Genetic fusions of heat-labile (LT) and heat-stable (ST) toxoids of porcine enterotoxigenic Escherichia coli elicit neutralizing anti-LT and anti-STa antibodies

Abstract

Enterotoxigenic Escherichia coli (ETEC) strains are a major cause of diarrheal disease in humans and farm animals. E. coli fimbriae, or colonization factor antigens (CFAs), and enterotoxins, including heat-labile enterotoxins (LT) and heat-stable enterotoxins (ST), are the key virulence factors in ETEC diarrhea. Unlike fimbriae or LT, STa has not often been included as an antigen in development of vaccines against ETEC diarrhea because of its poor immunogenicity. STa becomes immunogenic only after being coupled with a strongly immunogenic carrier protein. However, native or shorter STa antigens either had to retain toxic activity in order to become antigenic or elicited anti-STa antibodies that were not sufficiently protective. In this study, we genetically mutated the porcine LT (pLT) gene for a pLT(192(R-->G)) toxoid and the porcine STa (pSTa) gene for three full-length pSTa toxoids [STa(11(N-->K)), STa(12(P-->F)), and STa(13(A-->Q))] and used the full-length pLT(192) as an adjuvant to carry the pSTa toxoid for pLT(192):pSTa-toxoid fusion antigens. Rabbits immunized with pLT(192):pSTa(12) or pLT(192):pSTa(13) fusion protein developed high titers of anti-LT and anti-STa antibodies. Furthermore, rabbit antiserum and antifecal antibodies were able to neutralize purified cholera toxin (CT) and STa toxin. In addition, preliminary data suggested that suckling piglets born by a sow immunized with the pLT(192):pSTa(13) fusion antigen were protected when challenged with an STa-positive ETEC strain. This study demonstrated that pSTa toxoids are antigenic when fused with a pLT toxoid and that the elicited anti-LT and anti-STa antibodies were protective. This fusion strategy could provide instructive information to develop effective toxoid vaccines against ETEC-associated diarrhea in animals and humans.

FIG. 1.

Construction of a porcine LT₁₉₂:STa-toxoid genetic fusion. PCR primers 1(184EcoRV-F) and 6 (LT-R) amplified the entire porcine eltAB genes (without a stop codon), and primers 5 (STa-F) and 4 (pBREagI-R) amplified the full-length porcine estA gene (without a signal peptide). Primers 7 (LT₁₉₂-R) and 8 (LT₁₉₂-F; complementary to primer 7) mutated eltAB genes for LT₁₉₂, and primers 10 (mSTa12-R) and 9 (mSTa12-F; complementary to primer 10) mutated the STa gene to produce an STa mutant. Primers 2 (pLT:STa-R) and 3 (pLT:STa-F) added a Gly-Pro linker and genetically fused the mutated LT genes and the mutated STa gene. The gene sizes are not proportional. The lower right panels show Western blot detection of toxoid fusions using anti-CT and anti-STa antibodies, and total protein samples from TOPO cells were used as a negative control.

FIG. 2.

STa competitive ELISA to detect expression of STa proteins in the STa recombinant 8330 (STa) and mutant strains 8413 (STa₁₁), 8415 (STa₁₂), and 8417 (STa₁₃). A 1.25-ng portion of STa-ovalbumin conjugate was applied to each well; anti-STa serum (1:10,000) was used as the primary antibody and horseradish peroxidase-conjugated goat anti-rabbit immunoglobulin (IgG; 1:10,000) was used as the secondary antibody. Optical densities were measured at 405 nm. Error bars indicate standard deviations.

FIG. 3.

Cyclic GMP ELISA to detect toxicity of STa toxoids. Culture growth supernatant from STa toxoid strains was used to stimulate T-84 cells to produce an increase of intracellular cGMP levels by using a cGMP EIA kit (Assay Design). Error bars indicate standard deviations.

FIG. 4.

Porcine ligated gut loop assay to detect STa toxoid toxic activity. A total of 2 × 10⁹ CFU of 8330 (STa), 8413 (STa₁₁), 8415 (STa₁₂), 8417 (STa₁₃), or negative control strain 8331 (−) was incubated in each loop (three repeats). After 8 h of incubation, fluid accumulated in each loop was measured, and a ratio of the accumulated fluid (g) to the loop length (cm) was used as an index. Error bars indicate standard deviations.

FIG. 5.

Antibody titration from serum and fecal samples of rabbits immunized with pLT₁₉₂:pSTa₁₂ or pLT₁₉₂:pSTa₁₃ fusion antigen. The titers (in log₁₀) of anti-LT were detected in a LT GM1 ELISA using purified CT and antigen, with rabbit antiserum samples (1:50) as the primary antibody. To determine the titers of anti-STa antibody, we used STa ovalbumin conjugates as the antigen and rabbit antiserum and antifecal samples (1:50) as the primary antibodies. HRP-conjugated IgG and IgA antibodies (1:5,000) were used as the secondary antibodies. Optical densities of greater than 0.4 (after subtracting the background reading) were used to calculate antibody titers (in log₁₀). Error bars indicate standard deviations.

FIG. 6.

Anti-LT and anti-STa antibody neutralization. Serum and fecal samples (1:50) from rabbits immunized with pLT₁₉₂:pSTa₁₂ or pLT₁₉₂:pSTa₁₃ fusion antigen were used to neutralize purified CT (10 ng) or STa (2 ng). The mixture was added to T-84 cells to test for an increase of intracellular cGMP (for STa; an ELISA kit from Assay Design was used) or cAMP (for CT; an ELISA kit from Invitrogen was used) levels. Cell culture medium alone was included as a negative control. CT or STa toxin alone or incubated with a serum or a fecal sample from the control rabbit was also included as a negative control. Error bars indicate standard deviations.

FIG. 7.

ELISA detection of anti-STa IgA antibodies in colostrum samples from the sow immunized with LT₁₉₂:STa₁₂ antigen and a negative control sow and in serum samples from the piglets. We coated each well with 1.25 ng STa-ovalbumin conjugates and then added 100 μl of each sow colostrum sample (1:10) and piglet serum sample (1:50). HRP-conjugated goat anti-porcine IgA (1:3,000) was used as the secondary antibody. Optical densities were measured at 405 nm. Error bars indicate standard deviations.