Development and immunogenicity evaluation of bivalent virus-like particles against bovine nebovirus and bovine norovirus in mice.

Bovine Nebovirus (BNeV) and Bovine Norovirus (BNoV) are major causes of viral diarrhea in calves, leading to significant economic losses. Currently, no commercial vaccines exist for these pathogens. Here, we developed bivalent virus-like particles (VLPs) co-expressing BNeV and BNoV VP1 proteins using a baculovirus-insect cell system and evaluated their immunogenicity in mice. Both monovalent (BNoV or BNeV VLPs) and bivalent VLPs induced peak IgG titers by day 14 post-booster, with the bivalent VLPs triggering faster antibody production. Histo-blood group antigen (HBGA) blocking assays (BT50) showed that bivalent VLPs induced higher blocking antibody titers, peaking at day 14 and remaining elevated at day 28. Additionally, bivalent VLPs enhanced IFN-γ secretion and increased CD3+CD8+ T-cell proportions. These findings demonstrate that BNeV-BNoV-VP1 bivalent VLPs effectively stimulate cellular immunity and blocking antibodies, supporting their potential as vaccines against BNeV and BNoV.