Bdbv gp

Human NK cells were enriched from peripheral blood by negative selection using RosetteSep negative selection kit (Stem Cell Technologies) followed by Ficoll separation. NK cells were rested overnight in the presence of 1 ng/ml recombinant IL-15 (PeproTech). 3 μg/ml of BDBV GP (IBT Bioservices) was coated on a Maxisorp ELISA plate (Nunc) at 4°C overnight, and plates were blocked with 5% BSA prior to addition of antibodies (5 μg/ml) in PBS for 2 hours at 37°C. The control EBOV-specific mAb c13C6 was purchased from IBT Bioservices. Unbound antibodies were removed by washing wells 3X with PBS prior to addition of NK cells. The NK cells were added at 5 x 10⁴ cells/well in the presence of brefeldin A (Sigma Aldrich), GolgiStop (BD Biosciences), and anti-CD107a PE-Cy5 antibody (BD Biosciences clone H4A3) and incubated for 5 hours at 37°C. NK cells were stained with flow cytometry antibodies for the following surface markers: CD3 AlexaFluor700 (BD Biosciences clone UCHT1), CD56 Pe-Cy7 (BD Biosciences clone B159), and CD16 APC-Cy7 (BD Biosciences clone 3G8), followed by intracellular staining for IFNγ (FITC, BD Biosciences clone B27) and MIP-1β (PE, BD Biosciences clone D21-1351) to detect the production of cytokines and chemokines. Cells were analyzed by flow cytometry on a BD LSRII flow cytometer and data was analyzed using FlowJo software.

Aliquots of thermolysin-treated or mock-treated purified virions were heated for 10 min at 95ºC and separated in Nu-PAGE 4 to 12% Bis-Tris gel with Novex Sharp Pre-Stained Protein Standard used as a molecular weight marker. Proteins were transferred to a nitrocellulose membrane using the iBlot Gel transfer system (Life Technologies). The membrane was incubated with primary rabbit polyclonal antibodies against BDBV GP (1:500; IBT Bioservices) and secondary goat anti-rabbit IgG antibodies conjugated with horseradish peroxidase (1:500; KPL). Protein bands were visualized using the chromogenic 4CN two-component peroxidase substrate system (KPL).