Adjuvant effect of enterotoxigenic Escherichia coli (ETEC) double-mutant heat-labile toxin (dmLT) on systemic immunogenicity induced by the CFA/I/II/IV MEFA ETEC vaccine: Dose-related enhancement of antibody responses to seven ETEC adhesins (CFA/I, CS1-CS6)

Abstract

Double-mutant heat-labile toxin (dmLT, LT_R192G/L211A) of enterotoxigenic Escherichia coli (ETEC) is an effective mucosal adjuvant. Recent studies have shown that dmLT also exhibits adjuvanticity for antigens administered parenterally. In this study, we subcutaneously (SC) immunized mice with the ETEC adhesin-based vaccine, CFA/I/II/IV MEFA (multiepitope fusion antigen), adjuvanted with dmLT and examined the impact of dmLT on antibody responses specific to the seven adhesins in the vaccine construction [CFA/I, CFA/II (CS1, CS2, CS3) and CFA/IV (CS4, CS5, CS6)]. Mice were immunized with a fixed dose of CFA/I/II/IV MEFA and ascending doses of dmLT adjuvant (0, 0.05, 0.1, 0.5 or 1.0 µg) to assess the potential dmLT dose response relationship. Data showed that dmLT enhanced systemic antibody responses to all seven antigens (CFA/I, CS1-CS6) targeted by MEFA in a dose-dependent way. The adjuvant effect of dmLT on the MEFA construct plateaued at a dose of 0.1 µg. Results also indicated that dmLT is an effective parenteral adjuvant when given by the SC route with the ETEC adhesin MEFA vaccine and that antibody enhancement was achieved with relatively low doses. These observations suggest the potential usefulness of dmLT for parenteral ETEC vaccine candidates and also perhaps for vaccines against other pathogens.

Keywords: CFA/I/II/IV MEFA; Dmlt; adjuvant; antibody response; dose effect; enterotoxigenic Escherichia coli (ETEC).

Figure 1.

Anti-LT, -CFA/I, -CS1, -CS2, -CS3, CS4, CS5 and anti-CS6 IgG antibody titers (log₁₀) in the serum samples of mice immunized with CFA/I/II/IV MEFA with or without dmLT adjuvant. Mice (n = 10) in each group were SC immunized with 16 µg CFA/I/II/IV MEFA and 0, 0.05, 0.1, 0.5 or 1 µg of dmLT. Bars in each group represent the means and standard deviations of IgG titers. Each dot indicates the antibody titer of a mouse. *, **, and *** represent p-value of <0.05, <0.01, and <0.001, respectively.

Figure 2.

Mouse serum antibody neutralization activity against cholera toxin (CT). Serum samples pooled from each immunization group (n = 10) or the control group mixed with 10 ng CT toxin were added to T-84 cells. Cells were lysed after 3 h incubation, and cell lysates were measured for intracellular cAMP levels by using cAMP EIA kit (Enzo Life Sciences). The mean and standard deviation of each group represented as columns and bars. *** indicates a p-value of <0.001.

Figure 3.

Mouse serum antibody adherence inhibition activity against ETEC or E. coli bacteria expressing CFA/I, CS1-CS6 adhesins. ETEC or recombinant E. coli expressing CFA/I, CS1, CS2, CS3, CS4/CS6, CS5/CS6, or CS6 adhesin (Table 1), after incubated with mouse serum samples pooled from each immunization group (n = 10) or the control group, were transferred to Caco-2 cells. Incubated for 1 h, cells were washed to remove non-adherent bacteria and lysed. Adherent bacteria were collected, diluted, and plated on LB agar plates. Bacteria were counted for CFUs after overnight growth at 37 ◦C. The number of adherent bacteria in the control group was referred as 100%. ** and *** indicate p-value of <0.01 and <0.001, respectively.