Immunogenicity and post-challenge virological outcomes of a multivalent subunit vaccine in sheep challenged with cytopathic BVDV-1a.

Bovine viral diarrhea virus (BVDV) continues to threaten the global cattle industry and can also infect other ruminants, including sheep. Currently available vaccines still have limitations in terms of safety, durability of immunity, and protective efficacy. To investigate a candidate multivalent BVDV subunit vaccine, four recombinant BVDV proteins (NS5A, E0, E2, and mE2T) were formulated as monovalent subunit vaccines, and a multivalent combined subunit vaccine (CSV) was further constructed by mixing the four antigens in equal proportions. Using a susceptible sheep model, we evaluated BVDV-specific antibody responses, lymphocyte proliferation, serum cytokine concentrations, neutralizing antibody activity, and whole-blood BVDV RNA loads following experimental challenge. The results showed that CSV significantly increased BVDV-specific IgG levels and the IgG2a/IgG1 ratio, enhanced peripheral blood lymphocyte proliferation, and increased serum concentrations of IFN-γ, TNF-α, and IL-4. Virus neutralization assays showed that sera from CSV-immunized sheep exhibited measurable neutralizing activity against the BVDV-1a NADL strain. Following intraperitoneal challenge with the cytopathic BVDV-1a NADL strain, the CSV group showed lower whole-blood BVDV RNA loads than the PBS group at the evaluated time points, while reduced RNA loads were also observed in the IB.V group. These findings show that CSV induced measurable immune responses and was associated with lower whole-blood BVDV RNA loads during the 2-week post-challenge observation period. This study supports the feasibility of a multivalent combination strategy for BVDV subunit vaccine development and provides an experimental basis for subsequent evaluation in cattle, including assessment of fetal protection and prevention of persistent infection.