While modified vaccinia virus Ankara (MVA) is currently in clinical development as a safe vaccine against smallpox and heterologous infectious diseases, its immunogenicity is likely limited due to the inability of the virus to replicate productively in mammalian hosts. In light of recent data demonstrating that vaccinia viruses, including MVA, preferentially infect antigen-presenting cells (APCs) that play crucial roles in generating antiviral immunity, we hypothesized that expression of specific cytokines and chemokines that mediate APC recruitment and activation from recombinant MVA (rMVA) vectors would enhance the immunogenicity of these vectors. To test this hypothesis, we generated rMVAs that express murine granulocyte-macrophage colony-stimulating factor (mGM-CSF), human CCL20/human macrophage inflammatory protein 3α (hCCL20/hMIP-3α), or human fms-like tyrosine kinase 3 ligand (hFlt3-L), factors predicted to increase levels of dendritic cells (DCs), to recruit DCs to sites of immunization, or to promote maturation of DCs in vivo, respectively. These rMVAs also coexpress the well-characterized, immunodominant lymphocytic choriomeningitis virus nucleoprotein (NP) antigen that enabled sensitive and quantitative assessment of antigen-specific CD8+ T-cell responses following immunization of BALB/c mice. Our results demonstrate that immunization of mice with rMVAs expressing mGM-CSF or hCCL20, but not hFlt3-L, results in two- to fourfold increases of cellular immune responses directed against vector-encoded antigens and 6- to 17-fold enhancements of MVA-specific antibody titers, compared to those responses elicited by nonadjuvanted rMVA. Of note, cytokine augmentation of cellular immune responses occurs when rMVAs are given as primary immunizations but not when they are used as booster immunizations, suggesting that these APC-modulating proteins, when used as poxvirus-encoded adjuvants, are more effective at stimulating naïve T-cell responses than in promoting recall of preexisting memory T-cell responses. Our results demonstrate that a strategy to express specific genetic adjuvants from rMVA vectors can be successfully applied to enhance the immunogenicity of MVA-based vaccines. Copyright © 2006, American Society for Microbiology. All Rights Reserved.