Fimbriae-mediated initial adherence is the initial and critical step required for enterotoxigenic Escherichia coli (ETEC) infection. Therefore, vaccine candidates have been developed that target these fimbriae and induce specific antifimbriae antibodies to block initial ETEC attachment. While this vaccine effectively protects against ETEC-associated post-weaning diarrhea (PWD), developing a broadly effective vaccine against initial ETEC attachment remains a challenging problem, owing to the immunological heterogeneity among these antigens. Here, we applied multi-epitope fusion antigen (MEFA) technology to construct a FaeG-FedF-FanC-FasA-Fim41a MEFA using the adhesive subunits of predominant fimbriae K88 and F18 as the backbone, which also integrated epitopes from adhesive subunits of the rare fimbriae K99, 987P, and F41; we then generated a MEFA computational model and tested the immunogenicity of this MEFA protein in immunized mice. We next evaluated the potential of the fimbriae-targeted MEFA as a vaccine candidate to effectively prevent PWD using in vitro assessment of its anti-fimbriae, antibody-directed inhibition of bacterial adherence. Computational modeling showed that all relevant epitopes were exposed on the MEFA surface and mice subcutaneously immunized with the MEFA protein developed IgG antibodies to all five fimbriae. Moreover, anti-fimbriae antibodies induced by the MEFA protein significantly inhibited the adhesion of K88+, F18+, K99+, 987P+, and F41+ ETEC strains to piglet small intestinal IPEC-1 and IPEC-J2 cell lines. Taken together, these results indicate that FaeG-FedF-FanC-FasA-Fim41a MEFA protein induced specific anti-fimbriae neutralizing antibodies against the five targeted fimbriae. Critically, these results show the potential of fimbriae-targeted MEFA and indicate their promise as a broad, effective vaccine against PWD.

As one of the crucial enterotoxins secreted by enterotoxigenic Escherichia coli (ETEC), heat-labile enterotoxin (LT) enhances bacterial adherence both in vivo and in vitro; however, the underlying mechanism remains unclear. To address this, we evaluated the adherence of LT-producing and LT-deficient ETEC strains using the IPEC-J2 cell model. The expression levels of inflammatory cytokines and chemokines, and tight-junction proteins were evaluated in IPEC-J2 cells after infection with various ETEC strains. Further, the levels of adhesins and enterotoxins were also evaluated in F4ac-producing ETEC (F4 + ETEC) strains after treatment with cyclic AMP (cAMP). The adherence of the ΔeltAB mutant was decreased compared with the wild-type strain, whereas adherence of the 1836-2/pBR322-eltAB strain was markedly increased compared with the 1836-2 parental strain. Production of LT up-regulated the expression of TNF-α, IL-6, CXCL-8, and IL-10 genes. However, it did not appear to affect tight junction protein expression. Importantly, we found that cAMP leads to the upregulation of adhesin production and STb enterotoxin. Moreover, the F4 + ETEC strains treated with cAMP also had greater adhesion to IPEC-J2 cells, and the adherence of ΔfaeG, ΔfliC, and ΔestB mutants was decreased. These results indicate that LT enhances the adherence of F4 + ETEC due primarily to the upregulation of F4 fimbriae, flagellin, and STb enterotoxin expression and provide insights into the pathogenic mechanism of LT and ETEC.

Fimbriae mediate the initial adherence of ETEC to the piglet small intestine and play an important role in development of ETEC-driven post-weaning 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 anti-fimbriae 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 multi-epitope 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 anti-fimbriae 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 co-administration of our MEFAs in mice successfully induced five fimbriae 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 adhere to piglet small intestinal IPEC-1 and IPEC-J2 cells. Our findings indicate that the anti-fimbriae antibodies induced by our FaeG-Fim41a-FanC-FasA and FedF-FasA-Fim41a-FanC fimbriae MEFAs blocked adherence of five ETEC fimbriae, suggesting these multivalent fimbriae MEFAs may be useful for developing broadly protective anti-fimbriae vaccines against PWD caused by ETEC infections. Importance Enterotoxigenic Escherichia coli (ETEC)-associated post-weaning 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+ and F18+ ETEC challenge models. However, they will not provide cross-protection against F5+, F6+, or F41+ ETEC-associated PWD cases due to lack of expressing these fimbriae antigens. Thus, a multivalent vaccine containing all ETEC five fimbriae would be more effective in preventing against ETEC-driven PWD. In this study, we designed two fimbriae-targeted MEFAs using the MEFA technology, further study demonstrated co-administrated these MEFAs in mice can induced protective antibodies against 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.

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There is increasing evidence indicating that the production of heat-labile enterotoxin (LT) enhances bacterial adherence within in vitro and in vivo models. However, which subunit plays the main role, and the precise regulatory mechanisms remain unclear. To further elucidate the contribution of the A subunit of LT (LTA) and the B subunit of LT (LTB) in LT-enhanced bacterial adherence, we generated several LT mutants where their ADP-ribosylation activity or GM1 binding ability was impaired and evaluated their abilities to enhance the two LT-deficient E. coli strains (1836-2 and EcNc) adherence. Our results showed that the two LT-deficient strains, expressing either the native LT or LT derivatives, had a significantly greater number of adhesions to host cells than the parent strains. The adherence abilities of strains expressing the LT mutants were significantly reduced compared with the strains expressing the native LT. Moreover, E. coli 1836-2 and EcNc strains when exogenously supplied with cyclic AMP (cAMP) highly up-regulated the adhesion molecules expression and improved their adherence abilities. Ganglioside GM1, the receptor for LTB subunit, is enriched in lipid rafts. The results showed that deletion of cholesterol from cells also significantly decreased the ability of LT to enhance bacterial adherence. Overall, our data indicated that both subunits are equally responsible for LT-enhanced bacterial adherence, the LTA subunit contributes to this process mainly by increasing bacterial adhesion molecules expression, while LTB subunit mainly by mediating the initial interaction with the GM1 receptors of host cells.