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 and antigen expression. In mice, the resulting recombinant virus elicited potent systemic antibody responses against both RV and CPA following oral administration. When tested in a maternal immunization model, intramuscular immunization of cows generated high titers of neutralizing antibodies in colostrum, demonstrating its potential utility for passive immunization. By integrating a key bacterial antigen into a licensed RV vaccine backbone, we establish a versatile strategy to combat viral-bacterial co-infections and polymicrobial enteric diseases, offering a promising new approach to reduce the global burden of diarrheal mortality.IMPORTANCEThe lack of vaccines targeting polymicrobial enteric infections represents a critical gap in the fight against diarrheal diseases, a leading cause of infant mortality worldwide. To bridge this gap, we engineered a novel bivalent vaccine designed to provide dual protection against rotavirus and Clostridium perfringens alpha-toxin (CPA)-mediated disease. Leveraging an enhanced reverse genetics system, we successfully utilized the commercialized rotavirus vaccine strain LLR as a viral vector to express the key C. perfringens virulence factor, CPA. This strategy not only offers a path to broader protection against diarrheal disease but also establishes a versatile platform for developing vaccines against other viral-bacterial co-infections.

Keywords: Clostridium perfringens; enteric pathogens; enteric viral vector; rotavirus; vaccine.