Spatially restricted antigen expression by an ionizable lipid nanoparticle enhances mRNA vaccine immunity against...

Pseudomonas aeruginosa is a major cause of hospital-acquired infections, yet no licensed vaccine is available. Although mRNA-LNP vaccines offer a flexible platform for antibacterial immunization, conventional formulations are primarily optimized for gene expression and often result in systemic antigen distribution. Here, we developed a novel ionizable lipid, LC3, that enables spatially confined antigen expression following intramuscular administration while enhancing immunostimulatory activity. Compared with SM102-based LNPs, mRNA-LNP_LC3 achieved preferential antigen expression at the injection site and draining lymph nodes, with reduced hepatic expression. Mechanistic studies revealed a pronounced decoupling between cellular uptake and productive gene expression, where LC3-based LNPs showed comparable uptake but cell-context-dependent expression differences between hepatocytes and muscle cells, indicating that intracellular processing rather than uptake efficiency governs functional expression and underlies the liver-sparing profile. Single-cell RNA sequencing demonstrated that LNP_LC3 vaccination induced a coordinated early immune activation program. Functionally, LNP_LC3 elicited stronger humoral and cellular immune responses than LNP_SM102 and conferred improved protection against P. aeruginosa, while maintaining a favorable safety profile. Collectively, these results demonstrate that LC3 enables spatially restricted antigen expression through cell-context-dependent intracellular processing, linking localized expression to enhanced immunostimulatory activity. LC3-based LNPs therefore integrate spatial control of antigen expression with improved immune activation, providing a rational strategy for mRNA-LNP vaccine design.