Molecular approaches to understand vector-host and vector-pathogen interactions

了解载体-宿主和载体-病原体相互作用的分子方法

• 批准号: 8156952
• 负责人: Jesus Valenzuela
• 金额: $ 142.2万
• 依托单位: NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/8156952
• 关键词: Molecular; pathogen; vector

The accomplishment from the two main projects of the unit are: 1) Discovery of two sand fly salivary vaccine candidates that produce a strong and long lasting cellular immune response in dogs. Dogs are the main reservoir of visceral leishmaniasis in Europe and South America. We developed an experimental model that mimics the natural exposure of dogs to sand fly bites and explored the immune responses to salivary proteins from the sand fly Lutzomyia longipalpis, the vector of visceral leishmaniasis in South America. Lu. longipalpis salivary proteins induced a DTH response at the bite site in the skin of dogs following repeated exposures to sand flies. This skin response is a surrogate of cellular immune response and a first screen marker of protection against Leishmania infection in rodent models. Two of 35 salivary proteins (LJM17 and LJL143) from the vector sand fly Lutzomyia longipalpis, identified using a novel approach termed reverse antigen screening, elicited strong cellular immunity in dogs. Immunization with either molecule induced high IgG2 antibody levels and significant IFN- production following in vitro stimulation of PBMC with salivary gland homogenate. Importantly, saliva stimulated lymphocytes from immunized dogs efficiently killed Leishmania infantum chagasi within autologous macrophages. These findings suggest that inclusion of these salivary molecules in anti-Leishmania canine vaccines would enhance their efficiency in protecting dogs from visceral leishmaniasis. 2) Identification of biological activities from novel salivary molecules of unknown sequence or function. Most sand fly salivary proteins are novel and do not have an assigned biological function. Soluble recombinant proteins were produced in a mammalian expression system to test potential biological activities. We identified a potent serotonin binding protein, a potent anticoagulant, a tryptase inhibitor and an anti-complement protein from the saliva of L. longipalpis. These molecules have the potential for use in the treatment of a variety of human ailments. This work not only demonstrates that the anticoagulant inhibitor of sand flies is a novel prothrombinase inhibitor, but also makes it a potential active drug to correct pro-coagulant disorders in human medicine. C3b-inhibitors could be potential new drugs that correct human diseases related to complement such as auto-immune diseases and post-surgery complications due to complement. 3) Discovery of Markers of Exposure Specific to Bites of Lutzomyia longipalpis, the Vector of Leishmania infantum chagasi in Latin America Leishmania parasites are transmitted by the bite of an infected vector sand fly that injects salivary molecules into the host skin during feeding. Certain salivary molecules can produce antibodies and can be used as an indicator of exposure to a vector sand fly and potentially the disease it transmits. To identify potential specific markers of vector exposure, we produced nine different recombinant salivary proteins from Lu. longipalpis and tested for their recognition by individuals exposed to another human-biting sand fly, Lu. intermedia, that transmits cutaneous leishmaniasis and commonly occurs in the same endemic areas as Lu. longipalpis. Two salivary proteins (named LJM17 and LJM11) were recognized only by humans exposed to Lu. longipalpis, suggesting they are immunogenic proteins and may be useful in epidemiological studies. The identification of specific salivary proteins as potential markers of exposure to vector sand flies will increase our understanding of vector human interaction, bring new insights to vector control, and in some instances act as an indicator for risk of acquiring disease. 4) Characterization of the salivary proteins from the sand fly Phlebotomus arabicus. We performed a transcriptomic analysis of the old world sand fly Phlebotomus arabicus and analyzed the repertoire of molecules in the saliva of this insect, performed bioinformatics analyses and some biological assays to validate the presence of powerful biological activities and antigenic molecules.

该单位的两个主要项目的成果是： 1) 发现两种白蛉唾液候选疫苗，可在狗体内产生强烈且持久的细胞免疫反应。 狗是欧洲和南美洲内脏利什曼病的主要宿主。我们开发了一个实验模型，模拟狗自然接触白蛉叮咬的情况，并探索了对长须白蛉（南美洲内脏利什曼病的传播媒介）唾液蛋白的免疫反应。 鲁。在反复接触白蛉后，长须唾液蛋白在狗的皮肤咬伤部位诱导了 DTH 反应。这种皮肤反应是细胞免疫反应的替代品，也是啮齿动物模型中预防利什曼原虫感染的第一个筛选标志。 使用一种称为反向抗原筛选的新方法鉴定出来自载体白蛉 Lutzomyia longipalpis 的 35 种唾液蛋白中的两种（LJM17 和 LJL143），它们在狗中引发了强大的细胞免疫。在用唾液腺匀浆体外刺激PBMC后，用任一分子进行免疫均诱导高IgG2抗体水平和显着的IFN-产生。重要的是，唾液刺激免疫狗的淋巴细胞，可有效杀死自体巨噬细胞内的婴儿利什曼原虫。这些发现表明，将这些唾液分子包含在抗利什曼原虫犬疫苗中将提高其保护狗免受内脏利什曼病的效率。 2) 鉴定序列或功能未知的新型唾液分子的生物活性。 大多数白蛉唾液蛋白都是新颖的，没有指定的生物学功能。在哺乳动物表达系统中产生可溶性重组蛋白以测试潜在的生物活性。我们从长须乳杆菌唾液中鉴定出一种有效的血清素结合蛋白、一种有效的抗凝剂、一种类胰蛋白酶抑制剂和一种抗补体蛋白。这些分子具有用于治疗多种人类疾病的潜力。这项工作不仅证明了白蛉抗凝抑制剂是一种新型凝血酶原酶抑制剂，而且使其成为人类医学中纠正促凝血障碍的潜在活性药物。 C3b抑制剂可能是纠正与补体相关的人类疾病的潜在新药，例如自身免疫性疾病和补体引起的手术后并发症。 3) 利什曼原虫媒介 Lutzomyia longipis 叮咬特异性暴露标记的发现 拉丁美洲的恰加西婴儿 利什曼原虫通过受感染的白蛉叮咬传播，白蛉在进食时将唾液分子注入宿主皮肤。某些唾液分子可以产生抗体，可以用作暴露于白蛉媒介以及其传播的疾病的指标。为了识别载体暴露的潜在特异性标记，我们从 Lu 中产生了九种不同的重组唾液蛋白。 longipalpis 并测试了暴露于另一种咬人沙蝇 Lu 的个体的识别能力。中间型，传播皮肤利什曼病，通常发生在与 Lu 相同的流行地区。长触须。两种唾液蛋白（名为 LJM17 和 LJM11）仅能被暴露于 Lu 的人类识别。 longipalpis，表明它们是免疫原性蛋白质，可能在流行病学研究中有用。识别特定唾液蛋白作为接触媒介沙蝇的潜在标记将增加我们对人类媒介相互作用的理解，为媒介控制带来新的见解，并在某些情况下充当感染疾病风险的指标。 4)白蛉白蛉唾液蛋白的表征。 我们对旧世界沙蝇阿拉伯白蛉进行了转录组分析，并分析了这种昆虫唾液中的分子库，进行了生物信息学分析和一些生物测定，以验证强大的生物活性和抗原分子的存在。