The Effect Of Different Cultivation Conditions On The Immunogenicity Of HA Protein Of Influenza B Virus Based On Vero Cells

Objective:Study on the immunogenicity of the hemagglutinin（HA）protein of influenza B virus under different cultivation conditions based on Vero cell matrix and its correlation with glycosylation sites of HA protein.Methods:（1）Optimization of culture conditions for influenza B virus:Single-factor experiments were conducted to explore the effects of various factors,including the multiplicity of infection（MOI）（0.05,0.10,0.15,0.20）,basal culture media（M199,MEM,DMEM）,culture time（48 h,72 h,96 h）,temperature（33℃,34℃,35℃,and 37℃）,sodium bicarbonate concentration in the maintenance fluid（0.5%,1.0%,1.5%,2.0%）,and TPCK trypsin concentration（0.5μg/m L,1.0μg/m L,1.5μg/m L,2.0μg/m L）on the Vero cell culture of influenza B virus.The conditions with high virus yield were screened.（2）Glycosylation study under two cultivation conditions:Two cultivation conditions（Group A:M199 medium,1%Na HCO₃,1%glutamine,1%fetal bovine serum,1μg/m L TPCK trypsin,MOI=0.1,35℃,72 h culture time;Group B:DMEM medium,3%Na HCO₃,2%glutamine,1%fetal bovine serum,1μg/m L TPCK trypsin,MOI=0.1,37℃,96 h culture time）were used to culture influenza B virus.The HA protein was isolated from the virus and subjected to reduction,alkylation,and enzymatic digestion.The enzymatic products were analyzed for glycosylation using high-performance liquid chromatography（HPLC）and capillary electrophoresis systems.（3）Immunogenicity evaluation:Eight-week-old female ICR mice were selected and intraperitoneally immunized with influenza B virus cultured under two different conditions.The hemagglutination inhibition（HI）antibody titer and neutralizing antibody titer of the mice in each group were compared using the hemagglutination inhibition test and in vitro cell neutralization assay,respectively.Safety and immunogenicity evaluations of the mice were conducted.Results:（1）The optimal cultivation conditions for high production and immunogenicity of influenza B virus are:MOI=0.10,TPCK enzyme concentration of1.0μg/m L,virus cultivation for 72 hours,and a cultivation temperature of 35°C.（2）HPLC analysis revealed that HA protein obtained under cultivation condition A had higher values for RT（min）,peak area（m AU·s）,peak area（%）,peak height（m AU）,start time（min）,end time（min）,and peak width（min）than HA protein obtained under cultivation condition B;further results of capillary electrophoresis analysis showed that the molecular weight and HA protein after enzymatic hydrolysis in A culture condition were higher than those in B culture condition;culture condition A has a higher level of glycosylation.（3）After immunization of mice,it was found that the hemagglutination inhibition（HI）titers in the serum of mice from cultivation condition B were higher than those from cultivation condition A.Conclusion:Cultivation conditions can affect the glycosylation of the HA protein of the virus,which in turn affects the immunogenicity of the influenza B vaccine.