Fig. 5
Detection of serum antibody biological functionality in vitro. (A, B) Serum antibody-mediated complement deposition. Serum samples from the identical group of female mice obtained earlier on day 49 after primary vaccination were blended and inactivated at 56°C for 30 min. Wild-type E. coli O111 (A) or E. coli O157 (B) strain of 1 × 107 CFU was resuspended in 80 µl of PBS with 1% BSA and mixed with 20 µl of heat-inactivated pooled serum from the relevant group. The mixtures were incubated at 37°C for 30 min. The treated wild-type E. coli O111 or E. coli O157 cells were washed with PBS and exposed to 100 µl of PBS-1% BSA solution containing rabbit complement and incubated at 37°C for 30 min. Then, the E. coli strains were stained with FITC-conjugated goat anti-rabbit complement polyclonal antibody and examined using flow cytometry. Sera for negative controls were obtained from mice injected with OMV vector. The experiment was conducted three times, a representative result is displayed. (C, D) Serum bactericidal antibody assay. Serum samples from the identical group of female mice obtained earlier were added to 96-well U-bottom plates in triplicate and serial 2-fold ratio dilutions in TSB broth. 1000 CFU/10 µl of wild-type E. coli O111 (C) or O157 (D) and 25 µl of baby rabbit complement were added to the wells. The total reaction volume was 100 µl and the initial serum dilution was 1:2 in this study. The plates were placed in a shaker (180 rpm) and incubated at 37°C for 1 h. The quantification of viable CFU was performed by titrating 10 µl of the reaction mixture onto TSB agar plates, followed by colony counting after an overnight incubation at 37°C. As negative controls, wells with bacteria and baby rabbit complement were added. The proportion of killed bacteria (per well) was calculated using the formula [1 - (number of CFU in the test well/number of CFU in the negative control well)] × 100. The standard differences between the mice in each group were shown by the error bars. Data are presented as the means ± SEM (n = 3). Statistical differences were assessed by a two-way ANOVA test followed by Tukey’s multiple comparison test; superscript letters a indicate P < 0.05 for comparison with the OMV vector group. (E, F) Adherence inhibition assay. The INT407 cells were suspended in DMEM medium added with 10% FBS (without penicillin-streptomycin) and plated in 24-well plates (~ 5 × 105 cells/well). The wild-type E. coli O111 (E) or O157 (F) strain of 1 × 107 CFU was suspended in 1 ml DMEM medium or 800 µl of DMEM medium supplemented with 200 µl of previously heat-inactivated pooled serum (1:5) from the relevant group. The mixtures were then incubated at 37°C for 30 min. After that, the treated bacteria of 1 × 106 CFU/100 µl were used to infect INT407 cells for 2 h at 37°C with 5% CO2. Three washes with PBS eliminated non-adherent bacteria, while adherent bacteria were retrieved using lysis with 0.1% SDS, serially diluted in PBS, and subsequently plated on MacConkey agar for quantification. The results were calculated as the percentage of adhering bacteria relative to the initial quantity of introduced bacteria, normalized to the proportion of bacterial adhesion when incubated in DMEM medium (without serum). The standard differences between the mice in each group were shown by the error bars. Data are presented as the means ± SEM (n = 3). Statistical differences were assessed by a one-way ANOVA test followed by Bonferroni’s multiple comparison test; superscript letters a indicate P < 0.05 for comparison with the OMV vector group, while ns means no significant difference