Some studies suggest that the risk of cardiovascular problems, such as a heart attack or stroke, remains high even many months after a SARS-CoV-2 infection clears up. Researchers are starting to pin down the frequency of these issues and what is causing the damage.

SARS-CoV-2 infection can result in the development of a constellation of persistent sequelae following acute disease called post-acute sequelae of COVID-19 (PASC) or Long COVID[1][1]–[3][2]. Individuals diagnosed with Long COVID frequently report unremitting fatigue, post-exertional malaise, and a variety of cognitive and autonomic dysfunctions[1][1]–[3][2]; however, the basic biological mechanisms responsible for these debilitating symptoms are unclear. Here, 215 individuals were included in an exploratory, cross-sectional study to perform multi-dimensional immune phenotyping in conjunction with machine learning methods to identify key immunological features distinguishing Long COVID. Marked differences were noted in specific circulating myeloid and lymphocyte populations relative to matched control groups, as well as evidence of elevated humoral responses directed against SARS-CoV-2 among participants with Long COVID. Further, unexpected increases were observed in antibody responses directed against non-SARS-CoV-2 viral pathogens, particularly Epstein-Barr virus. Analysis of circulating immune mediators and various hormones also revealed pronounced differences, with levels of cortisol being uniformly lower among participants with Long COVID relative to matched control groups. Integration of immune phenotyping data into unbiased machine learning models identified significant distinguishing features critical in accurate classification of Long COVID, with decreased levels of cortisol being the most significant individual predictor. These findings will help guide additional studies into the pathobiology of Long COVID and may aid in the future development of objective biomarkers for Long COVID.

### Competing Interest Statement

In the past three years, H.K. received expenses and/or personal fees from UnitedHealth, Element Science, Aetna, Reality Labs, Tesseract/4Catalyst, F-Prime, the Siegfried and Jensen Law Firm, Arnold and Porter Law Firm, and Martin/Baughman Law Firm. He is a co-founder of Refactor Health and HugoHealth, and is associated with contracts, through Yale New Haven Hospital, from the Centers for Medicare & Medicaid Services and through Yale University from Johnson & Johnson. A. I. consults for 4BIO Capital, BlueWillow Biologics, Healthspan Technologies, Revelar Biotherapeutics, RIGImmune, and Xanadu Bio. A.M.R. is an inventor of a patent describing the REAP technology. A.M.R. is the founder of Seranova Bio and holds equity in Seranova Bio.

### Funding Statement

This work was supported by grants from National Institute of Allergy and Infectious Diseases (R01AI157488 to A.I.), FDA Office of Womens Health Research Centers of Excellence in Regulatory Science and Innovation (CERSI) (to A.I.), Fast Grant from Emergent Ventures at the Mercatus Center (to A.I.), RTW Foundation (to D.P.), the Howard Hughes Medical Institute Collaborative COVID-19 Initiative (to R.M. and A.I.), and the Howard Hughes Medical Institute (to A.I. and R.M.).

### Author Declarations

I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained.

Yes

The details of the IRB/oversight body that provided approval or exemption for the research described are given below:

This study was approved by the Mount Sinai Program for the Protection of Human Subjects (IRB #20-01758) and Yale Institutional Review Board (IRB #2000029451 for MY-LC; IRB #2000028924 for enrollment of pre-vaccinated Healthy Controls). Informed consent was obtained from all enrolled participants.

I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals.

Yes

I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance).

Yes

I have followed all appropriate research reporting guidelines and uploaded the relevant EQUATOR Network research reporting checklist(s) and other pertinent material as supplementary files, if applicable.

Yes

All data produced in the present study are available upon reasonable request to the authors.

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