Abstract Rationale Healthcare-associated bacterial infections (HAI), caused by multidrug-resistant pathogens, such as Pseudomonas aeruginosa (Pa), Klebsiella pneumoniae (Kp) and Acinetobacter baumannii (Ab), pose a threat to public health. Vaccines represent a pivotal intervention in combating HAI, and bacterial membrane vesicles (BMVs) are potential candidates for such vaccines. Herein, calcium phosphate nanoparticles (CaP) are utilized as an adjuvant for BMVs to enhance antibacterial immune responses to prevent HAI. Methods Calcium phosphate (CaP) was precipitated by mixing and stirring a solution containing NaH2PO4, Na2HPO4, and CaCl2. BMVs from Pa, Kp or Ab were respectively isolated using gradient centrifugation. BMVs@CaP were prepared via ultrasonic oscillation. The characterization of BMVs@CaP was verified using transmission electron microscopy, Fourier transform infrared spectroscopy and etc. C57BL/6 mice were subcutaneously injected with BMVs@CaP and then were infected with different bacteria via intranasal administration. The efficacy was evaluated through lung colony-forming unit, enzyme-linked immunosorbent assay and etc. Flow cytometry and immunofluorescence were used to investigate BMVs@CaP enhancing immune responses through the expression of co-stimulatory molecules and major histocompatibility complex (MHC) in dendritic cells (DCs), T cell differentiation, and IgG production. Transcriptomic analysis was performed to identify differentially expressed genes in DCs. Western blotting (WB) and other molecular experiments were conducted to validate that BMVs@CaP modulate maturation of DCs via activating Nod-like receptor protein 1 (NLRP1) inflammasome. Results BMVs@CaP had an average particle size of approximately 150 nm, with a stable structure and successful load of CaP nanoparticles. CaP-modified BMVs derived from Kp, Ab, and Pa had significant prophylactic efficacy in vivo. BMVs@CaP reduced the release of pro-inflammatory cytokines (IL-1β and TNF-α) and alleviated pathological damage to lung tissues. Taking Pa-derived BMVs@CaP as an example, compared with the PBS group, the bacterial load was decreased by 46.45% in the BMVs group and by 82.77% in the BMVs@CaP group. Additionally, BMVs@CaP remarkably upregulated the expression of CD80, CD86, and MHCII molecules on the surface of DCs, promoted Th2 cell differentiation, and increased the production of BMV-specific antibodies. Transcriptomic analysis and WB results revealed that BMVs@CaP activated NLRP1 inflammasome in DCs. Conclusions CaP significantly enhanced the specific prophylactic effect of BMVs. BMVs@CaP activated NLRP1 inflammasome in DCs, thereby promoting antigen presentation and humoral immune responses. This study highlights the application potential of CaP as an adjuvant for BMV-based vaccines against HAI. This abstract is funded by: This work was supported by grants from the National Natural Science Foundation of China (82102190).
Chen et al. (2026) studied this question.