Self-destructing attenuated adjuvant Salmonella serves as a safe and potent live vaccine adjuvant.

Next-generation vaccine adjuvants, including live bacterial platforms such as Salmonella, must enhance antigen-specific immune responses while maintaining an optimal safety profile, particularly for mucosal delivery. We report a rationally engineered live attenuated Salmonella adjuvant platform, self-destructing attenuated adjuvant Salmonella (SDAAS), designed for effective innate immune stimulation with built-in biosafety. Three candidate strains, χ12612, χ12621, and χ12626, were developed incorporating programmed auxotrophy for self-limiting growth, lipid A and lipopolysaccharide carbohydrate modifications to reduce endotoxin activity, and engineered flagellin. The SDAAS strains exhibited self-limiting growth, high LD₅₀ values across injection routes, and complete safety following parenteral and mucosal administration. In vitro assays showed minimal cytotoxicity, while functional analyses demonstrated robust TLR4 activation with reduced endotoxin activity from lipid A engineering and strong TLR5 signaling mediated by modified flagellin. Using ovalbumin as a model antigen, transcriptomic analysis revealed broad activation of proinflammatory, pattern recognition receptor, and antigen presentation pathways, most prominently in strain χ12621. These innate signatures correlated with elevated IL-6 and TNF-α, low IL-10, and strong antigen-specific IgG responses comparable to or exceeding those induced by alum and other commercial adjuvants. Collectively, these findings establish SDAAS strains as safe, tunable, and broadly applicable adjuvant platforms for mucosal and systemic vaccine delivery.