Characterization of Respiratory Synctial Virus (RSV) Fusion (F) Proteins on Viral-Like Particles Open Access

Grace, Jillian (Spring 2019)

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The Respiratory Syncytial Virus (RSV) fusion (F) glycoprotein is an extracellular surface protein recognized by Th2 cells and expressed on the surface of RSV viruses. The F protein contains two variants that differ in conformation and antigenic sites available: the metastable prefusion conformation and the more stably expressed postfusion conformation that becomes stabilized after fusion of the virus with the host cell membrane. Artificially stabilized prefusion conformations possess antigenic sites that are critical to elicit neutralizing antibodies. This study is focused on generating RSV F prefusion variants expressed on virus-like particles (VLPs). VLPs are nanostructures that resemble viruses but do not contain the RNA viral genome and thus lack pathogenicity. We developed multiple VLP variants and characterized them by enzyme-linked immunosorbent assay (ELISA), immunoblotting, and electron microscopy (EM) analysis. Based on prior research evaluating anti-F antibodies in human sera, prefusion F protein elicits a significantly higher neutralization response compared to postfusion F due to availability of epitopes that are readily accessible and recognized by humoral immune cells. However, prefusion F protein is highly unstable and easily converts to postfusion F. We compared DS-Cav 1, SC-TM, and wildtype variants to determine which variant most stably produced prefusion F protein and was most readily recognized by anti-F prefusion-specific antibodies. We found DS-Cav 1 and SC-TM to have substantially greater recognition of anti-F antibodies via ELISA. This could indicate a need to create variants that contain mechanisms to prevent release of the fusion peptide, reduced charge repulsion in the refolding region 2 (RR2), and increased packing in the hydrophobic region of the prefusion conformation.

Table of Contents

Introduction                                                                                                                        1

Results                                                                                                                                  9

Discussion                                                                                                                           19

Works Cited                                                                                                                         23

Methods                                                                                                                               26

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