Epitopes involved in antibody-mediated protection from Ebola virus

Science

Volumn 287, Issue 5458, 2000, Pages 1664-1666

  Wilson, Julie A ^a   Hevey, Michael ^a   Bakken, Russell ^a   Guest, Shawn ^a   Bray, Mike ^a   Schmaljohn, Alan L ^a   Hart, Mary Kate ^a  

^a UNITED STATES ARMY MEDICAL RESEARCH INSTITUTE OF INFECTIOUS DISEASES   (United States)

Author keywords

[No Author keywords available]

Indexed keywords

EPITOPE; MONOCLONAL ANTIBODY; NEUTRALIZING ANTIBODY; VIRUS GLYCOPROTEIN;

ANIMAL EXPERIMENT; ANIMAL MODEL; ANTIBODY RESPONSE; ARTICLE; CONTROLLED STUDY; EBOLA VIRUS; ENZYME LINKED IMMUNOSORBENT ASSAY; HEMORRHAGIC FEVER; IMMUNOPRECIPITATION; MOUSE; NONHUMAN; PRIORITY JOURNAL; RECEPTOR BINDING; RNA EDITING; VACCINE PRODUCTION; VIRION; VIRUS CELL INTERACTION; VIRUS IMMUNITY; VIRUS NEUTRALIZATION;

ANIMALS; ANTIBODIES, MONOCLONAL; ANTIBODIES, VIRAL; ANTIBODY AFFINITY; ANTIGENS, VIRAL; BINDING, COMPETITIVE; COMPLEMENT SYSTEM PROTEINS; EBOLAVIRUS; EPITOPES; FEMALE; HEMORRHAGIC FEVER, EBOLA; HUMANS; IMMUNOGLOBULIN G; MICE; MICE, INBRED BALB C; MICE, INBRED C57BL; NEUTRALIZATION TESTS; PLAQUE ASSAY; SPECIFIC PATHOGEN-FREE ORGANISMS; VIRAL ENVELOPE PROTEINS;

ANIMALIA; EBOLA VIRUS; RNA VIRUSES;

EID: 0034051595     PISSN: 00368075     EISSN: None     Source Type: Journal    
DOI: 10.1126/science.287.5458.1664     Document Type: Article

References (26)

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12. 7 replicons in the tail vein 21 days later. Spleens were removed 3 days after the final injection and fused (11) to P3X63Ag8.653 myeloma cells to produce hybridomas. Hybridoma supernatants were assayed for reactivity with Ebola Zaire GP in ELISA, immunofluorescence, and radioimmunoprecipitation assays.
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15. mAbs were biotinylated with the use of an EZ-Link Sulfo-NHS-LC-Biotinylation kit (Pierce) according to the manufacturer's instructions. Competitive binding was performed by reacting a 10-fold excess of unlabeled mAbs with biotin-labeled mAbs and sucrose-purified Ebola Zaire 1995 virus. The results were evaluated by ELISA (13). Monoclonal antibodies that reduced absorbance values similar to those obtained in positive control wells (containing the same unlabeled and biotin-labeled mAb) were considered competitive. The concentrations of biotinylated mAbs used in the competition assays were determined by prior titration to be in the linear portion of their binding curve to Ebola.
16. ELISAs were performed as described (14) with plates coated with sucrose-purified, irradiated virions prepared from the following Ebola strains: Zaire 1995, Zaire 1976 (isolate Mayinga), Sudan (isolate Boniface), and Ivory Coast (#807212, obtained from the Centers for Disease Control). In some experiments, the secondary antibodies were conjugated with alkaline phosphatase, and ρ-nitrophenyl phosphate was used as the substrate. ELISA values were considered positive if they exceeded background values by 0.2 absorbance units.
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20. Fourfold serial dilutions of mAbs (starting at 100 μg/ml) were mixed with 100 plaque-forming units of mouse-adapted Ebola Zaire at 37°C for 1 hour in the presence or absence of 5% guinea pig complement (Accurate Scientific) and used to infect Vero E6 cells. Cells were overlaid with agarose (18), and a second overlay containing 5% neutral red was added 6 days later. Plaques were counted the next day. Neutralization titers were determined to be the last dilution of mAb that reduced the number of plaques by 80% compared with control wells.
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24. Specific pathogen-free 5- to 8-week-old female BALB/c or C57BL/6 mice (National Cancer Institute, Frederick, MD) were housed in cages equipped with microisolators and were provided food and water ad libitum. Research with animals was done in accordance with the Guide for the Care and Use of Laboratory Animals, prepared by the Committee on Care and Use of Laboratory Animals of the Institute of Laboratory Animal Resources, National Research Council (NIH Publication No. 86-23, revised 1996). Mice were transferred to a Biosafety Level 4 containment area and challenged by intraperitoneal inoculation of mouse-adapted Ebola Zaire 1976 virus (16).
25. On the basis of the Ebola Zaire GP sequence (5), peptides were synthesized directly on SPOTS membranes by Genosys. Binding of the mAbs to the peptides was performed according to the manufacturer's instructions.
26. We thank P. B. Jahrling at the U.S. Army Medical Research Institute of Infectious Diseases for providing the Ebola seed stocks used in the characterization of mAbs and S. Messer, M. Azarion, S. Lewis, and J. Kondig for technical assistance. J.A.W. was supported by a National Research Council fellowship. The views of the authors do not purport to reflect the positions of the Army or the Department of Defense.