California National Primate Research Center Development of a Nonhuman Primate Model of Long COVID

修订后的标题： 加州国家灵长类动物研究中心长冠状病毒非人类灵长类动物模型的建立 修订摘要： SARS-CoV-2是一种引起冠状病毒病19(新冠肺炎)的病毒，是一种急性呼吸道感染，90%以上的感染者可治愈。然而，据报道，疾病的第二个延迟期与多器官受累。无论年龄或感染严重程度如何，近三分之一的康复者报告了一系列症状，通常以持续性疲劳和脑雾为特征。被称为“长冠状病毒病”或“新冠肺炎长时间拖拉机综合症”，在临床上被称为SARSCoV-2感染的急性后遗症，症状涉及肺、心脏、血管系统和中枢神经系统。潜在的机制尚不清楚，但免疫激活/失调加上SARS-CoV-2病毒残留物可能在慢性炎症中发挥作用，导致对原本健康的细胞、组织和器官的伤害。这项应用的总体目标是确定感染SARS-CoV-2的恒河猴是否能够发展出持续的病理生理学，概括为人类的长期COVID。建议进行这项研究的理由是，有报道称，无论SARS-CoV-2急性感染的严重程度如何，患者都会出现慢性症状，而且对这种长期疾病状态的生物学基础了解有限。因此，迫切需要了解慢性冠状病毒病，并开发一种可用于研究这种综合征的治疗策略的传染病动物模型。我们的次要目标是描绘病毒在呼吸道和胃肠道中脱落的动力学，以便于在受限的BSL3设施外对恒河猴模型进行长期研究。 修订后的具体目标： 新冠肺炎猕猴模型的研究是补充临床数据的关键，因为肺、心和脑内的免疫病理可以通过纵向功能测试和组织分析进行严格的评估。我们假设成年恒河猴在急性SARS-CoV-2感染后会出现慢性炎症和与心肺、免疫和神经系统相关的功能缺陷。为了验证这一假设，我们将给一群老年雄性恒河猴接种SARS-CoV-2病毒，并逐步监测动物是否有病毒脱落。在确认病毒核酸清除后，将使用高灵敏的分析方法进行心血管和肺功能的评估。为了阐明SARS-CoV-2感染后的病毒学和免疫学反应对心血管机能和肺功能参数的影响，以指导Long CoVID的临床表现，我们将完成以下具体目标：目的1：建立SARS-CoV-2感染后上下呼吸道和胃肠道病毒核酸持续和免疫激活的时空动力学。我们将在鼻道、远端肺和直肠进行SARS-CoV-2的连续采样，以使用经过验证的RT-PCR方法和一种基于CRISPR的新方法来量化病毒随时间的脱落程度。将在血液和肺灌洗中进行免疫分析。目的2：评价SARS-CoV-2感染对心肺功能的影响。我们将评估动物在基线和感染SARSCoV-2后10周和24周病毒核酸清除后的心血管测量(超声心动图、多普勒)和肺功能(静态肺力学)。目的3：确定SARS-CoV-2感染后接种疫苗对免疫、心血管和肺功能的影响。在病毒核酸清除后，我们将在感染SARS-CoV-2后10周接种疫苗并监测免疫激活/调节失调、心血管和肺功能。

Revised Title: California National Primate Research Center Development of a Nonhuman Primate Model of Long COVID Revised Abstract: SARS-CoV-2, the virus that causes coronavirus disease 19 (COVID-19), is an acute respiratory infection which resolves in more than 90% of infected individuals. However, a second protracted phase of disease with multiorgan involvement has been reported. Regardless of age or infection severity, nearly one-third of recovered individuals report a constellation of symptoms often characterized by persistent fatigue and brain fog. Referred to as “Long COVID” or the “COVID-19 long-hauler syndrome” and clinically as Post-Acute Sequelae of SARSCoV-2 infection, symptoms involve the lungs, heart, vasculature, and the central nervous system. The underlying mechanisms are unknown, but immune activation/dysregulation coupled with SARS-CoV-2 viral remnants may play a role in chronic inflammation resulting in injury to otherwise healthy cells, tissues, and organs. The overall goal of this application is to determine if the SARS-CoV-2 infected rhesus macaque can develop persistent pathophysiology that recapitulates Long COVID in humans. The rationale for the proposed study is based upon reports of patients who develop chronic symptoms regardless of acute SARS-CoV-2 infection severity and a limited understanding of the biological basis for this prolonged disease state. There is therefore an urgent need to understand Long COVID and develop an animal model of infectious disease that can be used to investigate therapeutic strategies for this syndrome. Our secondary objective is to delineate the kinetics of virus shedding in the respiratory and gastrointestinal tract to facilitate long-term studies in the rhesus model outside restricted BSL3 facilities. Revised Specific Aims: Studies in the COVID-19 rhesus macaque model are critically needed to complement clinical data since the immunopathology within the lung, heart, and brain can be rigorously assessed by longitudinal functional testing and tissue analysis. We hypothesize that adult rhesus macaques will develop chronic inflammation and functional deficits associated with the cardiopulmonary, immune and nervous systems following recovery from acute SARS-CoV-2 infection. To test this hypothesis, we will inoculate a cohort of aged male rhesus macaques with SARS-CoV-2 and progressively monitor animals for viral shedding. Assessment of cardiovascular and lung function will be conducted following confirmation of viral nucleic acid clearance using highly sensitive assays. To delineate how virological and immunological responses following SARS-CoV-2 impact parameters of cardiovascular fitness and lung function to direct the clinical manifestations of Long COVID, we will complete the following Specific Aims: Aim 1: Establish spatiotemporal kinetics of viral nucleic acid persistence and immune activation in the upper and lower respiratory tract, and gastrointestinal tract following SARS-CoV-2 infection. We will conduct serial sampling for SARS-CoV-2 in nasal passages, distal lung, and rectum to quantify the degree of viral shedding over time using validated RT-PCR methods as well as a novel CRISPR based assay. Immune profiling will be conducted in blood and lung lavage. Aim 2: Assess the impact of SARS-CoV-2 infection on cardiovascular and pulmonary function. We will assess animals for cardiovascular measures (echocardiogram, doppler) and lung function (static lung mechanics) at baseline and following viral nucleic acid clearance at 10- and 24-weeks post-infection with SARSCoV-2. Aim 3: Determine the impact of vaccination following SARS-CoV-2 infection on immune, cardiovascular and pulmonary function. Following viral nucleic acid clearance, we will vaccinate and monitor immune activation/dysregulation, cardiovascular and lung function at 10-weeks post-infection with SARS-CoV-2.