Porcine Adenoviral Vector System For Transgene Expression And Ligand Display

Porcine adenovirus type 3(PAd V-3) has many qualities which make it an ideal choice for using as a delivery vector. PAd V-3 is a low grade pathogen, presented in swine populations almost world-wide. It is species specific having only been isolated from swine, which does not infect human and is lack of cross-neutralization with preexisting anti-HAd V immunity in humans. When engineered to contain a foreign gene, recombinant PAd V3(r PAd V3) can be grown to high titers in tissue culture cells making it cheap to be produced. Knowledge of the complete nucleotide sequence of the PAd V-3 genome has enabled rationally directed insertions of foreign genes which remain stably inserted in the genome and can be expressed at high levels following delivery to the target host. So PAd V-3 vector is an effective delivery system in pigs, and also presents great potential as effective gene delivery vehicle in human.Despite these advantages, there are still many steps necessary before using the PAd V-3 in the clinics, especially in human. The first challenge is to construct an efficient approach for producing recombinant porcine adenoviruses for transgene expression and a robust and universal platform for the presentation of polypeptides on the surface of the viral capsid. In this study, a novel porcine adenoviral system for transgene expression has been developed. A series of PAd V-3 mutants with deletions in the whole or partial or with chimeric p Ⅸgene were generated and evaluated to get more insight in the functional domain of p IX in PAd V-3 life cycle and to present polypeptides on the surface of the viral capsid.1. Construction of a novel and simple method for rapid generation of recombinant porcine adenoviral vectors for transgene expressionIn this chapter, we herein reported a new and simple cloning approach for the rapid generation of a porcine adenovirus(PAd V-3) vector which showed promise for gene transfer to human cells and evasion of human adenovirus type 5(HAd V-5) immunity. Based on the final cloning plasmid, p FPAV3-Ccd B-Cm, and our modified SLi CE strategy(SLi CE cloning and lethal Ccd B screening), the process for generating recombinant PAd V-3 plasmids required only one step in 3 days, with a cloning efficiency as high as 620±49.56 CFU/ng and zero background(100% accuracy). The recombinant PAd V-3 plasmids could be successfully rescued in porcine retinal pigment epithelium cells(VR1BL), and the foreign genes were highly expressed at 24 h after transduction into swine testicle(ST) cells. These results revealed that our modified SLi CE cloning system was rapid and cost-efficient, which could be used as universal cloning method for modification the other regions of PAd V-3 genome as well as other adenoviral genomes.2. Chimeric protein IX-mcherry fusion influenced the replication of porcine adenovirus type 3The minor protein IX(p IX) represents a versatile platform for the presentation of polypeptides on the surface of the viral capsid. In order to evaluate the ability of p IX for the presentation of polypeptides on the PAd V-3’capsid, the r PAd V3-p IX-mcherry was rescued and evaluated by different assay. Though the r PAd V3-p IX-mcherry containing p IX-mcherry fusion was rescued successfully in VR1 BL cells, the infectivity and overall production of the mutant virus progeny were significantly reduced compared with the wild virus. In addition, the fusion protein p IX-mcherry was mainly located in the cytoplasm. Our data indicated that p IX was more important to the replication of PAd V-3, and may be not an effective platform for presentation of large targeting polypeptides on the surface of the virus capsid.3. p IX was not essential for porcine adenovirus type 3 virions productionThe minor protein IX(p IX) has multiple roles in the Ad life cycle. In order to insight the function of p IX in PAd V3’ life cycle, the virus r PAd V3-△ p IX with deletion of the whole p IX of PAd V-3 was rescued and evaluated by different assays. Though the r PAd V3-△ p IX without p IX was rescued successfully in VR1 BL cells, this recombinant virus was more susceptible to heat than the r PAV219 with native p IX. The deletion of p IX decreased the titers of PAd V-3 in VIDOR1 cells and inhibited the spread from cell to cell of PAd V-3. Those results showed that(1) p IX was not essential for formation of progeny virions in PAd V3;(2) there were big differences between the p IX-deleted PAd V3-△ p IX and wild virus in virus characteristics, for instance thermostablity, replication and plaques formation, which indicated p IX may play more important roles in viral life cycle.4. The C-terminal domain deletion of minor protein IX decreased the replication and spread from cell to cell of porcine adenovirus type 3In this study, we aligned the amino acid sequences of PAd V-3 p IX with different human adenoviruses using CLUSTAL W program and identified three domains: the conserved N-terminus domain, coiled-coil domain(similar to the C-terminus of HAd Vs) and C-terminus domain. Then the r PAd V3-NMp IX with deletion of the C-terminus domain was rescued and evaluated by different assays. Though the r PAd V3-NMp IX was rescued successfully in VR1 BL cells, and the thermostablity of this virus was similar to r PAV219, the infectivity, overall production and the spread from cell to cell of the mutant virus were significantly reduced compared with r PAV219. Those results showed that the C-terminus domain of PAd V3’ p IX was not concerned with the stability of capsid, but it was the key domain of p IX in the replication of PAd V-3.5. Co-expression of native p IX and p IX-mcherry enhances p IX-mcherry incorporation in the PAd V-3’ capsidIn this chapter, the r PAd V3-E1-p IX-mcherry with native p IX and p IX-mcherry inserted in E1 region was rescued and evaluated by different assays. The r PAd V3-E1-p IX-mcherry was rescued successfully in VR1 BL cells, and the thermostablity, the infectivity and overall production of this virus were similar to r PAV219. A small proportion of fusion protein p IX-mcherry reached the nucleus and incorporated into the capsid. Those results showed that co-expression of native p IX and p IX-mcherry enhances p IX-mcherry incorporation in the PAd V-3’ capsid.