Assay Of The Recombinant Vaccine Against Pandemic Influenza (Bombyx Mori Baculovirus)

Recombinant Pandemic Influenza Vaccine (Silkworm Baculorvirus) is a novel human vaccine against bird flu. The vaccine is silkworm baculovirus ,which displaying the HA Protein of A/Zhejiang/16/06(H5N1)virus on the surface of the baculovirus envelope. After propagating in the pupaes of Bombyx.mori, the baculorviruses were collected and purified to produce vaccine. Evaluation of the vaccine in the mice and monkeys suggested that the vaccine is safe and effective. Mice and monkeys immunized with this recombinant virus induced production of neutralizing antibodies, which provided protection against H5N1 infections. SFDA has no quality standard for this innovative vaccine. So we conducted preliminary docimasy of the vaccine to lay the foundation for the establishing of quality standard. Vaccine virus seeds, Bmgp64HA, are passaged in BmN cells. We isolate the genome DNA of the viruses and using PCR technique to identify GP-HA sequence.We find that the viruses before 10 generations have full fragment of GP-HA with no mutation. This information helpes the choosing of working seed viruses. The total protein of vaccine sample are digested with trypsin. After that, we perform MALDI-TOF MS analysis. 7 fragments of HA were detectd and sequence coverage is 24%. The result shows that vaccine contains HA protein. Hemagglutination test showes that this vaccine has HA activity, the hemagglutination titer is up to 1:16. The vaccine samples are identified by Western Blotting with the HA antibody, resulting in a 3 bands. This result agree with our expectations that vaccines contain three forms of HA: integrated HA,HA1 and HA2. We use SRID, DOT-BLOT and indirect ELISA method to determinate the content of HA in vaccine. In our study, SRID method and the DOT-BLOT method can not determine the HA content of the vaccine. measured by indirect ELISA, the HA content of the vaccine is 4.6‰. Virus particles can be detectd by the electron microscope.