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

非洲马病病毒(AHSV)是呼肠孤病毒科环状病毒属的一种双链RNA病毒，包括蓝舌病毒(BTV)。至于BTV，AHSV是通过库蚊传播的，这些媒介以斑马、骡子、驴和马为食，但只在马身上造成严重影响和高死亡率。病毒感染表现为急性肺型和心型相关，两者的病死率都很高(60%-90%)。目前还没有有效的治疗方法，非洲使用的减毒活疫苗有时会导致疾病，九种不同血清型的存在使灭活病毒疫苗的使用复杂化。无论是LAV疫苗还是灭活病毒疫苗都不能对抗体进行诊断试验，以区分感染动物和接种疫苗的动物，这阻碍了控制措施。因此，需要进一步的研究来开发新的AFH疫苗。研究背景AHSV包含七个结构蛋白(VP)，其中两个的部分三维结构被命名为VP2和VP7。衣壳蛋白VP2决定了血清型，并已被证明可诱导保护性反应；VP7也是一种有效的抗原，已被证明可保护小鼠免受致死量病毒的攻击，并可诱导特异性单抗(MAbs)。因此，VP2和VP7是甲型单纯疱疹病毒疫苗的主要候选疫苗，但关于它们的免疫原性的分子细节仍不清楚。博士项目旨在根据最近获得专利的方法(PCT/GB2017/052608)，利用结构生物学和病毒学技术相结合的方法开发一种新的甲型单纯疱疹病毒疫苗原型。学生将使用生物物理技术在体外量化抗VP2和抗VP7单抗/纳米抗体的结合亲和力，同时将建立与AHSV并行的基于细胞的分析，以选择那些对病毒进入具有抑制作用的抗体。目标2：确定VP2和VP7与单抗/纳米抗体的复合体的晶体结构。学生将继续测定VP2和VP7的晶体结构，以及与目标1中确定的最佳单抗/纳米体的复合体的晶体结构，这将揭示抗体表位识别的分子细节。目标3：设计广谱AFHV疫苗原型学生将基于序列分析设计VP2和VP7片段的免疫原，以扩大不同血清型的序列覆盖范围，并测试其热稳定性，以满足在炎热国家交付的要求。这些免疫原将被克隆、表达、纯化并与病毒样颗粒结合，以测试它们增强广泛中和单抗的能力，这些单抗将作为治疗工具进行测试，以阻止AHSV的入侵，从而为合成广谱疫苗原型铺平道路。个人目标的成功独立于其他目标的成功，但集体信息对PHD项目的总体目标具有协同影响。

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.