Coimmunization with Two Enterotoxigenic Escherichia coli (ETEC) Fimbrial Multiepitope Fusion Antigens Induces the Production of Neutralizing Antibodies against Five ETEC Fimbriae (F4, F5, F6, F18, and F41)

ABSTRACT

Fimbriae mediate the initial adherence of enterotoxigenic Escherichia coli (ETEC) to the piglet small intestine and play an important role in development of ETEC-driven postweaning diarrhea (PWD). PWD inflicts huge economic losses on the swine industry each year, making development of alternative treatment and prevention measures for PWD essential. Vaccine candidates that induce antifimbria antibodies that block the initial attachment and colonization of ETEC pathogens with fimbriae are one approach that could help prevent PWD. In this study, we constructed two multiepitope fusion antigens (MEFAs) that carried, expressed, and displayed representative epitopes of F4, F5, F6, F18, and F41 ETEC fimbriae. These MEFAs used either the F4 major subunit FaeG or the F18 adhesive subunit FedF as a backbone. To assess the potential of these MEFAs as antifimbria vaccine candidates that could help prevent PWD, we generated computational models of the MEFAs, constructed them, and then tested their immunogenicity by using them to immunize mice. Computational modeling showed that all relevant epitopes were exposed on the MEFA surface. We found that coadministration of our MEFAs in mice successfully induced five fimbria-specific antibodies in accordance with the epitopes included in the MEFA constructs. Furthermore, the induced antibodies can significantly inhibit the ability of ETEC strains that express F4, F5, F6, F18, and F41 fimbriae to adhere to piglet small intestinal IPEC-1 and IPEC-J2 cells. Our findings indicate that the antifimbria antibodies induced by our FaeG-Fim41a-FanC-FasA and FedF-FasA-Fim41a-FanC fimbria MEFAs blocked adherence of five ETEC fimbriae, suggesting these multivalent fimbria MEFAs may be useful for developing broadly protective antifimbria vaccines against PWD caused by ETEC infections.

IMPORTANCE Enterotoxigenic Escherichia coli (ETEC)-associated postweaning diarrhea (PWD) is still a leading disease in recently weaned piglets. Vaccination is considered to be the most ideal and efficacious strategy for preventing PWD. Recently, a commercialized live monovalent F4 oral vaccine and a bivalent F4/F18 oral vaccine have been demonstrated to effectively protect piglets in the F4-positive (F4⁺) and F18⁺ ETEC challenge models. However, they will not provide cross-protection against F5⁺, F6⁺, or F41⁺ ETEC-associated PWD cases, as they lack all five fimbria antigens. Thus, a multivalent vaccine containing all five ETEC fimbriae would be more effective in preventing ETEC-driven PWD. In this study, we designed two fimbria-targeted MEFAs using the MEFA technology, and further study demonstrated that these coadministered MEFAs in mice can induce protective antibodies against the five fimbriae expressed by ETEC. These MEFAs could be used as an efficient PWD vaccine candidate; furthermore, MEFA-based structural technology provides an alternative and promising strategy for the development of vaccines against pathogens with heterogeneous virulence factors.