LACK OF EVIDENCE OF PHENOTYPIC COMPLEMENTATION OF E1A E1B-DELETED ADENOVIRUS TYPE-5 UPON SUPERINFECTION BY WILD-TYPE VIRUS IN THE COTTON RAT

The safety of replication-defective viruses used as vectors is based o n the deletion of essential gene(s). Adenovirus vector safety relies o n the deletion of the E1A/E1B region. This region encodes the immediat e-early proteins that trans activate all other early regions, so DNA r eplication in these deletion mutants is dramatically reduced. We have previously shown that E1A deletion is efficient in vivo and significan tly reduces the dissemination of adenovirus in mice and cotton rats. H owever, the pattern of dissemination of E1A-deleted and wild-type viru ses showed that both could be localized in the same tissues, thus invo lving a theoretical risk of phenotypic complementation if a recipient of E1A-deleted adenovirus is infected after adenovirus-mediated gene t herapy by a wild-type adenovirus. In this report, we show that complem entation can be evidenced in vitro in Vero cells infected with E1A/E1B -defective adenovirus vectors expressing reporter genes (either beta-g alactosidase or luciferase), passaged three times until no infectious virus can be recovered by plating on 293 cells, and then infected with wild-type adenovirus 5. A mixed virus population was maintained at a stable state for at least 10 passages. In contrast, no evidence of com plementation was found in cotton rats inoculated intravenously or intr amuscularly with Ad-beta-gal-nls and Ad-luc and infected 24 h later in tranasally with wild-type adenovirus 5. No increase in the level of lu ciferase expression was found in these animals, compared with that in controls, nor was any viral population expressing beta-galactosidase o r luciferase isolated from various organs or any animal excretion or s ecretion.