Molecular Mechanisms Regulating Early Stage Tick Feeding

描述(由申请人提供)：在美国，报告的人类媒介传播疾病主要是由壁虱传播的。若要在自然界中持续循环传播硬虱传播的疾病病原体，则必须成功喂食硬蜱。调节早期扁虱摄食的分子机制尚不清楚。它们对于了解壁虱获取和传播病原体至关重要，而这反过来又对设计控制壁虱和壁虱传播疾病的新方法非常重要。这项建议的目标是对美洲Amblyomma americanum tick唾液蛋白进行分子鉴定和靶标验证，这些蛋白在最初的48小时内被注射到宿主体内。关注这一时间点的理由是，它先于蜱类进食、吸食血液和壁虱传播疾病的关键环节。假设在摄食的最初48小时内，对摄食成功和宿主的病原体感染起关键作用的唾液蛋白被注射到宿主体内，阻断它们的功能将保护动物免受扁虱摄食和病原体的感染。这一假说是基于初步的实验证据，即若虫或成虫反复感染兔48小时，可产生保护性的壁虱免疫，表现为壁虱未能附着或保持附着在宿主皮肤上的死亡率。以前通过用单一的硬虱唾液重组蛋白免疫动物来增强硬虱保护性免疫的努力取得了好坏参半的结果。在这项研究中，我们提出了一种以前从未探索过的方法，用多表位嵌合的硬虱唾液蛋白疫苗抗原免疫动物。这个想法是，对这些嵌合抗原的免疫力将模仿在反复感染期间赋予扁虱唾液蛋白抗原的保护性扁虱免疫。有三个具体目标。第一种方法是克隆编码美洲按蚊唾液蛋白的cDNA，这些唾液蛋白在取食扁虱的最初48小时内被注射到宿主体内。第二个目的是验证硬蜱唾液蛋白中可能的抗原肽区域的免疫原性。经过验证的免疫原肽将代表在反复取食硬蜱时介导硬蜱抵抗的硬蜱唾液蛋白区域，因此将代表潜在的硬蜱疫苗抗原。三是验证免疫原肽-鸡尾酒免疫的兔对蜱虫的免疫力和记忆抗体反应。其基本原理是，如果用免疫原肽-鸡尾酒免疫兔子可以获得与反复扁虱感染相媲美的扁虱免疫力，那么来自这一应用的数据将为设计嵌合的扁虱唾液蛋白奠定基础。

DESCRIPTION (provided by applicant): In the United States reported human vector-borne diseases are primarily tick-borne. For continued cycling of tick-borne disease pathogens in nature, successful tick feeding is required. Molecular mechanisms that regulate early stage tick feeding are poorly defined. They are critical to understanding the acquisition and transmission of pathogens by ticks, which in turn will be important in designing novel approaches to control ticks and tick- borne diseases. The goal of this proposal is molecular identification and target validation of Amblyomma americanum tick saliva proteins that are injected into the host during the first 48 h of tick feeding. The rationale to focus on this time point is that, it precedes the key facets of tick feeding, blood meal up take and tick borne disease agent transmission. The hypothesis is that tick saliva proteins critical to feeding success and pathogen infection of the host are injected into the host during the first 48 h of feeding and blocking their functions will protect animals against tick feeding and pathogen infection. This hypothesis is based on preliminary experimental evidence that repeated tick infestation of rabbits for 48 h with nymph or adult ticks conferred protective tick immunity as revealed by mortality failure of ticks to attach or remain attached onto host skin. Previous efforts to confer protective tick immunity by immunizing animals with single tick saliva recombinant proteins have produced mixed results. In this research we have proposed a previously unexplored approach, to immunize animals against tick feeding with multi-epitope chimeric tick saliva protein vaccine antigens. The idea is that immunity to these chimeric antigens will mimic protective tick immunity conferred tick saliva protein antigens during repeated infestations. There are 3 specific aims. The first is to clone cDNAs that encode A. americanum tick saliva proteins that are injected into the host during the first 48 h of tick feeding. The second is to validate immunogenicity of putative antigenic peptide regions in tick saliva proteins. Validated immunogenic peptides will represent tick saliva protein regions that mediate tick resistance in repeated tick feeding and will thus represent potential tick vaccine antigens. The third is to validate tick immunity and anamnestic antibody response of immunogenic peptide-cocktail immunized rabbits to tick infestation. The rationale is that if immunization of rabbits with immunogenic peptide-cocktails confers tick immunity that is comparable to that in repeated tick infestation, data from this application will set the foundation to design chimeric tick saliva proteins.