Arthropod saliva /midgut transcripts for vector vaccines

用于载体疫苗的节肢动物唾液/中肠转录本

• 批准号: 6987110
• 负责人: Jesus Valenzuela
• 金额: --
• 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/6987110
• 关键词: Leishmania; Psychodidae; antigen antibody reaction; arthropod borne communicable disease; biotechnology; complementary DNA; disease vectors; enzyme linked immunosorbent assay; flow cytometry; functional /structural genomics; hamsters; high throughput technology; host organism interaction; human tissue; laboratory mouse; microorganism antigen; nucleic acid sequence; polymerase chain reaction; proteomics; salivary glands; ticks; vaccine development; vector vaccine; western blottings

The Vector Molecular Biology Unit focuses on the molecular aspects of salivary and midgut transcripts and proteins in vector/host and vector/parasite interactions. We are interested in the use of salivary genes as vaccines to block parasite or pathogen transmission and in the use of midgut proteins as transmission blocking vaccines. We have previously identified and isolated a salivary gene from the sand fly Phlebotomus papatasi that confers protection against Leishmania major infection in mice. The approach to use vector salivary proteins or genes to block parasite infection may be applicable to other species of Leishmania and also to other vector borne diseases. By using high-throughput approaches we have isolated and sequenced a large number of cDNAs from various species of sand flies and from the tick Ixodes scapularis, the vector of Lyme disease. We have selected a number of these salivary genes and prepared DNA vaccines using a high-throughput cloning approach. We tested salivary DNA constructs from the sand flies P. argentipes, P. ariasi and L. longipalpis in animals and identified salivary proteins that produced a strong delayed skin response, salivary proteins that produce a strong antibody response and salivary proteins that produced both types of immune responses. Future work is directed to test these molecules for protection against pathogen transmission and to understand the mechanism of this protection. Another interest of the unit is the identification of the Leishmania major attachment site on the Phlebotomus papatasi sand fly midgut. Classical biochemical attempts have failed to identify and isolate this attachment site in the vector midgut. We have isolated and sequenced a large set of full-length genes expressed on the midgut of P. papatasi and identified a transcript coding for a tandem repeat galectin (galactose binding protein) and named PpGalec. Confocal microscopy studies demonstrated that PpGalec is expressed in the midgut surface of P. papatasi. Invitro and invivo binding experiments show that PpGalec is used by Leishmania major as a receptor for mediating specific binding to the insect midgut, an event that is crucial for parasite survival and that accounts for species-specific vector competence. These studies demonstrate the feasibility of using midgut receptors for parasite ligands as target antigens for transmission-blocking vaccines.

载体分子生物学单元专注于载体/宿主和载体/寄生虫相互作用中唾液和中肠转录物和蛋白质的分子方面。我们对使用唾液基因作为疫苗来阻止寄生虫或病原体传播以及使用中肠蛋白作为传播阻断疫苗感兴趣。我们之前已经从沙蝇白蛉（Phlebotomus papatasi）中鉴定并分离出一种唾液基因，该基因可以保护小鼠免受利什曼原虫感染。使用媒介唾液蛋白或基因来阻止寄生虫感染的方法可能适用于利什曼原虫的其他物种以及其他媒介传播的疾病。通过使用高通量方法，我们从各种白蛉和莱姆病媒介肩突蜱中分离出大量 cDNA，并对其进行了测序。我们选择了一些唾液基因，并使用高通量克隆方法制备了 DNA 疫苗。我们测试了动物体内白蛉 P. argentipes、P. ariasi 和 L. longipalpis 的唾液 DNA 构建体，并鉴定了产生强烈延迟皮肤反应的唾液蛋白、产生强烈抗体反应的唾液蛋白以及产生两种类型的唾液蛋白。免疫反应。未来的工作旨在测试这些分子对病原体传播的保护作用，并了解这种保护的机制。该单位的另一个兴趣是识别白蛉白蛉中肠上的利什曼原虫主要附着位点。经典的生化尝试未能识别和分离载体中肠中的这个附着位点。我们分离并测序了 P. papatasi 中肠上表达的大量全长基因，并鉴定了编码串联重复半乳糖凝集素（半乳糖结合蛋白）的转录本，并将其命名为 PpGalec。共聚焦显微镜研究表明，PpGalec 在 P. papatasi 的中肠表面表达。体外和体内结合实验表明，大型利什曼原虫使用 PpGalec 作为介导与昆虫中肠特异性结合的受体，这一事件对于寄生虫的生存至关重要，并且决定了物种特异性载体的能力。这些研究证明了使用寄生虫配体的中肠受体作为阻断传播疫苗的靶抗原的可行性。