| Live attenuated influenza vaccine(LAIV)is administered intranasally,induces serum neutralizing antibody and elicits local mucosal immunoglobulin A(IgA)antibody response.Currently LAIV in market are cultured in hen embryo.Hen embryo as vaccine production substrate,aside from its supply period restricts vaccine production capacity,there is risk of introducing contaminant exogenous factor considering inoculated viruses are still alive.Vero cell is a common cell substrate applied for human biological product,while when influenza virus cultured in Vero cells,the yield differs greatly or many viruses could not be serial passaged in Vero cells.For now,there is only one research on Vero cell adapted influenza A,there is no research on influenza B in Vero cells.In this study,we conducted reverse genetic technology to build a reassortment with 2 circulating BV antigen and 6 internal gene segments of influenza B master donor strain B/Yunnan/2011Vca(BVca).Through the process,progress including amplify 8 gene segments of influenza B in a single reaction with primer cocktail,clone gene segment to pHW2000 applying homologous recombination and construct a positive plasmid screening system for influenza B,were all employed for achieving a rapid and efficient reverse genetic rescue technology for influenza B.We rescued reassortant virus BWVca with 2 circulating surface antigen and 6 internal gene segments of BVca.BWVca could be serial passaged in Vero cells at 25? with cold adapted(ca),temperature sensitivity(ts)and attenuation(att)phenotype characteristics.Under optimized culture condition,the infectious titer could reach 7.5 LgTCID50/mL.All the results indicated reassortant virus BWVca can be a promising candidate vaccine strain for live attenuated influenza vaccine.The reassortment possibilities between LAIV and wild-type virus concerns people.With 8 segments in influenza virus genome,two different virus co-infect one cell,the combinations of reassortant can be as many as 256.In this study,since the virus strains we used have possess same surface antigen,we focused on genes encoding the viral ribonucleoprotein(RNP)complex(PB1,PB2,PA and NP).M and NS alone or both were also added into combinations.Eventually we reconstructed 34 different strains of virus by swapping gene segment of BWVca and BV-W.Att phenotype were used to evaluate the bio-safety of recombinant viruses.Among the 34 strains viruses,11 strains showed partial att character(met att determination criteria but viral load was detected in lung tissue),23 strains showed complete att character.Through these results,we proved the vaccine strain candidate BWVca possesses bio-safety,and for the first time we proved the master donor strain BVca can be used as a donor strain for B type live attenuated influenza vaccine.In 2016,the advisory committee of immune practice(ACIP)suggested to not recommend Flumist(?)in 2016-2017 influenza season for its poor vaccine efficacy compared to trivalent influenza vaccine.Researches aiming enhance LAIV efficacy have became a focus.In the present study,we combined Vero cell based LAIV with immuno-potentiating adjuvant PolyI:C,Flagellin,BW006 or CpG2395,found when we combine flagellin or PolyI:C with LAIV and immunize mice,vaccine associated cross protection was detectable,protective rate for BV lineage antigen drifted virus enhanced by 30%and 20%,protective rate for BY lineage enhanced by 40%and 30%,respectively.This study is based on the optimization of reverse genetic technology,obtained a reassortant virus contains surface antigen of circulating strain and ca,ts,att phenotypic characteristics of master donor strain,could be a Vero cell influenza LAIV seed virus;we further partly random recombined attenuated strain and wild-type virus,proved the use of LAIV will not lead to virulence reversion;with certain adjuvant addition to LAIV,cross protection could be improved. |
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