Research Of A/H1N1 Influenza Vaccine Derived From MDCK Cells

Influenza is acute respiratory infections caused by the influenza virus. The incidence of influenza was first in a variety of infectious diseases. Influenza is one of the important infectious diseases among a serious hazard to human health. According to the notice issued by the World Health Organization, there are about 600 million ~ l200 million people infected with influenza per year in the world, and there are about 500 thousand ~ l million people died from the flu. Influenza A virus often cause a worldwide influenza pandemic, and make a great threat to human. In the last century, the "Spanish flu"," Asian flu","Hong Kong flu " and other large influenza pandemic broke out, Which is caused by the influenza A H1N1 virus, influenza A H2N2 virus and influenza A H3N2 virus. The three influenza pandemic caused thousands of people dead, and caused tremendous damage and loss to human health and the economy. With the changes in global climate and environment, influenza cause increasingly serious harm on human health and the public health security. In recent years, highly pathogenic H5N1 avian influenza and Influenza A H1N1 outbroke in Mexico in 2009 sounded the alarm to the people once again. Remind the human that influenza prevention and control is extremely important.Vaccination is the most economical and effective means for the prevention and control of influenza. Currently, the main medium for influenza vaccine production is chick embryo. However, it is difficult to control the quality of the vaccine producted by chick embryo, and it is a long cycle to product vaccine by chick embryo, and the vaccine producted by chick embryo is allergic. Once flu outbreaks, the source of chick embryo will become a bottleneck, and it can not be able to cope with flu pandemic. Therefore, development of new influenza vaccine production media for human use have become the key to vaccine production. WHO recommends that cells is a fine media for the production of vaccine. Production influenza vaccine using cells is easy to control the quality, and the closed production system is facilitate and safe. Mass products can be made according to demand. Currently, the main approved cell lines for the production of influenza vaccines for human use are Vero cells and MDCK cells. According to reports, there are already companies using Vero cells and MDCK cells to product influenza vaccine for human use by the micro-carrier systems. Baxter company has produced avian flu vaccine-CELVAPAN by Vero cells in 2009. Novartis company has produced the flu vaccine-Optaflu by MDCK cells in 2007. Novartis company also produced the A/H1N1 influenza vaccine by MDCK cells in 2009.The main problem of Influenza vaccine production using cells is the low influenza virus yield. Human influenza viruses adsorb to and infect cells through binding to sialic acid oligosaccharides on the cell surface. The end of N-acetyl sialic acid of the sialic acid oligosaccharides is connected to the galactose byα2, 6 glycosidic bond(NeuAcα2,6 Gal). Both of MDCK cells and Vero cells have this type of sialic acid oligosaccharides, but the abundance of sialic acid oligosaccharides is low. Receptors of influenza virus on cell surface have individual differences of type and amount. The replication efficiency of influenza virus is different in different cells. So, we can screen monoclonal cell lines to obtain cell line adapted to influenza virus. Influenza virus have better adaptability on MDCK cells than Vero cells, so we can screen MDCK monoclonal cell lines to obtain the cell line which can support influenza virus grow to high titers.In order to produce influenza vaccine by cells instead of chicken embryos, we can screen the cell line and the virus strain which have high yield. And then optimize the culture conditions of cells and virus. Then product influenza vaccine by large-scale. We screen the cell lines and virus strains by two ways: On the one hand, we prepare monoclonal cell lines by limited dilution, and then Infected with influenza virus. So, we can obtain a monoclonal cell line adapted to influenza virus. On the other hand, we screen the virus strains. MDCK monoclonal cell lines is infected by different influenza virus strains. And we can obtain a influenza virus strain which have high yield. Screening by this two ways, we can obtain the cell lines and virus strains which have high yield. This technology can establish the basis for large-scale production of influenza vaccines useing mammalian cells. When the influenza outbreak, this technology can provide technical assurance for large-scale production of influenza vaccines. so, it is significant.The purpose of this research is preparing MDCK monoclonal cell lines by limited dilution, and screening monoclonal cell lines for a cell line adapted to influenza viruses, and infecting monoclonal cell lines by different Influenza virus strains, and screening Influenza virus strains for a virus strain which have high yield. And then optimize the culture conditions of the monoclonal cell line and Influenza virus strain. Then product influenza vaccine by large-scale and prepare influenza vaccine. This technology can establish the basis for producing influenza vaccine by cells instead of chicken embryos.This study includes the following three parts:First, screening and identifying the A/H1N1 influenza virus strain and MDCK monoclonal cell line and optimizing the culture conditions. We prepare MDCK monoclonal cell lines by limited dilution and establish bank of MDCK monoclonal cell lines. And then infect MDCK monoclonal cell lines by different A/H1N1 influenza virus strains. Then we can obtain the cell line and the virus strain which have high yield according to HAU titer. The cell line and the virus strain we have obtained is M77 monoclonal cell line and A/H1N1 influenza virus A/Beijing/501/2009. Then we test the TCID50 titers, Plaque forming units, the quantity of NeuAcα2,6 Gal and the stability of TCID50 titers. All the results show that the influenza virus titer cultured by M77 cells were significantly higher than that of normal MDCK cells. We established M77 cell bank and tested M77 cells. We established A/Beijing/501/2009 virus database and tested the virus. The results show that the M77 cells and the A/Beijing/501/2009 virus meet the requirements. We optimized the culture conditions: cell abundance, viral dilution, multiplicity of infection, incubation temperature, the virus harvest time. The optimal culture conditions: cell abundance=100%, serum-free DMEM medium(containing 1μg/ml of TPCK-trypsin), MOI=0.001, incubation temperature=37℃, virus harvest time=72h ~ 96h after infection. Optimization of the culture conditions has provided technical ensure for the expansion of A/H1N1 influenza vaccine.Second, the expansion culture of A/H1N1 influenza virus, concentration and purification of the virus, detection and identification of the virus. We infected M77 monoclonal cell line with A/H1N1 influenza virus A/Beijing/501/2009 using the optimal culture conditions. We cultured the virus in cell culture bottles, cell factories and in the fermentation tank and the results were good. The virus solution was inactivated by 1: 4000 of formaldehyde, concentrated by 10KD film package, purified by sucrose density gradient centrifugation, lysised by 0.5% of Triton X-100. We observed the virus samples after purification using electron microscope. We tested the virus samples before and after purification for HAU, total protein and HA antigen content. We also tested the residual DNA. The results showed that the A/H1N1 influenza virus cultured by cells have typical features of influenza virus, the purity of the virus is 98.3235%, the miscellaneous protein removal rate is 98.30%, the HA recovery rate is 40%, DNA residues after treatment is lower than 100 pg/dose.Third, Evaluation of immune effectiveness of the A/H1N1 influenza vaccine cultured by cells. We purified the A/H1N1 influenza vaccine cultured by MDCK cells and made it to influenza lysis vaccine. Group the 6 weeks old female Balb/c mice randomly, 12 mice each group. We divided the mice into 3 groups: the PBS control group, chicken embryo-derived A/H1N1 influenza split vaccine immune group and MDCK cell-derived A/H1N1 influenza split vaccine immune group. We vaccinated the mice with HA antigen through intramuscular, 15μg per mice(the HA concentration measured by single immunodiffusion). The injected dose is 200μl per mice. A second immunization is two weeks after the first immunization. We collected blood from tail vein at one week after the first immunization, two weeks after the first immunization, one week after the second immunization, two weeks after the second immunization, respectively. We tested the antibody IgG, IgG1, IgG 2a and IgG 2b titers and HI titers in serum. And tested the cytokines IL-4 and IFN-γtiters in serum. We also tested the frequency of lymphocytes which secreted IL-4 and IFN-γin spleen. Virus attacking test had been done on the mice using influenza virus A/Beijing/501/2009 and A/PR/8/34 at two weeks after the second immunization. We evaluated the protection and cross-protection of A/H1N1 influenza split vaccine cultured by cells. The results show that both of the chicken embryo-derived vaccines and cell-derived vaccine can activate the humoral immunity to high level, and the cell-derived vaccine is better than chicken embryo-derived vaccines. The protection of chicken embryo-derived vaccines and cell-derived vaccine in mice is all 100%. Cross-protection experiments shows that cross-protection of cell-derived vaccine is better than that of chicken embryo-derived vaccines.Conclusion: Screening monoclonal cell lines, we can obtain a cell line which have high yield. Optimize the culture conditions and then product influenza vaccine by large-scale. We have obtained high virus yield. The animal experiment confirmed that cell-derived A/H1N1 influenza vaccine had good immunogenicity, higher immune protection and better cross protection. This research has established the basis for producing influenza vaccine by MDCK cells.