A multiantigenic antibacterial nanovaccine utilizing hybrid membrane vesicles for combating Pseudomonas aeruginosa infections

Abstract

Bacterial infections, especially those caused by multidrug-resistant pathogens, pose a significant threat to public health. Vaccines are a crucial tool in fighting these infections; however, no clinically available vaccine exists for the most common bacterial infections, such as those caused by Pseudomonas aeruginosa. Herein, a multiantigenic antibacterial nanovaccine (AuNP@HMV@SPs) is reported to combat P. aeruginosa infections. This nanovaccine utilizes the hybrid membrane vesicles (HMVs) created by fusing macrophage membrane vesicles (MMVs) with bacterial outer membrane vesicles (OMVs). The HMVs mitigate the toxic effects of both OMVs and bacterial secreted toxins (SP) adsorbed on the surface of MMVs, while preserving their stimulating properties. Gold nanoparticles (AuNPs) are utilized as adjuvant to enhance immune response without comprising safety. The nanovaccine AuNP@HMV@SPs induces robust humoral and cellular immune responses, leading to destruction of bacterial cells and neutralization of their secreted toxins. In murine models of septicemia and pneumonia caused by P. aeruginosa, AuNP@HMV@SPs exhibits superior prophylactic efficacy compared to control groups including OMVs, or MMVs@SPs and HMV@SPs, achieving 100% survival in septicemia and > 99.9% reduction in lung bacterial load in pneumonia. This study highlights AuNP@HMV@SPs as a safe and effective antibacterial nanovaccine, targeting both bacteria and their secreted toxins, and offers a promising platform for developing multiantigenic antibacterial vaccines against multidrug-resistant pathogens.

Keywords: antibacterial; bacterial toxins; hybrid cell membrane; outer membrane vesicle; vaccine.

SCHEME 1

Schematic illustration of the construction and application of multiantigenic hybrid cell membrane‐based vaccine (AuNP@HMV@SPs) against P. aeruginosa infection.

FIGURE 1

Characterization and toxicity evaluation of HMV@SPs. (a) Fluorescent spectra of OMVs labelled with a FRET dye pair (DiO and DiI) after sonication with unlabeled MMVs at different protein weight ratios. Levels of pro‐inflammatory cytokines (b) IL‐6 and (c) TNF‐α production from RAW 264.7 cells treated with HMVs at different protein weight ratios of MMVs to OMVs for 12 h (2 µg/mL OMVs, n = 3). (d) TEM images of HMVs (Scale bar = 100 nm). (e) Fluorescence colocalization images of HMVs fused by DiI‐labelled OMVs and DiO‐labelled MMVs (Scale bars = 3 µm). (f) RBC hemolysis level and (g) cytotoxicity toward RAW 264.7 cells of HMV@SPs at different protein weight ratios of HMVs to SP (80 µg/mL SP, n = 3). Levels of pro‐inflammatory cytokine (H) IL‐6 and (I) TNF‐α produced from RAW 264.7 cells treated with OMVs, SP, or HMV@SPs for 12 h (OMVs: 2 µg/mL, SP: 1.5 µg/mL, n = 3). (J) Schematic illustration of the experiment to evaluate the in vivo inflammatory toxicity of HMV@SPs. Mice were injected intraperitoneally with OMVs, SP or HMV@SPs, and serum samples were collected at 5 h after injection for measurement of pro‐inflammatory cytokines levels. Serum level of (K) IL‐6 and (L) TNF‐α (OMVs: 5 µg/mL, SP: 3.8 µg/mL, n = 3). Statistical analysis was conducted using the student's t‐test. *p < 0.05, **p < 0.01, ***p < 0.001.

FIGURE 2

Physiochemical characterization, in vitro toxicity and BMDCs activation of AuNP@HMV@SPs. (a) TEM images of AuNPs and AuNP@HMV@SPs. (b) Hydrodynamic size and zeta potential of AuNPs, HMVs, HMV@SPs, and AuNP@HMV@SPs (n = 3). (c) SDS‐PAGE protein analysis of OMVs, SP, MMVs, AuNP@HMV@SPs, HMVs, and MMV@SPs. (d) RBC hemolysis level and (E) Cytotoxicity level towards RAW 264.7 cells of SP, HMV@SPs, and AuNP@HMV@SPs (Concentration: 80 µg/mL SP, n = 3). The dashed line represented 5% hemolysis level. (f) CLSM images of BMDCs cultured with AuNP@HMV@SPs for 1 h or 3 h, where DiI (red) and FITC (green) were used to stain OMVs and SP, respectively (30 µg/mL OMVs and 22 µg/mL SP, Scale bars = 20 µm). (g) Percentage of CD80⁺CD86⁺ cells in BMDCs (CD11c⁺ cells) after different treatments for 24 h, and mean florescence intensity of (H) PE‐labeled CD80 and (I) APC‐labeled CD86 in BMDCs (OMVs: 2 µg/mL, SP: 1.5 µg/mL, n = 3). Statistical analysis was conducted using the student's t‐test. *p < 0.05, **p < 0.01, ***p < 0.001 and ns representing non‐significance.

FIGURE 3

In vivo biocompatibility of AuNP@HMV@SPs. (a) Schematic illustration of experimental design to evaluate the in vivo biocompatibility of AuNP@HMV@SPs (OMVs: 2 µg/mL, SP: 1.5 µg/mL) through subcutaneous injection. Mice without immunization were used as the control group. (b) Biochemical analysis of mouse serum at day 21 (n = 4). Normal ranges: ALT (alanine transaminase): 10.06–96.47 U/L, AST (aspartate transaminase): 36.31–235.48 U/L, UREA (blood urea nitrogen): 10.81–34.74 mg/dL, CREA (creatinine): 10.91–85.09 µM. (c) Histological analysis of heart, liver, lung, spleen, and kidney tissues. Scale bar = 200 µm.

FIGURE 4

In vivo immune responses activated by AuNP@HMV@SPs. (a) Schematic illustration of experimental design to evaluate the in vivo immune responses activated by AuNP@HMV@SPs (OMVs: 2 µg/mL, SP: 1.5 µg/mL) via subcutaneous injection. (b) Percentage of CD80⁺ and CD86⁺ cells (gated on CD11c⁺ cells) in the lymph nodes collected on day 21 (n = 4). (c) Percentage of GL7⁺ cells (gated on CD19⁺IgG⁻ cells) in the spleen collected on day 21 (n = 5). (d) Anti‐P. aeruginosa specific antibody IgG titers in serum collected on day 21 (n = 5). (e) CD3⁺ T cells, (f) CD3⁺CD4⁺ T cells, and (g) CD3⁺CD8⁺ T cells in the spleen collected on day 21 (n = 5). Naive mice without immunization were used as control. Statistical analysis was conducted using the student's t‐test or One‐way ANOVA with Tukey's post hoc test. *p < 0.05, **p < 0.01, ***p < 0.001 and ns representing non‐significance.

FIGURE 5

In vivo prophylactic effect of AuNP@HMV@SPs in a systemic P. aeruginosa infection model. (a) Schematic illustration of the experimental design to evaluate the in vivo prophylactic effect of AuNP@HMV@SPs (OMVs: 2 µg/mL, SP: 1.5 µg/mL) via subcutaneous injection against systemic P. aeruginosa infection. The number of bacteria in (b) blood, (c) spleen, (d) lung, and (e) kidney of the infected mice after different treatments (n = 5). (f) Survival curves of the mice intravenously infected with P. aeruginosa (2×10⁷ CFU/20 g) after different treatment (n = 7). Levels of inflammatory cytokines including (g) IL‐6, (h) TNF‐α, and (i) IL‐1β in the serum of the infected mice after different treatments (n = 3). (j) Histological analysis of heart, liver, lung, spleen and kidney tissues of the infected mice after different treatments. Scale bar: 200 µm. Infected mice without any treatment were used as control. Statistical analysis was conducted using the student's t‐test or One‐way ANOVA with Tukey's post hoc test. *p < 0.05, **p < 0.01, ***p < 0.001.

FIGURE 6

Prophylactic effects of AuNP@HMV@SPs in a pneumonia mouse model. (a) Schematic illustration of the experimental design to evaluate the in vivo prophylactic effects of AuNP@HMV@SPs (OMVs: 2 µg/mL, SP: 1.5 µg/mL) via subcutaneous injection against pneumonia. (b) The number of bacteria in the lungs of the mice immunized with different vaccines after one day after infection (n = 5). (c) Gram staining of lung tissue of the immunized mice one day after infection. Scale bar: 50 µm. Blue arrows point to bacteria. Levels of inflammatory cytokines including (d) IL‐6, (e) TNF‐α, and (f) IL‐1β in the serum of the immunized mice one day after infection (n = 3). (g) Histological analysis of lung tissue using H&E from the immunized mice four days after infection. Infected mice without vaccination were used as the control group. Scale bar: 500 µm (upper panel) or 200 µm (lower panel). (h) Lung injury score analyzed by the H&E staining images in (g) (n = 3). Statistical analysis was conducted using the student's t‐test or One‐way ANOVA with Tukey's post hoc test. *p < 0.05, **p < 0.01, ***p < 0.001 and ns representing non‐significance.