Immunogenicity and plasmid delivery pathways of non-invasive <i>Lactococcus lactis</i> -vectored mucosal DNA vaccination

Mucosal DNA vaccination using a non-invasive <i>Lactococcus lactis</i> (LL) vector has been investigated. However, its immunogenicity and plasmid transfer mechanisms remain largely unknown. In this study, we investigated the intranasal delivery of LL carrying a mammalian enhanced green fluorescent protein (EGFP)-expressing plasmid and the cellular pathways underlying DNA transfer. Intranasally administered LL was primarily localized on the nasal epithelial surfaces, and a smaller fraction penetrated the subepithelial tissues. Intranasal administration of LL-carrying pLEC-EGFP plasmid induces antigen-specific serum IgG and mucosal IgA responses. <i>In vitro</i> co-culture analyses demonstrated that plasmid delivery and expression occurred in phagocytic cell lines but not in epithelial cell lines. This transfer was inhibited by compounds specific for phagocytosis, consistent with the observed time course of DNA transfer and localization of LL within Lamp-1⁺ phagolysosomes. In contrast, compounds for bactericidal mechanisms, including lysosomal acidification, reactive oxygen species, and reactive nitrogen species, did not affect DNA transfer. As our findings suggest that phagocytosis is the primary pathway for plasmid delivery by non-invasive LL vectors in cell culture assays, further studies to confirm these findings in animal models are warranted to develop new strategies for improved LL-based mucosal DNA vaccines.