Preparation Process Study Of HIV-1 C Subtype Recombination MVA Vaccine

HIV-1 C subtype recombination MVA (Modified Vaccinia Ankara) vaccine is a recombinant vaccine modified vaccinia Angola which carries structure and regulation gene of HIV-1 to express Env, Gag, Pol, Nef and Tat. MVA lost host gene and large amounts of DNA fragments after proliferation in chick embryo fibroblast (CEF) for multi generation. Meanwhile, inserted exogenous recombination gene can be expressed in MVA genome in the cytoplasm. Compared with separated strain and attenuated vaccine, MVA is a safe and efficient vector which has been applied in vaccine research, gene therapy, biological active peptide production. At present, there is no similar vaccine in mass production and sales.This study is part of the HIV vaccine project of Medical Genetics Lab in Institute of Medical Biology, Chinese Academy of Medica Sciences. Vaccinia virus is now known as one of the most complex structure of the virus belonging to the vaccinia virus, pox virus. Based on structure of vaccinia virus and viral vaccine pharmacopoeia standards, proliferating MVA in CEF, combining with ultrasonic treatment, centrifugation, chromatography and ultrafiltration, finally we obtained the purification methods for large-scale generating MVA vaccine.First, we generated of CEF cells from chicken embryos and collected MVA virus after vaccination and proliferation. By centrifugal enrichment, suspension, ultrasound and ultrafiltration, we obtained the first step purified virus stock. Furthermore, the virus stock were purified by cushion centrifugation by 30% and 36% sucrose cushion, homogenate and affinity chromatography using Q Sephrose XL and SOURCE 30Q ion exchange liquid chromatography. In whole preparation process, including purification and aseptic processing are required to operate in accordance with the production standard. Finnaly, we conducted quality control experiments, including visual inspection, pH detection, identification, detection, titer test, sterility test, pyrogen test, the residual host cell protein detection, and assessment of the specific antibody and cellular immune response after MVA immunization.The results revealed that purified MVA reached viral vaccine quality standards. The target genes, gag, pol, Nef and Tat, were all amplified by PCR; WB showed that target protein expressed also, corresponding antibody were detected by ELISA from MVA immunized mice; cellular immune reaction were detected by ELISPOT. Together, in this study, we explored one MVA vaccine purification method, reaching recombinant virus vaccine standard, providing technical data for preparation of similar vaccines.