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.

OVERVIEW NARRATIVE The CNPRC is an established national resource that has as a primary mission to improve human health and quality of life through support of exceptional nonhuman primate research programs that advance our knowledge of the causes, preventions, treatments, and cures of debilitating diseases. This mission is pursued through robust and well-developed on-site research programs, facilities, and expertise that are significantly augmented by collaborative programs that serve the greater biomedical research community.