Optimization of Serum-Free Cultivation of Recombinant Sheeppox Virus SSPV (Tk) Omp16 in Vero Cells

Brucellosis remains a major zoonotic disease of global public health and veterinary importance. Recombinant capripoxvirus vectors have emerged as promising platforms for the development of next-generation vaccines owing to their favorable safety profile, genomic stability, and ability to induce robust immune responses. The aim of this study was to optimize serum-free cultivation conditions for the recombinant sheeppox virus strain SSPV (TK) OMP16 expressing the Brucella outer membrane protein OMP16 in Vero cell culture. The recombinant virus was propagated on Vero cell monolayers using Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 2% fetal bovine serum or serum-free OptiPro™ medium containing 1% L-glutamine. Adaptation to serum-free conditions was achieved by five sequential blind passages in Vero cells. Viral biomass accumulation was evaluated at multiplicities of infection (MOI) of 0.01, 0.05, 0.1, and 0.5 TCID50/cell during cultivation periods ranging from 12 to 120 h. Infectious activity was determined by TCID50 assay in Vero cell culture. Serum-free OptiPro medium supported efficient replication of the recombinant virus and produced infectious titers comparable to those obtained in conventional serum-containing DMEM. The highest infectious activity of recombinant strain SSPV (TK) OMP16 was 6.25 log TCID₅₀/cm³ at an MOI of 0.1 TCID₅₀/cell at 96 h post-infection. Sterility testing confirmed the absence of bacterial and fungal contamination in all virus suspensions. The optimized serum-free cultivation protocol provides a standardized approach for propagation of recombinant capripoxvirus vectors and represents a practical basis for further development and scale-up of recombinant vaccine production against brucellosis.