TRANSDUCTION OF BOVINE PERIPHERAL BLOOD CELLS WITH RECOMBINANT BOVINE ADENOVIRUS-3 EXPRESSING GREEN FLUORESCENT PROTEIN
Bovine adenovirus type 3 (BAdV-3), a non-enveloped icosahedral particle with a double-stranded DNA genome of 34,446 base pair, has been developed as a vaccine vector (Zakhartchouk et al., 1999). It belongs to Mastadenovirus genus in Adenoviridae family. Like other Mastadenovirus members, BAdV-3 genome is composed of early, intermediate and late regions (Reddy et al., 1998). A number of characteristics including lack of virulence and ability to grow to high titers have made BAdV-3 a vector of choice for further development as a vaccine delivery vehicle for cattle. One of the ways to improve the efficacy of immune response is by targeting recombinant BAdV-3 to immune cells. However little is known about the interaction of BAdV-3 with different immune cells. Determining the tropism of BAdV-3 for specific leukocyte subpopulations may help in devising ways to target BAdV-3 to appropriate leukocyte population for the induction of robust and efficient immune responses in calves immunized with recombinant BAdV-3 expressing vaccine antigens. The specific aim of this work is to determine the interaction of recombinant BAV304a (GFP expression cassette inserted in E3 deleted region of BAdV-3 (Du and Tikoo, 2010)) with different leukocyte populations in the blood. Different leukocyte populations present in bovine blood (consisting of monocytes, B-cells, T-cells, NK cells and dendritic cells) and PMNs (neutrophils) were transduced with BAV304a. The transduction of bovine peripheral blood mononuclear cells (PBMCs) with BAV304a revealed a GFP expression of 12-15%. Transduction efficiencies of bovine PBMC were shown to be highest with an MOI of 2. Further analysis of bovine PBMC subpopulations transduced with BAV304a at MOI 2 was completed by monoclonal antibody labeling of lineage specific proteins. The following subpopulations were analyzed: CD14+ & CD11c+ (monocytes); CD3 (T-cells); CD21 (B-cells); CD335 (NK cells) and CD209+ & CD14- (dendritic cells). I observed 100% transduction of monocytes, while 2%, 1% and 4% transduction was observed in B-cells, T-cells and NK-cells, respectively. Similar results were obtained following transduction of monocytes purified from PBMC cells using high-speed cell sorter. However, transduction of purified dendritic cells (CD209+CD14-) and purified PMNs revealed GFP expression of 1-2% and 9-18%, respectively. This result indicates that BAV304a does not transduce myeloid dendritic cells efficiently but both monocytes and PMNs can be transduced by BAV304a. Though monocytes and neutrophils showed 100% and 9-18% transduction by BAV304a, respectively, Western blot analysis detected a block in the expression of BAV304a specific proteins. While, expression of most of the late proteins could not be detected in transduced monocytes, no expression of any BAV304a specific protein (early or late) could be detected in transduced PMNs. Moreover, no progeny virions could be detected in BAV304a infected monocytes or PMNs confirming a block in the later stages of virus replication. The TCID50 assay showed no detectable viral CPE and GFP expression with infected lysates of monocytes and PMNs. These results demonstrate that BAV304a transduces some subpopulations of PBMCs primarily monocytes and PMNs without viral replication. In contrast, dendritic cells, the primary cells involved in the induction of T cell responses, were refractory to transduction by BAV304a.
Bovine adenovirus, leukocytes, transduction, replication
Master of Science (M.Sc.)
School of Public Health
Vaccinology and Immunotherapeutics