Chromosomal Integration Pattern of a Helper-Dependent Minimal Adenovirus Vector with a Selectable Marker Inserted into a 27.4-Kilobase Genomic Stuffer

Helper-dependent minimal adenovirus vectors are promising tools for gene transfer and therapy because of their high capacity and the absence of immunostimulatory or cytotoxic viral genes. In order to characterize this new vector system with respect to its integrative properties, the integration pattern of a minimal adenovirus vector with a neo(r) gene inserted centrally into a noncoding 27.4-kb genomic stuffer element derived from the human X chromosome after infection of a sex chromosome aneuploid (X0) human glioblastoma cell line was studied. Our results indicate that even extensive homologies and abundant chromosomal repeat elements present in the vector did not lead to integration of the vector via homologous or homology-mediated mechanisms. Instead, integration occurred primarily by insertion of a monomer with no or little loss of sequences at the vector ends, apparently at random sites, which is very similar to E1 deletion adenovirus vectors. It is therefore unlikely that the incorporation of stuffer elements derived from human genomic DNA, which were shown to allow long-term transgene expression in vivo in a number of studies, leads to an enhanced risk of insertional mutagenesis. Furthermore, our findings indicate that the potential of minimal adenovirus vectors as tools for targeted insertion and gene targeting is limited despite the possibility of incorporating long stretches of homologous sequences. However, we found an enhanced efficiency of stable neo(r) transduction of the minimal adenovirus vector compared to an E1 deletion adenovirus vector, possibly caused by the absence of potential growth-inhibitory viral genes. Complete integration of the vector and tolerance of the integrated vector sequences by the cell might indicate a potential use of these vectors as tools for stable transfer of (large) genes

Vaccine vectors based on Adenovirus 19a/64 exhibit broad cellular tropism and potently restimulate HCMV-specific T cell responses ex vivo

Abstract Human Cytomegalovirus (HCMV) remains a major health burden and the development of a vaccine is a global priority. We developed new viral vectors delivering the T cell immunogens IE-1 and pp65 based on Adenovirus 19a/64 (Ad19a/64), a member of subgroup D. In this ex vivo study, the novel vectors were compared side by side to Ad5 or modified Vaccinia Ankara (MVA) strains expressing the same transgenes. We found that unlike Ad5, Ad19a/64 vectors readily transduce a broad panel of immune cells, including monocytes, T cells, NK cells and monocyte-derived dendritic cells (moDCs). Both Ad19a/64- and MVA-transduced moDCs efficiently restimulated IE-1 or pp65-specific T cells but MVA induced a higher amount of cytotoxicity in this cell type. Ad5 and Ad19 induced upregulation of CD86 and HLA-DR in moDCs whereas expression of CD80 and CD83 was largely unaltered. By contrast, MVA transduction led to downregulation of all markers. Taken together, our data demonstrate that Ad19a/64 is a promising vector for the delivery of HCMV immunogens since it transduces dendritic cells with an efficiency that is comparable to MVA, but cytotoxicity and interference with dendritic cell maturation are less pronounced