ELISpot measurements of IFN-γ expression in spleen cells of naïve or HAd5-primed mice immunized with HAd-HA-NP.

<p>Naïve or HAd5-primed mice were immunized as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033428#s4" target="_blank">Materials and Methods</a>. At four weeks after final immunization, animals were euthanized, and the spleens were collected. Single cell suspensions were prepared by passage through screens, and 1×10⁶ cells were cultured in the presence of HA-518 or NP-147 peptide on anti-interferon-γ antibody-coated 96-well filter plates and developed according to an ELISpot protocol. Splenocytes cultured in presence of phorbol myristate acetate (PMA) and ionomycin served as positive controls in each group. The ELISpot plates were read using a Bioreader 5000 (<i>BIOSYS</i>, Miami, FL). (A) Number of IFN-γ-secreting NP-147-specific CD8 T cells in intranasally (i.n.) primed groups. (B) Number of IFN-γ-secreting NP-147-specific CD8 T cells in intramuscularly (i.m.) primed groups. (C) Number of IFN-γ-secreting HA-518-specific CD8 T cells in i.n. primed groups. (D) Number of IFN-γ-secreting HA-518-specific CD8 T cells in i.m. primed groups. The error bars represent Mean ± SD from five animals/group: *, <i>P</i>≤0.05; **, <i>P</i>≤0.01; ***, <i>P</i>≤0.005; †, <i>P</i>≤0.001; ‡, <i>P</i>≤0.0001.