Structural and functional studies on African horse sickness virus (AFHV) to engineer a protein-based vaccine prototype

点击翻译按钮获取中文摘要

ImportanceAfrican horse sickness virus (AHSV) is caused a double stranded RNA virus belonging to the genus Orbivirus, family Reoviridae, which includes bluetongue virus (BTV). As for BTV, AHSV is spread by Culicoides (midge) vectors, which feed on zebras, mules, donkeys and horses, but lead to severe effects and high mortality only in horses. Viral infection manifests as acute pulmonary and cardiac forms associated, which both have a high fatality rate (60-90%). There is no effective therapy available, the live-attenuated vaccine (LAV) used in Africa can sometimes cause disease, and the existence of nine different serotypes complicates the use of inactivated virus vaccines. Neither LAV nor inactivated virus vaccines allow the use of diagnostic tests for antibodies that can differentiate infected from vaccinated animals, which hinders control measures.Further research is therefore required to develop a novel AFH vaccine.Research BackgroundAHSV contains seven structural proteins (VPs) and partial three-dimensional structures of two of them, named VP2 and VP7, is available to date. Capsid protein VP2 determines the serotype and has been shown to induce a protective response; VP7 is also a validated antigen, which has been shown to protect mice from a lethal dose of the virus and to induce specific monoclonal antibodies (mAbs). VP2 and VP7 are therefore leading AHSV vaccine candidates, although the molecular details about their immunogenicity remain elusive.The PhD project aims to develop a novel AHSV vaccine prototype using a combination of structural biology and virology techniques following a recently patented approach (PCT/GB2017/052608).PhD objectives:Objective 1: Determine the effects of anti-VP2 and anti-VP7 mAbs and nanobodiesAntibodies against each of the nine VP2 serotypes and VP7 will be produced. The student will use biophysical techniques to quantify individual binding affinities of anti-VP2 and anti-VP7 mAbs/nanobodies in vitro, whilst in parallel cell-based assay with AHSV will be set-up to select those that have inhibitory effects on viral entry. Antigenic cartography will be used to map the relationships between the different serotypes.Objective 2: Determine the crystal structures of VP2 and VP7 in complex with mAbs/nanobodies The student will pursue the determination of the crystal structures of VP2 and VP7 and in complex with the best mAbs/nanobodies identified in objective 1, which will reveal the molecular details of antibody-epitope recognition. This information will be exploited to guide immunogen design.Objective 3: Engineering a broad-spectrum AFHV vaccine prototypeThe student will design immunogens of fragments of VP2 and VP7 based on sequence analysis to broaden the sequence coverage across serotypes and to test their thermostability, as required for delivery in hot countries. These immunogens will be cloned, expressed, purified, and conjugated to virus-like particles to test their ability to boost broadly neutralizing mAbs, which will be tested as therapeutic tools to block AHSV invasion, therefore paving the way for a synthetic broad-spectrum vaccine prototype.The success of the individual objectives is independent from the success of the other objectives but the collective information has a synergic impact on the overarching aim of the PhD project.