Characterization of the rhesus macaque (Macaca mulatta) scrub typhus model: Susceptibility to intradermal challenge with the human pathogen Orientia tsutsugamushi Karp

BackgroundScrub typhus is an important endemic disease in tropical Asia caused by Orientia tsutsugamushi for which no effective broadly protective vaccine is available. The successful evaluation of vaccine candidates requires well-characterized animal models and a better understanding of the immune response against O. tsutsugamushi. While many animal species have been used to study host immunity and vaccine responses in scrub typhus, only limited data exists in non-human primate (NHP) models.Methodology/Principle findingsIn this study we evaluated a NHP scrub typhus disease model based on intradermal inoculation of O. tsutsugamushi Karp strain in rhesus macaques (n = 7). After an intradermal inoculation with 106 murine LD50 of O. tsutsugamushi at the anterior thigh (n = 4) or mock inoculum (n = 3), a series of time course investigations involving hematological, biochemical, molecular and immunological assays were performed, until day 28, when tissues were collected for pathology and immunohistochemistry. In all NHPs with O. tsutsugamushi inoculation, but not with mock inoculation, the development of a classic eschar with central necrosis, regional lymphadenopathy, and elevation of body temperature was observed on days 7–21 post inoculation (pi); bacteremia was detected by qPCR on days 6–18 pi; and alteration of liver enzyme function and increase of white blood cells on day 14 pi. Immune assays demonstrated raised serum levels of soluble cell adhesion molecules, anti-O. tsutsugamushi-specific antibody responses (IgM and IgG) and pathogen-specific cell-mediated immune responses in inoculated macaques. The qPCR assays detected O. tsutsugamushi in eschar, spleen, draining and non-draining lymph nodes, and immuno-double staining demonstrated intracellular O. tsutsugamushi in antigen presenting cells of eschars and lymph nodes.Conclusions/SignificanceThese data show the potential of using rhesus macaques as a scrub typhus model, for evaluation of correlates of protection in both natural and vaccine induced immunity, and support the evaluation of future vaccine candidates against scrub typhus.背景 恙虫病（Scrub typhus）是热带亚洲地区重要的地方性流行疾病，其病原体为恙虫病东方体（Orientia tsutsugamushi），目前尚无有效的广谱保护性疫苗可供临床应用。疫苗候选株的成功评估，亟需建立经过充分表征的动物模型，并深化对恙虫病东方体宿主免疫应答的认知。尽管已有多种动物模型被用于恙虫病的宿主免疫及疫苗应答研究，但目前非人灵长类（NHP）模型的相关研究数据仍较为匮乏。 研究方法/主要结果 本研究以7只恒河猴为实验对象，构建了基于皮内接种恙虫病东方体卡普（Karp）株的非人灵长类恙虫病感染模型。其中4只恒河猴于前大腿皮内接种10^6小鼠半数致死量的恙虫病东方体，剩余3只接种空白对照接种液；在接种后第28天采集组织样本用于病理学及免疫组织化学检测前，我们对所有实验动物开展了涵盖血液学、生化、分子生物学及免疫学检测的系列时序性研究。在所有接种恙虫病东方体的非人灵长类动物中（空白对照组未出现相关异常），于接种后7~21天观察到典型的中央坏死性焦痂、区域淋巴结肿大及体温升高现象；接种后6~18天可通过实时荧光定量PCR（qPCR）检测到菌血症；接种后14天则出现肝功能酶学指标异常及白细胞计数升高。免疫学检测结果显示，接种组恒河猴的血清可溶性细胞黏附分子水平、抗恙虫病东方体特异性抗体应答（IgM与IgG）及病原体特异性细胞免疫应答均显著上调。qPCR检测可在焦痂、脾脏、引流及非引流淋巴结中检出恙虫病东方体；免疫双染色实验证实，焦痂及淋巴结的抗原呈递细胞内存在胞内寄生的恙虫病东方体。 结论与意义 本研究数据证实，恒河猴可作为恙虫病研究模型，用于评估自然感染与疫苗诱导免疫中的保护相关标志物，同时为后续抗恙虫病疫苗候选株的研发与评估提供了坚实的实验依据。