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

修订标题： 加州国家灵长类动物研究中心开发非人灵长类动物长COVID模型 修订摘要： SARS-CoV-2是导致冠状病毒病19（COVID-19）的病毒，是一种急性呼吸道感染，超过90%的感染者会痊愈。然而，已报告了多器官受累的疾病的第二个长期阶段。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，通常以持续疲劳和脑雾为特征。被称为“长COVID”或“COVID-19长-呼吸综合征”，临床上称为SARSCoV-2感染的急性后后遗症，症状涉及肺、心脏、血管和中枢神经系统。其潜在机制尚不清楚，但免疫激活/失调加上SARS-CoV-2病毒残留可能在慢性炎症中发挥作用，导致对其他健康细胞、组织和器官的损伤。本申请的总体目标是确定SARS-CoV-2感染的恒河猴是否可以发展出重现人类长期COVID的持续性病理生理学。拟议研究的基本原理是基于患者出现慢性症状的报告，无论急性SARS-CoV-2感染的严重程度如何，以及对这种长期疾病状态的生物学基础的有限理解。因此，迫切需要了解Long COVID并开发可用于研究该综合征治疗策略的传染病动物模型。我们的次要目的是描述呼吸道和胃肠道中病毒脱落的动力学，以促进在受限的BSL 3设施之外的恒河猴模型中的长期研究。 修订后的具体目标： 由于肺、心脏和脑内的免疫病理学可以通过纵向功能测试和组织分析进行严格评估，因此迫切需要对COVID-19恒河猴模型进行研究，以补充临床数据。我们假设成年恒河猴从急性SARS-CoV-2感染中恢复后，将发展与心肺、免疫和神经系统相关的慢性炎症和功能缺陷。为了验证这一假设，我们将用SARS-CoV-2感染一组老年雄性恒河猴，并逐步监测动物的病毒脱落。将在使用高灵敏度测定确认病毒核酸清除后进行心血管和肺功能评估。为了描述SARS-CoV-2后的病毒学和免疫学应答如何影响心血管健康和肺功能的参数以指导Long COVID的临床表现，我们将完成以下具体目标：目标1：建立SARS-CoV-2感染后上呼吸道和下呼吸道以及胃肠道中病毒核酸持续存在和免疫激活的时空动力学。我们将在鼻道、远端肺和直肠中对SARS-CoV-2进行连续采样，以使用经验证的RT-PCR方法以及基于CRISPR的新型检测来量化病毒随时间的脱落程度。将在血液和肺灌洗液中进行免疫分析。目的2：评估SARS-CoV-2感染对心血管和肺功能的影响。我们将在基线和SARSCoV-2感染后10周和24周病毒核酸清除后评估动物的心血管指标（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。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/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.