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

## TL;DR

This study developed a new bivalent vaccine against ETEC in piglets by combining epitopes from two strains, showing strong immune responses and protection.

## Contribution

A novel recombinant subunit vaccine design using K88-FaeG as a backbone with K99-FanC epitopes for improved cross-protection.

## Key findings

- FaeG-Ep3 induced the highest antibody titers and effectively blocked K88 and K99 adhesion.
- FaeG-Ep3-immunized mice showed no weight loss and preserved intestinal structure after infection.
- FaeG-Ep3 demonstrated 83% villus protection comparable to traditional vaccines.

## Abstract

Enterotoxigenic Escherichia coli (ETEC) is a major cause of piglet diarrhea and economic losses in swine farming. Traditional vaccines are limited by insufficient cross-protection due to diverse ETEC serotypes. This study developed a recombinant subunit vaccine by inserting K99-FanC epitopes into the K88-FaeG protein. The key findings include the following: FaeG-Ep3 showed the highest antibody titers and effectively blocked K88 and K99 adhesion; in challenge experiments, FaeG-Ep3 provided protection against intestinal damage. These results indicate that FaeG-Ep3 may serve as a potential vaccine candidate and provide useful insights for the development of multivalent ETEC vaccines.

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.