Preparation and Evaluation of Novel Epitope-Based ETEC K88-K99 Bivalent Vaccine

Abstract

Enterotoxigenic Escherichia coli (ETEC) is one of the primary pathogens causing diarrhea in piglets, causing significant economic losses in the swine farming industry. Due to the numerous serotypes of ETEC, traditional vaccines fail to provide sufficient cross-protection, and subunit vaccines based on epitope design have emerged as a safer and more effective approach for prevention and control. Unlike vaccine development strategies that involve the tandem arrangement of multiple antigenic epitopes, this study used the K88-FaeG protein as a backbone and incorporated the antigenic epitopes of K99-FanC to achieve a better immunogenicity. By using bioinformatics software to predict B-cell linear epitopes (score of over 0.6), B-cell epitopes from three-dimensional structures (50% amino acid score of ≥0.2), and B-cell epitope IgG antibody subtypes, as well as docking analysis with Sus scrofa aminopeptidase N (APN) receptors, six antigenic epitopes of K99-FanC were selected. Through Western blotting and competitive ELISA, we confirmed that all six recombinant proteins exhibited binding capabilities to K88- and K99-positive serum. The ELISA results showed that the serum levels of specific IgG and IgA antibodies increased after immunization, with FaeG-Ep3 and FaeG-Ep5 inducing the highest antibody titers against FanC-IgG (Log2 = 14.96) and FaeG-IgG (Log2 = 17.96), respectively. Bacterial adhesion assays revealed that only FaeG-Ep3 effectively blocked the adhesion of both K99 and K88 to IPEC-J2 cells. Immunization challenge experiments showed that, in the unimmunized group, mice infected with K88 and K99 experienced weight loss (p < 0.05) with intestinal villus shedding and intestinal wall structural damage. However, in the FaeG-Ep3-immunized group, no significant weight loss occurred after infection, and the villus protection rate (83%) was the same as that in the FaeG and FanC immunized groups. Overall, the FaeG-Ep3 recombinant protein identified in this study shows potential vaccine application value and provides new insights for developing multivalent vaccines against ETEC.

Keywords: Enterotoxigenic Escherichia coli; FaeG; FanC; epitope; vaccine.

Figure 1

Prediction of B-cell epitopes in ETEC K99-FanC. (A): The ABCpred software predicts continuous (linear) B-cell epitopes; (B): Parker hydrophilicity and Chou–Fasman algorithms from the IEDB database are used to predict B-cell epitopes; (C): the DiscoTope-3 software predicts three-dimensional conformational B-cell epitopes; (D): the IgPred software predicts the scores of IgG antibody produced by B-cell epitopes; and (E): FanC B-cell epitope displayed on three-dimensional structure, FanC PDB ID: P18103.1.A.

Figure 2

Docking studies of vaccine protein and APN receptors. (A): Display of 163–173AA and FaeG epitopes on the three-dimensional structure of FaeG. Molecular docking of FaeG and FaeG-Ep with APN (551 AA–920 AA) receptors analyzed by HDOCK software, FaeG PDB ID: 2j6g.1 and APN PDB ID: 5lds.1.A. (B): APN amino acid docking site.

Figure 3

Expression and identification of recombinant proteins. (A): SDS-PAGE analysis of FaeG-Ep fusion proteins, FaeG, and FanC expression; (B): Western blot analysis of FaeG-Ep fusion proteins and FanC expression; (C): Western blot analysis of FaeG-Ep fusion proteins and FaeG expression; and (D): ELISA competition assay was conducted utilizing K99-FanC as the encapsulated antigen and each FaeG-Ep protein as the competitor, with anti-K99 serum diluted from 1:2000.

Figure 4

Preparation of FaeG-Ep mouse immune serum and identification of FanC-B-cell epitopes. (A): ELISA detection of the FaeG-Ep, FaeG, and FanC sera at 14, 28, and 42 days specific IgG titer; (B): ELISA detection of the FaeG-Ep, FaeG, and FanC sera at 42 days specific IgA titer; (C): ELISA detection of FaeG-Ep serum specific anti-FanC IgG titer; (D): ELISA detection of FaeG-Ep serum specific anti-FaeG IgG titer; (E,F): neutralization experiment of anti ETEC K99 and ETEC K88 bacterial adhesion to IPEC-J2 cells; and (G): the chimeric schematic of the bivalent epitope vaccine FaeG-Ep3. All experimental results are based on three sets of repeated data.

Figure 5

The protective effect of FaeG-Ep antigen on mice. (A,B): The measurement results of the body weight of mice infected with ETEC K99 and ETEC K88 for three days and (C,D): bacterial counts performed on the cecal contents of all groups three days post-infection with ETEC K88 and ETEC K99. (E): Protective effects of Ep3 antigens on intestinal villi and intestinal mucosal structures in mice infected with ETEC K99 and ETEC K88. Black arrows: intestinal villi; Red arrows: muscular layer (bar = 100 μm and 50 μm). (F,G): ELISA was used to detect IgG antibodies against FaeG and FaeG-Ep3 proteins in K99 and K88 pig serum.