A multivalent subunit vaccine augments pre-existing immunity to protect against T3SS-positive Pseudomonas aeruginosa but reveals immunological interference against ExlA-positive strains

Pseudomonas aeruginosa (Pa) is a ubiquitous, opportunistic nosocomial pathogen that poses a significant threat due to its innate and acquired multidrug resistance. Novel vaccine strategies are urgently needed for vulnerable populations, many of which harbor pre-existing immunity from prior encounters with Pa. Here, we evaluated a multivalent subunit vaccine combining type III secretion system (T3SS) antigens and exolysin A (ExlA) in a nanoemulsion formulation using a clinically relevant murine pulmonary pre-exposure model. This approach allowed us to determine whether vaccination could overcome the limitations of the host’s initial, ineffective immune response. Vaccination fundamentally transforms suboptimal baseline memory into a potent, multi-faceted Th1/Th17-polarized response. This globally transformed signature was characterized by significantly enhanced antigen-specific IFN- γ and IL-17A production, both locally and systematically in the lung, with exceptionally large biological effect sizes (Cohen’s d values reaching 21.35). By utilizing log10 transformation to accurately reflect pathogen growth kinetics, we demonstrated that this vaccine-augmented immunity conferred statistically significant protection following heterologous challenge. In the twice-exposed cohort, vaccination promoted superior bacterial clearance of the T3SS-positive strain, though clearance of the ExlA-positive strain was not enhanced, potentially due to immunological interference from pre-existing T3SS memory. In the thrice-exposed cohort, a functional protective threshold was observed, where high levels of natural immunity matched the vaccine-induced clearance levels. Our findings established that our vaccine formulation can effectively boost and redirect pre-existing immunity, offering a promising approach to overcome the limitations of natural exposure and protect at-risk individuals from diverse Pa infections.