A NONHUMAN PRIMATE MODEL OF RICKETTSIA PROWAZEKII INFECTION (EPIDEMIC TYPHUS)

普瓦泽克立克次体感染（流行性斑疹伤寒）的非人类灵长类动物模型

• 批准号: 8173017
• 负责人: CHAD J. ROY
• 金额: $ 6.18万
• 依托单位: TULANE UNIVERSITY OF LOUISIANA
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-05-01 至 2011-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8173017
• 关键词: Accounting; Acute; Aerosols; Affect; Albumins; Animals; Anorexia; Appearance; Area; Arts; B-Lymphocytes; Biological; Blood Glucose; Blood Urea Nitrogen; Blood Vessels; Body Weight decreased; Brain; CD11 Antigens; CD14 gene; CD3 Antigens; CD8-Positive T-Lymphocytes; Cardiovascular system; Cell Count; Cells; Cerebral cortex; Characteristics; Chinese People; Chronic; Circadian Rhythms; Clinical; Clinical Chemistry; Computer Retrieval of Information on Scientific Projects Database; Data; Dendritic Cells; Development; Disease; Disease model; Dose; Drops; Edema; Environment; Epidemic; Euthanasia; Exposure to; Eye; Feces; Fever; Funding; Future; Globulins; Goals; Grant; Hematology; Homeostasis; Hour; Human; IL3RA gene; Immune; Immune response; Immunologics; Infection; Inflammation; Institution; Intravenous; Lesion; Lice; Liver; Lung; Lymphocyte; Lymphocyte Subset; MS4A1 gene; Macaca mulatta; Malaise; Measurement; Membrane; Mental Depression; Methodology; Microglia; Modeling; Monitor; Mucous body substance; Natural Killer Cells; Neuraxis; Nodule; Pathologic; Pathology; Physiological; Primates; Recording of previous events; Relative (related person); Reporting; Research; Research Personnel; Resources; Rickettsia prowazekii; Route; Safety; Source; Staining method; Stains; Symptoms; Syndrome; System; Temperature; Testing; Thick; Time; Tissues; Typhus; United States National Institutes of Health; Up-Regulation; Vaccines; Vasculitis; Weight; base; body system; calreticulin; dosage; experience; human disease; improved; lymphoid hyperplasia; model development; monocyte; neutrophil; new product development; nonhuman primate; peripheral blood; protective efficacy; response; trend; vascular inflammation; vector; weapons

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. Rickettsia prowazekii is the causative agent of epidemic typhus, a vector (louse)-borne disease characterized by a sudden onset of febrile symptoms with significant pathologic sequelae. In addition to causing epidemic disease in poor and unhygienic environments, R. prowazekii has also been identified as possessing the characteristics of a biological weapon. There presently is no vaccine available for protection against epidemic typhus, although development of new products is currently underway. A fully characterized primate model of disease is needed to determine immune response, safety, and protective efficacy of vaccine products developed to protect against R. prowazekii infection. The goal of this project is to develop a robust nonhuman model of disease for epidemic typhus in order to accelerate testing of newly developed vaccine products. The specific hypothesis is that use of the Rhesus macaque (Macaca mulatta) of Chinese origin will provide an optimal test system for an infection model of epidemic typhus. We base this hypothesis upon the observations from limited past studies that detailed 1) a clear dose-response relationship in prior model development efforts with the Breinl strain using Rhesus macaques when directly compared to experimental infection with avirulent strains, and 2) typical pathology in the form of disseminated vasculitis including typhus nodules in the central nervous system that emulated the human clinical syndrome. Based on these observations, the experimental focus of this proposal is on the model development of epidemic typhus in the Rhesus macaque. The specific aims are to: 1. Reestablish a nonhuman primate model for intravenous R. prowazekii infection. Studies over 25 years ago reported on model development of R. prowazekii infection in the nonhuman primate without the benefit of advances in immunologic and physiologic measurement. We will utilize the state of the art methodology during experimental infection to achieve precise account of biological response in infected animals. This will reestablish the model in the context of immune and pathologic response for future nonhuman primate studies involving R. prowazekii infection. 2. Develop an aerosol challenge model R. prowazekii challenge. Rickettsia prowazekii, in addition to causing epidemic disease, is considered a biological threat agent because of its low dose, aerosol infectivity and history of weaponization. As such, new products must provide protective efficacy against a realistic aerosol challenge. Clinical and immunologic parameters obtained from the IV Rickettsia prowazekii infections will serve as a guide to development of the aerosol model of disease. The aerosol disease model will provide an optimal system for testing of products developed expressly for protective efficacy against potential mucosal challenge with R. prowazekii. Six rhesus macaques were exposed intravenously to Breinl strain of Rickettsia prowazekii. Three groups (n=2) were administered graduated doses from 2.5E+04 to 1.0E+06 PFU. All animals became clinically ill two to three days postexposure; noted signs included depression, anorexia, weakness, and general malaise. Changes in stool were also noted. Local edema and thick discharge was observed in the mucus membranes (eyes) of three of the four animals 96 hours postexposure. Clinical signs improved over the next 5-7 days postexposure with a return to clinically normal appearance thereafter. Mild anorexia was the only lasting clinical sign that endured for up to 14 days postexposure. Subsequently, all animals experienced significant weight loss postexposure of between 10-15% of preexposure weight. Weights of three of the four animals gradually returned to preexposure weights. Telemetric monitoring of animals showed acute core temperature spikes of up to +2.5 degrees centigrade in all animals for up to 96 hours postexposure, gradually returning to homeostasis, although without a return to normal diurnal variation. Gradual increase in core temperature was observed thereafter (from day 10-30) postexposure; low grade (+1.0-1.5 degrees centigrade) fever was observed in all animals (4/4) to day 30. Similarly, disturbances in cardiovascular rhythm was recorded. Clinical chemistries of peripheral blood showed initial decreases of globulin, blood urea nitrogen, and blood glucose in all animals exposed. Increases in albumin, abumin-globulin ratio, and calreticulin were also observed. All animals initially showed a dramatic drop in circulating neutrophils and monocytes and concurrent increase in lymphocytes +5 days postexposure. Neutrophils gradually increased +20 days postexposure, with lymphocytes and monocytes declining to below normal levels by the time of euthanasia. Exposed animals also showed a dose-dependent increase in CD3+ and CD8+ lymphocytes +10 days postexposure. Both animals administered 1.0E+06 PFU showed dramatic increases in CD20+ B cells relative to the lower dosage group; CD14+ monocytes also increased in these animals +10 days postexposure. Total NK cell counts increased in all animals exposed; CD11+ dendritic cells also increased in all animals while the CD123+ count of dendritic cells decreased. Histopathological analysis of tissues from infected animals indicated mild lymphoid hyperplasia and mild chronic inflammation throughout the major organ systems. Animals exposed to the higher inoculating dose (1.0E+06 PFU) showed both lymphoid hyperplasia and chronic inflammation in the brain, lung, and liver that was consistent with previously described pathology associated with typhus infection. Chronic focal inflammation and vascular lesions in the cerebral cortex were noted in both of these animals. Further analysis of the brain showed the presence of R. prowazekii in areas with vascular lesions and upregulating of CD163 on microglia. These stains were colocalized with Ab-CD163 which also indicated upregulation of CD163 in many of the microglia affected. The increased presence of the double-stained positive cells was observed as a dose dependent phenomenon. Thus far, the results from the model development studies are positive in that we have observed dose-dependent changes associated with experimental infection of R. prowazekii in rhesus macaques. The preliminary results from the IV exposures showed clinical illness characterized by many of the signs associated with the human disease, including core temperature and cardiovascular system alternation during active infection. Similarly, clinical chemistry and hematology values changed dramatically when infected, showing many of the same trends observed in previous studies involving R. prowazekii and nonhuman primates. Advanced analysis of peripheral blood also showed increases in particular lymphocyte subpopulations in response to infection. Pathologically, animals at the highest inoculating dose (1.0E+06 PFU) showed disease-specific chronic focal inflammation in the form of vascular lesions in the brain; a noted hallmark of R. prowazekii infection. Taken together, these preliminary results have shown that the rhesus macaque is susceptible to IV infection with R. prowazekii. These data will provide baseline infection data in future studies where IV exposure is appropriate and serve as a basis for model development studies employing an alternative route of exposure to study infection.

该子项目是利用该技术的众多研究子项目之一 资源由 NIH/NCRR 资助的中心拨款提供。子项目和 研究者 (PI) 可能已从 NIH 的另一个来源获得主要资金， 因此可以在其他 CRISP 条目中表示。列出的机构是 对于中心来说，它不一定是研究者的机构。 普瓦泽基立克次体是流行性斑疹伤寒的病原体，流行性斑疹伤寒是一种媒介（虱子）传播的疾病，其特征是突然出现发热症状并伴有明显的病理后遗症。 除了在恶劣和不卫生的环境中引起流行病外，普氏罗氏菌还被认为具有生物武器的特征。 目前尚无疫苗可用于预防流行性斑疹伤寒，但新产品的开发正在进行中。 需要一个完全表征的灵长类疾病模型来确定为预防普罗瓦泽基罗氏菌感染而开发的疫苗产品的免疫反应、安全性和保护功效。 该项目的目标是开发一种强大的流行性斑疹伤寒非人类疾病模型，以加速新开发的疫苗产品的测试。 具体假设是，使用中国原产的恒河猴（Macaca mulatta）将为流行性斑疹伤寒感染模型提供最佳的测试系统。 我们的这一假设基于过去有限研究的观察结果，这些研究详细说明了 1) 在使用恒河猴的 Breinl 菌株的先前模型开发工作中，与无毒菌株的实验感染直接比较时，存在明确的剂量反应关系；2) 典型的病理学形式为播散性血管炎，包括模拟人类临床综合征的中枢神经系统中的斑疹伤寒结节。 基于这些观察，本提案的实验重点是恒河猴流行性斑疹伤寒的模型开发。 具体目标是： 1. 重建普罗瓦泽基罗氏菌静脉感染的非人灵长类动物模型。 25 多年前的研究报告了非人类灵长类动物中普罗瓦泽基氏菌感染模型的开发，但没有受益于免疫学和生理学测量方面的进步。 我们将在实验感染期间利用最先进的方法来精确描述受感染动物的生物反应。 这将为未来涉及普罗瓦兹基菌感染的非人灵长类动物研究在免疫和病理反应的背景下重新建立模型。 2. 开发 R. prowazekii 挑战的气溶胶挑战模型。普瓦泽基立克次体除了引起流行病外，还因其低剂量、气溶胶传染性和武器化历史而被认为是生物威胁剂。 因此，新产品必须提供针对实际气溶胶挑战的防护功效。 从 IV 普瓦泽基立克次体感染获得的临床和免疫学参数将作为疾病气溶胶模型开发的指南。 气溶胶疾病模型将为测试专门开发的产品提供最佳系统，以防止普罗瓦泽基菌潜在的粘膜挑战的保护功效。 六只恒河猴通过静脉注射接触普氏立克次体 Breinl 株。 三组（n=2）接受从 2.5E+04 到 1.0E+06 PFU 的分级剂量。 所有动物在暴露后两到三天内都出现临床症状；值得注意的症状包括抑郁、厌食、虚弱和全身不适。 还注意到粪便的变化。 暴露后 96 小时，四只动物中的三只的粘膜（眼睛）观察到局部水肿和浓稠分泌物。 暴露后的 5-7 天内，临床症状有所改善，随后恢复到临床正常外观。 轻度厌食是唯一持续的临床症状，在暴露后可持续长达 14 天。 随后，所有动物在暴露后均经历了显着的体重减轻，为暴露前体重的 10-15%。 四只动物中三只的体重逐渐恢复到暴露前的体重。 对动物的遥测监测显示，在暴露后长达 96 小时内，所有动物的核心温度急剧峰值高达 +2.5 摄氏度，逐渐恢复稳态，但没有恢复正常的昼夜变化。 暴露后（第 10-30 天）观察到核心温度逐渐升高；到第 30 天，所有动物 (4/4) 均观察到低度发热（+1.0-1.5 摄氏度）。同样，记录了心血管节律紊乱。 外周血的临床化学显示所有暴露的动物的球蛋白、血尿素氮和血糖最初下降。 还观察到白蛋白、白蛋白-球蛋白比率和钙网蛋白增加。 所有动物最初都表现出循环中性粒细胞和单核细胞的急剧下降，并且在暴露后+5天淋巴细胞同时增加。 暴露后+20天，中性粒细胞逐渐增加，而在安乐死时淋巴细胞和单核细胞下降至正常水平以下。 暴露后10天后，暴露的动物还表现出CD3+和CD8+淋巴细胞的剂量依赖性增加。 与较低剂量组相比，给予 1.0E+06 PFU 的两只动物的 CD20+ B 细胞均显着增加；暴露后 10 天以上这些动物的 CD14+ 单核细胞也有所增加。 所有暴露的动物的 NK 细胞总数均增加；所有动物中的 CD11+ 树突状细胞也有所增加，而树突状细胞的 CD123+ 计数则减少。 对受感染动物组织的组织病理学分析表明，整个主要器官系统有轻度淋巴增生和轻度慢性炎症。 暴露于较高接种剂量（1.0E+06 PFU）的动物在脑、肺和肝脏中显示出淋巴增生和慢性炎症，这与先前描述的与斑疹伤寒感染相关的病理学一致。 在这两只动物的大脑皮层中都发现了慢性局灶性炎症和血管病变。 对大脑的进一步分析表明，在血管病变区域存在普罗瓦泽基菌，并且小胶质细胞上的 CD163 上调。 这些染色剂与 Ab-CD163 共定位，这也表明许多受影响的小胶质细胞中 CD163 上调。 观察到双染阳性细胞的存在增加是剂量依赖性现象。 到目前为止，模型开发研究的结果是积极的，因为我们观察到与恒河猴中普罗瓦泽基氏菌实验性感染相关的剂量依赖性变化。 静脉注射的初步结果显示，临床疾病的特征是许多与人类疾病相关的体征，包括活动性感染期间的核心温度和心血管系统变化。 同样，感染后临床化学和血液学值发生巨大变化，显示出许多与之前涉及普罗瓦泽基人和非人类灵长类动物的研究中观察到的相同趋势。 外周血的高级分析还显示，特定淋巴细胞亚群因感染而增加。 病理学上，最高接种剂量（1.0E+06 PFU）的动物表现出大脑血管病变形式的疾病特异性慢性局灶性炎症；普罗瓦泽基立克次体感染的一个显着特征。 综上所述，这些初步结果表明恒河猴容易受到普罗瓦泽基罗氏菌的静脉感染。 这些数据将为未来的研究提供基线感染数据，其中静脉暴露是适当的，并作为采用替代暴露研究感染途径的模型开发研究的基础。