Fig. 4.

Intranasal immunization with PA-Q11 generated functional PA-specific CD8+ T cells. PA-specific CD8+ T cells in DLNs of mice immunized by the indicated materials (A), quantified by PA tetramer staining and flow cytometry. DLNs were collected 10 d after primary vaccination. Antigen-specific IFN-γ producing cells were elevated in harvested splenocytes from PAQ11-immunized mice (B, C57BL/6 mice were immunized and boosted intranasally with PAQ11 or Q11. On day 10 after boost, splenocytes were harvested and quantified with ELISPOT). In (C), targeted killing of PA+ cells was measured in vaccinated mice. Target cells were pulsed with PA peptide or irrelevant peptide control and injected i.v. into PAQ11 I.N., Q11 I.N. vaccinated or naive mice. After 16 h, the mice were sacrificed and specific lysis of PA+ cells was analyzed. In (D), reduced viral loads were observed in the lung after PAQ11 I.N. vaccination, based on qRT-PCR quantification of viral M1 mRNA of mice sublethally infected with PR8 influenza virus (data were normalized to β-actin and expressed as the fold change in relative mRNA expression over Q11 I.N. control). Each data point indicates 1 mouse, with 6 mice per group from at least two independent experiments. Means ± SD indicated, ****P < 0.0001, ns: not significant by two-tailed t-test. Dose for A: one i.n. administration of 50 μL of 2 mM peptide at day 0; Dose for B, C, D: i.n. administration of 50 μL of 2 mM peptide at day 0 and day 28.