B subunit of the type 2 Shiga toxin e variant (Stx2e) bundled by a five-stranded α-helical coiled coil protects piglets from porcine edema disease

Porcine edema disease (ED) is caused by infection with Shiga toxin type 2 e variant (Stx2e)-producing Escherichia coli (STEC). To develop a new ED vaccine, we engineered a fusion protein, in which the Stx2e B subunit (Stx2eB) was fused to the five-stranded α-helical coiled coil domain of cartilage oligomeric matrix protein (COMP), based on our recent finding that the coiled coil strongly stabilizes the B subunit pentamer of Stx2 ( Tamaki Y, Harakuni T, Arakawa T. Shiga toxin type 2 B subunit protects mice against toxin challenge when leashed and bundled by a stable pentameric coiled-coil molecule. Vaccine. 2024 Mar 7;42(7):1757–1767 ). Purified Stx2eB–COMP fusion protein administered to mice conferred complete protection against a lethal dose of Stx2e. However, unfused Stx2eB conferred only 10 % protection. Furthermore, when Stx2eB was fused to a trimeric or tetrameric coiled coil, a marked reduction in protective efficacy was observed, indicating the importance of “five-to-five” fusion stoichiometry. Next, we immunized weaned piglets twice with 100 μg or 10 μg of the Stx2eB–COMP fusion protein, and then orally challenged the animals with a lethal dose of STEC. The piglets immunized with the high dose were almost completely free from clinical symptoms of ED, whereas three of the six piglets administered adjuvant-only died, and the remaining surviving piglets exhibited severe ED symptoms. Although all piglets immunized with the low dose survived, they exhibited mild to moderate ED symptoms. Our findings indicate that Stx2eB is highly protective only when bundled and molecularly stabilized by the coiled coil molecule. • Shiga toxin type 2e (Stx2e) was fused to cartilage oligomeric matrix protein (COMP). • Fusion was between the Stx2eB subunit and the COMP pentameric α-helical coiled coil. • Stx2eB–COMP fusion protein, expressed in E. coli inclusion bodies, refolded in vitro. • Only the “five-to-five” chimera was protective in mice against Stx2e challenge. • Fusion protein protected piglets against orally infected Stx2e-producing E. coli.