Studies On Production Of Live Attenuated Influenza Vaccine(LAIV) Using MDCK Cell Line And Validation Of An Egg-based Potency Assay

Madin-Darby canine kidney (MDCK) cells using Cytodex(?)3microcarrier beads culture systems were established to produce A/17/California/2009/38and B/56/Brisbane/60/08influenza virus in this study. The culture conditions such as concentration of TPCK-Trypsin, pH and multiplicity of infection (MOI) were investigated using an orthogonal array design. By this means, we established optimal parameters for culture conditions:pH7.2and0.1MOI.We performed the test for three times to confirm the repeatability of the parameters. Both viruses reach the top titer after72hours of culture, and with the titer of7.6±0.151gEID50and7.1±0.261gEID50respectively, and there were no any cytopathic effect of MDCK cells obverved when we added1.Oμg/ml TPCK-Trypsin. Currently, influenza vaccines were mainly produced using egg-based substrates, Ovalbumin introduced from eggs might cause severe allergic reaction, so the WHO recommends using mammalian cell lines as a substitute to egg-based substrates in the manufacture of influenza vaccine. This study demonstrates the feasibility of the development of MDCK cell-based Live attenuated influenza vaccine in microcarrier culture systems and could be valuable to reduction of culture time, improvement of productivity.Live attenuated influenza vaccines (LAIVs) preventing seasonal influenza virus are produced in egg embryo, and formulated as trivalent live attenuated vaccine with three strains-A(H1N1), A(H3N2), and B strain. Since estimates of the titer may be different between EID50and TCID50, when viruses were adapted for growing in mammalian cells, we describe an egg-based potency assay to test the titer of trivalent live attenuated influenza vaccine produced by MDCK substrates, and to validate the test in specificity, linearity, accuracy, precision, repetitiveness and ruggedness in this study. We use50%egg infectious dose (EID50) to indicate of the titer of LAIV. This experiment is of high specificity, there is neither adjuvant interference nor cross interference of heterologous antisera observed in the testing of monovalent titers after neutralizing the other two strains in the trivalent live attenuated influenza vaccine. The mean correlation coefficient of the regression lines for titer testing of H1N1, H3N2and type B strains are ranging from0.994to0.999; the inter-assay coefficients of variance for monlvalent strains is ranging from1.65%to2.58%; the mean coefficient of recovery is ranging from97.3%to103.8%. This study demonstrates EID50assay is suitable for estimate the potency of LAIV manufactured using MDCK cell culture system. Estimation of individual titres is important in understanding the immunogenicity and stability of individual strains.In order to understand the correlation between the two assays, we compared the estimates of the titer of A/17/California/2009/38, A/17/Perth/09/87and B/56/Brisbane/60/08obtained by TCID50and EID50. Date sugest TCID50titer for A/17/California/2009/38, A/17/Perth/09/87and B/56/Brisbane/60/08was.2±0.151gTCID50,7.5±0.171gTCID50and7.0±0.101gTCID50respectively, and TCID50titer was7.3±0.201gEID50,7.5±0.151gEID50and7.0±0.121gEID50. There were no differences between the two assays.