Immune responses to Flu antigen after pretreatment with denileukin diftitox and vaccination with rV-Inf(A)NP34/TRICOM (+ rF-GM-CSF) in a foreign antigen system. C57BL/6 mice were subcutaneously vaccinated on day 0 with 5 × 107 pfu rV-Inf(A)NP34/TRICOM (admixed with 1 × 107 pfu rF-GM-CSF) and boosted on day 14 with 5 × 107 pfu rF-Inf(A)NP34/TRICOM (admixed with 1 × 107 pfu rF-GM-CSF) (vaccine alone). One group additionally received an intraperitoneal injection of 0.75 μg denileukin diftitox 1 day before the vaccine prime (vaccine + denileukin diftitox, day −1). Immune assays were performed on day 35, 3 weeks after the boost. Background-subtracted values are shown. (A) CD4+ T-cell proliferation to the indicated concentrations of inactivated Influenza A virus was measured by 3H-thymidine incorporation. Values are represented as mean proliferation (± SD) of triplicate wells. Statistical evaluation was performed by repeated-measures one-way ANOVA using GraphPad Prism. All values for the group pretreated with denileukin diftitox on day −1 relative to vaccine prime were statistically significant (P < .01) compared with vaccine alone. (B) CD4+ T cells were stimulated with 32.5 μg/mL inactivated Influenza A virus for 72 hours. Mean cytokine concentration in supernatants (± SD) was measured by Cytometric Bead Array analysis of triplicate samples. Statistical evaluation was performed by repeated-measures one-way ANOVA using GraphPad Prism. (C) CD8+ T cells were stimulated with the indicated concentrations of NP34 peptide for 48 hours. IFN-γ concentration in supernatants was measured by ELISA. (D) Mean serum levels of anti-Flu IgG (± SD) were measured by ELISA.
