VLPs were prepared by expressing 1) VP40, 2) VP40 and GP and 3) VP40, VP24, VP35, VP30, NP and GP (cVLPs). A, B) VLPs were negatively stained and observed by electron microscopy. Shown in A) is an example of VLP filovirus morphology. A representative VLP picture shown in B) demonstrates the pleimorphic morphology of all the VLP preparations. All line bars represent a length of 500nm. C) Western blot analyses of purified VLP preparations using antibodies against EBOV proteins GP, NP, VP40, VP35 and VP24. D) Silver stains were performed on SDS-PAGE-separated purified VLPs. The first lane, marked control, shows the contents of mock VLPs. In the second lane is an example of the proteins found in cVLPs. Each of the next lanes shows the contents of cVLPs produced with exclusion of a single EBOV protein (protein excluded indicated above lane). Labeled arrows point to the indicated EBOV proteins. E) Shows silver stain of mock, VP40 and VP40+GP VLPs.

A) DCs treated with TNFα (50ng/mL), LPS (10ng/mL) and VP40+GP VLPs (10μg/mL) for 10, 30, 60 and 120 minutes were harvested and total cell lysates (∼10⁵ cellular equivalents/ lane) tested for the presence of IκBα and the loading control tubulin by western blotting. B, C) DCs were pretreated for 15 minutes with SN50 NF-κB inhibitor and control peptide and 1 hour with IKK2 inhibitor. Then DCs were treated with VLPs for 12−16 hours and spent media tested for the presence of B) IL-6 and C) lactate dehydrogenase. In C) 100% represents the amount of lactate dehydrogenase released into the media from DCs treated with 1% Triton X-100.

A) Western blot analyses of purified VLP preparations using antibody against EBOV protein VP40. B) ELISA, performed on equivalent amounts of VP40, VP40+GP, VP40+GPΔmuc VLPs using an EBOV-neutralizing anti-GP antibody (AZ52). C) Shown is one blot of one representative experiment where equivalent protein concentrations (15 μg/mL) of VP40+GP and VP40+GPΔmuc VLPs and 10 ng/mL of control LPS were used to treat DCs for 10, 30, 60 and 120 minutes and the total cell lysates (∼10⁵ cellular equivalents/ lane) were tested for the presence of IκBα, phosphorylated ERK1/2 (pERK1/2) and total ERK1/2. D) Shown is a representative experiment where equivalent protein concentrations (15 μg/mL) of VP40+GP and VP40+GPΔmuc VLPs and 10 ng/mL of control LPS were used to treat DCs (5×10⁵ cells) for 20 hours and supernatant was tested for levels of IL-8. The control in this experiment is unstimulated DCs (unstim). E) Association of GFP-tagged VLPs with DCs was assayed by flow cytometry. Control (top left dot plot) depicts DCs stained with isotype controls. Equivalent concentrations of (15 μg/mL) GFP-VP40+GP (bottom right dot plot), GFP-VP40+GPΔmuc (bottom left dot plot) and mock VLPs purified from GFP-transfected cells (top right dot plot) were incubated with DCs for 30 minutes at 37°C, washed, stained for the DC marker CD11c and assayed by flow cytometry.

A) Equivalent protein concentrations (10 μg/mL) of VP40, VP40+GP and cVLPs along with LPS (10 ng/mL) were added to DCs. Spent media, harvested 24 hours post-treatment, was tested for the presence of IL-8. B) Mock, VP40+GP and LPS were used to stimulate DCs for 16−18 hours. Spent supernatant was tested for the presence of 22 cytokines listed in Table 1. A select number of cytokines are shown, including all cytokines present at levels >200pg/mL from VLP-stimulated DC supernatants.

DCs treated with LPS (10 ng/mL) and VLP (VP40+GP VLPs (10 μg/mL)) for 10, 30, 60 and 120 minutes were harvested and total cell lysates (∼10⁵ cellular equivalents/ lane) were tested for the presence of phosphorylated ERK1/2 (pERK1/2), total ERK1/2, phosphorylated-p38 (pp38), total p38, phosphorylated-SAPK/JNK (pSAPK/JNK), total SAPK/JNK and tubulin. The same membranes blotted and tested for pERK1/2, pp38 and pSAPK/JNK were stripped and tested for total levels of ERK1/2, p38 and SAPK/JNK, respectively. The membrane western blotted for SAPK/JNK was stripped and tested for levels of tubulin.