C) Anti-adenovirus serotype 5 neutralizing antibody (serum). Pre-existing immunity against recombinant adenovirus 5 was assessed by determining the amount of neutralizing antibody present in serum according to established methods [30] . In brief, serum was incubated at 56uC for 30 minutes and then diluted in DMEM in twofold increments starting from a 1:20 dilution. Each dilution (100 ml) was mixed with an aliquot of a standard stock of adenovirus type 5 expressing E. coli beta-galactosidase (10 6 pfu), incubated for 1 hour at 37uC, and applied to HeLa cells in 96-well plates (2610 4 cells/well). One hundred microliters of DMEM supplemented with 20% FBS was then added to each well. Cells were incubated at 37uC for 24 hours. Neutralizing antibody titers were calculated as the highest dilution at which 50% of the cells stained blue by visual inspection.