AbstractFunctional avidity of T cells is a critical determinant for clearing viral infection and eliminating tumor. Understanding how functional avidity is maintained in T cells is imperative for immunotherapy. However, studies systematically characterize T cell with high functional avidity induced in vivo are still lacking. Previously, we and others found vaccinia vectored vaccine (VACV) induced antigen-specific CD8⁺ T cells with relatively high functional avidity to those from DNA vaccine. Herein, we used functional, immune phenotyping and transcriptomic studies to define the immune signature of these CD8⁺ T cells with high functional avidity. Antigen-specific CD8⁺ T cells induced by VACV executed superior in vivo killing activity and displayed a distinct transcriptional profile, whereas no significantly differences were found in composition of memory sub-populations and cytokine poly-functionality. Transcriptional analyses revealed unique features of VACV induced CD8⁺ T cells in several biological processes, including transport, cell cycle, cell communication and metabolic processes. In summary, we characterize CD8⁺ T cells of high functional avidity induced in vivo by VACV, which not only improves our understanding of adaptive T cell immunity in VACV vaccination, but also provides clues to modulate functional avidity of CD8⁺ T cells for T cell based immunotherapy.