| Human infection with avian influenza A(H7N9)has been documented in some countries and areas since 2013.The mortality in patients of A(H7N9)virus infection can be as high as 30%.According to some research on H7N9,because of the virus's increasing affinity of a-2,6-sialic acid,which is the main receptor of epithelial cell in human respiratory tract,and some H7N9 virus strain has shown resistance of anti-virus drugs,all these led WHO list H7N9 influenza virus as a potential pandemic virus.Thus,vaccines could be a promising and effective way to deal with pandemic of H7N9 influenza virus.To date,all virus seed batch for domestic vaccine production are purchasing from abroad.Given the H7N9 vaccine seed must be brought from the foreign countries,we might lose the optimum timing for control and prevention of H7N9 influenza virus.Besides,nearly all of the influenza vaccine are chicken embryonated egg dependent.Chicken embryonated egg is an extremely time consuming system with many disadvantages with respect to selection of virus variants and presence of adventitious viruses.Compared to chicken embryonated egg,Vero cell can be cultured in fermentation cylinder to easily realize large scale production,quality control with a superior safety level,which makes Vero cell suitable for vaccine production.However,not all influenza virus can prolife in Vero cells at a stable yield is a main obstacle for Vero cell based vaccine development.Therefore,by addressing unstable yield of influenza virus in Vero cells,we developed a Vero cell derived H7N9 pandemic influenza vaccine with independent intellectual property.Firstly,we used A/Kunming/1/2005Va(H3N2)as a donor strain to afford Vero cell high yield characteristic from six internal genes and A/Shanghai/2/2013(H7N9)to provide surface antigen gene HA,NA.With the 6+2 combination coinfection of 293T cell,we obtained the reassortant virus A/Shanghai/2/2013(H7N9)Va.Then,we evaluate the bio-safety and biological characteristics of H7N9Va virus in different ways.Results show that H7N9Va virus can be passage at least 15 times in Vero cells without any mutation.All of the tested BALB/c mice can survive totally after H7N9Va virus intranasal inoculation and all the SPF chicken embryonated egg were 100%survived after injection H7N9Va virus in a high TCID50 value.There is no significant difference of virus loads between the H7N9Va group mice's lungs and H7N9 group's.Only mild inflammatory reaction can be find in the lungs of mice infected by H7N9Va.Besides,we find that the H7N9Va virus had good immunogenicity on mice.Secondly,we cultured H7N9Va virus in Vero cells through formentation cylinder.We optimized the culture method of H7N9Va virus.We added TPCK-treat trypsin at a concentration of 1?g/mL into the virus maintain medium.Then we optimized the inoculated quantity of H7N9Va virus.In a single cycle of H7N9Va virus culture,only O.O1MOI virus was needed.After that,we purified H7N9Va virus though filtration clarification,ultra-filtration concentration and chromatography.The purified virus confirmed by Chinese pharmacopeia was inactivated and prepared into a H7N9Va inactivated whole virus vaccine,and the H7N9Va inactivated whole virus vaccine were safe and effective.Lastly,a dose sparing test fond that AS03 adjuvant can reduce the antigen of the inactivated whole virus vaccine into 3.75 ?g HA.In conclusion,a Vero cell-derived H7N9 influenza candidate vaccine with Vero cell high yield characteristic and superior safety was obtained in this study through a reverse genetic method.Thereafter,we optimized virus culture condition and explored downstream purification strategy.We also applied adjuvant to spare the effective dose by 7/8.Thus,we are able to produce pandemic vaccine for emergent use in a short time,which is critical in handling influenza pandemic. |
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