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

克氏锥虫寄生虫集中在锥蝽昆虫的粪便中，并通过在昆虫叮咬处附近的沉积进入血液。当寄生虫进入血液时，它们会分化成一种复制形式，这种形式会将80kDa酶脯氨酸寡肽酶（Tc80）分泌到细胞外血液中，从而通过降解细胞外基质成分侵入哺乳动物细胞。重组Tc80最近已被证明在免疫小鼠中引起强烈的体液反应，这些小鼠也免受致命剂量的克鲁兹锥虫的侵害。Tc80在入侵过程中的绝对需要，以及它在不同克鲁兹锥虫菌株中的保守性，使该蛋白成为开发恰加斯阻断疫苗的令人兴奋的候选蛋白。目标1：确定抗Tc80对Tc80活性和寄生虫入侵的影响学生将使用生物物理技术来量化体外抗Tc80单抗的个体结合亲和力和动力学特性，同时使用克氏T.荧光菌株进行平行细胞检测，这将允许选择对寄生虫入侵有抑制作用的单抗。该项目的这一部分是与伦敦卫生和热带医学学院（LSHTM）的寄生虫学家John Kelly教授合作完成的。目标2：用特异性单克隆抗体确定Tc80和Tc80复合物的晶体结构。学生将利用目标1中筛选到的最佳单克隆抗体对Tc80及其复合物进行晶体结构的测定，从而揭示抗体表位识别的分子细节。这些信息将用于指导未来的免疫原设计（Campeotto等人，专利申请中）。Campeotto的团队可以定期访问钻石光源同步加速器设施（DLS，牛津，英国），学生将有可能接受PI和他在DLS的合作者Juan Sanchez-Weatherby博士的进一步培训。学生将基于从目标2中获得的结构信息，并基于生物信息学和建模分析，设计Tc80的硅免疫原。这些免疫原将被克隆、表达、纯化并结合到病毒样颗粒上，以促进小鼠体内广泛中和的单克隆抗体的产生2。随后，老鼠将被注射致死剂量的寄生虫。了解更多信息的参考资料：Bivona, a.e.等人（2018）。克氏锥虫80 kDa脯氨酸寡肽酶（Tc80）作为恰加斯病疫苗的新型免疫原热带病杂志，2012(3),444 - 444。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机会：粘，点击和粘合”。Front.Immunol, 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