Dissecting the neutralising antibody response to the porcine reproductive and respiratory syndrome virus to identify novel vaccine targets

猪繁殖与呼吸综合征（PRRS）是影响全球养猪业最重要的传染病。猪呼吸综合征病毒（PRRSV）是对动物福利和粮食安全的主要威胁，正如在东南亚迅速蔓延的猪高热病暴发所证明的那样，造成了毁灭性的后果。据估计，美国和欧洲每年因PRRSV造成的损失分别超过6亿美元和15亿欧元。在英国，几乎一半的猪群感染了PRRSV，每头母猪的成本超过100英镑。PRRSV存在于两个基因和抗原性不同的物种，即PRRSV-1和PRRSV -2，它们都在快速进化。来自这两个物种的高致病性毒株的出现，以及目前的减毒活疫苗未能提供广泛的保护，以抵御不断扩大的病毒毒株多样性，对全球范围内的有效疾病控制构成重大挑战。因此，迫切需要探索疫苗开发的替代方法，以对抗PRRSV。中和抗体对PRRSV具有保护作用，最近的研究表明，抗体反应可以中和多种PRRSV毒株。对保守抗原靶点的进一步了解将有助于设计新型疫苗。在许多高度可变的人类病毒的背景下，广泛中和抗体识别的表位的鉴定是最近研究的一个热点领域。其核心是生成和分析天然单克隆抗体（mab）特异性的方法。抗原特异性B细胞及其免疫球蛋白基因分析方法的最新进展为设计新的免疫原提供了大量的人单克隆抗体。新一代测序技术的出现为分析免疫个体的功能性抗体库开辟了新的途径。已经建立了将来自单个B细胞的免疫球蛋白重链和轻链的可变序列配对的方法，从而提供了天然单克隆抗体的信息。然后，生物信息学可用于创建B细胞反应的综合视图，并鉴定重组单克隆抗体的克隆或克隆家族及其功能表征。然而，这些方法尚未在猪的环境中建立和利用。该项目旨在采用这些尖端技术来鉴定和表征能够中和PRRSV的天然存在的猪单克隆抗体，并绘制它们对病毒的次要（GP2a， GP3和GP4）和主要（GP5和M）包膜蛋白的反应性。拟议的项目解决了探索PRRS疫苗开发新方法的迫切需要。这与BBSRC战略高度相关；针对动物卫生的优先事项，生物科学的系统方法和管理动物的福利。提议的工作结合了前沿技术和互补学科；具有PRRS免疫学（Graham），免疫遗传学（Hammond），结构病毒学（Grimes）和蛋白质表达（Owens）所需的专业知识。该项目得到了全球最大的动物保健公司Zoetis （Raue）的支持，Zoetis将提供工业视角，以确保项目完成后能够顺利进入下一个开发阶段。

Porcine reproductive and respiratory syndrome (PRRS) is the most important infectious disease affecting the global pig industry. PRRS viruses (PRRSV) are a major threat to both animal welfare and food security, as demonstrated by the pig high fever disease outbreak that rapidly spread across Southeast Asia with devastating consequences. Annual losses to PRRSV in the USA and Europe are estimated to exceed US$600 million and 1.5 billion euros, respectively. In the UK almost half of pig herds are infected with PRRSV with costs exceeding £100/sow. PRRSV exists as two genetically and antigenically distinct species, PRRSV-1 and -2, which are both rapidly evolving. The emergence of highly pathogenic strains from both species and the failure of current live attenuated vaccines to provide broad protection against an ever-expanding diversity of viral strains pose significant challenges to effective disease control world-wide. There is therefore an urgent requirement to explore alternative approaches to vaccine development to combat PRRSV. Neutralizing antibodies confer protection against PRRSV and recent studies have shown antibody responses can neutralize a wide diversity of PRRSV strains. An improved understanding of conserved antigenic targets would enable the design of novel vaccines.Identification of the epitopes recognised by broadly neutralising antibodies is an area of intense recent research in the context of a number of highly variable human viruses. Central to this are methods to generate and analyse the specificity of naturally occurring monoclonal Abs (mAbs). Recent advances in methodologies to analyse antigen-specific B cells and their immunoglobulin genes are now providing large numbers of human mAbs for potential application in the design of novel immunogens.The advent of next-generation sequencing technologies has opened new approaches to analyse the functional antibody repertoire from immune individuals. Methods have been established that pair the variable sequences of immunoglobulin heavy and light chains from individual B cells thus providing information on native mAbs. Bioinformatics can then be used to create a comprehensive view of the B cell response and to identify clones or clonal families for expression of recombinant mAbs and their functional characterisation. However, these approaches have yet to be established and exploited in the context of the pig. This project aims to adapt these cutting edge technologies to identify and characterise naturally occurring porcine mAbs capable of neutralising PRRSV and to map their reactivity to the minor (GP2a, GP3 and GP4) and major (GP5 and M) envelope proteins of the virus.The proposed project addresses the urgent need to explore novel approaches to PRRS vaccine development. It is highly relevant to BBSRC strategy; targeting the priorities of animal health, systems approaches to the biosciences and welfare of managed animals. The proposed work combines cutting edge techniques and complimentary disciplines; with the required expertise in PRRS immunology (Graham), immunogenetics (Hammond), structural virology (Grimes) and protein expression (Owens). The project is support by Zoetis (Raue), the largest global animal health company, who will provide an industrial perspective to ensure that upon completion the project is well placed to proceed to the next stages of development.