Fig. 5

Ad5 neutralizing antibodies limit GUCY2C responses in mice. BALB/c mice were naïve or preconditioned by immunizing 2 times with 10⁸ IFU control Ad5 to induce High Ad5 neutralizing antibody (NAb) titers (n = 10 mice/group). a, b two weeks after preconditioning, serum was collected and an in vitro assay quantifying inhibition of A549 cell infection by an Ad5-GFP reporter virus in the presence of serum titrations (a) was used to calculate Ad5 NAb titers (b). The Ad5 NAb titer was calculated as the dilution of serum that produced 50% inhibition of GFP reporter expression. c, d mice were then immunized with 10⁸ IFU of Ad5-mGUCY2C-S1 expressing mouse GUCY2C fused to the CD4⁺ T-helper cell epitope S1. GUCY2C-specific antibody (c) and CD8⁺ T-cell responses (d) were quantified two weeks later by ELISA and IFNγ-ELISpot, respectively. Bars in (c) indicate serum dilutions of 1/20, 1/40, 1/80, and 1/160 (left to right). NS = not significant, ** P < 0.01, **** P < 0.0001, T-test (b) or Two-way ANOVA (c and d)