A rotavirus-vectored bivalent vaccine elicits protective immunity against bovine coronavirus and rotavirus

Bovine rotavirus (BRV) and bovine coronavirus (BCoV) are major causes of neonatal calf diarrhea and frequently occur as co-infections. Here, we engineered the Lanzhou lamb rotavirus vaccine strain LLR to express either the N-terminal domain (NTD) or the C-terminal domain (CTD) of the BCoV spike S1 subunit from a modified genome segment 8. The resulting viruses, rLLR-GSG-P2A-BCoV NTD and rLLR-GSG-P2A-BCoV CTD, replicated efficiently in cell culture, stably retained the inserted sequences through serial passage, and expressed the corresponding BCoV antigens. In a mouse model, oral inoculation of neonatal mice induced systemic antibody responses against both RV and BCoV. Maternal immunization in mice protected suckling mice against RV challenge and conferred partial protection against BCoV-associated diarrhea. In pregnant cows, intramuscular immunization elicited RV- and BCoV-specific serum antibodies, and the rLLR-GSG-P2A-BCoV CTD immunization modestly increased neutralizing antibody titers in colostrum. These findings establish the LLR strain as a genetically stable vector for the delivery of heterologous coronavirus antigens and support further evaluation of rotavirus-vectored vaccines for controlling viral coinfections that contribute to neonatal calf diarrhea. IMPORTANCE: Neonatal calf diarrhea is frequently caused by more than one enteric pathogen, yet most vaccines target individual agents. We developed recombinant rotaviruses that express neutralizing-antibody targets from the bovine coronavirus spike protein. The vaccine candidates rLLR-GSG-P2A-BCoV CTD maintained efficient replication and stable antigen expression, induced antibodies against both viruses in mice, and increased neutralizing activity in the serum and colostrum of immunized cows. This study provides proof of concept for using a licensed rotavirus vaccine backbone as a bivalent platform against two major viral causes of calf diarrhea and identifies a strategy that could be adapted to other polymicrobial enteric diseases.