Metabolic-engineered probiotic-OMVs as nanovaccine mediating sequential immunomodulation for chronic bone infection immunotherapy

Chronic, drug-resistant bone infection caused by methicillin-resistant Staphylococcus aureus (MRSA) features an immunosuppressive niche enabling persistent infection and impaired bone healing. Specific treatment requires initial antibacterial immune activation against infection, followed by reshaping an anti-inflammatory microenvironment for later bone repair. Here, we screen out Lactococcus lactis outer membrane vesicles (Lac-OMVs) with first-stage dendritic cells (DCs) activation and later-stage inflammatory macrophage repolarization potential. Mechanistically, enrichment of the nicotinamide metabolism pathway within Lac-OMVs is discovered, with nicotinamide adenine dinucleotide (NAD+) as a key anti-inflammatory mediator. NAD+-enriched Lac-OMVs (NAD+-Lac-OMVs) is thus metabolically engineered via targeted culture condition optimizing, exerting biphasic immunomodulatory effects: (i) Early-stage DCs activation establishes robust humoral immunity, protecting against primary and recurrent MRSA challenge (99.35 % and 98.07 % bacteria clearance each); (ii) Later-stage targeted NAD+ delivery reprograms inflammatory macrophages at defect site, resolving inflammation and establishing a pro-osteogenic microenvironment (~10 times higher bone-repair rate).