Work

131. Design and Construction of an Adenovirus Vector Containing Fiber Fibritin/Canine CD40L Fibers Directed to Canine CD40-Expressing Cells

Year: 2006

Type: article

Abstract: Dendritic cells (DCs) are a central element in the development of antigen-specific immune responses. On this basis, antigen loading of dendritic cells via transduction with viral vectors in vivo has been proposed as a standardized and easily clinically applicable approach for tumor vaccination strategies. For genetic vaccine approaches to be effective, it is of vital importance to achieve a selective and high transduction efficiency of the DC present in situ, while also ensuring their effective activation and migration. Towards this goal, we have reported that an adenovirus (Ad) serotype 5 vector targeted to human (hCD40) via genetic capsid incorporation of human CD40 ligand (hCD40L) achieves selective transduction of DC in vitro (Belousova et al. Journal of Virology, 2003). We have sought further evaluation of this CD40-targeted vector system in vivo. Of note, certain dog breeds are subject to spontaneous cancers such as osteosarcoma and lymphoma, thereby providing large animal models as stringent in vivo models of human immunotherapy. We have therefore adapted our CD40 targeting system for use in canine systems. We have constructed an E1-deleted, luciferase transgene Ad5 vector encoding a genetically modified fiber that displays canine CD40L, utilizing similar technique as our hCD40L-incorporating Ad5 vector. Viral particles were harvested from Ad5 fiber included- 293F28 cells and 293 cells, purified by standard cesium chloride gradient centrifugation and yielded titers in excess of 3.0 × 10e12 vp/ mL. Characterization of AdFFcCD40L vector infectivity in canine CD40-positive 293 cells (293cCD40) showed 6.9-to-14.6 fold greater luciferase reporter gene activities versus cCD40-negative 293 cells. Herein, we have demonstrated the construction, purification, and initial infectivity characterization of a novel Ad vector that exploits the native canine CD40-CD40L pathway. These data support our use of our cCD40-targeted Ad vectors in novel canine models of immunotherapy.

Source: Molecular Therapy

DOI: 10.1016/j.ymthe.2006.08.152