Figure 3.

CAP1 immunogenicity and antigen-specific CD8 frequency and phenotype in CB6 mice

(A) In vivo immunogenicity of Ad5-CAP1 vector in CB6 mice. Mice were injected intramuscularly with 5 × 10⁸ vp of Ad5-CAP1 at day 0. At 28 and 100 days after, distinct groups of vaccinated mice were sacrificed for spleen removal. T cell responses were measured by IFN-γ ELISpot on splenocytes. Responses against individual CAP1 antigens, a peptide pool covering the major Ad5 capsid protein (Hexon) and HA tag are shown; peptide diluent DMSO and concanavalin A were used as negative and positive controls, respectively. Bars show mean ± SEM, n = 12. (B) Kinetics of the frequency of the major CAP1-specific CD8 T cells (OVA SIINFEKL; MCMV M45 and M139, >200 IFNγ SFCs/million splenocytes) in blood, as detected by fluorescent pentamers. Blood was collected at days 21, 50, and 100 postimmunization, pooled, and processed for staining and FACS analysis. Blood from naive, unvaccinated age-matched control mice was collected as control for pentamer background. (C) Phenotype of Ad5-CAP1 vaccine-induced T cells analyzed by flow cytometry. Antigen-specific CD8⁺ T cells in the blood were identified by pentamer staining and analyzed for effector (E), central memory (CM), and effector memory (EM) phenotype, based on the expression of memory markers CD62L and CD127.