Immunogenicity of a bivalent recombinant adenovirus expressing VP1 proteins of bovine norovirus and bovine nebovirus in mice and calves

Bovine norovirus (BNoV) and bovine nebovirus (BNeV) are emerging pathogens responsible for severe diarrhea in calves worldwide. In this study, we constructed a bivalent recombinant adenovirus vector vaccine, designated rAd5-BNoV + BNeV_VP1 encoding the genes for BNoV-VP1 and BNeV-VP1 based on replication-deficient human adenovirus type 5 (rAd5), and evaluated its immunogenicity in mice and calves. Serum antibody responses were assessed by enzyme-linked immunosorbent assay (ELISA) and serum blocking titer 50 (BT50). Results demonstrated that mice immunized with rAd5-BNoV + BNeV_VP1 produced significant VP1-specific IgG titers as early as two weeks post-vaccination. BNoV-VP1-specific IgG antibody titers peaked at 1:10⁵ in both intramuscular (I.M.) and oral groups (n = 6), while BNeV-VP1-specific IgG titers peaked at 1:10⁵ in the I.M. groups (n = 6) and 1:10⁴ in the oral groups (n = 6). The BT50 values peaked at 640 in the I.M. groups and 576 in the oral groups (n = 6). Furthermore, the percentages of IL-4-producing cells and CD3⁺CD8⁺ T cells in splenocytes were significantly elevated in mice immunized via the I.M. route. The vaccine also elicited a robust humoral immune response in calves. Following a booster I.M. injection with rAd5-BNoV + BNeV_VP1, serum BNoV-VP1-specific and BNeV-VP1-specific IgG antibody titers were significantly higher (P ≤ 0.001) compared to controls. The BT50 in immunized calves reached 1:80 at 21 and 35 days post-immunization. These results collectively indicate that rAd5-BNoV + BNeV_VP1 effectively induces immunity against BNoV and BNeV infection.