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 3alpha (hCCL20/hMIP-3alpha), 
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.