Heat-labile- and heat-stable-toxoid fusions (LTR₁₉₂G-STaP₁₃F) of human enterotoxigenic Escherichia coli elicit neutralizing antitoxin antibodies

Abstract

Enterotoxigenic Escherichia coli (ETEC) strains are a major cause of diarrheal disease in humans and animals. Adhesins and enterotoxins, including heat-labile (LT) and heat-stable (STa) toxins, are the key virulence factors. Antigenic adhesin and LT antigens have been used in developing vaccines against ETEC diarrhea. However, STa has not been included because of its poor immunogenicity and potent toxicity. Our recent study showed that porcine-type STa toxoids became immunogenic and elicited neutralizing anti-STa antibodies after being genetically fused to a full-length porcine-type LT toxoid, LT(R₁₉₂G) (W. Zhang et al., Infect. Immun. 78:316-325, 2010). In this study, we mutated human-type LT and STa genes, which are highly homologous to porcine-type toxin genes, for a full-length LT toxoid (LT(R₁₉₂)) and a full-length STa toxoid (STa(P₁₃F)) and genetically fused them to produce LT₁₉₂-STa₁₃ toxoid fusions. Mice immunized with LT₁₉₂-STa₁₃ fusion antigens developed anti-LT and anti-STa IgG (in serum and feces) and IgA antibodies (in feces). Moreover, secretory IgA antibodies from immunized mice were shown to neutralize STa and cholera toxins in T-84 cells. In addition, we fused the STa₁₃ toxoid at the N terminus and C terminus, between the A1 and A2 peptides, and between the A and B subunits of LT₁₉₂ to obtain different fusions in order to explore strategies for enhancing STa immunogenicity. This study demonstrated that human-type LT₁₉₂-STa₁₃ fusions induce neutralizing antitoxin antibodies and provided important information for developing toxoid vaccines against human ETEC diarrhea.

Fig. 1.

Construction of LT₁₉₂-STa₁₃ fusions and detection of expressed fusion proteins. (A) Construction of LT₁₉₂-STa₁₃fusions with STa₁₃ toxoid genetically fused to the LT₁₉₂ toxoid at different locations or with a different linker. Fusions 1b and 2b had STa₁₃ fused at the C terminus of LT₁₉₂ with a Gly-Pro linker or an L linker, fusion 3b had STa₁₃ fused at the N terminus of LT₁₉₂ with a Gly-Pro linker, fusion 4b had STa₁₃ fused between the A1 and A2 peptides, and fusion 5b had Sta₁₃ fused at the end of the A subunit. Each fusion was expressed as a single 6His-tagged peptide. The drawing is not to scale with regard to peptide sizes. (B) Expressed fusion proteins were detected with rabbit anti-CT antiserum (1:3,300; Sigma) and HRP-conjugated goat anti-rabbit IgG (1:5,000; Sigma). (C) Expressed fusion proteins were detected with purified rabbit anti-STa antiserum (1:5,000) and HRP-conjugated goat anti-rabbit IgG (1:5,000; Sigma). Lanes M, marker; lanes (-), total protein extract from strain 8955.

Fig. 2.

Anti-STa and anti-LT antibody titration of mouse serum and fecal resuspension samples. (A) Anti-STa antibody titration of serum and fecal samples from immunized mice in an STa ELISA. STa-ovalbumin conjugate (1.25 ng) was used as the coating antigen, and HRP-conjugated goat-anti-mouse IgG and IgA (1:3,300) were used as the secondary antibodies. (B) Anti-LT antibody titration of serum and fecal resuspension samples from immunized mice in a GM1 ELISA. CT was used as the antigen, and HRP-conjugated goat-anti-mouse IgG and IgA (1:3,300) were the secondary antibodies. Serum and fecal samples from the control mice were included as negative controls. Optical densities greater than 0.4 (after subtracting the background reading) were used to calculate antibody titers (log₁₀). Bars and error bars indicate means and standard deviations.

Fig. 3.

Neutralizing antibody activity against STa and CT in T84 cells. (A) Serum and fecal resuspension samples from mice immunized with 6His-tagged fusion 1b to 5b proteins were used to neutralize 2 ng STa toxin in a cGMP ELISA using a cyclic GMP ELISA kit (cGMP EIA; Assay Design). Serum and fecal samples from the control group were included and treated in the same way as other samples. (B) Serum and fecal resuspension samples from immunized mice and control mice were used to neutralize 10 ng CT (Sigma) in cAMP ELISAs using a cyclic AMP ELISA kit (cAMP EIA; Assay Design). Intracellular cGMP and cAMP concentrations were calculated by following the manufacture's protocol. The P value inside each bar was calculated by comparing the value to that obtained with the negative control with Student's t test. Bars and error bars indicate means and standard deviations.