Research On The Preparation Of Inactivated Influenza Vaccines By Culturing Vero Cells And Influenza A Virus In A Microcarrier Bioreactor

Influenza virus is an acute respiratory infectious disease pathogen that is a serious threat to human health.New viruses are produced through antigen drift,causing high morbidity and mortality worldwide every year.Vaccines are the primary and effective means of preventing influenza.Currently listed influenza vaccines mainly include inactivated vaccine,live attenuated vaccine and recombinant HA vaccine.And the majority of the vaccine would choose chicken embryo as the production medium.Chicken embryos have the risk of contamination of exogenous factors as medium.Meanwhile,the source of chicken embryos will be affected because of the prevalence of avian influenza accompanied with a pandemic situation.In view of this,WHO encourages research on the use of mammalian cells instead of chicken embryos as the production medium of influenza vaccines.Vero cells are approved as the continuous cell line for human vaccine production by WHO,which could be cultured by bioreactor in large-scale.But the cells are not sensitive to influenza virus,which has been reduced to zero for successive viral titer.On the other hand,due to the high variability of influenza virus,WHO infers the next year’s epidemic strains according to the information provided by the global influenza virus monitoring points every year.In general,the epidemic strains are low in chick embryo and Vero cells,and the selected strains needed to reproduce the new strains with the high-yielding parent plants.Therefore,time is very tense to prepare vaccine seeds,produce vaccine and finish all the tests before the high incidence of influenza.Overall,it is particularly important to produce an influenza strain which high-yielding in Vero cell stably and rapidly.In this study,one Vero cells adapted virus strain named "A/kunming/1/2005va" is chosen as the parent strain.A rapid preparation method of Vero cell influenza vaccine candidate strains is established using one-step cloning method and the transfection reagent called "Effectent Transfection Reagent",through comparing two transfection techniques and two virus rescue methods,which are point mutation and one step rapid cloning.In this way,a new vaccine candidate,with a high yield phenotype on Vero cells,named"A/Shanghai Jiading/SWL1970/2015(H1N1)Va" is rescued by the combination between the six core fragments PB2,PB1,PA,NP,NS,M in virus strain A/kunming/1/2005va and the surface antigenic fragment HA and NA in the epidemic strain A/Shanghai Jiading/SWL1970/2015(H1N1).In view of the deficiency of the enzyme that cleaves influenza virus HAO into HA1 in Vero cells,the virus can be assembled only if the trypsin is added to the culture medium.In addition,residual serum in conventional cell culture will lead to the inactivation of the trypsin.Currently,serum-free medium for cell culture is a hotspot in vaccine production.It can overcome the serum interference to the virus culture,and eliminate the potential pollution source and facilitate the separation and purification.By optimizing the bioreactor culture method and fermentation process parameters,the production technology of bioreactor and serum-free culture virus were established in this research.Some stable post-treatment processes were established by exploring technology of vaccine,including multi-stage filtration clarification,ultra-filtration concentration,gel filtration and ion exchange chromatography,sterilization filtration and formaldehyde inactivation.Using different gel media for purification process comparison study,it is ultimately determined to use Sepharose 4 Fast Flow and Capto Q to get the high virus recovery rate,the vaccine protein removal rate of 99%or more,the vaccine protein content of not more than lOug/agent and the DNA residue ≤100 pg/agent.These several key verification indicators all meet or exceed the current Chinese Pharmacopoeia and WHO standards.In conclusion,we established a platform for the stable and rapid preparation of Vero cell influenza vaccine candidate strains by reverse genetics,a technology for the Vero cell bioreactor microcarrier serum-free culture and some post-treatment processes with high virus recovery rate.The influenza-inactivated vaccine prepared by this technique was safe and effective.It will promote the upgrading of global influenza vaccine production technology.