Comparative Study: Influenza Virus And Vaccines Prepared By MDCK Cells And By Eggs

Objective: To establish a method for purification of cell-cultured influenza vaccine.The differences in the biological characteristics of the HA protein prepared by MDCK cells and eggs were studied from the molecular level,protein level and glycosylation level,and the effects of these changes on the immunogenicity of the virus and the efficacy of the vaccine were systematically evaluated.Method: The MDCK suspension cell platform technology was applied to culture influenza viruses H1N1,H3N2,BV and BY,respectively,and obtain relatively pure antigens through multi-step purification.The cell-cultured monovalent H1N1 vaccine and quadrivalent influenza vaccine were prepared and compared with the monovalent H1N1 vaccine and quadrivalent influenza vaccine prepared by eggs.The monovalent H1N1 vaccine prepared by two methods were further processed and deglycosylated to prepare the deglycosylated monovalent H1N1 vaccine.The differences in immunogenicity between monovalent and tetravalent vaccines were compared by animal experiments and the related immunological effects were evaluated.Meanwhile,nucleic acid sequencing was applied to compare the differences in the HA nucleic acid sequences of the H1N1 virus cultured in the two methods,and the differences in the amino acid sequences,glycosylation sites and modification of the HA protein were compared by mass spectrometry.Through animal experiments the differences in serum cytokines and differentiation of immune cells in spleen cells of mice after vaccination were compared,the effects of the differences in biological characteristics on the immunogenicity of vaccines were analyzed,and the ability of the two vaccines to induce different types of immune responses and the effect of dose on vaccine effectiveness were systematically compared.Result: The purification results showed that the purification strategy was applicable to four viruses,the removal rate of host proteins in the virus venom was higher than 90%,the removal rate of host DNA was higher than 90%,the recovery rate of hemagglutinin was higher than 40%,and the addition of benzonase nuclease after a series of purification could further remove the residual DNA.In the biological characteristics test,the HA nucleic acid sequences prepared by the two methods were consistent.The coverage of amino acid sequence of HA protein prepared by two methods and theoretical sequence were higher than 94%.HA protein amino acid composition and theoretical sequence coverage were higher than 94%.Glycosylation sites were found at N27,N39,N103,N178,N303 and N497 of the H1N1 virus HA proteins prepared by eggs,and glycosylation sites were found at N27,N39,N178,N292 and N497 of the H1N1 virus HA proteins prepared by the cells,the missing glycosylation sites were caused by deamidation.In addition,a glycosylation site was also found at N292,and the glycosylation site deletion at N103 and N303 was caused by deamination.Glycosylation modification of the HA protein of H1N1 virus cultured in the cells were more complex,and multi-antenna glycosylation modification accounted for a higher proportion.The glycosylation modification of the HA protein of H1N1 virus cultured in the eggs were relatively simple,mainly polymannose type.The results of the cellular or innate immune-related multiplex cytokine detection assay showed that the concentrations of MCP-1,IP-10 and IL-6 in the serum of mice were significantly increased within 6 hours after vaccination with the cell-cultured vaccine,which indicated that the innate immune response of mice was activated.On the contrary,there was no significant difference in the cytokine changes between embryo matrix influenza vaccine and deglycosylated influenza vaccine,indicating that glycosylation of HA protein is one of the factors inducing innate immunity.The results of animal experiments on humoral immunity-related multiplex cytokine detection assay showed no significant difference in serum concentrations of IL-4,IL-5 and IL-10 in each group after 10 days of vaccination,indicating that the humoral immunity induced by influenza vaccine with different methods and the decarcosylated influenza vaccine may be relatively consistent.The intracellular cytokine staining test results showed no significant difference in the proportion of B cells among different vaccine groups,which further indicated that the humoral immunity induced by each types influenza vaccine was relatively consistent.And cell-cultured vaccine can effectively activate CD8+ T cell differentiation,and T cells secreting TNF-?,IFN-? and IL-2 were significantly higher than the proportion of egg-based and to deglycosylaed vaccine group,suggests that cell-cultured vaccine can induce strong cellular immune response,at the same time also shows that removing of glycosylation could significantly reduce the immunogenicity and immune effect of vaccine.In the in vitro neutralization test,the neutralization activity of antibody in the serum of mice increased significantly after the second injection,and the serum neutralization value of mice in the cel-based vaccine group were significantly higher than that in the egg-based vaccine group and the deglycosylated vaccine group.However,in the hemagglutination inhibition test,the serum protective ability of the egg-based vaccine group was slightly higher than that of the cell-cultured vaccine group.The contradiction between the two results may be related to the culture methods of the virus.Four different dose groups were set in the cell-cultured vaccine group,and the results showed that the ability to induce innate immunity,cellular immunity and humoral immunity in the high-dose group was significantly stronger than that in the low-dose group,indicating that the increased dose had a significant effect on the protective results of the cell-cultured vaccine.Conclusion: This experiment explored a purification strategy for cell-cultured influenza vaccine,which could effectively remove host proteins and DNA while the host DNA content,without the addition of benzonase nuclease,could reach the European pharmacopoeia requirements for nucleic acid residue in vaccine.This means the strategy may be applied to actual vaccine production.In the study of biological characteristics of HA protein showed the glycosylation sites and modification were influenced by culture methods.A unique glycosylation site N292 was found in the cellcultured H1N1 virus,but the site was located in the HA protein stem,not in the receptor binding region neither in the antigen epitope region,so the effect of the this site on immunogenicity still needs to be further determined.There were significant differences in glycosylation modification of several glycosylation sites in receptor-binding regions of H1N1 virus HA in different culture methods.The globular heads of HA glycoprotein plays a key role of identification when viruses infect cells,so the differences between glycosylation in these locations may lead to changes in the immunogenicity of virus,suggesting that the multi-antenna glycosylation modification and complex glycosylation on the HA protein may be an important reason for the immunogenicity difference of influenza virus.Glycosylation sites lost at N103 and N303 is closely related to the pH value,ion species,and ion strength of culture medium,which indicates that the intracellular environment of mammalian cells may be the cause of viral glycoprotein conservation.As a result of deamidation,the local hydrophobicity of the protein might be changed,and the spatial structure of the protein might be efeected.In the animal experiments we found that the presence of glycosylation had significant effect on vaccine-induced innate immunity and cellular immunity,indicating that glycosylation of HA protein played a certain role in inducing innate immunity and cellular immunity,this may be because the glycosylation modification of the cellcultured influenza virus were more easily recognized by innate immunity and cellular immune-related cells.The secretion of humoral immune-related cytokines and the differentiation of B cells indicated that the humoral immunity levels of influenza vaccines prepared with different methods were consistent.Although,the results of in vitro neutralization test are inconsistent with the results of hemagglutination inhibition test,the reason for this phenomenon may be that the virus used in the in vitro neutralization test were cultured from MDCK,so the antibody specificity in the serum of the mice is stronger,resulting in a higher neutralization value of the serum.In contrast,Red the blood cells used in the hemagglutination inhibited test were made from chicken,which were more capable of binding to the antibody induced by eggsbased vaccine,leading to better results in egg-based vaccine group in the hemagglutination inhibition test.The results of these two experiments indicate that the detection method should be distinguished in the identification of the two types influenza vaccines so as to obtain more accurate identification results.