BALB/c mice were immunized intramuscularly or intranasally with SuOMV, sOMV, or nOMV. or mucosal sIgA. Taken together, the preparation method had a significant effect on the yield, morphology, and composition of AbOMVs, that further influenced the protective effect against infection. (has emerged as one of the most challenging pathogens to effectively treat (5, 6). The mortality rates associated with infection has reported to be 26.0C55.7% (7), and thus effective prevention or treatment procedures are urgently needed. Vaccines are the most efficient and cost-effective way to control infectious diseases. Different kinds of vaccines have been formulated aiming at protecting against infection. Inactivated whole cells (IWC) (8), outer membrane complex (OMC) (9), outer membrane vesicles (OMVs) (10, 11), and several outer membrane proteins including OmpA (12), Omp22 (13), and OmpK (14) have been identified as U 95666E vaccine candidates through active and passive immunizations of experimental animals. OMVs are highly immunogenic nano-sized spherical structures enriched with outer membrane proteins, and are considered as vaccine candidates against derived pathogenic bacteria (15, 16). OMV vaccines against serogroup B strains of (MenB) have been licensed in Norway, Cuba, and New Zealand, which led to the substantial reduction of local cases of invasive meningococcal disease (17, 18). Several methods have been developed to prepare OMVs. Gram-negative bacteria secrete OMVs U 95666E spontaneously to the culture medium in the process of growth, and OMVs could be enriched and isolated from the cell culture supernatant (19). Other methods to obtain OMVs from bacterial cells included using detergents and/or chelating agents to promote the production of U 95666E the OMVs (20, 21), always with a higher yield than isolation from the culture supernatant (22). In addition to yield, it was proven that the MenB OMVs obtained by varied methods exhibited differences in morphology, protein composition, toxicity, immunogenicity, and even in serum bactericidal activity (SBA) (23C25). The most studied method to obtain OMV (AbOMV) so far has been isolation by the combination of ultrafiltration and ultracentrifugation from the cell culture supernatants (10, 26, 27). Though AbOMV isolated from the above method was immunogenic and protective, the yield of AbOMV is generally low. In the current study, novel methods were established to prepare AbOMV from bacterial cells. The AbOMVs obtained from different methods were analyzed for yield, morphology, surface charge, protein composition, and immunogenicity. The protective efficacy against the infection of was also evaluated and the mechanism was explored with the objective to provide a basis for U 95666E the selection of the best approach for AbOMV vaccine development. Materials and Methods Ethics Statement All animal care and protocols were performed in accordance with the Regulations for the Administration of Affairs Concerning Experimental Animals approved by the State Council of People’s Republic of China. All animal experiments were approved by the Animal Ethical and Experimental Committee of the Third Military Medical University. Well-trained and skilled animal care personnel participated in the current study to minimize the suffering of animals. Animals and Bacteria Strains SPF female C57BL/6J mice of 6C8 weeks were purchased from the Experimental Animal Center of the Third Military Medical University. All mice were kept under pathogen-free conditions in the animal center of the Third Military Medical University (Chongqing, China). ATCC17978 and LAC-4 were kindly provided by Prof. Yun Shi (West China Hospital, Sichuan University). Reagents RPMI 1640(SH30809.01), TRYPSIN 0.25% Solution (SH30042.01), and PBS buffer (SH30256.01B) were purchased from HyClone; Penicillin-streptomycin solution (100 , C0222), PE anti-mouse CD40(124609), APC anti-mouse CD86 (105011), PerCP/Cy5.5 anti-mouse CD80 (104721), and FITC anti-mouse CD11c(117305) were purchased from Beyotime; Murine M-CSF(315-02-10 g) and IL-4(214-14-20 g) were purchased from PEPROTECH; FBS (P30-3302) was purchased from U 95666E PAN; Goat Anti-Mouse IgA alpha chain (HRP) (ab97235), Goat Anti-Mouse IgG1 heavy chain (HRP) (ab97240), and Goat Anti-Mouse IgG2a heavy chain (HRP) (ab97245) were purchased from Abcam; Mouse TNF- ELISA kit (1217202), Mouse IL-12p70 Elisa kit (1211202), Mouse IL-10 ELISA kit (1211002), and Mouse IL-6 ELISA kit (1210602) were purchased from Dakewe; Lowry protein concentration assay kit (PC0030), DAB color development kit (DA1010), and KDO (2-keto-3-deoxyoctane, K2755) was purchased from Solarbio; Quant-iT? PicoGreen? dsDNA Reagent (P7581) was purchased from Thermo (Invitrogen). Mouse monoclonal to TYRO3 Preparation of AbOMVs sOMV (spontaneously released OMV) were prepared as described previously with slight modification (26). Briefly, the 17978 was grown to late log phase in.