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

非洲马瘟病毒（African horse sickness virus，AHSV）是呼肠孤病毒科环状病毒属（Orbivirus）的一种双链RNA病毒，其中包括蓝舌病毒（bluetongue virus，BTV）。至于BTV，AHSV通过库蠓（蠓）媒介传播，以斑马、骡子、驴和马为食，但仅在马中导致严重影响和高死亡率。病毒感染表现为相关的急性肺和心脏形式，两者都具有高死亡率（60-90%）。没有有效的治疗方法，非洲使用的减毒活疫苗（LAV）有时会引起疾病，九种不同血清型的存在使灭活病毒疫苗的使用变得复杂。由于LAV和灭活疫苗均不允许使用抗体诊断试验来区分感染动物和接种动物，这阻碍了控制措施的实施，因此需要进一步研究开发新型AFH疫苗。研究背景AHSV含有7种结构蛋白（VP），其中两种VP 2和VP 7的部分三维结构是已知的。衣壳蛋白VP 2决定血清型，并已显示诱导保护性反应; VP 7也是一种经验证的抗原，已显示保护小鼠免受致死剂量的病毒感染，并诱导特异性单克隆抗体（mAb）。因此，VP 2和VP 7是主要的AHSV疫苗候选者，尽管关于其免疫原性的分子细节仍然是难以捉摸的。博士项目旨在根据最近获得专利的方法，使用结构生物学和病毒学技术相结合来开发一种新型AHSV疫苗原型（PCT/GB 2017/052608）.博士目标：目标1：确定抗VP 2和抗VP 7单克隆抗体和纳米抗体的作用。将产生针对九种VP 2血清型和VP 7中的每一种的抗体。学生将使用生物物理技术来量化体外抗VP 2和抗VP 7 mAb/纳米抗体的个体结合亲和力，同时将建立平行的基于细胞的AHSV测定，以选择那些对病毒进入具有抑制作用的抗体。目标2：确定与单克隆抗体/纳米抗体复合的VP 2和VP 7的晶体结构学生将继续确定与目标1中鉴定的最佳单克隆抗体/纳米抗体复合的VP 2和VP 7的晶体结构，这将揭示抗体-表位识别的分子细节。这些信息将被利用来指导免疫原design.Objective 3：工程广谱AFHV疫苗prototypeThe学生将设计免疫原的片段的VP 2和VP 7的序列分析的基础上，以扩大跨血清型的序列覆盖范围，并测试其热稳定性，需要在炎热的国家交付。这些免疫原将被克隆、表达、纯化并与病毒样颗粒缀合，以测试它们增强广泛中和mAb的能力，这些mAb将被测试为阻断AHSV入侵的治疗工具，从而为合成宽-单个目标的成功独立于其他目标的成功，但集体信息对疫苗原型具有协同影响。PhD项目的主要目标。

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.