Molecular insights into long COVID: serum proteome reveals dominant signaling pathways

In a recent study published in the Nature Communications Journal, researchers examined the serum proteome of unvaccinated adults who had post-acute sequelae of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) (PASC) and were infected with the SARS-CoV-2 ancestral strain.

Study: Persistent serum protein signatures define an inflammatory subcategory of long COVID. Image Credit: DmitryDemidovich/Shutterstock.com

Background

Long coronavirus disease (COVID), also known as PASC, is a clinical condition characterized by a range of symptoms that can last for several months after the initial SARS-CoV-2 infection. The causes of this condition may be due to ongoing inflammation, unrepaired tissue damage, or slow clearance of ribonucleic acid (RNA) or viral protein.

However, the exact biological distinctions are not yet comprehensively comprehended. Therefore, the present study compared the serum proteome of 55 PASC individuals with symptoms lasting 60 days or more after acute infection onset to samples from recovered SARS-CoV-2 infected and uninfected persons.

About the study

The study included 55 adults with persistent symptoms lasting at least 60 days after being infected with SARS-CoV-2, 24 who had recovered from the infection, and 22 who tested negative.

The team enrolled participants during the initial outbreak of the COVID-19 pandemic. Blood was drawn from uninfected individuals once at the beginning of the study. At the same time, those who had recovered from COVID-19 provided one or more blood samples at various time points ranging from 60 days or later to 379 days post-symptom onset (PSO).

The team analyzed the serum proteome and identified specific proteins that differentiate uninfected, PASC, and recovered participants in the cohort. The study utilized the initial samples collected after 60 days or more from PASC participants, the final post-60-day sample of recovered participants, and a single sample from uninfected persons.

The researchers utilized canonical pathways from the Molecular Signatures Database (MSigDB). They employed a rule-in statistical method for different pathways that distinguished between PASC and uninfected and recovered individuals.

Results

Most participants experienced mild symptoms during acute SARS-CoV-2 infection. Three individuals were hospitalized and needed oxygen therapy. Remdesivir was administered to two of them. One participant was administered steroids. The study participants were not vaccinated at the time of enrollment.

Participants in the cohort who underwent PASC reported experiencing various symptoms at or beyond 60 days post-onset. These symptoms ranged from fatigue, fever, and chills to more severe symptoms such as arrhythmia and brain fog. The authors divided individual PASC symptoms into symptom groups based on affected organs, such as pulmonary, cardiovascular, and neurologic systems.

A total of 275, 25, and 14 proteins were found to have significant differential expression between the PASC, recovered, and uninfected groups, respectively, in pairwise comparisons.

The PASC group showed differing levels of serum proteomic signature expression, indicating that some individuals have an inflammatory signature while others do not. This suggests that the disorder is heterogeneous.

Almost 85 pathways were identified with significant rule-in performance. Several clusters were analyzed, and two of them exhibited a significant presence of inflammatory modules such as type I and type II interferon (IFN) signaling, tumor necrosis factor (TNF) signaling, and nuclear factor kappa B (NF-κB) signaling. However, three clusters did not display a clear inflammatory protein signature.

Cluster 1 included solely uninfected or recovered individuals, while clusters 2 and 3 comprised a combination of uninfected, recovered, and PASC individuals.

The heterogeneity of PASC is highlighted by the distribution of participants in inflammatory and non-inflammatory proteomic clusters. This distribution indicates that only a portion of PASC participants experiences ongoing inflammation.

The study discovered that individuals with an inflammatory protein signature during PASC maintained the signature over time, and most persons were in the same cluster across the longitudinal study period. It is uncertain whether the two clusters involving inflammatory PASC are distinct with separate molecular drivers or if they indicate a range of diseases.

The study analyzed 54 modules that were categorized into five clusters of participants. The researchers used the single-sample Gene Set Enrichment Analysis (ssGSEA) score for each module across samples to identify the modules that significantly differentiated each cluster.

Elevated levels of IFN-γ, interleukin (IL)-12 p40, and IFN-γ-driven chemokines were observed in inflammatory PASC compared to the uninfected and recovered cohorts.

The study found that in the inflammatory PASC cluster 5, there was a protein signature indicating persistent type I interferon signaling. This included an increase in type I IFN-induced proteins during acute SARS-CoV-2 infection.

Type I IFN-associated proteins were increased in inflammatory PASC at the earliest sampling point and remained elevated for around 180 days post-infection. The study also found that the inflammatory PASC exhibited pathway enrichment for regulating IFN-α signaling, as indicated by the ssGSEA analysis.

Conclusion

A comprehensive screening process discovered a serum proteomic signature that detected individuals with PASC and persistent inflammatory disease symptoms. Around 60% of PASC in our cohort displayed an inflammatory signature.

The presence of persistent inflammation in individuals with this condition was associated with various clinical features that were not distinguishable, highlighting the need for biological and clinical assessments in this heterogeneous population.

The study's results shed light on the molecular mechanisms behind persistent inflammation in PASC and propose potential therapeutic targets such as JAK inhibitors or cytokine blockade for individuals with the persistent inflammatory protein signature.

Written by

Bhavana Kunkalikar

Bhavana Kunkalikar is a medical writer based in Goa, India. Her academic background is in Pharmaceutical sciences and she holds a Bachelor's degree in Pharmacy. Her educational background allowed her to foster an interest in anatomical and physiological sciences. Her college project work based on ‘The manifestations and causes of sickle cell anemia’ formed the stepping stone to a life-long fascination with human pathophysiology.