Inhalable DNA nano-adjuvant elicits robust lung-resident memory immunity against pneumonic plague

Pneumonic plague, caused by Yersinia pestis, remains a deadly threat due to its high mortality and rapid progression. In our previous study, Yersinia pestis antigens formulated with CpG oligodeoxynucleotides show great potential against pneumonic plague, but these agonists display low TLR9 affinity, poor stability, limited cellular uptake, and weak induction of tissue-resident memory immunity. Here, we introduce AdjCRU, a cruciform DNA nano-adjuvant built on a four-way junction with each arm presenting looped CpG motifs (LoDNA) for TLR9 engagement. Guided by computational design and molecular dynamics simulations, the four-way junction is chosen over other nanostructures for superior TLR9 binding. AdjCRU is nuclease-resistant, prepared by one-step annealing of four single-stranded oligonucleotides. When administered via aerosolized intratracheal inoculation alongside Yersinia pestis recombinant antigen rV10, AdjCRU is effectively internalized into lysosomes of antigen-presenting cell (APC), inducing markedly enhanced APC maturation, germinal center reaction, systemic and mucosal antibody titers, and T/B cell activation. Notably, rV10+AdjCRU drives robust lung-resident memory T/B cell immunity, and significantly improves survival by 40 % over free LoDNA in a lethal pneumonic plague mouse model. By integrating programmability, biocompatibility, and enhanced TLR9 stimulation into a single, modular platform, AdjCRU offers a versatile strategy for next-generation mucosal adjuvants against respiratory pathogens.