Abstract
The increasing burden of antibiotic-resistant Pseudomonas aeruginosa infections has highlighted the need for alternative preventive strategies, including the development of vaccines. Accordingly, the present study aims to evaluate the humoral and cellular immune response in mice induced by OMV preparations from P. aeruginosa. Before immunization, a preliminary acute toxicity evaluation was performed, and no obvious toxicity or mortality was observed at the selected dose. Twenty-two female mice were randomly divided into treatment groups, each containing eight mice, and a control group containing six mice. The treated groups were immunized subcutaneously with thermal stress-derived OMVs (H-OMVs) or ultrasonication derived OMVs (S-OMVs), respectively, at a dose of 5 µg of OMV protein in 0.2 mL. While, the control group received PBS only. A primary dose was administered on day 1, followed by a booster dose on day 14. Serum levels of IgM, IgG, IL-17, and IFN-γ were measured on days 14 and 28 post-immunization using the sandwich ELISA technique. The results showed that the outer membrane vesicles induced a clear humoral immune response compared to the control. IgM increased early on the 14th day, with the highest value recorded in the S-OMVs group (4.123 ± 0.021 mg/ml,) followed by H-OMVs group (3.962 ± 0.089 mg/ml) and the control group (1.755 ± 0.153 mg/ml). On the 28th day, IgM levels remained higher than the control particularly in S-OMVs. IgG level showed strong increase on the 28th day, reaching 11.825 ± 0.950 mg/ml in the H-OMVs group and 11.391 ± 0.079 mg/ml in the S-OMVs group, compared to 6.046 ± 0.355 mg/ml in the control group, indicating the development of a more mature and persistent humoral response after immunization. As for the cellular immune response, H-OMVs showed a higher ability to stimulate IL-17, particularly on the 28th day, whereas S-OMVs showed a greater ability to stimulate IFN-γ, with levels on the 28th day. These results indicate that heat stress-derived vesicles were associated with a more obvious activation of the IL-17/Th17 axis, whereas ultrasonication was associated with stronger activation of the IFN-γ/Th1 axis. The findings of this study suggest that Pseudomonas aeruginosa OMVs can induce humoral and cellular immune responses in mice, with the response pattern varying according to the preparation method. This highlights the immunological importance of P. aeruginosa OMVs and supports their potential use in future immune-based studies and vaccine-related applications.

Key words: Pseudomonas aeruginosa, outer membrane vesicles, immune response, IgM, IgG, IL-17, IFN-γ