Adaptation Of Modified MDCK Cell Suspension Culture For Production Of Avian Influenza Virus Vaccine

Based on their pathogenecity,two types of AIV have been described,namely a highly pathogenic type(HPAIV)that causes severe disease with high mortality,and low pathogenic type(LPAIV)inducing asymptomatic or mild infection.AIV subtypes,H5,H7 and H9,currently endemic in poultry in some regions of the world,have been shown to be capable of causing zoonotic transmission to humans,posing the threat of influenza pandemics in humans.Vaccination is the major strategy to prevent influenza pandemics.Several cell lines are used for influenza virus productlion such as MDCK and Vero cell.Some advantages of cell cultures include the simple production process,rapid proliferation and high virus yields.However,two problems faced in MDCK cell culture,one is that MDCK cell attachment is too strong to adapt industrial production;another is lower yield of avian influenza virus in MDCK cell culture compared with the production using fertilized chicken eggs.Some research demonstrated that the biological characteristics would change by modifying the gene of cells.The TIGAR(TP53-induced glycolysis and apoptosis regulator)gene could reduce apoptosis induced by various stresses and increase cell survival time.Others concluded that Sialyltransferase(siat7e)gene is one of the important genes that control the degree of cell attachment,and the product encoded by the ?-galactoside ?-2,3-sialyltransferase I(ST3GAL4)gene might enhance the sensitivity of host cells to AIVs.In previous study,we have generated two modified MDCK cells,MDCK cells(TG-418-E5)sTablely expressing the TIGAR gene,and MDCK cells(M54)expressing the Siat7e and the ST3GAL4 gene.We will make the adaptation of the two modified MDCK to suspension culture especially in serum-free medium.1.Suspension adaptation of two modified MDCK cellsBecause no adaptation was done to two modified MDCK cells,TG-418-E5 cells were used to suspension culture with microcarriers.We analyzed the influence of initial cell density,microcarrier concentration and liquid exchange method on TG-418-E5 cell growth to determine the optimal culture conditions in the use of microcarrier suspension culture.Additionally,we also investigate the effect of reducing the concentration of serum in the medium on the state of the cells in shake flask system.TG-418-E5 cell was cultured with microcarrier of 3 g/L,30�104 cells/mL initial cell seeding density,in the medium semi-changed at 48 hours and entire-changed at 72 hours,the viable cell density(VCD)reached 310 x 104 cells/mL at 96 hours.Compared with MDCK cells,the peak viable cell density of TG-418-E5 cells increased nearly 23.5 percent(about 40�104 cells)with microcarriers culture under 5%serum or lower in the media.However,when serum concentration in medium was reduced,the morphology of both cells changed,the cell outline became unclear,and the growth tended to be relatively slow.Thus,TG-418-E5 cell is difficult to adapt to the serum-free.At the same time,we used the method of gradually reducing serum concentration to adapt the M54 cell line by single cell suspension adaptation.We compared the effect of three cell seeding densities on the number of viable cells and glucose and lactate concentrations in the cell supernatant.We found that the initial seeding concentration of 60�104 cells/mL was the optimal inoculation density for M54x cells,which would bring the highest growth density,the fastest growth rate,the higher concentration of glucose and the lower lactate concentration in the medium.The peak cell density of 420 � 104 cells/mL was achieved 96 hours after seeding with suspension culture of M54x cells.At 72 h,the cells were in the logarithmic growth phase when the cell grew fast and well.After 72 hours,the glucose concentration in the cell supernatant became lower and the lactic acid concentration was higher.72h was a critical time point when the virus and cells were in the better condition,and when the concentration of glucose and the lactic acid in the cell supernatant was suiTable for the virus growth.Therefore,72 hours is an ideal time point for M54x cells infectiong with AIV.Since the use of microcarriers greatly increases the cost,and the most economical and practical method is low serum suspension culture,M54x cell is selected for low serum suspension culture and adaptation.2.AIV production in suspension culture by two modified cellsIn this study,the production of three candidate vaccine strains,YA-5(Clade 2.3.2.1 H5),R2344(Clade 2.3.4.4)and rGW(H7N9)in two modified cell cultured in shake flask or BIOSTAT A bioreactor were investigated.The best time of virus infection,multiplicity of infection(MOI),TPCK-trypsin concentration and the best harvest time were determined in different culture system.In microcarriers suspension culture of TG-418-E5 cells with the above optimal conditions,the peak hemagglutination(HA)titer of the virus was reached 72h post infection.Compared production in MDCK cells,the HA titer of the virus increased 1-2(log2)titer,but the TG-418-E5 cell with microcarrier suspension culture cannot adapt to a low serum,so we do not make further scale-up culture.We then focused on the suspension culture of M54x cell.We compared the effects of MOI,TPCK-trypsin concentration and culture temperature on HA titer of avian influenza virus in the M54x cells with the flask under serum-free suspension culture.We found that an MOI of 0.0001 and a TPCK-trypsin concentration of 5?g/mL were the best vaccine strain infection condition.Avian influenza virus vaccine strains reached a high virus yield at 33 ? in M54x cell culture.Peak HA titer of three kinds of AIV strains,YA-5,R2344 and rGW,reached 9,8,and 9 log2/50?l when harvested 72-96h post infection.We also tried to culture M54x cell with reactor,in which the optimal process parameters for breeding of avian influenza virus in M54x cells were initially explored.After 72 hours of cell culture,the medium was replaced with half volume of fresh medium,and TPCK-trypsin was added to a final concentration of 5?g/mL.Three kinds of AIV strains(H5 subtypes YA-5,R2344 and H7 subtypes rGW)were inoculated according to the MOI of 0.0001 with the set parameters of bioreactor.The virus was harvested between 72h and 96h(the HA titer was measured for every 6h sample to observe the lesion)post infection(p.i.).HA titer of H5 vaccine strain YA-5 harvested 76 h p.i.reached 9 log2,HA titer of r2344 harvested at 66 h p.i.reached 9 log2;and HA titer of rGW of H7 subtype harvested at 72 h reached 9 log2.In this study we successfully esTablelished the M54x suspension cell lines and is capable of production of avian influenza virus with sTable and high virus yield.These results demonstrate the great potential of this suspension MDCK cell line in serum-free medium for influenza vaccine production.It provided experimental basis of the large-scale cultivation of M54x cells to produce AIVs with bioreactors under serum-free,protein-free,animal-free medium.