Fimbriae-mediated initial adherence is the initial and critical step required for enterotoxigenicEscherichia coli(ETEC) infection. Therefore, vaccine candidates have been developed that target these fimbriae and induce specific anti-fimbriae 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.

EnterotoxigenicEscherichia 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.

Enterotoxigenic Escherichia coli (ETEC) producing type Ib heat-stable toxin (STa) are a top cause of children's diarrhea and travelers’ diarrhea, thus STa needs to be targeted in ETEC vaccine development. However, since this 19-amino acid STa is poorly immunogenic, the effort to genetically fuse or chemically couple to carrier proteins has been made to enhance STa immunogenicity. In this study, we selected one genetic fusion and one chemical conjugate to comparatively evaluate STa immunogenicity. The genetic fusion is 3xSTa_N12S-mnLT_R192G/L211A carrying three toxoid (STa_N12S) genetically fused to a double mutant LT monomer (mnLT_R192G/L211A); the chemical conjugate is BSA-STa_A14T which has toxoid STa_A14T chemically coupled to bovine serum albumin (BSA). We immunized mice with the STa toxoid fusion and chemical conjugates, and examined antibody responses. Furthermore, we immunized pigs and evaluated derived antibodies for efficacy to passively provide protection against ETEC diarrhea using a piglet model. Data showed that mice subcutaneously immunized with BSA-STa_A14T or 3xSTa_N12S-mnLT_R192G/L211A developed a strong anti-STa antibody, and the induced antibodies exhibited equivalent toxin neutralizing activities. Pigs immunized with 3xSTa_N12S-mnLT_R192G/L211A or BSA-STa_A14T developed similar levels of anti-STa antibodies; piglets with passively acquired antibodies induced by the genetic fusion appeared better protected against STa + ETEC. Results from the current study indicate that the fusion and conjugate approaches are viable options for facilitating STa immunogenicity and developing ETEC vaccines.