Protective role of self-assembled nanoparticle vaccine of Pseudomonas aeruginosa in a mouse model of bronchiectasis with acute infection

To establish a mouse model of bronchiectasis with acute infection and evaluate the immunogenicity and protective effect of a self-assembling Pseudomonas aeruginosa (PA) nanoparticle vaccine rePO-FN based on fusion of PcrV-OprI (rePO) protein with self-assembling ferritin (Ferritin).

① SPF-grade female C57BL/6 mice (aged 6~8 weeks, weighing 18~20 g) were randomly allocated into normal saline group, and low-, medium- and high-dose elastase groups (n=6). A mouse model of bronchiectasis was established via intratracheal instillation of different doses of elastase (30 μL of normal saline containing 0.65, 1.30 and 2.60 IU elastase) for 3 consecutive days. At 14 and 21 d after modeling, ELISA and HE staining were performed respectively to detect the concentration of IL-6 and to observe pathological changes in lung tissue in order to confirm the modeling. ② A recombinant plasmid encoding the gene of fusion protein rePO-FN was constructed and expressed in E. coli. The target protein was purified via affinity chromatography and renatured to obtain the desired protein. The physicochemical properties of the rePO-FN protein were characterized using SDS-PAGE protein gel electrophoresis, dynamic light scattering, molecular sieve chromatography, and transmission electron microscopy. ③ C57BL/6J mice were randomly divided into PBS group, rePO group, rePO-FN group, and Ferritin group (n=10). The mice in the above groups were immunized intramuscularly with 100 μL PBS buffer alone or containing 10 μg of corresponding proteins on days 0, 7, and 14. ELISA was used to measure the specific antibodies in serum. In 7 d after the final immunization, an acute PA infection model was used to compare the survival rates and bacterial colonization among the PBS, rePO, and rePO-FN groups. After establishing a bronchiectasis model by intratracheal instillation of 2.60 IU of elastase in C57BL/6J mice as described above, the mice were randomly divided into bronchiectasis PBS group, bronchiectasis rePO group, and bronchiectasis rePO-FN group (n=10). Immunization was conducted at the same dose and procedure as described above, in 21 d after bronchiectasis modeling. At the 7^th d after the final immunization, an acute PA infection model was used to compare the survival rates and bacterial colonization among the groups.

① Repeated intratracheal instillation of elastase significantly increased the concentration of IL-6 in the lung tissue when compared to the content of the normal saline group (P<0.05). Pathological observations revealed varying degrees of bronchial wall destruction, alveolar fusion, edema, neutrophil infiltration, and hemorrhage, with the severity increasing with elastase dose, which confirming successful establishment of the mouse model of bronchiectasis. ② Well-dispersed rePO-FN nanoparticles were successfully prepared, with an average particle size of 91.28 nm, a Zeta potential of approximately-6.5 mV, and a polydispersity index (PDI) of 0.306. Molecular sieve chromatography determined the elution volume of rePO-FN protein to be 8.80 mL, corresponding to a molecular weight of approximately 1400 kDa. ③ Under acute PA XN-1 strain infection, the survival rate of the rePO-FN immunization group and the bronchiectasis rePO-FN immunization group were significantly higher than that of the PBS control group (P<0.05). Additionally, bacterial colonization in the lung tissues was significantly lower in the rePO-FN immune group and the bronchiectasis rePO-FN immune group under acute PA XN-1 strain infection than that in the rePO group and the bronchiectasis rePO group (P<0.05).

Our vaccine rePO-FN can effectively trigger a strong humoral immune response and provide significant protection against PA infection in a mouse bronchiectasis model.