Construction Of Hexon-chimaeric Recombinant Adenovirus Serotype5Vectors And Function Study

Recombinant adenovirus type5has been widely used in clinical study of cancergene therapy and vaccine of human immunodeficiency virus (HIV), hepatitis B virusand Mycobacterium tuberculosis. However, high prevalence of pre-existing anti-Ad5immunity in human populations will significantly reduce the transduction efficiencyand transgene expression of viral vector. In this paper, we firstly choose Ad26，Ad37and Ad43as source types of chimeric gene according to the current results of theseroprevalence of56human adenovirus serotypes and by analyzing genes and aminoacid sequences of the hexon hypervariable regions (HVR) in all rare adenovirusserotypes. Secondly, according to the related findings on the replacement of Ad5hexon from rare human adenovirus serotypes, we designed and constructed severalhexon chimeric rAd5vectors with full-length of HVR5and HVR7genes and Loop4gene from Ad26, Ad37and Ad43replacing the corresponding regions of rAd5.We finally successfully constructed six chimeric rAd5vectors with replacedhexon which were defined as rAd5HVR26(5+7), rAd5HVR26(5+7)Loop4,rAd5HVR37(5+7), rAd5HVR(5+7)Loop4, rAd5HVR43(5+7) andrAd5HVR43(5+7)Loop4. For the convenience of comparative evaluation of thepackaging, replication, transgene passage stability and expression level of thesechimeric vectors, we also cloned GFP and Luciferase report genes into the shuttlevector pDC316, respectively. And the shuttle vector and viral vector werecotransfected into293cells and Hela cells. By the observation of green fluorescenceor the detection of luciferase activity, we could confirm the activity of the chimericvectors in vitro.In this study, we finally obtained a chimeric vector, Ad5HVR43(5+7), whichwas easy to package, stable in passage, and significantly improved in its replicationand transgene expression level. It laid the foundation of our subsequent immune levelevaluation in vivo and the exploration of adenovirus immune mechanisms. Weexpected to find a new recombinant adenovirus vector which has higherimmunogenicity and better capacity to evade the pre-existing immunization, for theapplication in cancer gene therapy and the human immunodeficiency virus (HIV) andtuberculosis vaccine development. These results will help to explain the mechanisms of adenovirus packaging, and provide an important basis for the further modificationof adenovirus vectors.