Replication-competent adenovirus 11p vectors

The ADP gene, which is located from 29,491 to 29,772 nt in the Ad5 E3 region, was cloned into the Ad11pe1 shuttle vector (Ad5 genome accession no. {"type":"entrez-nucleotide","attrs":{"text":"M73260","term_id":"209842","term_text":"M73260"}}M73260, version {"type":"entrez-nucleotide","attrs":{"text":"M73260.1","term_id":"209842","term_text":"M73260.1"}}M73260.1). The gene encoding red fluorescence protein (RFP) was obtained from the pVisionRFP-N plasmid (https://www.biovision.com, Bio Vision Research Products, CA, USA). An RCAd11p vector carrying ADP or RFP under the control of the CMV promoter and the SV40 polyA sequence was constructed by inserting the gene into the Ad11p E1 shuttle vector at 451 nt upstream of the E1A region [11]. A linear shuttle vector carrying the ADP/RFP-expressing cassette as well as the full-length Ad11p genome was obtained by homologous recombination in BJ5183 Escherichia coli cells. The recombinant plasmid of the ADP/RFP-Ad11p genome was propagated in DH5α cells, and purified pAd11pADP/RFP was digested with the NotI restriction enzyme and transfected into A549/293 cell lines using Lipofectamine^TM 2000 (Invitrogen) for harvesting of infectious particles. To evaluate the infectivity of the vectors, a monolayer of A549 cells was infected at a tenfold dilution, and the cytopathic effect (CPE) was monitored daily. TCID₅₀ was calculated 3 and 5 days post-infection (p.i.).

To elucidate functional differences with ADP insertion at the E1 or E3 region of the adenovirus 11p genome, we also introduced a DNA fragment of CMV-ADP-SV40 into the E3 shuttle vector at 28356-29482 nt of the Ad11p genome [11]. The two arms of the shuttle vector were released by NaeI digestion and then cloned into the Ad11p genome through homologous recombination in BJ5183 cells [11]. pAd11pE3ADP can release the Ad11p genome expressing ADP from the E3 region by NotI digestion. The linearized Ad11p genome carrying the ADP cassette was transfected into A549 cells using Lipofectamine 2000 (Invitrogen). CPE was detectable after 4–7 days p.i., and the transfected cells were harvested and kept at −80°C as viral stocks. The ADP expression cassette in RCAd11pe3ADP was obtained by restriction enzyme digestion with KpnI and HindIII.