Heterologous prime-boost immunization of two-component vaccine candidate PWDVax protected pigs against F18 enterotoxigenic <i>Escherichia coli</i> post-weaning diarrhea

Post-weaning diarrhea (PWD) is associated predominantly with enterotoxigenic <i>Escherichia coli</i> (ETEC) and continuously causes significant economic losses to swine producers worldwide. Currently, there are no effective countermeasures against this significant swine disease. Challenges persist in developing vaccines against PWD since ETEC strains produce heterogeneous virulence factors, including F4 (K88) and F18 fimbria and heat-labile toxin (LT), heat-stable toxin type I (STa), heat-stable toxin II (STb), and Shiga toxin type 2e (Stx2e, also causes edema disease). An effective PWD vaccine would induce broadly protective immunity, ideally against two fimbriae and four toxins. In this study, by applying a novel epitope- and structure-based multiepitope-fusion-antigen (MEFA) vaccinology platform, we created a monomeric polyvalent fimbria-toxin protein (fimbria-toxin MEFA) and a holotoxin-structured protein to target PWD virulence determinants (F4 and F18 fimbriae and LT, STa, STb, and Stx2e toxins) and developed a two-component multivalent PWD vaccine candidate, PWDVax. We further applied a heterologous prime-boost immunization strategy and assessed vaccine protection against F18 ETEC-associated PWD. Piglets, after being primed intramuscularly with a fimbria-toxin MEFA monomer protein and boosted orally with live <i>Escherichia coli</i> bacteria producing GM₁-binding holotoxin-structured fimbria-toxin MEFA, developed IgG and secretory IgA responses to the target fimbriae and toxins. Challenged with an F18ac ETEC strain, the immunized piglets were protected against watery diarrhea (87.5%) or any diarrhea (66.7%). These data indicated that PWDVax protects against F18 ETEC-associated PWD and can become an effective PWD vaccine. The two-component vaccine and heterologous prime-boost immunization strategy may be instructive for developing neonatal vaccines in general.IMPORTANCEEnterotoxigenic <i>Escherichia coli</i> (ETEC)-associated post-weaning diarrhea (PWD) is a global swine disease, remains a major threat to pig health and well-being, and causes significant economic losses. Currently, there are no effective vaccines available against this disease because of challenges including heterogeneity among ETEC strains (or virulence factors) and difficulties in inducing protective immunity against some key virulence determinants. PWDVax, a two-component PWD vaccine candidate, unprecedentedly targeted two ETEC fimbriae (F4 and F18) and four toxins (LT, STa, STb, and Stx2e), the virulence factors associated with nearly all PWD clinical cases. Under a heterologous prime-boost immunization schedule, it induced broad systemic and mucosal antigen-specific antibodies but also protected weaned piglets against F18 ETEC diarrhea. This makes PWDVax potentially an effective vaccine to protect against PWD, particularly the current F18 ETEC-associated severe PWD outbreaks in the United States. Additionally, the two-component vaccine and heterologous prime-boost immunization strategy may also facilitate the development of effective neonatal vaccines for humans.