New approaches to a livestock trypanosomiasis vaccine: targeting the bite-site by immunization with novel metacyclic-stage parasite antigens

我们将表达新发现的针对感染期细胞表面的寄生虫蛋白的重组形式，并检测它们保护接种疫苗的小鼠免受非洲锥虫感染的能力。非洲动物锥虫病(AAT)是一种由媒介传播的血液寄生虫(锥虫和间日锥虫)引起的牲畜疾病，在37个撒哈拉以南国家流行。AAT会导致慢性贫血和肌肉萎缩，如果不治疗会导致死亡。联合国粮农组织认为，AAT是非洲与贫困作斗争的核心，每年有5000万头牛处于危险之中，农业生产力损失数十亿美元。解决AAT是一个巨大的挑战，因为杀锥虫的药物经常会引起寄生虫耐药性，媒介控制通常是不可持续的，而疫苗也是不可用的，因为锥虫的表面被可变表面糖蛋白(VSG)覆盖。通过抗原变异对活性VSG进行连续替换，可以使寄生虫种群无限期地逃避宿主免疫。抗原变异是联系AAT生理和流行病学的共同线索，也是了解和预防疾病的关键。然而，疫苗仍然是AAT最可持续的解决方案，因此，发现能够激发保护性免疫的不变寄生虫抗原是当务之急。十年的锥虫基因组学和基因表达分析彻底改变了我们对抗原图景的理解。我们的工作已经确定了在苍蝇口器的后循环生命阶段中唯一表达的寄生虫特异性蛋白。我们已经证明，亚环蛋白的结构是独特的，与VSG主导的血流表面不同，它形成了锥体。这些阶段特定的蛋白质通常是不变的，并产生强烈的免疫反应。我们的假设是，用这些偏环抗原免疫可以为宿主免疫系统提供一个对抗感染的‘常备起点’，产生抗体，在偏环素迁移到血液中之前中和它们，更重要的是，在它们变得抗原性变异之前。我们的目标是通过评估一组新抗原的保护特性和保护相关性来测试这一点。我们的动机是将这些知识转化为有效的疫苗接种策略。在这个项目中，我们的方法有几个创新。首先，我们检查宿主和寄生虫在咬伤部位的相互作用，免疫反应就是在这里启动的。其次，我们关注在抗原变异开始之前的锥虫生命周期的后循环阶段。第三，我们使用了一个自然的挑战模型，该模型产生了真正的感染启动。我们有四个具体目标。1)重组蛋白在多种组合中的表达。2)用不同的抗原制剂给小鼠接种疫苗。3)通过采采虫感染小鼠，比较接种和对照动物的感染情况。4)利用小鼠白细胞和寄生虫的转录组分析咬合部位周围的基因表达，以确定保护的相关性，同时提供感染最初几天宿主-寄生虫相互作用的首次全球分析。该项目将为实验性多价疫苗识别抗原，同时建立保护的免疫学关联。除了将这一新知识立即应用于AAT疫苗的疗效试验外，它还将改变我们对叮咬部位宿主-寄生虫相互作用的理解。AAT疫苗的潜在商业应用是巨大的，可能对世界各地的动物健康和牲畜生产力产生的积极影响将是深远的。作为世界上最严重的兽医疾病之一，AAT的可持续解决方案将是一项开创性的突破，有助于改善世界上最贫穷国家的健康和财富。

We will express recombinant forms of newly-discovered, parasite proteins that are specific to the surface of infective-stage cells, and examine their ability to protect vaccinated mice against infection by African trypanosomes.African animal trypanosomiasis (AAT) is a livestock disease caused by vector-borne, blood parasites (Trypanosoma congolense and T. vivax), and endemic in 37 sub-Saharan countries. AAT causes chronic anaemia and muscle wastage, resulting in death if untreated. The UN FAO considers AAT to "lie at the heart of Africa's struggle against poverty" with 50 million cattle at risk and billions of dollars lost in agricultural productivity annually. Resolving AAT is an enormous challenge because trypanocidal drugs frequently solicit parasite resistance, vector control is typically unsustainable, while vaccines are unavailable because the trypanosome surface is enveloped by a variable surface glycoprotein (VSG) coat. Serial replacement of the active VSG through antigenic variation allows the parasite population to evade host immunity indefinitely. Antigenic variation is the common thread linking the physiology and epidemiology of AAT, and key to understanding and preventing disease.Nevertheless, a vaccine remains the most sustainable solution to AAT, and thus, discovering invariant parasite antigens that elicit protective immunity is a priority. A decade of trypanosome genomics and gene expression analysis has revolutionized our understanding of the antigenic landscape. Our work has identified parasite-specific proteins expressed uniquely during the metacyclic life stage in the fly mouthparts. We have shown that metacyclic protein architecture is distinct, and unlike the VSG-dominated surface of bloodstream form trypanosomes. These stage-specific proteins are typically invariant and produce strong immune responses. Our hypothesis is that immunizing with these metacyclic antigens can provide the host immune system with a 'standing-start' in fighting infection, producing antibodies that neutralize metacyclics before they migrate into the bloodstream and, critically, before they become antigenically variant. We aim to test this by evaluating a panel of novel antigens for their protective properties and correlates of protection.Our motivation is to translate this knowledge into an effective vaccination strategy. In this project, our approach has several innovations. First, we examine host-parasite interactions at the bite site, where the immunological response initiates. Second, we focus on the metacyclic stage of the trypanosome life cycle prior to the onset of antigenic variation. Third, we use a natural challenge model that produces an authentic initiation to infection. We have four specific objectives. 1) Recombinant protein expression in a range of combinations. 2) Vaccinate mice using different antigen formulations. 3) Infect mice via infected tsetse bite and compare the infection in vaccinated and control animals. 4) Profile gene expression around the bite site using transcriptomics of mouse leucocytes and parasites to identify correlates of protection, while providing the first global analysis of host-parasite interactions during the first days of infection.This project will identify antigens for an experimental multivalent vaccine against metacyclic T. congolense and T. vivax, while establishing the immunological correlates of protection. Besides the immediate application of this new knowledge to efficacy trials of an AAT vaccine, it will also transform our understanding of host-parasite interactions at the bite site. The potential commercial application of AAT vaccines is enormous, and the likely positive effect on animal health and livestock productivity across the world would be profound. As one of the world's foremost veterinary diseases, a sustainable solution to AAT would be a seminal breakthrough leading to improved health and wealth in the world's poorest countries.