A NON-HUMAN PRIMATE MODEL OF TICK-IMMUNITY

这个子项目是许多研究子项目中的一个 由NIH/NCRR资助的中心赠款提供的资源。子项目及 研究者（PI）可能从另一个NIH来源获得了主要资金， 因此可以在其他CRISP条目中表示。所列机构为 研究中心，而研究中心不一定是研究者所在的机构。 肩突硬蜱是一种重要的节肢动物传播媒介，主要传播莱姆病、立克次体病、无形体病、巴贝虫病和蜱传脑炎等病原体。对于针对这些病原体的安全有效的疫苗存在未满足的需求。针对蜱虫载体的疫苗可能会针对蜱虫传播的多种病原体。脊椎动物宿主在反复蜱虫侵染后，在12-24小时内排斥蜱虫，并阻断病原体传播。针对关键蜱唾液抗原的宿主免疫力可能在蜱排斥和随后的病原体传播障碍中起着关键作用。虽然获得性蜱免疫的现象提供了一个机会，以确定关键的蜱喂养和病原体传播的唾液蛋白，动物模型，可以证明蜱免疫和可行的病原体传播是不可用的。 以莱姆病病原体伯氏疏螺旋体为重点，目前的试点提案评估了非人灵长类动物（NHP）是否可以作为这种动物模型，并加强了确定疫苗以阻断蜱虫喂养和病原体传播的努力。在特定目标1中，用10-20个无病原体I重复感染两个NHP（Macaca mulatta）三次。肩胛蜱，每次蜱虫侵染之间有三周的休息期。在杜兰国家灵长类动物研究中心，在资助期的前6个月进行了蜱虫放置优化和蜱虫侵染研究。 尽管受动物数量的限制，但该初步研究并未揭示获得性蜱免疫的特征。在所有侵染中，蜱虫附着和蜱虫重量相当，可见蜱虫成功充血。反复蜱虫侵染后，在蜱虫喂食部位未观察到显著发红。这些观察结果表明，NHP与小鼠一样，可能不会引起对蜱的抗性。然而，蜱喂食部位皮肤活检的组织学分析和从这些蜱感染动物获得的血清对蜱唾液抗原的反应性将是必不可少的。这些分析尚待进行，目前正在耶鲁大学比较医学和传染病科进行。在特定目标2中，我们用10只疏螺旋体感染的肩突硬蜱攻击来自目标1的两种反复蜱虫侵染的NHP。用疏螺旋体感染的蜱对两个未处理的NHP进行类似的攻击。在两组中观察到相当的蜱虫充血。两组中喂食蜱的中肠和唾液腺中的疏螺旋体负荷也相当。 然而，在第2、4、6和8周的NHP的皮肤活检培养物显示，在反复感染蜱虫的实验组中没有活的螺旋体。在对照组中，1只动物显示出活螺旋体。这表明，在反复感染，NHP可能会引发体液免疫对蜱唾液抗原的病原体传播的关键。在8周结束时处死所有动物，并对与莱姆病相关的组织进行尸检以进行RNA和DNA分析。这些组织正在耶鲁大学传染病科进行螺旋体负荷的定量评估，研究即将完成。 这些结果将证实NHP对蜱虫侵染的反应，并揭示NHP模型在检查蜱虫取食和病原体传播的关键蜱虫抗原方面的实用性。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Ixodes scapularis is an important arthropod vector for pathogens responsible for Lyme disease, rickettsial disease, anaplasmosis, babesiosis, and tick borne encephalitis. There is an unmet need for safe and effective vaccines against these pathogens. Vaccines directed against the tick vector would potentially target multiple pathogens transmitted by the tick. The vertebrate host, upon repeated tick infestation, rejects ticks within 12-24 h and also blocks pathogen transmission. Host immunity directed against crucial tick salivary antigens presumably plays a pivotal role in tick rejection and in the consequent impairment of pathogen transmission. While the phenomenon of acquired tick immunity provides an opportunity to define salivary proteins critical for tick feeding and pathogen transmission, animal models that can demonstrate both tick immunity and viable pathogen transmission are not available. With a focus of Borrelia burgdorferi, the agent of Lyme disease, the current pilot proposal assessed if non-human primates (NHP) might serve such an animal model and enhance efforts to identify vaccines to block tick feeding and pathogen transmission. In Specific Aim 1, two NHPs (Macaca mulatta) were repeatedly infested three times with 10-20 pathogen-free I. scapularis ticks with a three-week resting period between each tick infestation. Optimization of tick placement, and tick infestation studies were conducted over the first 6 months of the funding period at the Tulane National Primate Research Center. The pilot study, albeit limited by the numbers of animals, did not reveal the hallmarks of acquired tick immunity. The ticks successfully engorged as seen by comparable tick attachment and tick weights at all infestations. No significant redness was observed at tick feeding sites upon repeated tick infestations. These observations suggested that NHPs, like mice, might not elicit resistance to ticks. However, histological analysis of the skin biopsies of the tick-feeding sites and reactivity of the sera obtained from these tick-infested animals to tick salivary antigens will be essential. These analyses are pending and are being conducted at the Sections of Comparative Medicine and Infectious Diseases, Yale University. In Specific Aim 2, we challenged the two repeatedly tick-infested NHPs from Aim 1 with 10 Borrelia-infected I.scapularis ticks. Two na¿ve NHPs were similarly challenged with Borrelia-infected ticks. Comparable tick engorgements were observed in both groups. Borrelia burden in the midguts and salivary glands of the fed ticks was also comparable in both groups. However, culture of skin biopsies of the NHPs at 2, 4, 6 and 8 weeks showed no viable spirochetes in the experimental group that was repeatedly infested with ticks. In the control group, one animal demonstrated viable spirochetes. This suggested that upon repeated infestation, NHPs might elicit humoral immunity against tick salivary antigens critical for pathogen transmission. All the animals were sacrificed at the end of 8 weeks and tissues relevant for Lyme disease necropsied for RNA and DNA analysis. These tissues are being processed for quantitative assessment of spirochete burden at the Section of Infectious Diseases, Yale University and studies nearing completion. These results will confirm the NHPs response to tick infestations and reveal the utility of the NHP model to examine tick antigens critical for tick feeding and pathogen transmission.