Voprosy virusologii · 2026
The influence of cultivation parameters on the reproduction of recombinant influenza viruses expressing immunodominant proteins of Mycobacterium bovis.
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Abitayev R T, Issabek A U, Nurpeisova A S, Bulatov Y A, Khudaibergenova B M
Аннотация
INTRODUCTION: Influenza A virus is considered a promising vector platform for the creation of recombinant vaccines due to its high immunogenicity and ability to induce a pronounced humoral and cellular immune response. The aim of the study is to investigate the optimal cultivation parameters for recombinant influenza A virus strains expressing Mycobacterium bovis ESAT-6 and TB10.4 antigens.
MATERIALS AND METHODS: Cultivation conditions for recombinant influenza viruses A/Ast/PR8-NS124-ESAT6 and A/Ast/PR8-NS124-TB10.4, expressing the ESAT6 and TB10.4 proteins of M. bovis, were optimized. The viruses were produced by reverse genetics using an eight-plasmid system. Cultivation was performed in MDCK and Vero cell lines at varying temperatures (30-37 °C), incubation times, and trypsin concentrations.
RESULTS: It was established that maximum infectious and hemagglutinating titers were achieved when cultured in Vero cells at 37 °C for 72 hours. A decrease in temperature to 30-35 °C was accompanied by a decrease in viral replication. The Vero cell line demonstrated higher productivity compared to MDCK. Hemagglutinating activity correlated with the infectious titer. RT-PCR confirmed the stability of genetic inserts in the NS segment of the genome throughout the entire cultivation period. Temperature and cell line type significantly influence the reproduction of recombinant viruses. The advantage of Vero cells is due to their higher virus accumulation rate. The stability of the genetic inserts confirms the reliability of the resulting strains as vector systems.
CONCLUSION: Optimal conditions for culturing recombinant influenza viruses, ensuring high reproduction rates and genetic stability, have been identified. The results confirm the potential of using influenza vectors for developing tuberculosis vaccines and the feasibility of scaling up their production.
MATERIALS AND METHODS: Cultivation conditions for recombinant influenza viruses A/Ast/PR8-NS124-ESAT6 and A/Ast/PR8-NS124-TB10.4, expressing the ESAT6 and TB10.4 proteins of M. bovis, were optimized. The viruses were produced by reverse genetics using an eight-plasmid system. Cultivation was performed in MDCK and Vero cell lines at varying temperatures (30-37 °C), incubation times, and trypsin concentrations.
RESULTS: It was established that maximum infectious and hemagglutinating titers were achieved when cultured in Vero cells at 37 °C for 72 hours. A decrease in temperature to 30-35 °C was accompanied by a decrease in viral replication. The Vero cell line demonstrated higher productivity compared to MDCK. Hemagglutinating activity correlated with the infectious titer. RT-PCR confirmed the stability of genetic inserts in the NS segment of the genome throughout the entire cultivation period. Temperature and cell line type significantly influence the reproduction of recombinant viruses. The advantage of Vero cells is due to their higher virus accumulation rate. The stability of the genetic inserts confirms the reliability of the resulting strains as vector systems.
CONCLUSION: Optimal conditions for culturing recombinant influenza viruses, ensuring high reproduction rates and genetic stability, have been identified. The results confirm the potential of using influenza vectors for developing tuberculosis vaccines and the feasibility of scaling up their production.