OPTIMIZATION OF SERUM-FREE CULTIVATION OF RECOMBINANT SHEEPPOX VIRUS SSPV (TK) OMP16 IN VERO CELLS
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Authors
Ye.B. Burashev
LLP "Research Institute for Biological Safety Problems", Gvardeyskiy settlement, Kordai district, Zhambyl region, Kazakhstan, 080409
R.A. Rystaeva
LLP "Research Institute for Biological Safety Problems", Gvardeyskiy settlement, Kordai district, Zhambyl region, Kazakhstan, 080409
Z.D. Omarova
LLP "Research Institute for Biological Safety Problems", Gvardeyskiy settlement, Kordai district, Zhambyl region, Kazakhstan, 080409
T.U. Argimbaeva
LLP "Research Institute for Biological Safety Problems", Gvardeyskiy settlement, Kordai district, Zhambyl region, Kazakhstan, 080409
N.A. Aubakir
LLP "Research Institute for Biological Safety Problems", Gvardeyskiy settlement, Kordai district, Zhambyl region, Kazakhstan, 080409
A.B. Tulendibayev
LLP "Research Institute for Biological Safety Problems", Gvardeyskiy settlement, Kordai district, Zhambyl region, Kazakhstan, 080409
T.T. Yermekbai
LLP "Research Institute for Biological Safety Problems", Gvardeyskiy settlement, Kordai district, Zhambyl region, Kazakhstan, 080409
S.O. Sadikaliyeva
LLP "Research Institute for Biological Safety Problems", Gvardeyskiy settlement, Kordai district, Zhambyl region, Kazakhstan, 080409
K.D. Zhugunissov
LLP "Research Institute for Biological Safety Problems", Gvardeyskiy settlement, Kordai district, Zhambyl region, Kazakhstan, 080409
O.V. Chervyakova
LLP "Research Institute for Biological Safety Problems", Gvardeyskiy settlement, Kordai district, Zhambyl region, Kazakhstan, 080409
Abstract
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.
Keywords
recombinant sheeppox virus, SSPV (TK) OMP16, Vero cells, serum-free cultivation, recombinant viral vector, brucellosis vaccine
Article Details
References
Corbel M.J. Brucellosis in Humans and Animals // WHO, FAO, OIE. – Geneva, 2006.
Pappas G., Papadimitriou P., Akritidis N., Christou L., Tsianos E.V. The new global map of human brucellosis // Lancet Infectious Diseases. – 2006. – № 6(2). – P. 91–99.
Franco M.P., Mulder M., Gilman R.H., Smits H.L. Human brucellosis // Lancet Infectious Diseases. – 2007. – № 7(12). – P. 775–786.
Dorneles E.M.S., Sriranganathan N., Lage A.P. Recent advances in Brucella abortus vaccines // Veterinary Research. – 2015. – № 46. – Article 76.
Ashford R.T., Muchowski J., Koylass M.S., et al. Application of Brucella vaccines in humans and animals: current status and future perspectives // Frontiers in Cellular and Infection Microbiology. – 2020.
De Figueiredo P., Ficht T.A., Rice-Ficht A., Rossetti C.A., Adams L.G. Pathogenesis and immunobiology of brucellosis: review of Brucella-host interactions // American Journal of Pathology. – 2015.
Baldi P.C., Giambartolomei G.H. Immunopathology and vaccine development against brucellosis // Expert Review of Vaccines. – 2020.
Volz A., Sutter G. Modified Vaccinia Virus Ankara: History, Value in Basic Research, and Current Perspectives for Vaccine Development // Advances in Virus Research. – 2017.
Gilbert S.C. Viral vectored vaccines // Nature Reviews Immunology. – 2013.
Babiuk S., Bowden T.R., Boyle D.B., Wallace D.B., Kitching R.P. Capripoxviruses: an emerging worldwide threat to sheep, goats and cattle // Transboundary and Emerging Diseases. – 2008.
Pasquevich K.A., Estein S.M., García Samartino C., Zwerdling A., Coria L.M., Barrionuevo P., et al. Immunization with recombinant Brucella outer membrane proteins Omp16 and Omp19 confers protection against Brucella abortus infection in mice // Vaccine. – 2009. – № 27(30). – P. 3983–3988.
Chervyakova O., Tailakova E., Kozhabergenov N., Sadikaliyeva S., Sultankulova K., Zakarya K., et al. Engineering of Recombinant Sheep Pox Viruses Expressing Foreign Antigens // Microorganisms. – 2021. – № 9(5). – Article 1005.
Tibor A., Decelle B., Letesson J.J. Outer membrane proteins Omp10, Omp16, and Omp19 of Brucella spp. are lipoproteins // Infection and Immunity. – 1999. – № 67(9). – P. 4960–4962.
Van der Valk J., Bieback K., Buta C., Cochrane B., Dirks W.G., Fu J., et al. Fetal bovine serum (FBS): Past, present and future // ALTEX. – 2018. – № 35(1). – P. 99–118. Crossref
Yao T., Asayama Y. Animal-cell culture media: History, characteristics, and current issues // Reproductive Medicine and Biology. – 2017. – № 16(2). – P. 99–117. Crossref
Gallo-Ramírez L.E., Nikolay A., Genzel Y., Reichl U. Bioreactor concepts for cell culture-based viral vaccine production // Expert Review of Vaccines. – 2015. – № 14(9). – P. 1181–1195. Crossref
Alfano R., Pennybaker A., Halfmann P., Huang C.Y.H. Formulation and production of a blood-free and chemically defined virus production media for VERO cells // Biotechnology and Bioengineering. – 2020. – № 117(11). – P. 3277–3285. Crossref
Kiesslich S., Vila-Chã Losa J.P., Gélinas J.F., Kamen A.A. Serum-free production of rVSV-ZEBOV in Vero cells: Microcarrier bioreactor versus scale-X™ hydro fixed-bed // Journal of Biotechnology. – 2020. – № 310. – P. 32–39. Crossref
Kiesslich S., Kim G.N., Shen C.F., Kang C.Y., Kamen A.A. Bioreactor production of rVSV-based vectors in Vero cell suspension cultures // Biotechnology and Bioengineering. – 2021. – № 118(7). – P. 2649–2659. Crossref
Fang Z., Lyu J., Li J., Li C., Zhang Y., Guo Y., Wang Y., Zhang Y., Chen K. Application of bioreactor technology for cell culture-based viral vaccine production: Present status and future prospects // Frontiers in Bioengineering and Biotechnology. – 2022. – № 10. – Article 921755. Crossref
Kuncorojakti S., Delaiah D., Aswin A., et al. Development of peptone-based serum-free media to support Vero CCL-81 cell proliferation and optimize SARS-CoV-2 viral production // Heliyon. – 2024. – № 10(24). – Article e41077. Crossref
Amanova Z., Sametova Z., Chervyakova O., et al. Comparative production of recombinant capripoxvirus in Vero cells using microcarrier- and macrocarrier-based dynamic cell culture systems under serum-free conditions // Applied Microbiology and Biotechnology. – 2026. – № 110(1). – Article 191. Crossref