Results of in vitro antibody adherence inhibition assaysa, using serum samples of mice immunized with CFA/I/II/IV-STaN12S-dmLT, co-administrated with CFA/I/II/IV MEFA and toxoid fusion 3xSTaN12S-dmLT, or the negative control mice.

a: ETEC field isolates and E. coli recombinant strains expressing CFA/I, CS1, CS2, CS3, CS4/CS6, CS5/CS6 and CS6 (3.5x106 CFUs) were individually incubated with serum samples (20 μl) pooled from mice in the group immunized with CFA/I/II/IV-STaN12S-dmLT, co-immunized with the CFA/I/II/IV MEFA and 3xSTaN12S-dmLT, or the control mice on a shaker (50 rpm) for 1 hour at room temperature. The serum-bacteria mixture was added to Caco-2 cells (7x105 cells; 1 ml final volume) and incubated in a CO2 incubator for 1 h. After washing off non-adherent bacteria, ETEC or E. coli bacteria adhered to Caco-2 cells (in 1 ml PBS) were serial diluted, plated, cultured overnight, and counted (CFUs).b: serum samples pooled from mice of the group immunized the CFA/I/II/IV-STaN12S-dmLT, the group co-immunized with CFA/I/II/IV and 3xSTaN12S-dmLT, or the control group. These serum samples were used in the antibody adherence inhibition assay.c: p values were calculated by using a Student t test comparing numbers of ETEC or E. coli bacteria adhered to the Caco-2 cells incubated with mouse serum of each immunization group vs. bacteria adherent to the cells treated with serum of the control group.The number of ETEC or E. coli bacteria adhered to Caco-2 cells was used to indicate activity of anti-CFA antibodies against bacteria adherence.