Structural and functional characterisation of Trypanosoma cruzi antigens for drug design and vaccine applications

克氏锥虫的寄生虫集中在三叶虫的粪便中，并在昆虫叮咬附近沉积后进入血液。当寄生虫进入血液时，它们会分化成复制形式，分泌80 kDa的酶Pro寡肽酶(Tc80)到细胞外血液中，这允许哺乳动物细胞通过降解细胞外基质成分来入侵。重组Tc80最近被证明在免疫的小鼠中引起了强烈的体液反应，这些小鼠也受到了致死剂量的Tc80的保护。在入侵过程中对Tc80的绝对需求，以及它在不同品系中的保守性，使这种蛋白质成为开发查加斯阻断疫苗的一个令人兴奋的候选。博士目标：目标1：确定抗Tc80对Tc80活性和寄生虫入侵的影响学生将在体外使用生物物理技术来量化抗Tc80单抗的结合亲和力和运动特性，同时将使用T.ruzi荧光菌株进行平行的基于细胞的检测，这将允许选择单抗，它们对寄生虫的入侵有抑制作用。该项目的这一部分是与伦敦卫生与热带医学院(LSHTM)的寄生虫学家约翰·凯利教授合作完成的。目标2：确定Tc80和Tc80与特定单抗形成的复合体的晶体结构。学生将继续确定Tc80的晶体结构，并与目标1中筛选出的最佳单抗形成复合体，这将揭示抗体表位识别的分子细节。这些信息将被用来指导未来的免疫原设计(Campretto等人，正在申请专利)。卡普雷托的团队可以例行访问钻石光源同步加速器设施(DLS，牛津，英国)，学生将有可能在DLS接受PI及其合作者Juan Sanchez-Weatherby博士的进一步培训。目标3：根据从目标2获得的结构信息，基于生物信息学和模型分析，设计用于疫苗应用的新型免疫原分子，学生将在Tc80的电子免疫原中设计。这些免疫原将被克隆、表达、纯化并连接到病毒样颗粒上，以促进MICE 2中广泛中和单抗的产生。小鼠随后将被致死剂量的寄生虫挑战。参考了解更多信息：1.Bivona，A.E.等人。(2018)。克氏锥虫80 kDa脯氨酰寡肽酶(Tc80)作为恰加斯病疫苗的新免疫原。《公共科学图书馆·被忽视的热带病》，12(3)，e0006384-23。DOI：http://doi.org/10.1371/journal.pntd.00063842.Brune D.K.和Howarth M.(2018)。颗粒疫苗模块化构建的新途径和新机遇：棒状、点击和粘合Front.免疫，https://doi.org/10.3389/fimmu.2018.01432

T.cruzi parasites concentrate in the faeces of the triatomine insects and gain access to the blood stream upon deposition near the insect bite. When the parasites enter the bloodstream, they differentiate into a replicative form, which secretes the 80kDa enzyme prolyl oligopeptidase (Tc80) into the extracellular blood, which allows the invasion of mammalian cells by degrading their extracellular matrix components. Recombinant Tc80 has recently been shown to elicit a strong humoral response in immunised mice, which were also protected from a lethal dose of T. cruzi1 The absolute requirement for Tc80 in the invasion process, together with its conservation across different strains of T. cruzi, make this protein an exciting candidate for the development of a Chagas blocking vaccine.PhD objectives:Objective 1: Determine the effects of anti-Tc80 on Tc80 activity and parasite invasion The student will use biophysical techniques to quantify individual binding affinities and kineticproperties of anti-Tc80 mAbs in vitro, whilst in parallel cell-based assays will be performed using a T. cruzi fluorescent strain, which will allow to select mAbs, which have inhibitory effects on parasite invasion. This part of the project is in collaboration with parasitologist Prof. John Kelly at the London School of Hygiene and Tropical Medicine (LSHTM) in London.Objective 2: Determine the crystal structures of Tc80 and Tc80 in complex with specific mAbs The student will pursue the determination of the crystal structures of Tc80 and in complex with the best mAbs screened in objective 1, which will reveal the the molecular details of antibody-epitope recognition. This information will be exploited to guide future immunogen design (Campeotto et al., patent pending). Campeotto's group has routine access to Diamond Light Source synchrotron facilities (DLS, Oxford, UK) and the student will have the possibility of receiving further training at by the PI and by his collaborator at DLS, Dr Juan Sanchez-Weatherby. Objective 3: Design novel immunogenic molecules for vaccine applications The student will design in silico immunogens of Tc80 based on the structural information gained from objective 2 and based on bioinformatics and modelling analysis. These immunogens will be cloned, expressed, purified and conjugated to Virus-Like-Particles to boost the production of broadly neutralizing mAbs in mice2. Mice will be subsequently challenged with a lethal dose of the parasite.References to learn more:1. Bivona, A. E. et al. (2018). "Trypanosoma cruzi 80 kDa prolyl oligopeptidase (Tc80) as a novel immunogen for Chagas disease vaccine". PLOS Neglected Tropical Diseases, 12(3),e0006384-23. doi: http://doi.org/10.1371/journal.pntd.00063842. Brune D. K. and Howarth M. (2018). "New Routes and Opportunities for Modular Construction of Particulate Vaccines: Stick, Click, and Glue". Front.Immunol,https://doi.org/10.3389/fimmu.2018.01432