579. Generation of Potent Cellular Immune Responses Induced by Intradermal Delivery of Plasmid DNA Coexpressing Flt3 Ligand and GM-CSF

Abstract

The protective efficacy of DNA vaccine has been demonstrated in murine models against infectious diseases or tumor growth. Recently, Fms-like tyrosine kinase 3-ligand (Flt3L) has been identified as an important cytokine for the generation of professional antigen-presenting cells (APCs), particularly dendritic cells (DCs). Administration of Flt3L protein or recombinant DNA encoding Flt3L into mice results in the expansion of the DC population in vivo. Granulocyte-macrophage colony-stimulating factor (GM-CSF) is also critical for DC survival and differentiation in vitro. Previous studies have shown that codelivery of Flt3L-encoding and GM-CSF-encoding plasmids with antigen results in increase in DC numbers in comparison with codelivery of either plasmid alone. In this study, we constructed a bicistronic plasmid vector, Flt3L-IRES-GM-CSF plasmid, coexpressing murine Flt3L lacking intracellular domain and GM-CSF by using an internal ribosome entry site (IRES). Expressions of GM-CSF and Flt3L could be detected in COS-7 cells transfected with the plasmid. The immune stimulatory activity of plasmid vector coexpressing Flt3L and GM-CSF (coexpressed DNA vaccine) in comparison with codelivery of Flt3L and GM-CSF plasmids (codelivered DNA vaccine) was studied in mice. We first immunized mice by intradermal injection of various plasmid vectors, including 40 μg Flt3L-IRES-GM-CSF plasmid, 40 μg Flt3L plasmid plus 40 μg GM-CSF plasmid, 40 μg Flt3L plasmid alone, 40 μg GM-CSF plasmid alone, or 40 μg control plasmid. Expressions of Flt3L and GM-CSF were not detected in the sera of mice receiving intradermal injection of the plasmids. Next, we showed the enhanced recruitment of DCs to the immunization site and proliferation of DCs and T cells in the spleen and lymph node. The populations of DC, CD4+, and CD8+ T cells were increased significantly by approximately 50% to 60% in mice receiving coexpressed DNA vaccine compared with those in mice receiving control plasmid. Compared to mice treated with codelivered DNA vaccine, there were 15% and 41% increases in the populations of DC and CD4+ T cells, respectively. The CD4+ but not CD4+CD25+ T cells were found to be the major group in the increased T-cell population in the spleen and lymph node. We will investigate the function of the DCs harvested from mice receiving coexpressed DNA plasmid by mixed lymphocyte reaction and antigen-specific T cell proliferation. In addition, we found that there was an increase, ranging from 20% to 40%, in the populations of MHC class II+, CD4+, and CD8+ T cells in the spleen of mice bearing syngeneic bladder tumor that received coexpressed DNA vaccine compared with those that received codelivered DNA vaccine or control plasmid. Immunization of mice with coexpressed DNA vaccine delayed bladder tumor growth. Taken together, regarding the application of Flt3L and GM-CSF, our results indicate that coexpressed DNA vaccine induces at least equal or higher immune stimulatory response compared with codelivered DNA vaccine, while only half the amount of DNA is needed. Currently, we are using the coexpressed DNA vaccine in combination with plasmid DNA encoding tumor epitopes or tumor-specific oncolytic virus for immune-gene therapy of cancer.