Design and characterization of nonhuman adenoviral vectors

Dinesh Bangari Singh, Purdue University

Abstract

The goal of this research was to examine the feasibility of designing gene delivery vectors based on nonpathogenic bovine adenovirus serotype 3 (BAd3) and porcine adenovirus serotype 3 (PAd3). We hypothesized that these vectors would not be prevalent in the human population and thus offer attractive alternatives to human adenovirus serotype 5 (HAd5) vectors by evading preexisting HAd5 immunity. Using virus neutralization assays, we examined whether preexisting HAd5 immunity in humans would cross-neutralize PAd3 or BAd3. We developed a system for generating replication-defective PAd3 and BAd3 vectors. Recombinant HAd5, BAd3 or PAd3 vectors expressing green fluorescent protein (GFP) were generated. These vectors were tested for their transduction efficiencies in a variety of human, murine, bovine and porcine cells in culture. Using antibody-mediated and recombinant fiber knob-mediated blocking assays, we also investigated the involvement of known primary receptor or co-receptors of HAd5 in PAd3 or BAd3 entry. Based on virus neutralization tests, we found that BAd3 and PAd3 were not prevalent in humans, and HAd-specific antibodies in humans did not cross-neutralize PAd3 or BAd3. PAd3 and BAd3 vectors efficiently transduced a variety of human as well as nonhuman cells in culture. Transduction by the HAd5 vector in the majority of human cell lines correlated with the expression levels of coxsackievirus-adenovirus receptor (CAR), the primary HAd5 receptor; while transduction by PAd3 and BAd3 vectors was CAR-independent. HAd5, BAd3 and PAd3 appeared to involve distinct receptors for cell entry. Overall, these results suggest that PAd3 and BAd3 vectors are promising gene delivery vehicles for human gene therapy as well as for recombinant vaccines for humans and animals. These studies also provided a platform for further in vitro and in vivo characterization of these nonhuman adenoviral vectors.

Degree

Ph.D.

Advisors

Mittal, Purdue University.

Subject Area

Microbiology|Molecular biology

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