Immunogenicity of Ad35K⁺⁺. (A) Analysis of Ad knob-specific antibodies in serum from patients with cancer. Recombinant Ad5 hexon, Ad5 knob, Ad35K, and Ad35K⁺⁺ as well as denaturated Ad5 particles were separated by polyacrylamide gel electrophoresis and stained with Coomassie blue (left). The proteins were plotted to nitrocellulose filter, which were incubated with human serum and subsequently with mouse anti–human immunoglobulin G–HRP conjugates (right). Human serum reacted with multimeric Ad5 hexon, trimeric Ad5 knobs, and virion pentons (∼ 70- and 80-kDa bands) but not with Ad35K or Ad35K⁺⁺. Shown is a representative sample. (B) Analysis of knob antibodies in serum from mice that were injected subcutaneously with Ad35K⁺⁺ (Ad35K⁺⁺ vacc) or injected intravenously with Ad35K⁺⁺ (Ad35K⁺⁺ i.v.). Shown are representative samples. Photographs were taken with a digital Panasonic Lumix TZ-3 camera and processed in Adobe Photoshop. (C) Effect of anti-Ad35K⁺⁺ serum on rituximab-mediated CDC. Raji cells were preincubated with mouse serum at a final dilution of 1:20 for 10 minutes at room temperature. Control serum was from normal mice. Test serum was from vaccinated mice that received subcutaneous Ad35K⁺⁺ vaccinations. Then, 25 ng/mL Ad35K or Ad35K⁺⁺ was added for 10 hours, followed by incubation with 15 μg/mL rituximab. CDC was induced 30 minutes later by adding NHS. Viable cells were counted 3 hours later. Shown are the mean values and SDs; n = 5.