Caux. fourfold increases of cellular immune responses directed against vector-encoded antigens and 6- to 17-fold enhancements of MVA-specific ALK inhibitor 1 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 na?ve T-cell responses than in promoting recall of preexisting memory T-cell responses. Our results demonstrate Mouse monoclonal to LPL that a strategy to express specific genetic adjuvants from rMVA vectors can be successfully applied to enhance the immunogenicity of MVA-based vaccines. The majority of successful vaccines have been derived from either inactivated organisms or live-attenuated organisms (69). Modified vaccinia virus Ankara (MVA) is an example of a live-attenuated vaccine that is currently in phase I clinical trials as a smallpox vaccine and in development as a vaccine vector for human immunodeficiency virus, malaria, and tuberculosis (2, 5, 23, 50, 53, ALK inhibitor 1 54, 59, 62, 86, 94). MVA was originally derived through extensive serial passaging ( 500 passages) of vaccinia virus Ankara in chicken embryo fibroblasts, which resulted in multiple deletions and mutations within the MVA genome (3, 49, 56, 81). As a result, MVA exhibits a severely restricted host range phenotype that includes an inability to replicate productively in primary human cells (8, 11, 20, 97). The safety of MVA has been demonstrated through administration of this virus to 120,000 individuals during the smallpox eradication campaign (48, 81, 95) and, more recently, to immunocompromised hosts including immunosuppressed macaques and immune-deficient mice (82, 98). MVA has been shown to abortively infect professional antigen-presenting cells (APCs), including dendritic cells (DCs), B cells, and macrophages (14; unpublished data), cells that play central roles in eliciting antiviral immune responses by mediating effective direct and cross-presentation of microbial antigens to na?ve CD4+ and CD8+ ALK inhibitor 1 T cells in the secondary lymphoid organs to initiate adaptive antiviral immune responses (4, 12, 16, 27, 31, 45, 51, 70). However, the immunogenicity of MVA is likely limited, despite its tropism for APCs, because of an inability to replicate in mammalian hosts that restricts viral gene (antigen) expression to cells infected at the site of immunization. We therefore proposed to enhance the immunogenicity of MVA vectors by generating recombinant viruses that express cytokines or chemokines that have known activities to increase the frequency and/or activation state of APCs, including granulocyte-macrophage colony-stimulating factor (GM-CSF), macrophage inflammatory protein 3 (MIP-3/CCL20), and fms-like tyrosine kinase 3 ligand (Flt3-L). GM-CSF acts on many immune cells during their early differentiation and ALK inhibitor 1 plays a major role in the development of immature macrophages from their hematopoeitic precursors (7). Administration of GM-CSF or induction of GM-CSF expression has been reported to induce the differentiation of ALK inhibitor 1 macrophages and DCs (43, 89). Other studies have demonstrated that GM-CSF, in combination with other cytokines, is capable of driving ex vivo differentiation of DCs from human peripheral blood monocytes (30, 40, 68, 91), whereas in vivo studies have shown that administration of recombinant GM-CSF increases the numbers of myeloid DCs in the spleen, bone marrow, and lymph nodes of na?ve mice (18, 72). As a result, GM-CSF has been codelivered as a recombinant protein, or as a gene product encoded by plasmid DNA or poxvirus (avipox and vaccinia virus) vectors, as an adjuvant to enhance the immune responses elicited by vaccines (6, 38, 39, 74, 77, 88, 96). CCL20 is a chemokine that recruits immature APCs expressing its cognate receptor CCR6 to peripheral sites where they may encounter microbes or infected cells (12, 13, 19, 35). While CCL20 has not been widely studied as a vaccine adjuvant (25), we sought to explore the notion that expressing CCL20 from recombinant MVA (rMVA) would recruit APCs to the site of immunization and result in enhanced antigen presentation and consequent increased vector immunogenicity. Flt3-L acts to generate DC precursors by modulating hematopoeitic cell differentiation in the bone marrow (17, 36, 37). Several published studies have.