The segmented genome of live-attenuated rotavirus (RV) vaccines provides an ideal platform for developing multivalent vaccines against enteric pathogens. Here, we engineered the commercial human RV vaccine strain LLR as a vector to deliver Clostridium perfringens alpha-toxin (CPA), a critical virulence factor associated with gastroenteritis. A screen of four 2A peptide sequences identified that porcine teschovirus-1 2A (P2A) preceded by an N-terminal GSG spacer enabled optimal cleavage an