Rescue and immunogenic evaluation of attenuated vaccine candidate from A/California/07/2009

Six internal protein gene segments of attenuated, cold-adapted(ca), temperature -sensitive (ts) influenza A/Ann Arbor/6/60 ca (H2N2) and HA, NA gene segments of A/California/07/2009ca were introduced to plasmid pAD3000 carrying pol I and pol II promoters, and rescued the reassortant virus from Vero cell using reverse genetics technology The reassortant has the attenuate characters, ca and ts, the TCID5(0) is 7 5, HA titer maintain at 1 256 and EID50 is 8 which was detected using SPF eggs The stable of reassortant is determined by RT-PCR gene segments from virus which were propagated in eggs The morphology of reassortant conform the wild type virus In order to test the immunogenicity, the reassortant viruses were purified Mice were intranasally immunized and intramuscular injected with inactive whole virus as control The high HI titer can be detected in both groups, seems hinger in i m (intramuscular) immunized groups (P=0 044), but higher IgA titer can only detected in in (intranasal) immunized group Compared with i m immunized group, higer pro-inflammation cytokines IL-1 beta, TNF alpha, IFN-alpha were tested in in groups, means faster and stronger mucosal immune response was induced Virus load were detected in lung, brain, spleen 4 days after immunization to determine the safety of reassortant as vaccine candidate, no detectable virus were found All results show that the vaccine candidate can be used for vaccine production