Bovine viral diarrhea virus (BVDV) is an important animal pathogen that affects cattle. Infections caused by the virus have resulted in substantial economic losses and outbreaks of BVDV are reported globally. Virus-like particles (VLPs) are promising vaccine technology largely due to their safety and strong ability to elicit robust immune responses. In this study, we developed a strategy to generate BVDV-VLPs using a baculovirus expression vector system (BEVS). We were able to assemble BVDV-VLPs composed of dimerized viral proteins E2 and E^rns, and the VLPs were spherical particles with the diameters of about 50 nm. Mice immunized with 15 μg of VLPs adjuvanted with ISA201 elicited higher levels of E2-specific IgG, IgG1, and IgG2a antibodies as well as higher BVDV-neutralizing activity in comparison with controls. Re-stimulation of the splenocytes collected from mice immunized with VLPs led to significantly increased levels of CD3⁺CD4⁺T cells and CD3⁺CD8⁺T cells. In addition, the splenocytes showed dramatically enhanced proliferation and the secretion of Th1-associated IFN-γ and Th2-associated IL-4 compared to that of the unstimulated control group. Taken together, our data indicate that BVDV-VLPs efficiently induced BVDV-specific humoral and cellular immune responses in mice, showing a promising potential of developing BVDV-VLP-based vaccines for the prevention of BVDV infections.

The Madin–Darby bovine kidney (MDBK) cell line is currently used for the production of bovine alphaherpesvirus-1 (BoHV-1) vaccine. For the purpose of vaccine manufacturing, suspension cells are preferred over adherent ones due to simplified sub-cultivation and an easier scale-up process, both of which could significantly reduce production cost. This study aimed to establish a procedure for the culture of BoHV-1 in the suspended MDBK cell line in serum-free medium. We screened several commercially available serum-free media and chose ST503 for subsequent experiments. We successfully adapted the adherent MDBK cells to suspended growth in ST503 in the absence of serum. The maximum density of suspension-adapted MDBK cells could reach 2.5 × 10⁷ cells/mL in ST503 medium with optimal conditions. The average size of suspension-adapted cells increased to 18 ± 1 µm from 16 ± 1 µm. Moreover, we examined tumorigenicity of the suspended cells and found no sign of tumorigenicity post adaptation. Next, we developed a protocol for the culture of BoHV-1 in the cell line described above and found that ultrasonic treatment could facilitate virus release and enhance virus yield by 11-fold, with the virus titer reaching 8.0 ± 0.2 log₁₀TCID₅₀/mL. Most importantly, the prototype inactivated BoHV-1 vaccine we generated using the suspension cultures of MDBK cells induced neutralizing antibodies to a titer comparable to that of the commercial inactivated BoHV-1 vaccine. Overall, we established and optimized a protocol for the production of inactivated BoHV-1 vaccine in MDBK cells adapted for suspension culture, which provides insights for future large-scale manufacturing of BoHV-1 vaccine.