MDCK That Express Trypsin Provide A Cell System To Improve The Production Of Human Influenza Viruses

Influenza epidemics are major health concern worldwide. Vaccination is the primarystrategy to protect the general population from a pandemic. Currently, most humaninfluenza vaccines are manufactured using chicken embryonated eggs, but thismanufacturing method has potential limitations, such as inflexibility, allergic reactionsinduced by egg proteins and inadequate supply of substrate especially in case of apandemic. Most importantly, the virus strains’ mutation during the adaption to chickenembryonated eggs could change the antigenicity of the virus, thus may affect theeffectiveness of the vaccines. Cell-based vaccines, however, offer a number ofadvantages over the traditional method to avoid these problems, and become the choiceof option for future vaccine. Some of the cell-culture-derived vaccines have beenapproved or currently under clinical evaluation. The results of clinical trials in manycountries showed that these vaccines are safe and highly immunogenic. However, lowviral titer is one of the crucial bottlenecks during cell culture for influenza vaccineproduction. Proteolytic cleavage of the influenza virus HA by host cell proteases iscrucial for virus infectivity and propagation. The fact that MDCK cells lack theseproteases is the main reason for low virus yield in this substrate cells. Some studies showthat an optimized trypsin addition led to high virus titer in manufacturing process. In thisstudy, we aim to introduce the bovine trypsin gene into MDCK cell by means of geneticengineering, thus to establish the cell lines expressing trypsin by itself in order topromote virus production.According to the cDNA sequence of bovine trypsinogen(pro-trypsin) in Genbank, wecloned the trypsinogen gene through gene synthesis. To mimic the natural states ofhuman respiratory epithelial cell protease cleavage of HA,6kinds of gene fragmentsincluding genes for secretory pro-trypsin, secretory trypsin, intracellular pro-trypsin,intracellular trypsin, transmembrane pro-trypsin and transmembrane trypsin wereobtained by PCR and overlap PCR and cloned into pMD18T vector. After identificationand sequencing, the6target fragments were jointed to the Eukaryotic expression plasmidpcDNA3.1by enzyme digestion and ligation. The ORF sequencing results show nodifference from the genome sequence in GenBank.Highly purified expression plasmids were extracted by removal of endotoxin andtransfected into MDCK cells using lipofectamine2000, respectively, thus the transient expression system was established. Western blotting proved that various proteins weresuccessfully expressed and the molecular sizes were in line with expectations; Indirectimmunofluorescence experiments showed the correct subcellular distribution. Using theoptimized method of specific fluorescent substrate to test the trypsin activity, thesecreted pro-trypsin was found to exhibit the highest enzyme activity about10.72×10-2U/ml. The NA activity of vaccine strain A/California/7/2009(H1N1) growing on MDCKtransient expressing secreted pro-trypsin were the highest among6kinds of cells. So thepcDNA3.1-S.pro-trypsin plasmid is the best one for further study.After screening with G418and limiting dilution method, stable cell line of MDCKexpressing secreted pro-trypsin was successfully established, named MT21. The PCR ofgenome DNA and RT-PCR of cell RNA have confirmed that the foreign sequence wasinserted into MDCK DNA. In addition, the foreign insert was very stable during the15round of passage, and no statistical changes were found on the enzyme activity duringthe passage.Monoclonal MT21and normal MDCK were infected with H1N1,H3N2,B type wildvirus strain and H1N1vaccine strain respectively, in combination with or without0.5μg/ml TPCK-trypsin. After incubation for24h,48h and72h, NA activity of theharvested virus were tested, virus growth curve were established and the TCID50of72h-grow virus were tested. The results exhibited that all tested virus titers growing onMT21are higher than the same virus on normal MDCK and the differences are moresignificant when0.5μg/ml TPCK-trypsin was added. So the stable monoclonal cell MT21expressing secreted pro-trypsin could provide a powerful tool for the cell-based vaccineproduction and other related researches.