An alternative to the adenovirus inverted terminal repeat sequence increases the viral genome replication rate and provides a selective advantage in vitro

During the development of human adenovirus 35-derived replication-incompetent (rAd35) vaccine vectors for prevention of infectious diseases, we detected mutations in the terminal 8 nt of the inverted terminal repeats (ITRs) of rAd35. The switch from the plasmid-encoded sequence 5′-CATCATCA-3′ to the alternative sequence 5′-CTATCTAT-3′ in the ITRs was found to be a general in vitro propagation phenomenon, as shown for several vectors carrying different transgenes or being derived from different adenovirus serotypes. In each tested case, the plasmid-encoded ITR sequence changed to exactly the same alternative ITR sequence, 5′-CTATCTAT-3′. The outgrowth of this alternative ITR version should result from a growth advantage conferred by the alternative ITR sequence. Indeed, replication kinetics studies of rAd35 harbouring either the original or alternative ITR sequence confirmed an increase in replication speed for rAd35 vectors with the alternative ITR sequence. These findings can be applied to generate recombinant adenoviral vectors harbouring the alternative ITR sequence, which will facilitate the generation of genetically homogeneous seed virus batches. Moreover, vector production may be accelerated by taking advantage of the observed improved replication kinetics associated with the alternative ITR sequence.