17. Human Adenovirus D17 Has Tropism for Endothelium Cells and Can Use Both hCAR and CD46 as Receptors

Abstract

Human adenovirus type 5-derived vectors (HAdV5) from species C were broadly explored for gene therapeutic approaches and vaccination. However, disadvantages associated with this vector type are the strong liver tropism in vivo in mice, preexisting immunity and induction of robust immune responses. There is growing interest in exploring other adenovirus types of which >69 were identified. Here we constructed a new first generation adenovirus labelled with green fluorescent protein marker based on human adenovirus D17 which was first isolated from conjunctival scrapings in 1955 and aimed at characterizing the vector in vitro and in vivo. We applied a new homologous recombineering technology to construct GFP labelled early E1 gene deleted HAdV17 and HAdV5, rescued viruses in complementary stable cell lines, and then screened a panel of different cell lines by FACS analyses and quantitative PCR. Competition assays based on soluble recombinant fiber knob blocking reagents (5knob, 17knob, JO4, Augmab) were used to characterize the receptor interaction in vitro. In vivo biodistribution analyses were performed after intravenous injection of recombinant viruses into normal and CD46 transgenic mice. We observed that HAdV17 has tropism for endothelium cells which are normally refractory to HAdV5 infection. This finding was further verified using primary human umbilical vein endothelial cells (HUVEC). Moreover, after performing competition assays we found that HAdV17 can utilize both CD46 (a membrane cofactor protein which is expressed on all nucleated cells) and CAR (coxsackievirus and adenovirus receptor) as cell attachment receptors. The endothelium tropism was CD46-dependent and could be blocked by the CD46 blocking reagent Ad35K++/Augmab. In vivo biodistribution studies showed significantly increased vector genome copies (VCN) in various organs of human CD46 transgenic mice compared to normal mice indicating involvement of CD46 as a receptor. Immunohistological analyses using cell-specific marker are ongoing. Neutralizing antibody assays revealed that there was less seroprevalence with HAdV17 compared to HAdV5. In total, we believe that HAdV17-based vectors, which can use both hCAR and CD46 as receptors, hold great promise for gene therapy in endothelial disease. The understanding of the molecular interaction between virus and host will be beneficial for vaccination and drug development.