Immunogenicity Of Recombinant Adenovirus Vaccine Against Multi-subtype Influenza Virus Prepared By Bioreactor Preparation And Purification

Influenza is an acute respiratory infectious disease caused by influenza virus, which is a serious threat to human health. In the past 100 years, influenza virus has caused multiple flu pandemics, leading to serious public health problems and huge economic losses. Vaccination is the most effective way to prevent the flu, while the existing vaccine is not enough to cope with the rapid variation of influenza viruses, the study of new influenza vaccine has become a pressing matter of the moment. Influenza recombinant adenovirus vaccine is one of the hotest research topics, with the advantages of excellent stability, broad range of host cells and high level of exogenous gene expression..Bioreactor microcarrier system increased the culture surface area becomes provided a single growth environment, and easily monitored training parameters, with becoming a new and attractive alternative technology. Mass clinical recombinant adenovirus vaccines should be produced by highly safe and efficient production and purification methods. Traditional CsCl density gradient centrifugation is suitable for a small amount of adenovirus purification, while the most effective method in large-scale production is chromatography. Anion exchange chromatography purification has the advantage of high capacity and low cost, and molecular sieve chromatography is characterized by mild condition and high recovery rate. Therefore, adenovirus is generally isolated and purified by the combinatrom of the above two methods.In the early days, lab constructed adenovirus vaccine mainly on seasonal influenza H3 subtype and pandemic influenza H1 subtype HA gene combined to avian influenza H5,H7,H9 subtype HA antigen Th and B cell epitopes cassette(EHA) which have a breakthrough species barrier to infect humans. In this experiment, H1,H3 and other multi subtype influenza recombinant adenovirus were produced on a large scale were by 7L biological reactor and Cytodex 1 type microcarrier. The fermentation products were purified by anion exchange chromatography and molecular sieve chromatography with the chromatography medium of Source 30 Q and Sepharose 4FF. The purified recombinant adenovirus of H1,H3 and other multi subtype influenza virus identified right was tested for immunogenicity in mice.HEK-293 cells were cultured in the bioreactor system. 2.8×108cells(1.6×104cells/mL) were inoculated. The training parameters optimized through multiple tests are: 37℃, 5% CO2, p H 7.0, 50% dissolved oxygen and stirring speed of 30 rpm. More than 80% cells adhesion on microcarrier surface, and amount of calls began to signigacantly grow after 24 hrs. Growth rate was highest in 48 hrs–60 hrs, slowing down after 72 hours. The amount of cells reached 1.4×109(8×104cells/cm2), increasing by 5 times. After 96 hrs culture, the number of cells began to decline, and gradually began to appear in the case of necrosis. H1,H3 and other multi subtype influenza recombinant adenovirus were cultured in bioreactor with 0.5 MOI. The virus was collected after 48 hours with the titer of 1.0×1010TCID50/ml.Titer of H1,H3 and other multi subtype influenza recombinant adenovirus preparation of biological reactor was 1.0×1010TCID50/ml, and it rose to 1.1×1010TCID50/ml after two steps chromatography purification. The recovery rate of the fermentation products by the biological reactor, molecular sieve chromatography and the two steps purification were 31.5%, 81.4% and 25.6%.The purified product was identified as complete genes with correct protein expression by PCR and Western blot. By the absorbance at 260 nm and 280 nm is A260/A280=1.26, indicary that the preliminary proof meet the requirement of purity.The immunological evaluation of recombinant adenovirus that meets the requirements was carried out in mice. Humoral immunity and cellular immunity were detected in each group. The results showed that H1,H3 and other multi subtypes of recombinant adenovirus after two steps of pufication had the highest level of IgG stimulated by the specific H1, H3 antigen stimulation, while the level of the non purified groups were equivalent. The results of IL-4 and IFN-γ cells factor level test showed that the levels of cytokines expressed in the two purified groups were siginificanttly higher than those in the non-purified groups.Overall, H1,H3 and other multi subtype influenza recombinant adenovirus after two steps purification have strong ability to stimulate the production of cellular and huoral immunity in mice. Immunity of recombinant adenovirus purified by one step purification was slightly inferior. Both were higher than that of the non purified group. H1,H3 subtype specific humoral and cellular immune responses of H1,H3 and other multi subtype influenza recombinant adenovirus produced by biological reactor were the basically same to that produced by cell culture bottles.In summary, using bioreactor micrarrier system H1 and H3 subtypes of influenza recombinant adenovirus were successfully prepared. Then fermentation products were separated and purified by the two step chromatography. This paper lays a preliminary foundation for the large-scale production of the recombinant adenovirus vaccine.