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.

Keywords: ETEC; MEFA; PWD; fimbriae; vaccine.

FIG 1

Construction and detection of fimbria multiepitope fusion antigens (MEFAs). (A) Schematic of the ETEC fimbrial MEFA protein constructs. Either the F4 fimbria major subunit FaeG or the F18 fimbria minor subunit FedF was used as the backbone, with 3 surface-exposed but less-antigenic epitopes substituted for the most antigenic epitopes of FanC (F5 fimbriae), FasA (F6 fimbriae), and Fim41a (F41 fimbriae) subunits, respectively. Amino acid sequences of epitopes representing each fimbrial subunit are shown in the top panel with different colors. (B) Expression of the FaeG-F41-FanC-FasA fimbria MEFA was detected using an anti-FaeG (1:1,000) monoclonal antibody (MAb). (C) The expression of FedF-FasA-F41-FanC was detected using mouse anti-FedF (1:4,000) antiserum. HRP-labeled goat anti-mouse IgG (1:5,000; Sigma) was used as the secondary antibody.

FIG 2

Computational models of the fimbrial MEFA proteins. (A) Rosetta was used to construct the FaeG-Fim41a-FanC-FasA MEFA model using the F4 major subunit FaeG model (PDB identification 2J6R) as the template. (B) Rosetta was used to construct the FedF-FasA-Fim41a-FanC MEFA model using the F18 minor subunit FedF model (PDB identification 4B4R) as the template. Epitopes of the structural subunits of the five fimbriae are highlighted in different colors as follows: F4 (green), F5 (orange), F6 (blue), F18 (pink), and F41 (purple).

FIG 3

Mouse serum anti-FaeG, FedF, FanC, FasA, and F41 IgG antibody titers (log₁₀). A total of 500 ng per well of FaeG, FedF, FanC, FasA, or Fim41a subunit purified recombinant proteins was used as coating antigen in ELISA to measure antifimbria-specific antibodies of each mouse serum sample from both the immunized and control group. Solid circles represent the titers of mice subcutaneously immunized with two fimbrial MEFA proteins; open circles represent the titers of the control mice. Each circle represents the IgG titer from a single mouse. Bars indicate the mean titers of the group specific to each fimbria. Antibody titers are presented in log₁₀.