Transduction of Bovine Peripheral Blood Mononuclear Cells with Recombinant Bovine Adenovirus-3 Expressing Chimeric pIX
| dc.contributor.advisor | Tikoo, Suresh | |
| dc.contributor.committeeMember | Griebel, Philip | |
| dc.contributor.committeeMember | Wilson, Heather | |
| dc.contributor.committeeMember | Godson, Dale | |
| dc.creator | Bravo Araya, Maria 1986- | |
| dc.date.accessioned | 2018-12-18T19:50:52Z | |
| dc.date.available | 2019-12-18T06:05:09Z | |
| dc.date.created | 2018-12 | |
| dc.date.issued | 2018-12-18 | |
| dc.date.submitted | December 2018 | |
| dc.date.updated | 2018-12-18T19:50:52Z | |
| dc.description.abstract | BAdV-3, like many other members of the family Adenoviridae, has been developed and evaluated as a vaccine delivery vehicle in cattle (Ayalew et al., 2014). However, protective immune responses using recombinant BAdV-3 based vaccines are achieved after two immunizations (Kumar et al., 2014; Zakhartchouk et al., 1999). One way to increase the efficiency of BAdV-3 based vectors is by increasing the transduction to antigen presenting cells (APCs). Since C-terminus of BAdV-3 minor capsid protein pIX is exposed to the exterior of virion capsid, efforts have been made to use it to add targeting ligands, so that recombinant BAdV-3 expressing chimeric pIX can be targeted to a particular cell (Zakhartchouk et al., 2004). In the present study, we constructed and characterized a recombinant BAdV-3, designated as BAV888, expressing chimeric minor capsid protein pIX fused to RGD. The RGD motif, which is not present in the BAdV-3 virion, is an important pathway for the internalization of the virus into integrin-positive cells, such as APCs. In vitro studies have demonstrated that at MOI 1, both BAV888 and BAV304a (BAdV-3 expressing GFP inserted in E3 region of the genome) can transduce PBMCs without a significant difference in the percentage of cells transduced. However, BAV888 transduced cells exhibited a mean fluorescence intensity significantly higher than BAV304a transduced cells. Further, analysis in different PBMC subpopulations (T-cells, B-cells, monocytes, NK-cells and dendritic cells) showed that BAV888 and BAV304a have tropism mainly for monocytes and NK-cells. Furthermore, the mean fluorescence intensity of GFP+ monocytes is up to four times higher in BAV888-transduced cells, suggesting that our recombinant virus is more efficiently internalized by monocytes. To prove this, we compared viral copy number of transduced monocytes and transcriptional analysis of viral proteins using qPCR. The copy number of BAV888 is significantly higher than BAV304a in transduced monocytes at the same MOI. Moreover, the expression of GFP and early and late viral proteins is higher in BAV888 transduced monocytes. To determine the expression of costimulatory molecules in BAV888 and BAV304a transduced monocytes, flow cytometry, and transcriptional analyses were performed. Although there is no significant difference in the expression of MHC class I and MHC class II between viruses using flow cytometry, analysis of the mRNA expression of showed a decrease in CD40 and CD86 expression in BAV888-transduced monocytes compared to BAV304a and mock transduced cells. In terms of cytokine production, BAV888-transduced monocytes show an increased production of TNFα and IL-12, compared to BAV304a transduced monocytes. In mRNA analysis, there was a significant increase in expression of IFNβ in BAV888-transduced monocytes relative to BAV304a-transduced and mock cells, which could suggest an anti-viral response against the virus. Our results demonstrate that, although there is no change in tropism of the virus, the addition of an RGD motif in the C-terminus of pIX of BAdV-3 can increased its uptake in PMBCs and, in particular, monocytes. | |
| dc.format.mimetype | application/pdf | |
| dc.identifier.uri | http://hdl.handle.net/10388/11669 | |
| dc.subject | Bovine adenovirus | |
| dc.subject | tropism | |
| dc.subject | adenovirus vector | |
| dc.subject | PBMCs | |
| dc.subject | pIX | |
| dc.title | Transduction of Bovine Peripheral Blood Mononuclear Cells with Recombinant Bovine Adenovirus-3 Expressing Chimeric pIX | |
| dc.type | Thesis | |
| dc.type.material | text | |
| local.embargo.terms | 2019-12-18 | |
| thesis.degree.department | School of Public Health | |
| thesis.degree.discipline | Vaccinology and Immunotherapeutics | |
| thesis.degree.grantor | University of Saskatchewan | |
| thesis.degree.level | Masters | |
| thesis.degree.name | Master of Science (M.Sc.) |