Establishment Of A Novel Replication-defective Adenovirus Vector System

Viral vectors play an important role in genetic engineering vaccine research. The currently used vaccine vectors are adenovirus vectors, adeno-associated virus vectors, herpes simplex virus vectors, retroviral vectors and so on. Among these viral vectors, adenovirus vectors are mostly employed in vaccine research and development. According to ability to produce progeny virus, adenovirus vectors are classified into two groups:replicating and replication-defective （non-replicating）. While replicating adenoviral vectors entered cells, they could produce a large number of progeny viruses, with the increased copies of foreign genes which induce a strong and lasting immune response. But the proteins of adenovirus, especially late proteins （particularly penton） are of toxicity. Hence, the safety of replicating adenovirus vector vaccines need to be improved. After infected cells, replication-defective adenovirus vector vaccines cannot produce progeny virus, and thereby safe. Because of the relatively low expression of foreign genes, the immune effects are poorer than replicating adenovirus vector vaccines. Therefore, to develop a novel adenovirus vector with replicating genome but non-progeny virus will promote the applications of adenovirus vectors in vaccine research, since it is safe and has high expression and immune effects of foreign genes.In the present work we attempted to construct a novel adenovirus vector with replicating genome but non-progeny virus by providing partial or complete E1region in-cis to replication-defective vectors with E1/E3deletion and by deleting Ⅳa2gene. These vectors, as well as a cell line which supports the proliferation of recombinant virus established eventually a novel replication-defective adenovirus vector system. Firstly, partial or complete E1region cassette was inserted into different locations of adenovirus genome. The structure with highest replication capacity was selected as the basement of the novel replication-defective adenovirus vector. Secondly, we established a PCR targeting method based on λ-RED recombinase system, and deleted Ⅳa2genes from the adenovirus infectious clone pFG140by this method, so that to rescue the recombinant adenovirus with IVa2deletion, and investigate the influence of IVa2deletion on expression of adenovirus late proteins and progeny virus. According to the above, we modified AdMax system by inserting E1region and deleting Ⅳa2gene, and then established a universal novel replication-defective adenovirus vector. At last, we constructed a transgenic cell line expressing IVa2protein to support the replication of recombinant adenovirus with IVa2deletion. The major findings are as follows:1. Established a novel replicating adenovirus vector based on inserting E1cassette into E1deletion region of adenovirus.The recombinant adenovirus Ad5E1A+（IE3） was obtained by inserting PGK-EIA cassette into E3-deleted region of adenovirus genome. It produced no progeny virus in HeLa cells. However, the viral genome can reach1.6E8copies/μg at48h.p.i （hours post-infection）. Late proteins can be barely detectable. Even in293cells, progeny viruses of Ad5El A+（IE3） reached only1.5E7FFU/mL at72h.p.i, which was50times lower than that of the positive control （Ad5El-E3-₂93, about7.2E8FFU/mL）. We obtained the recombinant adenovirus Ad5El+（IE3） by inserting PGK-E1cassette into adenovirus E3-deletion region. Ad5El+（IE3） produced progeny viruses in both HeLa and A549cells and viral genome reach4.5E8copies/μg at48h.p.i., but the progeny viruses only reached3.6E6FFU/mL at72h.p.i. The recombinant adenovirus Ad5E1+（IE1） which was obtained by inserting PGK-E1into E1deletion region produced about3.3E8FFU/mL progeny viruses at72h.p.i in HeLa cells. The level of progeny viruses was comparable to those of the positive control. Therefore, the construction was selected as the basement of the novel replication-defective adenovirus vector. 2. Constructed a novel replication-defective adenovirus vector based on Ⅳa2gene deletion.A PCR targeting method was established based on the λ-RED recombinase system. The recombinant pFG140-△Ⅳa2（1104） was obtained by homologous recombination between a linear fragment and adenovirus infectious clone pFG140with the help of λ-RED recombinase. Endonucleases digestion and DNA sequencing confirmed the precise deletion of1104bp fragment of IVa2. With the same method, four copies of miRNA122target sequence were inserted into3’untranslated region of Ⅳa2gene. Endonucleases digestion and DNA sequencing confirmed the precise insertion at expected position. These two examples demonstrate that the PCR targeting strategy can manipulate adenovirus genomic DNA on a plasmid promptly and accurately by λ-RED recombinase system.We obtained recombinant adenovirus Ad5△Ⅳa2（1104） with Ⅳa2deletion from pFG140-△Ⅳa2（1104）. It could not produce progeny virus without Ⅳa2gene. Viral genome reached4.5E8copies/μg at48h.p.i, but the expression of late proteins was inhibited. On the base of the structure of Ad5E1+（IE1） and Ad5AlVa2（1104）, we obtained recombinant adenovirus Ad5E1+（IE1）△Ⅳa2（1104）, which the complete E1region was inserted into E1-deleted region and Ⅳ a2gene was deleted by modifying AdMax system. The rescue of the recombinant virus successfully proved that a novel replication-defective adenovirus vector was established.3. Obtained a transgenic cell lines expressing IVa2protein.With G418selection pressure, we obtained a G418-resistant cell line from pAAV2neo-Ⅳa2（2）-transfected293cells. Six clones were able to support the replication of recombinant adenovirus Ad5E1+（1El）△Ⅳa2（1104） and generated progeny viruses in the dozens of monoclonal picked. Western Blot showed that six monoclonal cells had Ⅳa2protein expression, but the expression levels were different. We chose the highest level of IVa2expression clones No.4to passage. IVa2can be detected at15passages, and no significant change of IVa2expression was found. We thereby established a novel replication-defective adenovirus vector system base on IVa2gene deletion adenovirus, together with the transgenic cell line expressing IVa2proteins.All the above results indicated that base on a replication-defective adenovirus vector with both E1/E3deletions, we constructed a replicating adenovirus vector by inserting a complete E1region of adenovirus PGK-E1into the El deleted region. PCR-targeted adenovirus late regulatory gene Ⅳa2by λ-RED recombinase system, obtained a recombinant adenovirus with replicating genome but non-progeny virus. We established the transgenic cell line expressing IVa2proteins, which can complement the reproduction of recombinant viruses. Both the viral vectors and transgenic cell lines established a novel replication-defective adenovirus system, and it will promote the application of adenovirus vectors in vaccine development.