Project Summary/Abstract At least 7 million people have died from COVID-19 with > half the world likely infected and a staggering burden of Post-Acute Sequelae of COVID-19 (PASC) affecting ~20M Americans with long-term, often crippling, symptoms including neurological symptoms (“brain fog”, memory and/or concentration disruptions), autonomic dysfunction, fatigue, flu-like symptoms, post-exertional malaise, and cardiovascular issues. PASC may develop in 50-70% of hospitalized and ~10-12% of non-hospitalized COVID-19 patients, with considerable personal, medical and economic burden. Although PASC is now well-described clinically, the immune mechanisms are poorly-understood and treatments are limited. One hypothesis is that persisting SARS-CoV-2 virus and/or antigen is involved. Alternative hypotheses that are not mutually exclusive include: (i) autoreactivity; (ii) persisting immune dysregulation; (iii) reactivation of latent viruses including EBV. Indeed, viral RNA/antigen is more likely to be found in PASC than healthy recovered individuals. Some groups have also reported EBV reactivation in PASC suggesting perhaps a connection between persisting SARS-CoV-2 and reactivation of other latent viruses. Persisting viral infections can cause immunopathogenesis and ongoing innate or adaptive immune stimulation. However, how SARS-CoV-2 persistence or reactivating latent viruses are involved and the role of persisting immune stimulation in PASC is unclear. We postulate that chronic stimulation of antigen-specific CD8 T cells, GC-dependent CD4 T cells and B cells, and/or myeloid cells occurs in PASC and drives pathogenesis, but also that these perturbations can be harnessed like immune biosensors to dissect disease mechanisms. Examining immune perturbations of chronic viral infections has been a hallmark of this CCHI U19 Program since its inception and is a strong foundation to address these questions. We hypothesize that PASC is associated with durable immune dysregulation linked to viral/antigen persistence, reactivation of latent viruses, and/or chronic inflammation. We will address these questions through deep interrogation of immune responses. In Aim 1, we will test whether altered CD8 T cell responses in PASC reflect exposure to persistent antigen, long-lasting inflammation or altered CD8 T cell activation during acute infection. In Aim 2, we will examine whether altered or misdirected germinal center (GC) responses in PASC implicate persisting antigen or “distraction” associated with altered inflammation and/or autoimmunity. In Aim 3, we will identify PASC-associated alterations in the myeloid cell compartment connected with changes in adaptive immune responses. This work will reveal new concepts about persisting/chronic infections with implications for therapeutics for PASC and possibly other post- acute infection sequelae. Project 1 capitalizes on a long history of expertise in our U19 Program on chronic viral infections and will thus draw on Proj...