Orally active bivalent V_HH construct prevents proliferation of F4⁺ enterotoxigenic Escherichia coli in weaned piglets

A major challenge in industrial pig production is the prevalence of post-weaning diarrhea (PWD) in piglets, often caused by enterotoxigenic Escherichia coli (ETEC). The increased use of antibiotics and zinc oxide to treat PWD has raised global concerns regarding antimicrobial resistance development and environmental pollution. Still, alternative treatments targeting ETEC and counteracting PWD are largely lacking. Here, we report the design of a pH, temperature, and protease-stable bivalent V_HH-based protein BL1.2 that cross-links a F4⁺ ETEC model strain by selectively binding to its fimbriae. This protein inhibits F4⁺ ETEC adhesion to porcine epithelial cells ex vivo and decreases F4⁺ ETEC proliferation when administrated as a feed additive to weaned F4⁺ ETEC challenged piglets. These findings highlight the potential of a highly specific bivalent V_HH-based feed additive in effectively delimiting pathogenic F4⁺ ETEC bacteria proliferation in piglets and may represent a sustainable solution for managing PWD while circumventing antimicrobial resistance development.