Persistent complement dysregulation with signs of thromboinflammation in active Long Covid

Abstract

Long Covid is a debilitating condition of unknown etiology. We performed multimodal proteomics analyses of blood serum from COVID-19 patients followed up to 12 months after confirmed severe acute respiratory syndrome coronavirus 2 infection. Analysis of >6500 proteins in 268 longitudinal samples revealed dysregulated activation of the complement system, an innate immune protection and homeostasis mechanism, in individuals experiencing Long Covid. Thus, active Long Covid was characterized by terminal complement system dysregulation and ongoing activation of the alternative and classical complement pathways, the latter associated with increased antibody titers against several herpesviruses possibly stimulating this pathway. Moreover, markers of hemolysis, tissue injury, platelet activation, and monocyte–platelet aggregates were increased in Long Covid. Machine learning confirmed complement and thromboinflammatory proteins as top biomarkers, warranting diagnostic and therapeutic interrogation of these systems.

Editor’s summary

Some individuals can endure persistent, debilitating symptoms for many months after an initial severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. However, the factors underpinning these health issues, called Long Covid, are poorly understood. Comparing the blood of patients with confirmed SARS-CoV-2 infection with that of uninfected controls, Cervia-Hasler et al . found that patients experiencing Long COVID exhibited changes to blood serum proteins indicating activation of the immune system’s complement cascade, altered coagulation, and tissue injury (see the Perspective by Ruf). At the cellular level, Long Covid was linked to aggregates comprising monocytes and platelets. These findings provide a resource of potential biomarkers for diagnosis and may inform directions for treatments. —Sarah H. Ross

Abstract

Analysis of blood revealed that Long COVID is characterized by changes to complement proteins and platelet activation.

Abstract

INTRODUCTION

Acute infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes various clinical phenotypes, ranging from asymptomatic to life-threatening COVID-19. About 5% of all infected individuals do not recover from acute disease but develop long-term complications, called Long Covid. Current hypotheses on factors contributing to Long Covid include tissue damage, viral reservoirs, autoimmunity, and persistent inflammation. There are currently no diagnostic tests or therapeutic solutions for affected patients.

RATIONALE

We followed 39 healthy controls and 113 COVID-19 patients for up to 1 year after initial confirmation of acute SARS-CoV-2 infection to identify biomarkers associated with Long Covid. At 6-month follow-up, 40 patients had Long Covid symptoms. Repeated clinical assessments were paired with blood draws, resulting in a total of 268 longitudinal blood samples. We measured >6500 proteins in serum by proteomics. Top candidate biomarkers were identified using computational tools and further evaluated experimentally.

RESULTS

Long Covid patients exhibited increased complement activation during acute disease, which also persisted at 6-month follow-up. The complement system is part of the innate immune system and contributes to immunity and homeostasis by targeting pathogens and damaged cells, among other functions. Interestingly, blood complement levels normalized in Long Covid patients recovering before their 6-month follow-up. The complement system can be activated by various triggers, resulting in formation of the terminal complement complex (TCC), made of the complement components C5b-9. These complexes can integrate into cell membranes and induce cell activation or lysis. Long Covid patients showed imbalanced TCC formation, marked by increased soluble C5bC6 complexes and decreased levels of C7-containing TCC formations that can incorporate into cell membranes. This suggested increased membrane insertion of TCCs in Long Covid patients, contributing to tissue damage. Accordingly, Long Covid patients showed elevated tissue injury markers in blood and a thromboinflammatory signature, characterized by markers of endothelial activation, such as von Willebrand factor (vWF), and red blood cell lysis. Low antithrombin III levels in Long Covid patients were accompanied by signs of increased cleavage by thrombin, a driver of TCC formation. Furthermore, Long Covid patients had elevated platelet activation markers and monocyte–platelet aggregates at 6-month follow-up, particularly in cases where Long Covid persisted for 12 months or more. These patients also showed signs of antibody-mediated activation of the classical complement pathway, which was associated with increased anti-CMV (cytomegalovirus, also known as human herpesvirus 5) and anti-EBV (Epstein-Barr virus) immunoglobulin G (IgG) antibody levels.

CONCLUSION

Our data suggest that active Long Covid is accompanied by a blood protein signature marked by increased complement activation and thromboinflammation, including activated platelets and markers of red blood cell lysis. Tissue injury may also be complement-mediated and, in turn, activate the complement system. Moreover, complement activation may be driven by antigen–antibody complexes, involving autoantibodies and antibodies against herpesviruses, as well as cross-talk with a dysregulated coagulation system. In addition to offering a basis for new diagnostic solutions, our work provides support for clinical research on complement modulators for patients suffering from Long Covid.