Table 1.
A list of Escherichia coli strains used in this study.
Fimbrial E. coli and ETEC strains were used for fimbria extraction and in vitro antibody adherence inhibition assay. Recombinant E. coli strains expressing each tip subunit were used to express proteins as the coating antigen of ELISAs to titrate anti-tip antibodies.
Fig 1.
Serum anti-CFA/I, -CS1, -CS2, -CS3, -CS4, -CS5 and anti-CS6 (CssA) IgG antibody titers (log10) in the mice subcutaneously immunized with major subunit CFA/I/II/IV MEFA (●), major subunit CFA/I/II/IV MEFA combined with toxoid fusion 3xSTaN12S-mnLTR192G/L211A (■), or the control mice (Δ; no response detected).
Antibody titers were compared for differences between different treatment groups using One-way ANOVA with Tukey’s post hoc test. Bars represent the mean titers of each group. Each dot represents an antigen-specific titer from a mouse in the group. * indicates a p-value of <0.05, and ** shows a p-value of <0.01.
Fig 2.
Serum anti-CfaE (CFA/I), -CooD (CS1), -CotD (CS2), -CstH (CS3), -CsaE (CS4), -CsfD (CS5), -CssB (CS6), -LngA (CS21) and anti-EtpA IgG antibody titers (log10) in the mice subcutaneously immunized with tip MEFA (●), tip MEFA combined with toxoid fusion 3xSTaN12S-mnLTR192G/L211A (■), or the control mice (Δ; no response detected).
Titers (in log10) were compared for differences between two immunization groups by using One-way ANOVA with Tukey’s post hoc test. Bars indicate the mean titers of each group. Each dot indicates a titer specific to each tip adhesin from a mouse.
Fig 3.
In vitro mouse serum antibody adherence inhibition assays from the groups subcutaneously immunized with tip MEFA alone (A), tip MEFA combined with toxoid fusion 3xSTaN12S-mnLTR192G/L211A (B), major subunit CFA/I/II/IV MEFA alone (C), major subunit CFA/I/II/IV MEFA combined with toxoid fusion 3xSTaN12S-mnLTR192G/L211A (D), or the control group (E).
The line in each group represents the median number of each bacteria expressing CFA/I (panel a), CS1 (panel b), CS2 (panel c), CS3 (panel d), CS4/CS6 (panel e), CS5/CS6 (panel f), CS6 (panel g), CS21 (panel h) or EtpA (panel i) adhesin (CFUs) adherent to Caco-2 cells. Data were analyzed using Kruskal-Wallis test, followed by Dunn’s pairwise comparison. * indicates a p-value of <0.05, and ** refers a p-value of <0.01 (comparison between the control group and any of the immunization groups).
Fig 4.
Mouse serum antibody titers (log10) and in vitro antibody adherence inhibition against ETEC H10407 (LT/STa/CFA/I).
Panel A: anti-CfaB IgG titers from the mice subcutaneously immunized with CFA/I fimbriae or CfaB major subunit recombinant protein, or the control mice; with major subunit CfaB recombinant protein as the ELISA coating antigen. Panel B: anti-CfaE IgG titers from the mice subcutaneously immunized with CFA/I fimbriae or CfaE tip subunit protein, or the control mice; with CFA/I tip subunit CfaE recombinant protein as the ELISA coating antigen. Panel C: antibody adherence inhibition against ETEC H10407 bacteria (1x103 CFUs) to Caco-2 cells from the serum samples of the mice immunized with CFA/I fimbriae, CFA/I major subunit CfaB, or CFA/I tip subunit CfaE. Antibody titers (A and B) and antibody inhibition data (C) were compared for differences between groups using One-way ANOVA with Tukey’s post hoc test and Kruskal-Wallis test with Dunn’s pairwise comparison, respectively. The line in each group represents the mean with standard deviation (A and B) or median with range (C). ** indicates a p-value of <0.01, compared the control with each immunization group.
Fig 5.
Bacteria colonization from mouse ileum samples after challenge with ETEC strain H10407.
Mice were challenged with H10407 after SC immunized with PBS (control), heat-extracted CFA/I fimbria (CFA/I), recombinant major subunit CfaB (CfaB), or recombinant tip subunit CfaE (CfaE), orally inoculated with H10407 (primed w/H10407), or orally primed with H10407 followed with SC boosters with CFA/I fimbriae (H10407 + CFA/I). Numbers of H10407 bacteria collected from mouse ileum samples were cultured and counted (CFUs in log10). Data were normalized by log transformation and analyzed using One-way ANOVA with Tukey’s post hoc test. The line in each group represents the mean with standard deviation.