Purification And Immunological Evaluation Of Recombinant Adenovirus Vectored H1、H3Subtype Influenza Virus

Influenza, caused by influenza virus, is a contagious acute respiratory infection thatbrings a series of public-health issues today, and results in substantial economic burden everyyear. There are three type influenza viruses that are A, B and C. All of them can infect humansespecially influenza A viruses which are the most virulent types of responsible for pandemics.People divide influenza A viruses into different subtypes basing on antigenic differences in theirsurface glycoprotein hem agglutinin (HA) and neuraminidase (NA). Influenza A virusesgenome comprise eight single-stranded negative-sense RNA segments encoding eleven proteins.Evolution of these viruses through accumulated mutations (antigenic drift) and geneticreassortment (antigenic shift) can result in the emergence of new strains.Recombinant adenovirus has been now focused on to develop influenza virus vaccinesdue to numerous noteworthy advantages.1)The safety of recombinant adenovirus basedvaccines has been proved in many studies;2)There exist techniques allowing construction andlarge-scale production of recombinant Adenovirus in a very cost effective manner;3)novelformulations have allowed recombinant Adenovirus vectored vaccines to be stored easily;4)recombinant Adenovirus vectored vaccines are able to provide ‘self-adjuvanting’ activity byactivating innate immunity; and5) recombinant Adenovirus vectored vaccines can beadministered by many routes owing to their ability to infect a wide variety of cell types in bothdividing and non-dividing cells.Recently, recombinant adenoviral vectors have been utilized in numbers of gene deliveryapproaches. Demand for characterization, production and purification of recombinantadenoviral vector has increased due to their usage. The classical method of adenoviruspurification through density gradient centrifugation is effective though, chromatographicseparation is the most effective method to product and purifier recombinant adenovirus indeed.Column chromatography is the most powerful method for adenovirus purification.A two-step chromatographic procedure involving an anion-exchange Source30Q columnlowed by a molecular sieve Sepharose4FF column was successfully used for purification of therecombinant adenovirus vectored H3subtype influenza virus in the study. The recombinantadenoviruses resulting from purification described above were evaluated for theirimmunological level in rats through intramuscular injection.The purification results showed that the average recovery of recombinant adenovirusesresulting from the two-step purification utilized in the study is42%and43%separately, while the purity is A260/A280=1.24and A260/A280=1.21.The existence of target gene was proved by PCR.The acute cytotoxicity test demonstrated that the recombinant adenovirus is not harmful to ratsand both better cellular and humeral immunity are induced by purified recombinant adenoviruscompared with unpurified adenovirus indicated by the mice immunogenicity test. Norecombinant adenovirus residue was detected by PCR.The results above showed that the two-step chromatographic procedure utilized in thestudy is safe and effective.