resolution microscopy, we tracked OMV biodistribution and immune activation throughout the respiratory tract from 1 to 72h post vaccination. OMVs persisted in the nasal cavity for up to 48 h and rapidly recruited Ly6Ghi neutrophils and myeloid-derived suppressor cells, followed by activation of local T cells. In the lungs, alveolar macrophages and plasmacytoid dendritic cells emerged as early responders. OMV exposure also induced costimulatory molecule expression across multiple myeloid cell subsets. Together, these findings reveal distinct spatio-temporal patterns of innate and adaptive immune activation at mucosal sites. Our multimodal analysis provides mechanistic insight into OMV-induced mucosal immunity and underscores their potential as a versatile platform for vaccine development.