Immunogenicity and vaccine efficacy of Actinobacillus pleuropneumoniae-derived extracellular vesicles as a novel vaccine candidate

Description

Actinobacillus pleuropneumoniae (APP) is a significant pathogen in the swine industry, leading to substantial economic losses and highlighting the need for effective vaccines. This study evaluates the potential of APP-derived extracellular vesicles (APP-EVs) as a vaccine candidate compared to the commercial Coglapix vaccine. APP-EVs, isolated using tangential flow filtration (TFF) and cushioned ultracentrifugation, exhibited an average size of 105 nm and a zeta potential of -17.4 mV. These EVs demonstrated high stability under external stressors, such as pH changes and enzymatic exposure and were found to contain 86 major metabolites. Additionally, APP-EVs induced dendritic cell (DC) maturation in a Toll-like receptor 4 (TLR4)-dependent manner without cytotoxicity. APP-EVs predominantly elicited Th1-mediated IgG responses in immunized mice without significant liver and kidney toxicity. Contrarily, unlike Coglapix, which induced stronger Th2-mediated responses and notable toxicity. In addition, APP-EVs triggered robust APP-specific Th1, Th17, and cytotoxic T lymphocyte (CTL) responses and promoted the activation of multifunctional T-cells. Notably, APP-EV immunization enhanced macrophage phagocytosis and improved survival rates in mice challenged with APP infection compared to those treated with Coglapix. These findings suggest that APP-EVs are promising vaccine candidates, capable of inducing potent APP-specific T-cell responses, particularly Th1, Th17, CTL, and multifunctional T-cells, thereby enhancing the protective immune response against APP infection.

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1. Isolation of Actinobacillus pleuropneumoniae-derived extracellular vesicles (APP-EVs): APP-EVs isolated using tangential flow filtration (TFF) and cushioned ultracentrifugation 2. Mouse immunization methods: For immunization, APP-EV low dose (50 μg/mouse), APP-EV high dose (200 μg/mouse) and Coglapix (commercial vaccine; 100 μL) vaccines were given intramuscularly to the mice in two doses, separated by two-week intervals 3. Analysis of APP-specific IgG titters: Serum was collected from the mice two weeks after the final immunization to assess APP-specific IgG titers. IgG titters analyzed using biotin-conjugated Goat anti-Mouse IgG2c (SouthernBiotech), Rat anti-Mouse IgG2b (BD Bioscience), Rat anti-Mouse IgG1 (BD Bioscience), and Rat anti-Mouse IgG2a (BD Bioscience). 4. Analysis of APP-specific T-cell responses in spleen cells: APP-specific T-cell responses analyzed using flow cytometry via intracellular cytokine staining in condition of APP protein stimulation (5 μg/mL) for 12 hours 5. Analysis of liver and kidney toxicity factors in serums: Levels of alanine transaminase (ALT; Fujifilm global), aspartate transaminase (AST; Fujifilm global), alkaline phosphatase (ALP; Fujifilm global), and creatinine (CREA; Fujifilm global) in serum samples (10 μL per sample) from each immunized mice were measured using a fully automatic analyzer (Dri-Chem-NX500, Fujifilm global) for biochemical testing, in accordance with the manufacturer’s guidelines. 6. Flow cytometry-based opsonophagocytic assay: Bacteria (APP; 1×107 CFU) were suspended in 10 mL of 1X PBS and stained with CellTrace CFSE Cell Proliferation Kit reagent (5 μM) at 37°C for 30 minutes. After staining, the bacteria were washed three times with 1X PBS and resuspended in RPMI medium containing 10% fetal bovine serum. Subsequently, CFSE-stained APP (CFSE-APP, 2×105 CFU/well) was added to RAW 264.7 cells (mouse macrophage cell line; 2×105/well) in U-bottom 96-well plates and co-incubated in a shaking incubator at 37°C. Following 1 hour of co-incubation, the cells were labeled with a macrophage-specific antibody (anti-F4/80-PE from BD Bioscience) at 4°C for 30 minutes. Finally, to prevent the detection of extracellular bacteria during flow cytometry analysis, 0.2 mg/mL Trypan Blue was added, and CFSE-APP+F4/80+ cells were examined. 7. Measurement of defense effect through APP challenge inoculation: Mice were maintained in the BL-2 biohazard animal facility at the KRIVB. C57BL/6 mice received two intramuscular doses of APP-EVs and Coglapix vaccines at 2-week intervals. Two weeks following the final vaccination, the mice were intraperitoneally challenged with APP serotype 5 (1×108 CFU/200 μL). Following infection, the survival rate of the mice was monitored and recorded daily to analyze the protective effect of the immunizations.

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