Complete replication-competent adenovirus 11p vectors with E1 or E3 insertions show improved heat stability

•Replicating adenovirus 11p GFP vectors at the E1 or E3 region were generated.•RCAd11pE3 and RCAd11pE1 vectors manifested significantly improved heat stability.•RCAd11pE3 and RCAd11pE1 showed more full viral particles than Ad11pwt after heating.•We demonstrated that both genome size and the insertion site affect virion stability.

Conventional adenovirus vectors harboring E1 or E3 deletions followed by the insertion of an exogenous gene show considerably reduced virion stability. Here, we report strategies to generate complete replication-competent Ad11p(RCAd11p) vectors that overcome the above disadvantage. A GFP cassette was successfully introduced either upstream of E1A or in the E3A region. The resulting vectors showed high expression levels of the hexon and E1genes and also strongly induced the cytopathic effect in targeted cells. When harboring oversized genomes, the RCAd11pE1 and RCAd11pE3 vectors showed significantly improved heat stability in comparison to Ad11pwt;of the three, RCAd11pE3 was the most tolerant to heat treatment. Electron microscopy showed that RCAd11pE3, RCAd11pE1, Ad11pwt, and Ad11pE1 Delmanifested dominant, moderate, minimum, or no full virus particles after heat treatment at 47 °C for 5 h. Our results demonstrated that both genome size and the insertion site in the viral genome affect virion stability.