Production and Characterization of Pre-Fusion Ebola Virus GP2 Expressed in Drosophila S2 Cells for Analysis of Vaccine-Induced Immune Responses 3190

Description Introduction The Ebola virus (EBOV) glycoprotein (GP) contains two subunits (GP1 and GP2). Upon receptor binding of proteolytically cleaved GP1 in the endosome, pre-fusion GP2 undergoes conformational changes, facilitating membrane fusion to release viral RNA. Our lab has developed a recombinant EBOV GP subunit vaccine with highly protective efficacy in non-human primates (NHPs). However, the mechanism by which GP-specific antibodies confer protection requires further investigation. Our research focuses on investigating the role of GP2-specific antibody binding in vaccine-induced protection. Method: The EBOV GP2 MUT gene was designed incorporating pre-fusion conformation stabilizing mutations and containing a C-tag for purification. Expression plasmids were transfected into Drosophila S2 cells and stably transformed cell lines selected by adding hygromycin. Protein expression was induced by adding CuSO4 and purified using affinity chromatography before characterization by SDS-PAGE and Western blot. Antibody response to GP2 MUT from immunized NHP sera was analyzed using multiplex immunoassay. Results The EBOV GP2 gene was successfully cloned into the pUHM vector, and sequencing confirmed the presence of the targeted mutations and C-tag. GP2 was successfully purified using affinity chromatography and characterized by SDS-PAGE and Western Blot. Stable pre-fusion GP2 was able to detect IgG from immunized NHPs. Funding Sources This project was supported by grants from the Undergraduate Research Opportunities Program, the National Institute of Allergy and Infectious Diseases (R01AI132323), and the National Institute of General Medical Sciences IDeA Networks of Biomedical Research Excellence P20GM103466. The content is solely the responsibility of the authors and does not represent the official views of the NIH.