key: cord-0265635-aw36siwv
authors: Woodruff, M.; Walker, T.; Truong, A.; Han, J.; Dixit, A.; Ramonell, R.; Runnstrom, M.; Rudoloph, M.; Khosroshahi, A.; Lee, F. E.-H.; Sanz, I.
title: Evidence of Persisting Autoreactivity in Post-Acute Sequelae of SARS-CoV-2 Infection
date: 2021-09-26
journal: nan
DOI: 10.1101/2021.09.21.21263845
sha: 26abd13ea06820c4982b204a4e0d98b4c24862f9
doc_id: 265635
cord_uid: aw36siwv

Recent studies have demonstrated the significant breadth of emerging autoreactivity in severe SARS-CoV-2 infection. Importantly, we have identified a relaxation of peripheral tolerance within early antibody secreting cells that emerge in patients with COVID-19 as important drivers of those responses. While often viral-specific, these extrafollicular-derived cells also display cross reactivity to autoantigens present in the inflammatory lung environment, and despite resolution of most autoreactivity within 6 months, they persisted in some patients. These results raise questions regarding autoreactive antibodies that arise during acute SARS-CoV-2 infection and their persistence in patients with symptoms in Post-Acute Sequelae of SARS-CoV-2 infection (PASC). Through clinical autoreactive antibody screening of 95 patients with PASC and no history of autoimmune disease, we identify significant autoreactive profiles in patients with ongoing symptoms post-recovery, with 80% of patients returning positive tests for at least one autoantigen, and 40% showing breaks in tolerance to 2 or more. Anti-nuclear antigen positivity was most common, displaying positivity in 63% of patients, however, positive tests were broad and included reactivities against carbamylated protein responses, RNA polymerase III, and phospholipids. We also identify patients with reactivity against dsDNA in the PASC cohort -- a reactivity not observed in acute infection even in the critically ill. These results demonstrate evidence of serum autoantibodies in patients who present to PASC clinics with persistent symptoms up 14 months following SARS-CoV-2 infection, and further confirm the growing linkage between COVID-19 and observed clinical autoreactivity -- even into the recovery phase of disease.

Recent studies have demonstrated the significant breadth of emerging autoreactivity in severe SARS-CoV-2 infection [1] [2] [3] . More specifically, we have identified a relaxation of peripheral tolerance within early antibody secreting cells that emerge in patients with COVID-19 as important drivers of those responses 2 . While often viral-specific, these extrafollicular-derived cells 4 also display cross reactivity to autoantigens present in the inflammatory lung environment including glomerular basement membrane and naïve B cell reactivity. Antinuclear antibodies (ANA) and anti-carbamylated protein (CarP) responses were highly represented (42% and 44%, respectively), and despite resolution of most autoreactivity within 6 months, they persisted in some patients 2 . These results raise questions regarding autoreactive antibodies that arise during acute SARS-CoV-2 infection and their persistence in patients with symptoms in Post-Acute Sequelae of SARS-CoV-2 infection (PASC) 5,6 .

To explore the possibility that autoreactivity is over-represented in patients diagnosed with PASC, 95 patients with heterogeneous symptomology were consecutively recruited upon presentation to two academic PASC clinics in Atlanta, Georgia, USA during December 2020-June 2021. Enrollees had a median age of 50 years (range 21-81), 72 (76%) were female, and the majority were African American (58%) ( Table 1) . Fifty-five (58%) had mild acute COVID-19 with the remaining requiring hospitalization. At the time of sampling, patients were a median of 110 days (range 22-446) from COVID-19 onset, with the most common self-reported PASC symptoms included dyspnea (69%), fatigue (64%), and brain fog (47%) ( Table 1) . Patients who were suspected or diagnosed with rheumatic diseases prior to COVID-19 diagnosis were excluded from the cohort.

The ninety-five participants consenting to enrollment had a median age of 50 years (range 21-81), 72 (76%) were female, and the majority were African American (58%) ( Table 1) . Fifty-five (58%) had mild acute COVID-19 with the remaining requiring hospitalization. At the time of sampling, patients were a median of 110 days (range 22-446) from COVID-19 onset, with the most common self-reported PASC symptoms included dyspnea (69%), fatigue (64%), and brain fog (47%) ( Table 1) . Patients who were suspected or diagnosed with rheumatic diseases prior to COVID-19 diagnosis were excluded from the cohort.

Similar to the patients hospitalized with COVID-19, 80% of PASC patients tested positive for reactivity against at least one autoantigen, with elevated ANAs (63%) particularly prominent in the cohort. (Fig 1a) . Anti-CarP responses alongside reactivity against RNA polymerase 3 were also elevated with 16% and 8% of patients returning positive tests, respectively (Fig 1a,b) . Of interest, dsDNA was uniquely elevated in the PASC cohort, with 3 patients (3%) returning positive clinical tests and an additional 4 (7%, total) expressing titers exceeding 5 standard deviations of a matched healthy donor cohort (Fig 1a,c) . Multiple breaks in tolerance were common with more than a third of patients (40%) displaying tolerance breaks against 2 or more independent autoantigens (Fig 1d) .

In summary, these results demonstrate evidence of elevated serum autoantibodies in patients who present to PASC clinics with persistent symptoms up 14 months following SARS-CoV-2 infection. Whether these autoantibodies in patients early post-recovery with PASC correlate with long-term persistence of symptoms and eventual diagnosis of autoimmune disease will require further study. Nonetheless, these data further confirm the growing linkage between COVID-19 and observed clinical autoreactivity -even into the recovery phase of disease. Further, the identification of persistent autoreactive antibodies, identifiable through formal rheumatologic evaluation, should be routinely considered as possible contributing factors in the development and persistence of post-acute sequelae following COVID-19 recovery. 

Patients with PASC (n=95) were referred by primary care providers or by self-referral to Emory University Midtown, Emory University Executive Park, and Grady Memorial Hospital PASC Clinics. Adults aged ≥18 years with documented SARS-CoV-2 antigen or anti-nucleocapsid antibody (64%), or those meeting the CDC COVID-19 clinical case definition who were experiencing new or worsening symptoms and were >14 days from COVID-19 onset (36%) were eligible. Sociodemographic, comorbidity, acute COVID-19, and PASC symptom data were collected by patient report through a review of systems and confirmed through medical record review.

Peripheral blood was collected in either heparin sodium tubes (PBMCs) or serum tubes (serum; both BD Diagnostic Systems). Study data were collected and managed using REDCap electronic data capture tools hosted at Emory University.

For autoimmune biomarker analysis, frozen plasma was shipped on dry ice to Exagen, Inc. All rights reserved. No reuse allowed without permission. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this this version posted September 26, 2021. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this this version posted September 26, 2021. ; https://doi.org/10.1101/2021.09.21.21263845 doi: medRxiv preprint

Diverse functional autoantibodies in patients with COVID-19

Relaxed peripheral tolerance drives broad de novo autoreactivity in severe

New-onset IgG autoantibodies in hospitalized patients with COVID-19