Macaque Immunization and Challenge
Cynomolgus macaques were injected intramuscularly with 1.0 ml of an equal mixture of immunogens at the doses indicated. Viral challenge was performed by inoculation of animals in the left or right caudal thigh with 0.5 ml of viral stock that contained a target dose of ˜1,000 plaque-forming units (pfu) ZEBOV at 4 wk after the initial immunization. No adverse effects of the adenovirus vaccination were observed acutely. The Ebola virus stock used in this study was originally obtained from a fatally infected human from the former Zaire in 1995 [12]. Collection of serum and blood for viral load and ELISA titers was performed as previously described [2]. Surviving animals were followed for at least 4 wk postchallenge.
Flow Cytometry and Antibodies
Transfected cells were collected after incubation with PBS containing 3 mM EDTA and incubated with control Ig or rabbit anti-sGP/GP serum (generously provided by Dr. A. Sanchez, CDC) for 30 min on ice. The cells were washed twice with ice-cold PBS containing 2.5% fetal bovine serum, incubated with FITC- or PE-conjugated secondary antibodies (Jackson ImmunoResearch Laboratories, West Grove, Pennsylvania, United States; and Sigma-Aldrich, St. Louis, Missouri, United States, respectively) for 30 min on ice, followed by washing. Analysis was conducted using a Becton-Dickinson four-color Calibur flow cytometer and FlowJo analysis software (Tree Star, Ashland, Oregon, United States).
ELISA
Nunc-Immuno Maxisorp plates (Nunc, Rochester, New York, United States) were coated with 100 μl/well of 10 μg/ml lectin from Galanthus nivalis (Sigma-Aldrich) in PBS and incubated overnight at 4 °C. All further incubations were carried out at room temperature. Plates were then blocked for 2 h in PBS containing 10% fetal calf serum and then washed twice with 0.2% Tween 20 (Sigma-Aldrich) in PBS. Ebola GP was obtained from the supernatants of HEK293 cells transfected with the mammalian expression plasmid Ebola GP(ΔTM) and was added to the plates at a concentration of 0.8–1.3 mg/ml total protein in 100 μl/well. Plates were then washed six times with PBS containing 0.2% Tween 20. Test sera were diluted in PBS containing 0.2% Tween 20 and 1% fetal calf serum and allowed to react with the Ag-coated wells for 60 min. After washing plates six times, goat anti-human IgG (H+L; Chemicon, Temecula, California, United States) conjugated to horseradish peroxidase was used as a detection antibody. Bound IgG was detected by Sigma Fast O-phenylenediamine dihydrochloride tablet sets (Sigma-Aldrich), and the optical density was determined. A panel of normal sera was run each time the assay was performed.
Neutralizing Antibody Analysis
Ebola GP(Z) pseudotyped lentiviral virions were produced as previously described [13]. Briefly, 293T cells were plated at a density of 2 × 106 per 10-cm diameter tissue culture dish and transfected the next day by calcium phosphate reagent (Invitrogen) with pCMVΔR8.2, pHR′CMV-Luc, and CMV/R Ebola GP(Z) plasmid DNA. Cells were transfected overnight, washed, and replenished with fresh medium. Supernatants containing pseudotyped virus were harvested 48 h later, filtered through a 0.45-μm pore-size syringe filter, and stored in aliquots at −80 °C. Neutralization assays were performed on HUVECs (Cambrex, East Rutherford, New Jersey, United States; #CC-2517) plated in a 24-well plate 1 d prior to infection. Virus stocks titrated to the 90% infectious dose were incubated at 37 °C for 1 h in the presence of serum from immunized cynomolgus macaques. The culture media was removed from the cells and replaced with the virus/serum media in the presence of polybrene (Sigma-Aldrich, #107689) at a final concentration of 5 μg/ml. At 72 h postinfection, cells were lysed and assayed by the Luciferase Assay System (Promega, Madison, Wisconsin, United States; #E1501/E1531). Luciferase activity was determined using a Veritas Microplate Luminometer from Turner Biosystems (Sunnyvale, California, United States).