Genetic Fusions of Heat-Labile (LT) and Heat-Stable (ST) Toxoids of Porcine Enterotoxigenic Escherichia coli Elicit Neutralizing Anti-LT and Anti-STa antibodies

• Published in: Infection and Immunity 2010-01, Vol.78 (1), p.316-325
• Main Authors: Zhang, Weiping, Zhang, Chengxian, Francis, David H, Fang, Ying, Knudsen, David, Nataro, James P, Robertson, Donald C
• Format: Article
• Language: English
• Subjects: Animals; Antibodies, Bacterial - blood; Antibodies, Neutralizing - blood; Bacterial Toxins - genetics; Bacterial Vaccines - immunology; Bacteriology; Biological and medical sciences; Cyclic GMP; Enterotoxigenic Escherichia coli - genetics; Enterotoxigenic Escherichia coli - metabolism; Enterotoxins - genetics; Enzyme-Linked Immunosorbent Assay; Escherichia coli; Escherichia coli Infections - prevention & control; Escherichia coli Infections - veterinary; Escherichia coli Proteins - genetics; Fundamental and applied biological sciences. Psychology; Gene Expression Regulation, Bacterial; Genetic Engineering; Microbial Immunity and Vaccines; Microbiology; Miscellaneous; Rabbits; Recombinant Proteins; Swine; Swine Diseases - immunology; Swine Diseases - microbiology
• ISSN: 0019-9567; 1098-5522
• DOI: 10.1128/IAI.00497-09

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 pLT₁₉₂₍R[rightward arrow]G₎ toxoid and the porcine STa (pSTa) gene for three full-length pSTa toxoids [STa₁₁₍N[rightward arrow]K₎, STa₁₂₍P[rightward arrow]F₎, and STa₁₃₍A[rightward arrow]Q₎] and used the full-length pLT₁₉₂ as an adjuvant to carry the pSTa toxoid for pLT₁₉₂:pSTa-toxoid fusion antigens. Rabbits immunized with pLT₁₉₂:pSTa₁₂ or pLT₁₉₂:pSTa₁₃ 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 pLT₁₉₂:pSTa₁₃ 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.