Construction Of Ad35 Adenovirus Vector And Study On Immunogenicity Of Viral Protein

Adenovirus (Ads) is a nonenveloped double-stranded, linear DNA virus with icosahedral symmetry. The human adenovirus constitutes a large family with at least 51 identified serotypes, which are classified into six subgroup on the base of hemagglutination.Among the 51 serotypes, the conventional Ad vectors that are most widely used, including for human clinical trials, are constructed based on the subgroup C Ad serotype 5 (Ad5). Ad5 vector has several advantages as a gene delivery. For example, Ad5 can infect many different types of cells, the virus particle is more stable and easy to get high titer of adenovirus. But clinical and preclinical studies have revealed two major disadvantages of Ad5 vector. First, target cells that are important for gene therapy, including malignant tumor cells and dendritic cells, express nil or insufficient level of a cellular receptor for Ad5, the coxsackievirus and adenovirus receptor. Second, about 90% of adults are seropositive for Ad5 because natural infection with Ad5 is common. Pre-exsiting anti-Ad5 antibodies not only largely inhibit Ad5 vector-mediated transduction, but may also enhance the toxicities induced by Ad5 vector.In contrast, human species B Ad serotype 35(Ad35) utilizes human CD46 as a cellular receptor. Human CD46 is ubiquitously express on almost all human cells, leading to a wide tropism of Ad35 vector. In addition, pre-exsiting anti-Ad5 immunity does not hamper Ad35 vector mediated transduction, and seroprevalence for Ad35 is much lower than that for Ad5. All these demonstrate that Ad35 vector is a promising vector candidate. Till now, there have been very few successful cases regarding the development of the Ad35 vector. Thus, it is essential to develop the Ad35 vector In the paper, we compare the evolution relationship of the human six subgroup adenovirus and concluded that the species B and species C are far from each other on the evolution relationship and at the same time we detected anti-Ad (including Ad4?Ad5?Ad 35)antibody in 1157 normal people serum samples. The results show that people infected by Ad4 and Ad5 are about 70% and 90% respectively, while less than 5% by Ad35. After the detection, we successfully made the Ad5 vector packaging system, including backbone plasmid, shuttle plasmid and cell line expressing Ad35 E1B constructively. With vitro recombination, we get replication-defective Ad35, the titer of which is 106TCID50/ml. In addition, we found that protein pXI is important for the package of replication-defective Ad35.In order to optimize the packaging system we analysis the immunogen ability of the adenovirus protein with proteomic assay, and the results show that the non-structure protein E2A (DNA binding protein) has strong immunogen besides some structure protein such and hexon?penton and fiber.