Ad5<i>Py</i>CSP.H(43 m-43) induces T cells responses similar to Ad5<i>Py</i>CSP in naïve mice and is not inhibited by Ad5 NAb <i>in vivo</i>.

<p>Naïve BALB/c mice or mice pre-immunized with two injections (1×10¹⁰ pu each) of Ad5 vector or an Ad5 vector containing the Ad35 fiber, were immunized with Ad5<i>Py</i>CSP, or hexon-modified vector Ad5PyCSP.H(43 m-43) (n = 6 mice/group). <i>Py</i>CSP-specific T cell responses were assessed by Intracellular cytokine staining (ICS) and IFNγ ELISpot four weeks after immunization. Targets were MHC-matched A20.2J cells pulsed with synthetic peptides representing the immunodominant CD8⁺ T cell epitope (PyCSP280–288) or CD4⁺ T cell epitope with nested subdominant CD8⁺ T cell epitope (<i>Py</i>CSP57–70) from <i>Py</i>CSP or a defined CD8⁺ T cell epitope for the influenza virus hemagglutinin antigen (HA 332–340). (A) Schematic of the experiment. (B) ICS analysis of CD8⁺ IFNγ⁺ T cell responses from immunized mice assayed individually. Error bars indicate the standard error of the mean, n = 6. (C) IFNγ ELISpot responses from pooled splenocytes (250,000 cells/well). Error bars indicate the standard deviation of the mean from quadruplicate samples. (D) ELISA against recombinant <i>Py</i>CSP protein capture antigen. Box-and-whisker plot with values for individual mice shown (n = 6). The mean is indicated by the red line. * indicates groups where titers from all mice were <8.