polyUs21 induces potent T- and B-cell responses in vivo. C57BL/6 mice were immunized intravenously, and 7 days later T- and B-cell responses were analyzed. (A) Splenocytes were isolated and cultured in vitro for 72 hours in the presence or absence of OVA. IFN-γ levels in the culture supernatants were determined by bead-based ELISA assay. Data contain results from at least 2 independent experiments. (B) IFN-γ production by splenocytes cultured for 72 hours in the presence or absence of OVA was assessed by flow cytometry gating on CD4⁺ CD3⁺ T cells. One representative of 3 independent experiments is shown. (C) In vivo CTL killing assays were performed at day 7 after immunization. Pooled data of 2 independent experiments are shown. (D) The indicated doses of R848 or polyUs21 in form of complexes with DOTAP were administered intravenously in combination with egg white preparation (n = 3). In vivo CTL assay was performed 7 days after immunization. (E) OVA-specific antibody levels of IgM, IgG1, and IgG2c isotypes in serial dilutions of serum were determined by ELISA in mice treated with adjuvant (solid lines) or in OVA + DOTAP–treated control mice (dotted lines). Serum dilution curves represent the mean ± SEM of at least 3 independent experiments.