BVs but not ODVs share their fate in BMDCs with OVA.

<p>Immature BMDCs were incubated with either BVs or ODVs together with OVA-FITC at 37°C for (A) 10 min, (B) 40 min or (C) 75 min or (D) with DQ-OVA for 3 h. After the fixation and permeabilization they were marked with specific antibodies. VP39 is shown in red and OVA-FITC or DQ-OVA in green.</p

ODVs reach late and acidic endosomes in BMDCs.

<p>BVs or ODVs were incubated with immature BMDCs at 37°C (A) for 1 h or (B) for 1:15 h and then fixed, permeabilized and stained with specific antibodies. (C) Polyhedra were incubated with immature BMDCs at 37°C for 1 or 48 h, and treated in the same way. All the analyses were carried out by confocal microscopy. LysoTracker DND-90 is shown in red, VP39 in green and LAMP2 in blue.</p

Purified ODVs characterization.

<p>(A) Schematic representation of the structure of both baculovirus phenotypes. The main proteins of the capsid and envelope are indicated with arrows. (B) Comparative Western blot analysis between BVs and ODVs. The proteins were detected by specific monoclonal antibodies α-P74, α-GP64 and α-VP39. (C) Transmission electron microscopy of ODVs. Purified ODVs were adsorbed to a copper grid, stained with 2% uranyl acetate and observed at a magnification of x 85,000. The displayed virions are representative of all the ODVs examined. Bar = 100 nm.</p

ODVs do not induce BMDCs maturation.

<p>BMDCs were incubated for 18 h with RPMI medium alone, BVs, ODVs or polyhedra. Cells were labeled with anti-CD11c, MHCII and CD86, and analyzed on a FACSCalibur. The results are representative of two independent experiments and are expressed (A) in histogram and (B) in bars graph as the geometric mean of the fluorescence intensity (MFI) for each indicated maturation marker in total CD11c+ cells (conventional DCs). (C) IL-6 levels were determined in BMDC supernatants by ELISA.</p

ODVs do not promote antiviral activity in mice.

<p>ODVs do not promote antiviral activity in mice.</p

ODVs do not stimulate innate immune response in mice.

<p>C57BL/6 mice were i.v. injected with PBS, BVs, ODVs at two different concentrations or with polyhedra. Subsequently, sera were collected at 6 h. IL-6, IL-12 and IFN-γ levels were titrated by ELISA. The results are representative of two independent experiments. *, p<0.05.</p

ODVs do not elicit cellular response against a coadministered antigen.

<p>C57BL/6 mice were immunized by a single i.v. injection of PBS or OVA (1 mg) combined with BVs, ODVs or mock purification of ODVs. Seven days later, immunized mice received an i.v. injection of a mixture (1:1) of OVA_256-264peptide-loaded CFSE^high and unloaded CFSE^low splenocytes as target cells. (A) A representative histogram of remaining CFSE^high and CFSE^low cells in control, ODVs+OVA and BVs+OVA immunized mice 20 h after injection of target cells is shown. (B) Percentage of specific <i>in vivo</i> killing. ***, p<0.001. The results are expressed as mean +/− SEM (n = 4).</p

Anti-OVA CTL response in mice injected with BV-OVA is long-lasting and not affected by repeated immunization with the same vector.

<p>(A, B) C57BL/6 mice were immunized by a single i.v. injection of 5×10⁷ PFU BV-WT, BV-OVA or PBS. One hundred and ten days later, immunized mice received an i.v. injection of a mixture (1∶1) of OVA_256-264 peptide-loaded CFSE^high and unloaded CFSE^low splenocytes. Twenty hours later, spleen cells were recovered to determine (A) the percentage of specific <i>in vivo</i> killing by flow cytometry and (B) IFN-γ content by ELISA in supernatants of spleen cells from immunized mice, cultured for 48 hours in the presence of OVA protein. (C) C57BL/6 mice were immunized by a single i.v. injection of 5×10⁷ PFU BV-WT or PBS. Seven and fourteen days later, spleen cells were recovered and cultured with BV-WT (MOI 5) for 48 hours. The IFN-γ content in supernatants was determined by ELISA (D) C57BL/6 mice were immunized by a single i.v. injection of 5×10⁷ PFU BV-WT, BV-OVA or PBS. Fifteen days later, mice received a second i.v. injection of 5×10⁷ PFU BV-WT, BV-OVA or PBS, combined as shown in the figure. Seven days later, immunized mice received an i.v. injection of a mixture (1∶1) of OVA_256-264 peptide-loaded CFSE^high and unloaded CFSE^low splenocytes. Twenty hours later, spleen cells were recovered and the percentage of specific <i>in vivo</i> killing was determined by flow cytometry. *, p<0.05; ***, p<0.001. Results are representative of at least two independent experiments and are expressed as mean +/− SEM (n = 5).</p

Anti-OVA CTL response in mice injected with BV-OVA.

<p>C57BL/6 mice were immunized by a single i.v. injection of 5×10⁷ PFU BV-WT, BV-OVA, BV-OVAsur, BV-WT + OVA (30 ng or 1 mg), OVA alone (1 mg) or PBS. Seven days later, immunized mice received an i.v. injection of a mixture (1∶1) of OVA_256-264 peptide-loaded CFSE^high and unloaded CFSE^low splenocytes as target cells. (A) A representative histogram of remaining CFSE^high and CFSE^low cells in control and BV-OVA immunized mice 20 hours after injection of target cells is shown. (B) Percentage of specific <i>in vivo</i> killing of one representative experiment. (C) IFN-γ content in supernatants of spleen cells from immunized mice determined by ELISA. Spleen cells were recovered and cultured for 48 hours in the presence of OVA protein or BV-WT. As control, spleen cells without stimulus were also cultured. *, p<0.05; **, p<0.01. (D, E) IFN-γ intracellular staining on splenocytes from immunized mice with 30 ng OVA+BV-WT, BV-WT, BV-OVA, OVA alone or PBS, as indicated above. Spleen cells were recovered and cultured for 12 hours in the presence of OVA protein or OVA_256-264, and incubated in the presence of brefeldin A for 6 additional hours. Then, cells were labeled for CD4 or CD8 markers and intracellular IFN-γ. (D) A representative dot plot is shown. (E) Percentage of IFN-γ+ CD4 or CD8 T cells. **, p<0.01; ***, p<0.001. (F) Comparison of specific <i>in vivo</i> killing between mice immunized with BV-OVA vs BV-OVAsur. ***, p<0.001. (G) Comparison of the frequency of IFN-γ-producing CD8 T cells in splenocytes from BV-OVA vs BV-OVAsur immunized mice. **, p<0.01. Results are representative of at least two independent experiments and are expressed as mean +/− SEM (n = 4).</p

Characterization of BV-OVA.

<p>(A) Scheme of Baculovirus (BV) vectors. The Ovalbumin (OVA) (653–1222) and enhanced Green Fluorescent Protein (GFP) (1-717) sequences were cloned 5′ of the VP39 sequence. A linker sequence (GGGGS) was added in the N-terminus of VP39 to provide distance and flexibility for the N-terminal fusion proteins to fold correctly. Production of the fusion protein is driven by the strong polyhedrin promoter. (B) Western blot analysis of purified virions. The virions were purified by ultracentrifugation for 30 min at 131,000× g onto a 25% sucrose cushion. Fusion proteins OVAVP39 (MW 64 KDa), GFPVP39 (MW 67 KDa) and OVAGP64 (MW 89 KDa) were detected with anti-OVA or anti-GFP specific polyclonal antibodies. (C) Immunoelectron microscopy of BV-OVA. Recombinant BV-OVA purified by ultracentrifugation through a 25% sucrose cushion were treated with 1% of Triton 100. Virions capsids were adsorbed to Formvar-coated grids, and the presence and localization of OVAVP39 fusion protein on the capside of the virion was detected with an anti-OVA polyclonal antibody and an anti rabbit IgG-gold conjugate. The figure is representative of all fields examined. Bar = 100 nm. (D) Uptake of BV by Bone Marrow-derived Dendritic Cells (BMDCs). The BMDCs were incubated with BV-GFP for 2 hours at 37°C or 4°C. Then, cells were washed, stained with anti-CD11c and analyzed on a FACSCanto II. In all cases, a minimum of 2×10⁵ events was acquired. Results are representative of two independent experiments and are expressed as the geometric mean of the fluorescence intensity (MFI) of the FL1 channel in total DCs or the percentage of DCs which are positive for FL1 channel. (E) <i>In vitro</i> OVA presentation by DCs infected with BV-OVA. Spleen CD11c+ cells (2×10⁵) were incubated <i>in vitro</i> with BV-OVA (•) or BV-GFP (○), and cultured overnight with 10⁵ B3Z cells/well. Then, cells were washed and the presentation of the OVA_257-264 epitope to B3Z cells was monitored by the activity of β–galactosidase with a colorimetric assay. Results are representative of two independent experiments and are expressed as mean +/− SEM of the optical density at 595 nm (OD₅₉₅, n = 3).</p