Abstract
This study focused on utilizing the double-mutant heat-labile toxin (R192G/L211A) (dmLT) as a backbone protein, into which neutralizing epitopes of ETEC (FaeG, FedF, FanC, FasA, and Fim41a) were embedded. A combination of computational modeling and immunogenicity analysis was conducted to evaluate the dmLT((R192G/L211A)) multiepitope fusion antigen (MEFA). Both the computational modeling and experimental results confirmed that all relevant epitopes were clearly exposed on the surface of the MEFA. Subcutaneous immunizations of rabbits with the MEFA protein yielded the development of IgG antibodies that targeted all five fimbriae. Furthermore, these antibodies demonstrated significant inhibition of adhesion for K88+, K99+, 987P+, F18+, and F41+ ETEC strains to porcine small intestinal epithelial cell line IPEC-J2 cells. These results indicated that the dmLT toxoid-based MEFA protein effectively elicits high-titer, functional antibodies capable of neutralizing the attachment of multiple prevalent ETEC fimbrial types, highlighting its potential as a broad-spectrum vaccine candidate. Consequently, it shows promising potential as a broad and effective vaccine against ETEC.