Studying On Preparation And Safety Evaluation Of H5 And H1 Subtype Recombinant Adenovirus Vaccine

Avian influenza virus(AIV)is a kind of zoonotic infectious disease that economic and health problems in the world.Most of the avian influenza virus subtypes cannot even cause waterfowl disease,but the outbreak of avian influenza in poultry is closely related to high mortality.Although avian influenza virus rarely transmit to humans and dependent on degeneration,but the virus has the potential to cause life-threatening infection after mutation or recombination.The emergence of a new strain of influenza virus makes it challenging to predict its performance,transmission,virulence,or the potential for human to human transmission.It is difficult to get prepared in advance because it is hard to predict the mutation of which strains or locations will cause the next pandemic.Vaccination can play an effective role in prevention,so the study of new vaccines is particularly important.Influenza recombinant adenovirus vaccine which can stimulate the body to produce higher levels of humoral immunity attracted much attention in recent years with good prospects for development,and the recombinant adenovirus vector has good sTableility,high safety,wide host range and other advantages.Technology of micro carrier culture is widely used in the field of vaccine production because of its large surface area convenient detection and control of the cell in real time.Anion exchange chromatography has advantages of high sensitivity,good selectivity,fast separation speed.Molecular sieve chromatography has advantages of mild operating conditions,better separation of polymer material and so on.In view of these advantages,the two kinds of purification methods were commonly used.In this study,we constructed a recombinant adenovirus vaccine based on seasonal influenza H5 subtype and pandemic influenza H1 subtype HA(HA1)gene,which was supplemented by avian influenza H1?H7 and H9 subtype HA antigen Th epitope and supplemented by epitope.Through the 7L bioreactor culture system,H5 subtype influenza recombinant adenovirus were cultured in a fermentation product processing,and then chromatography and molecular sieve chromatography were utilized following anion exchange,and safety evaluation test wastaken after correct identification.The HEK-293 cells were cultured in the bioreactor.After several experiments,the parameters were determined as follows: temperature: 37 ?,pH:6.9,stirring rate: 30r/min,dissolved oxygen 50%,CO2 concentration: 5%.After culturing 24 h,more than 85% cells adherent the micro carrier,and the growth rate of cellsat 24-36 h was comparatively higher.The total number of cellsat 84 h increased by 4 times after culture medium replacementat 48 h.,and then the growth of cells showed a downward trend.A large number of H5?H1 subtype influenza recombinant adenovirus were produced by the bioreactor,H5 ? H1 subtype influenza recombinant adenovirus were inoculated by 5 MOI,when HEK-293 cells were covered with 80-90% of the micro carrier surface.The virus supernatant was received afer 48 h and the virus titer was 1.0×1010TCID50/mL.The virus titer of H5 ? H1 subtype of influenza recombinant adenovirus was1.0×1010TCID50/mL after bioreactor culture,the virus titer was 1.0×1010TCID50/mL after screen chromatography anion exchange chromatography and molecular purification,two step purification recovery rate were 30.8% and 89.3%,the total recovery rate was 27.5%.The purified samples were identified by PCR,and the results showed that the gene was not lost.OD260/OD280=1.25,that would fill the bill.Experimental study and safety evaluation of fermentation and purified recombinant adenovirus in mice.The results show that,the multi influenza recombinant adenovirus vaccine can stimulate production of antibody and specific cellular immune response in mice after two step purification,and the levels of cytokine in two step purification vaccine group were higher than that of the other vaccine group.Safety evaluation test results showed the two step purification of recombinant adenovirus subtype influenza vaccine has good security.In conclusion,this experiment demonstrated that the H5?H1 subtype influenza recombinant adenovirus vaccine can induce cellular and humoral immune responses in mice,which were cultured by bioreactor microcarrier system and purified by anion exchange chromatography and molecular sieve chromatography.