FIG. 1.

hCCL20, mGM-CSF, hFlt3-L, and LCMV-NP are expressed from their respective rMVA vectors in vitro and in vivo. DF-1 cells were infected with MVA, MVA-NP, MVA-NP-hCCL20, MVA-NP-mGM-CSF, or MVA-NP-hFlt3-L and cells (A) or supernatants (B) were harvested at 24 hpi for analysis of LCMV-NP expression (A) or cytokine expression (B). (A) Infected cells were permeabilized and stained with a primary anti-LCMV-NP mouse monoclonal antibody (1:100), followed by incubation with a phycoerythrin-conjugated secondary anti-mouse IgG antibody (1:400), and were analyzed by flow cytometry. The negative controls are the MVA-infected cells, and the positive controls are the MVA-NP-infected cells. (B) Expression of hCCL20 and mGM-CSF in supernatants from cells infected with MVA, MVA-NP, MVA-NP-hCCL20, or MVA-NP-mGM-CSF was measured using cytokine-specific Quantikine ELISA kits. ND, not detected. (C and D) Mice were immunized i.p. with MVA-NP-mGM-CSF (C) or MVA-NP-hCCL20 (D), and sera were analyzed at 1, 6, and 24 h following immunization for mGM-CSF (C) or hCCL20 (D) expression using Quantikine ELISA kits. Data were compiled from at least two independent experiments; symbols represent values determined from individual mice. Neither mGM-CSF nor hCCL20 was detected in sera from mice immunized with control MVA or mock-immunized with PBS (not shown). (E) Supernatants from MVA-NP- or MVA-NP-hFlt3-L-infected cells were incubated with SDS buffer, resolved by SDS-polyacrylamide gel electrophoresis, and prepared for immunoblot analysis. hFlt3-L was detected using a biotinylated anti-Flt3-L antibody (0.2 μg/ml) followed by incubation with neutravidin-HRP conjugate (1:10,000). The positive control is a purified hFlt3-L, and the negative control is the supernatant from cells infected with MVA-NP.