Human embryonic kidney cells expressing the SV40 large T antigen (HEK 293T) (a gift from T. Dull, Cell Genesys, CA), African green monkey kidney epithelial cells (Vero cells [ATCC CCL-81]) and African green monkey kidney fibroblast-like cells expressing the SV40 T antigen (Cos7 cells [ATCC CRL-1651]) were maintained in Dulbecco's Modified Eagle's Medium (DMEM, Lonza, Walkersville, MD) supplemented with 10% heat-inactivated fetal bovine serum (FBS, HyClone, Logan, UT), 2 mM glutamine, 100 U/ml penicillin, and 100 μg/ml streptomycin (Lonza, Walkersville, MD). Cells cultures were grown at 37°C in a humidified 5% CO2 incubator.
The pVR1012-EBOVGP, pVR1012-SUDVGP, and pVR1012-TAFVGP plasmids encode EBOV, SUDV, and TAFV GP1,2, respectively, and were kindly provided by Gary Nabel (Vaccine Research Center, NIH, Bethesda, MD) and Anthony Sanchez (CDC, Atlanta, GA). pVR1012-EBOVGPΔMLD encodes the EBOV MLD-deleted GP1,2 and was described previously . pBDBV GP1,2 encodes wild-type BDBV GP1,2 and was previously described . pMLV-GagPol is a Moloney murine leukemia virus (MLV)-based gag-pol expression plasmid and pRT43.2nlsβgal is a MLV-based packageable genome encoding β-galactosidase and a nuclear localization signal . pVSV-G is a commercial plasmid from Clontech (Mountain View, CA), encoding the G glycoprotein of Vesicular stomatitis virus (VSV).
The following EBOV GP1,2 peptides were synthesized and Reverse Phase HPLC-purified at the FDA CBER Core Facility: ZGP-1 (VSGTGPCAGDFAFHK, amino acid 141-155) , ZGP-4 (LYDRLASTV, amino acid 161-169) and ZGP-5 (EYLFEVDNL, amino acid 231-239) .
VLP and CpG production and characterization
VLPs were produced by cotransfecting 16 μg of pVR1012-EBOVGPΔMLD and 8 μg pMLV-GagPol into HEK 293T cells at a density of 5 × 106 cells/100 mm cell culture dish by using 60 μl per dish of Lipofectamine 2000 (Invitrogen, Carlsbad, CA). Supernatants were collected 48 and 72 h post transfection, clarified through 0.45 μm-pore size filters, and concentrated by Amicon Ultracel 100 k centrifugal filters (Millipore, Billerica, MA). The concentrated VLP-containing supernatants were centrifuged through a 20% (wt/vol) sucrose cushion in TNE buffer (10 mM Tris [pH 8.0], 1 mM EDTA, 100 mM NaCl) at 82,705 × g in a Beckman XL-90 ultracentrifuge using a Beckman SW28Ti rotor. The resulting pellets were resuspended in endotoxin-free phosphate-buffered saline (PBS) (Teknova, Hollister, CA) and stored at -80°C. A total of ten batches of VLPs were generated, pooled together and total protein concentration was measured using the Bio-Rad DC Protein Assay Kit (Bio-Rad, Hercules, CA). Western blot and silver stain analyses were performed to characterize the purity and makeup of the purified VLPs. Briefly, 10, 1, and 0.1 μg of the samples were denatured for 5 min at 95°C in 1× NuPAGE LDS Sample Buffer and 1× NuPAGE Sample Reducing Agent (Invitrogen, Carlsbad, CA), electrophoresed on a pre-cast NuPAGE 4-12% Bis-Tris gel at 200 V and transferred to a PVDF membrane (Invitrogen, Carlsbad, CA) for 90 min at 30 V. The membrane was probed with 35 ng/ml of an anti-GP1,2 rabbit polyclonal antibody R.F88-2, which was raised by injecting a conserved 38-mer GP1,2 peptide (amino acid residues 72-109) , followed by incubation with goat anti-rabbit IgG conjugated with horse radish peroxidase (HRP) diluted at 1:10,000 (Thermo Scientific, Rockford, IL), developed with Western Lightning™ Plus Chemiluminescence Reagent (PerkinElmer, Waltham, MA), and subsequently exposed to a Kodak BioMax MR Film (Carestream Health, Rochester, NY). Likewise, a silver stain analysis was performed using the SilverQuest™ Silver Staining Kit (Invitrogen, Carlsbad, CA) according to the manufacturer's instructions. Phosphorothioate CpG ODN 1555 (5'-GCTAGACGTTAGCGT-3', underlined portion represents the active CpG motif) was synthesized at the CBER core facility. Endotoxin was removed using the ToxinEraser™ Endotoxin Removal Kit and residual endotoxin was measured using the ToxinSensor™ Chromogenic LAL Endotoxin Assay Kit, following the manufacturers' manuals (GenScript, Piscataway, NJ).
Animals and vaccination experiment
Female BALB/c (H-2d) mice, aged 6-8 weeks, were obtained from Charles River Laboratories (Germantown, MD) and divided randomly into six vaccination groups. For VLP immunizations, mice were injected intraperitoneally (i.p.) with 33 μg of VLPs in 100 μl endotoxin-free PBS. For DNA immunizations, mice were injected intramuscularly (i.m.) with a combination of 50 μg of DNA (pVR1012-EBOVGPΔMLD + pMLV-GagPol at a 2:1 ratio) and 16 μg CpG in 100 μl endotoxin-free PBS. For CpG immunizations, mice were injected with 16 μg CpG in 100 μl endotoxin-free PBS via the i.m. route. The mice were either immunized with VLPs, DNA, a combination of DNA and VLPs, CpG or endotoxin-free PBS (Figure 2). Two weeks after each injection, blood samples were collected by nicking the tails with #10 Carbon Steel Surgical Blades (Braintree Scientific, Braintree, MA) and collecting blood in BD Microtainer tubes (Becton, Dickinson and Company, Franklin Lakes, NJ). Blood was allowed to clot for 1-2 h at room temperature, centrifuged at 8,600 xg for 3 min, and the resulting serum in the supernatant was collected and stored at -20°C. The animal protocol and procedures were approved by Institutional Animal Care and Use Committees at the Center for Biologics Evaluation and Research (protocol #2009-04) in animal facilities accredited by the Association for Assessment and Accreditation of Laboratory Animal Care International. All experiments were performed according to institutional guidelines.
Immuno 96 MicroWell plates (Nunc, Rochester, NY) were coated with 50 μl of 1 μg/mL anti-human Fc IgG antibody (Kirkegaard & Perry Laboratories, Gaithersburg, MD) in PBS overnight at 4°C. The next day, plates were washed once with PBS and blocked with 100 μl of 3% Bovine Serum Albumin (BSA) in PBS for 1 h at 37°C. Plates were then incubated with 50 μl of either Fc or GP1,2-Fc (1 μg/ml)  in TBS-T (Tris-buffered saline, 0.1% Tween-20) for 90 min at 37°C. After washing 2× with TBS-T, 50 μl of each serum sample diluted in TBS-T, as indicated in the figures and legends, was added and incubated for 1 h at 37°C. Plates were then incubated with 50 μl horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG diluted at 1:500 (Pierce, Rockford, IL) for 40 min at 37°C after 2 washes with TBS-T. After washing 4× with TBS-T, 100 μl/well of the ABTS substrate (Kirkegaard & Perry Laboratories, Gaithersburg, MD) was added for 3 min at room temperature and plates were read on a VICTOR3 V plate reader (Perkin Elmer, Shelton, CT) at 405 nm.
Cell lysates for western blots were prepared by transiently transfecting 5 × 106 HEK 293T cells/100 mm cell culture dish as previously described  except for the following modifications: cells were transfected with 24 μg per plate of either pVR1012-EBOVGP, pVR1012-SUDVGP, pVR1012-TAFVGP or pBDBVGP. A mock transfection was also performed with no plasmid DNA. Total protein concentration in the cell lysates was measured by using the Bio-Rad DC Protein Assay Kit (Bio-Rad, Hercules, CA) and 20 μg were loaded in each lane on a pre-cast NuPAGE 4-12% Bis-Tris gel, electrophoresed, and transferred to PVDF membranes, as previously described . The membranes were incubated with one of the following: pooled sera from the VLP group diluted at 1:2,000, pooled sera from the DNA/VLP group diluted at 1:2,000 or an anti-GP1,2 rabbit polyclonal antibody R.F88-2 diluted at 1:10,000  Secondary antibody incubation was performed using HRP-conjugated goat anti-mouse IgG (1:5,000) or goat anti-rabbit IgG (1:10,000) (Thermo Scientific, Rockford, IL) and developed following the same protocol as above. Another western blot was also performed to titrate the pooled sera from the VLP and DNA/VLP groups at the dilutions of 1:2,000, 1:5,000 and 1:10,000.
Neutralization of rVSV-ZEBOVGP
The recombinant VSV virus expressing the EBOV GP1,2 [rVSV-ZEBOVGP] or the wild-type VSV were generated as described previously . Briefly, BSR-T7 cells were co-transfected with pBS-N, pBS-P, pBS-L, and pVSV-EBOVGP or pVSVFL(+). After 48 h of incubation at 37°C, supernatants were collected, titrated on Vero E6, and stored at -80°C. For neutralization, VeroE6 cells were seeded at 50% confluency in 6-well plates and incubated at 37°C overnight. rVSV-ZEBOVGP or wild-type VSV (100 pfu) in 45 μl of medium, which was prepared with or without 5% guinea pig complement (Accurate Chemical Corp. Westbury, NY), was mixed with mouse serum at the final concentration of 1:25 dilution and incubated overnight at 4°C. Normal mouse serum pre-diluted at 1:25 was used as the negative control. On the next day, the virus-serum mixtures were added to the cells and incubated for 1 h at 37°C. Each serum was tested in duplicate samples. After washing two times with medium, the cells were overlaid with medium containing 1% Bacto-agar and incubated at 37°C. After 48 h, the cell monolayers were fixed with 10% TCA and stained with 1% crystal violet for 30 min. Plaque numbers were used to calculate the titer.
Neutralization of MLV pseudotyped virus bearing envelopes from different viruses
Retroviral vector pseudotypes were generated by cotransfecting HEK 293T cells as previously described  but modified by using the following plasmids: 10 μg of pRT43.2nlsβgal, 2.5 μg of pMLV-GagPol and 5 μg of the expression plasmid pVR1012-EBOVGP, pVR1012-SUDVGP, pVR1012-TAFVGP, pBDBVGP, or pVSV-G. One day prior to neutralization, 4 × 104 cells/well of Vero were seeded in 24-well cell culture plates. The next day, the vector pseudotypes were thawed on ice, incubated with serum at the final concentration of 1:25 dilution for 1 h at 37°C, supplemented with 8 μg/mL polybrene (American Bioanalytical, Natick, MA) and 200 μL of this mixture replaced the medium on Vero cells. After an overnight culture, supernatants were removed from the wells and replaced with 1 ml of complete culture media. 48 h after transduction, cells were fixed and histochemically stained for β-galactosidase activity, and the titer was quantified by microscopic enumeration of blue forming units (BFU), as previously described .
Interferon (IFN)-γ ELISPOT assay
Erythrocytes from mice splenocytes were depleted by incubating in 1× BD PharmLyse Buffer (Becton, Dickinson and Company, Franklin Lakes, NJ) for 5 min at room temperature according to manufacturer's instructions. The splenocytes were then used in an IFN-γ ELISPOT assay as previously described  except for the following modifications: 500,000 splenocytes per well were stimulated with 50 μl of 2 μg/ml ZGP-1, ZGP-4, ZGP-5 or a no-peptide negative control and 0.5 μg/ml biotinylated anti-mouse IFN-γ (Clone R4-6A2) (BD Pharmingen, Franklin Lakes, NJ) was added per well in the staining process. Three replicates were used for each combination.
To evaluate statistical significance of the ELISPOT results, we evaluated both within-group and between-group treatment difference. For the within-group comparison, three replicates for each sample were averaged and the difference between these averaged responses with respect to treatment was tested using the paired t-test. For the between-group comparison, the treatment difference for each mouse was obtained by taking the difference between treated vs. untreated averaged response (averaged over three replicates). This individual treatment difference was then used to compare the treatment effect between groups. Two-sample t-test was used for each two-group comparison.