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蜱传疾病。为了使蜱传疾病病原体在自然界中继续循环，需要成功地喂养蜱虫。调控早期蜱虫摄食的分子机制尚不明确。它们对于了解蜱虫获取和传播病原体至关重要，进而对设计控制蜱虫和蜱虫传播疾病的新方法也很重要。本研究的目的是在蜱取食前48小时注射到宿主体内的美洲双盲虫蜱唾液蛋白的分子鉴定和靶标验证。重点关注这一时间点的理由是，它先于蜱食、血食和蜱传疾病媒介传播的关键方面。假设蜱虫在进食的前48小时内将对宿主摄食成功和病原体感染至关重要的蜱虫唾液蛋白注射到宿主体内，阻断其功能可以保护动物免受蜱虫摄食和病原体感染。这一假设是基于初步的实验证据，即蜱虫或成年蜱虫在兔体内重复感染48小时，会产生保护性的蜱虫免疫，这是由蜱虫无法附着或保持附着在宿主皮肤上的死亡所揭示的。以前通过用单个蜱虫唾液重组蛋白免疫动物来获得保护性蜱虫免疫的努力产生了不同的结果。在这项研究中，我们提出了一种以前未开发的方法，用多表位嵌合蜱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将模仿在反复感染期间赋予蜱虫唾液蛋白抗原的保护性蜱免疫。有3个具体目标。首先是克隆编码美洲蜱唾液蛋白的cdna，这些蛋白在蜱虫进食后的48小时内被注射到宿主体内。二是验证蜱虫唾液蛋白中推定的抗原肽区域的免疫原性。经过验证的免疫原性肽将代表蜱虫唾液蛋白区域，该区域介导蜱虫在反复喂食蜱虫时的抗性，因此将代表潜在的蜱虫疫苗抗原。三是验证免疫原性肽鸡尾酒免疫家兔对蜱虫的免疫和免疫抗体反应。其基本原理是，如果用免疫原性肽鸡尾酒免疫兔子，可以获得与蜱虫反复侵扰相当的蜱虫免疫力，那么该应用程序的数据将为设计嵌合蜱虫唾液蛋白奠定基础。

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. PUBLIC HEALTH RELEVANCE: In the United States ticks transmit more vector borne disease agents than any other vector arthropod. Limitations associated with current acaricide based tick control strategies that threaten the future sustainability of containment programs for tick borne illnesses, have necessitated the need for development of alternative tick control strategies. Identification of important tick proteins that regulate tick physiology and facilitate tick feeding is important before alternative tick control methods can be developed.