Elicitation of antibodies broadly neutralizing the porcine reproductive and respiratory syndrome virus (PRRSV) using reverse vaccinology

猪繁殖与呼吸综合征病毒(PRRSV)是猪的重要病原。不幸的是，现有的减毒活疫苗和灭活疫苗对症状性疾病只能提供有限的保护，不能有效地防止强毒野毒在猪群中的传播。尽管疫苗接种后对同源病毒株产生了强烈的体液免疫反应，但对基因分化的病毒株的保护作用是不够的。传统的疫苗方法显然不能涵盖由两个不同物种(PRRSV-1和-2)组成的PRRSV的巨大遗传变异性。到目前为止，PRRSV糖蛋白的结构仍然难以捉摸，这使得基于结构的疫苗设计用于提高疫苗效力变得复杂。在这个项目中，我们将应用反向疫苗接种方法来填补这些空白。使用带有荧光标记的PRRSV糖蛋白和病毒粒子的多色流式细胞仪，然后进行最先进的单B细胞测序，我们将分析PRRSV特异性猪抗体谱系。单个抗体将被重组表达，重组抗体将根据它们的中和性和效价以及它们的同源抗原来表征。同时，我们将在我们成功确定PRRSV中和表位的基础上，将重点放在高度保守的表位上，这些表位针对的是对疫苗设计特别感兴趣的广谱中和抗体。为此，我们将依次用PRRSV-1和PRRSV-2病毒粒子免疫小鼠，然后分离与这两个物种的糖蛋白反应的记忆B细胞，并鉴定如上所述的bNAbs。在这项研究中，我们还将研究在猪或老鼠中产生的有趣的中和抗体，关于它们的中和机制，以及病毒是否可以通过突变来逃脱这些抗体，即所谓的“免疫逃逸”。最后，我们将从结构上表征中和抗体mAb18，我们已经在一项识别PRRSV GP2的概念验证研究中确定了mAb18与表位多肽的复合体，并使用所得到的结构来开发针对表位的疫苗。为此，我们将把这个表位转移到合适的“支架”蛋白上，以纳米颗粒的形式生产基于支架的候选疫苗，并在猪身上进行挑战试验验证该候选疫苗的有效性。综上所述，该项目将对PRRSV糖蛋白的免疫反应提供重要的见解，以促进知情的疫苗设计。

Porcine reproductive and respiratory syndrome virus (PRRSV) is an economically important pathogen of swine. Unfortunately, the available live attenuated and inactivated vaccines provide only limited protection against symptomatic disease and cannot effectively prevent the spread of virulent field viruses in swine herds. Although a strong humoral immune response against homologous viral strains is elicited after vaccination, the achieved protection against genetically divergent viral strains is insufficient. Traditional vaccine approaches apparently cannot cover the large genetic variability of PRRSV comprising two separate species (PRRSV-1 and -2). The structure of PRRSV glycoproteins remains elusive to date, complicating the use of structure-based vaccine design to improve vaccine efficacy. In this project, we will apply reverse vaccinology approaches to fill these gaps. Using multi-color FACS with fluorescently labeled PRRSV glycoproteins and virions followed by state-of-the-art single B cell sequencing we will analyze the PRRSV-specific porcine antibody repertoire. Individual antibodies will be expressed recombinantly and the recombinant antibodies characterized with respect to their neutralization profile and potency as well as their cognate antigen. In parallel, we will build on our successful pipeline to identify PRRSV neutralization epitopes to focus on highly conserved epitopes targeted by broadly neutralizing antibodies that are of particular interest for vaccine design. For this purpose, we will immunize mice sequentially with PRRSV-1 and PRRSV-2 virions followed by isolation of memory B cells reacting with glycoproteins of both species and characterization of bnAbs as described above. We will also investigate interesting neutralizing antibodies generated in pigs or mice in this study with respect to their neutralization mechanism and whether the virus can escape these antibodies by mutation in a so-called "immune escape". Finally, we will structurally characterize the neutralizing antibody mAb18, which we have already identified in a proof-of-concept study to recognize PRRSV gp2, in complex with an epitope peptide and use the resulting structure to develop an epitope-focused vaccine. To this end, we will transfer this epitope to suitable "scaffold" proteins, produce a scaffold-based vaccine candidate in the form of nanoparticles and validate this candidate in a challenge trial in pigs. Taken together, this project will provide important insights into the immune response to PRRSV glycoproteins to facilitate informed vaccine design.