Plasma proteomic signature predicts who will get persistent symptoms following SARS-CoV-2 infection - The Lancet
Discussion
We report plasma proteomics responses to SARS-CoV-2 infection during the first UK wave, made possible due to high-frequency serial biosampling of HCW at risk of infection during the peak of the first epidemic wave in London. Non-severe SARS-CoV-2 infection markedly perturbs the plasma proteome from the time of first infection, and for the ensuing 6 weeks. This plasma proteomics perturbation is related to the extent of symptoms and associates with the presence of persistent symptoms (Figure 6).
Figure 6Graphical abstract.
The sustained perturbation of the plasma proteome following non-severe SARS-CoV-2 uncovered in the current study, fits with similar findings from a pilot using the same assay in individuals infected with SARS-CoV-2 showing mild or absent symptoms.
“The long tail of Covid-19” - The detection of a prolonged inflammatory response after a SARS-CoV-2 infection in asymptomatic and mildly affected patients.
PCA of the plasma proteome in this independent cohort was most discriminant of infected cases (n = 10) compared to uninfected individuals (n = 10) at 6 weeks, in keeping with our PCA findings reported in Figure 2d. We go on to show that the plasma proteome of HCW in the week prior to PCR-confirmed SARS-CoV-2 infection is indistinguishable from that of uninfected HCW, that the earliest detectable perturbation coincides with the first PCR positive test result, and that it is progressively accentuated in subsequent weeks, resulting in complete separation of groups from week-five onwards.
A number of studies have previously undertaken a discovery proteomics approach to study the perturbation of the plasma proteome during acute infection,
Prognostic tools and candidate drugs based on plasma proteomics of patients with severe COVID-19 complications.
In line with previous works and using our assay that was enriched for neuroinflammatory biomarkers, we found that drivers of proteomic perturbation include oxidative stress markers
Elevated expression of serum endothelial cell adhesion molecules in COVID-19 patients.
(e.g., NCAM2). However, plasma proteome signatures of severe hospitalized patients are expectedly different from ours as a result of acute inflammatory changes that would occur not just from initial infection but from the subsequent cascade of events that are not COVID-specific, such as mechanical ventilation or prolonged immobilisation. This can overshadow what may be the subtle changes occurring from infection. A previous study that also looked at non-hospitalized patients with SARS-CoV-2 found that certain symptoms such as fatigue and shortness of breath were still present up to 4-7 months after infection.
Post-COVID syndrome in non-hospitalised patients with COVID-19: a longitudinal prospective cohort study.
Only antibody serological analysis was performed and results suggested that lower baseline IgG was related to persistence of symptoms. We found no significant association between anti-S1 and anti-NP levels at baseline in those with persistent symptoms out to 12 months. Within our assay, one of the proteins that most strongly predicted persistent symptoms is APP, which in serum can act as an anti-coagulant. About 90% of soluble APP is thought to be derived by secretion from activated platelets and it acts as an inhibitor of coagulation factors IXa and Xia.
Expression and processing of amyloid precursor protein in vascular endothelium.
The iron-sulfur cluster biogenesis protein (HSCB also known as HSC20) which is a mitochondrial co-chaperone, has a key role in red blood cell erythropoiesis and hematopoiesis
Mutations in the iron-sulfur cluster biogenesis protein HSCB cause congenital sideroblastic anemia.
and also predicted persistent symptoms. It is plausible that HSCB is being released into the blood secondary to mitochondrial disruption from COVID-mediated red blood cell destruction. Indeed, it has been amply shown
that acute COVID-19 infection causes multiple autoimmune heamolytic anaemia (AIHA) subtypes, beginning approximately 7 days after infectious symptoms. So what we may be capturing here by MS are milder subclinical forms of AIHA in non-severe SARS-CoV-2 infection which nevertheless cause measurable protein abnormalities in plasma. Extracellular HSP90 another heat shock protein which is functionally diverse but essential for maintaining healthy cells, could be indicative of vascular stress as it has been shown to be released by vascular smooth muscle cells under oxidative stress. Serum HSP90 is then thought to act as a cytokine stimulating IL-8
Plasma Hsp90 levels in patients with systemic sclerosis and relation to lung and skin involvement: a cross-sectional and longitudinal study.
PLD3 is an endo lysosomal protein with no detectable phospholipase activity. Along with PLD4 it has an anti-inflammatory function as it acts as an ssDNA exonuclease that breaks down Toll-like receptor 9 ligands mediating the TLR9 response.
“The long tail of Covid-19” - The detection of a prolonged inflammatory response after a SARS-CoV-2 infection in asymptomatic and mildly affected patients.
which we confirm in this larger cohort, however, it also appears to have utility to predict persistent symptoms. In mice, serum CST3 is controlled by the anti-inflammatory cytokine IL10 of which increasing levels suppress CST3 expression.
IL-10 controls cystatin C synthesis and blood concentration in response to inflammation through regulation of IFN regulatory factor 8 expression.
A longitudinal study looking at immune mediators show IL10 levels are significantly elevated at 4 weeks only in severe cases of SARS-CoV-2 infection and not in milder cases.
Longitudinal COVID-19 profiling associates IL-1RA and IL-10 with disease severity and RANTES with mild disease.
This corroborates what we observe for CST3 as mild infection has increased cystatin C that is not being suppressed by higher IL-10 levels. S100A9 forms part of calprotectin which is a marker of neutrophil-related inflammatory processes. Silvin et al. first described that elevated calprotectin can indicate abnormal myeloid cells driving severe infection in patients infected with SARS-CoV-2. Since then many studies have now determined calprotectin as a biomarker of severe SARS-CoV-2 infection
Plasma proteomics identify biomarkers and pathogenesis of COVID-19.
detectable in the blood and lungs. Whilst the levels we observed were vastly lower than those expected in severely affected patients, our data suggests that higher calprotectin levels in mildly symptomatic patients at the time of first infection could be a risk marker of persistent symptoms. Recent studies have shown that impaired pulmonary function is detectable several months after SARS-CoV-2 infection
Immuno-proteomic profiling reveals aberrant immune cell regulation in the airways of individuals with ongoing post-COVID-19 respiratory disease.
and this could be the underlying cause for persistent symptoms in some individuals. The perturbed proteins we describe here have anti-coagulation and anti-inflammatory functions in health, while other altered proteins have been implicated in erythropoiesis and hematopoiesis
Mutations in the iron-sulfur cluster biogenesis protein HSCB cause congenital sideroblastic anemia.
and have links with lung disease so it is plausible that we are detecting surrogate indicators of possible mild pulmonary function impairment.
Pathway enrichment analysis of samples from our HCW with non-severe SARS-CoV-2 infection revealed pathways previously reported including lipid atherosclerosis and cholesterol metabolism pathways,
Low serum level of apolipoprotein A1 may predict the severity of COVID-19: a retrospective study.
Host lipids play a vital role in the life cycle of viruses as they are required for viral genome replication, assembly, maturation and transport through cell membrane.
Cholesterol, lipoproteins, and COVID-19: basic concepts and clinical applications.
For example, SARS-CoV-2 infection relies on its spike protein binding to angiotensin converting enzyme 2 (ACE2) receptor. As the later resides into a cholesterol-rich environment, alteration in membrane cholesterol composition would interfere with membrane dynamics which can potentially result in failure of endocytosis.
Lipid homeostasis and mevalonate pathway in COVID-19: basic concepts and potential therapeutic targets.
In addition, apolipoproteins also modulate the immune response, inflammation and even haematopoiesis meaning that they have a pivotal role in viral responses.
A time-resolved proteomic and prognostic map of COVID-19.
) are more abundant in the non-severe infections. The interplay between SARS-CoV-2 and the coagulation cascade has become apparent in the early stages of the pandemic when increased rates of thromboembolic events were noted in acute infections.
Coagulation dysfunction in COVID-19: the interplay between inflammation, viral infection and the coagulation system.
However, little is known about the coagulation dysregulation in the convalescence period. We found greater abundance in plasma of molecules such as α2-macroglobulin. These might protect against thrombo-inflammation in the context of SARS-CoV-2 by enhancing antithrombin activity, and binding coagulation inducing proteases and inflammatory mediators.
Thromboinflammation in COVID-19: Can α(2) -macroglobulin help to control the fire?.
We report that the plasma trajectory of E-selectin displayed the strongest correlation with symptom burden which fits with previous data linking endothelial markers such as E-selectin to SARS-CoV-2 infection severity.
Between inflammation and thrombosis: endothelial cells in COVID-19.
Increased expression of selectins and other cell adhesion molecules in the pulmonary endothelium would recruit immune cells such as neutrophils and monocytes that then release pro-inflammatory cytokines which are associated with pro-coagulation processes and the generation of reactive oxygen species which can further damage the endothelium.
To infect target cells, SARS-CoV-2 relies on host serine or cysteine proteases to cleave its spike protein into an active conformation. Correlated with symptom severity in our study, the serine protease Cathepsin B has been previously identified as an efficient activator of the spike protein in-vivo and provides a novel therapeutic target for drug development.
Unraveling the mystery surrounding post-acute sequelae of COVID-19.
We show that signature biomarkers at the time of seroconversion for SARS-CoV-2 associates with the persistence of symptoms at 12 months (Figure 5). The most predictive biomarker according to the Gini coefficient was HSCB which facilitates the insertion of iron-sulfur clusters in many cytoplasmic and mitochondrial proteins.
Disease-causing SDHAF1 mutations impair transfer of Fe-S clusters to SDHB.
Its increased synthesis at seroconversion hints towards the need to handle higher iron levels as a hyperferritinimic state has been previously linked to tissue damage (e.g., through enhanced oxidative stress and lipid peroxidase) and impaired immunity
Iron and immunity: immunological consequences of iron deficiency and overload.
which could explain the noted association with persistent symptoms. The functional intersection between iron metabolism and SARS-CoV-2 was highlighted when high levels of ferritin were previously found to associate with admission to intensive care and mortality.
Persisting alterations of iron homeostasis in COVID-19 are associated with non-resolving lung pathologies and poor patients’ performance: a prospective observational cohort study.
This iron metabolism dysregulation in COVID-19 has a pathophysiological basis as the similarity between hepcidin and the SARS-CoV-2 spike protein has been recently discovered.
COVID-19 and iron dysregulation: distant sequence similarity between hepcidin and the novel coronavirus spike glycoprotein.
Some of our persistent symptoms signature biomarkers were persistently elevated at 6 weeks (Figure S3). These can be broadly divided into two categories: proteins related to inflammation and those related to the stress response. An ongoing elevated C-reactive protein suggests a residual systematic inflammatory response.
Long-term clinical and biochemical residue after COVID-19 recovery.
However, the upregulation of collagen (e.g., COL6A3) and metalloproteases genes (e.g., MMP3) suggests concomitant activation of the repair pathway via fibrotic remodelling. Increased expression of S100A9 was previously related to neurodegenerative amyloid disease.
Co-aggregation of pro-inflammatory S100A9 with α-synuclein in Parkinson's disease: ex vivo and in vitro studies.
Continued mitochondrial PRDX3 expression at 6 weeks suggests a continued oxidative stress response, while persistent upregulation of glycolysis-related genes such as ALDOA indicate a catabolic stress response,
Long-term clinical and biochemical residue after COVID-19 recovery.
) findings from other studies.
Limitations
Our study has some important limitations. This was a single-centre study and the generally low sample number as well as the low number of samples available from HCW in the week before their first PCR positive test, was small (n = 5). Though every attempt was made to minimise self-reporting bias, this could still have potentially survey results. External independent cohort validation of the multi-marker proteomics profile predictive of persistent symptoms was not undertaken but will be a key focus of our future work. To predict persistent symptoms, we used proteomics signatures at the time of first seroconversion (n = 52) due to smaller numbers available for first PCR positive test (n = 29). Given all data collection occurred early during the first wave, differences by variant or those caused by vaccination status, were beyond the scope of this study.
Contributors
GC and JCM equally contributed to this work and are co-first authors. KM and WH equally contributed to this work and are co-senior authors. GC, JCM, WH and KM conceptualized the study reported. KM, WH and GC designed and supervised the proteomics experiments. GC, WH, NP, ID and TB have verified the underlying data. WH, ID, TB, JS and JH conducted the proteomics experiments. GC analysed the data and wrote the manuscript with input from all the authors. TB, JCM, CM, ÁM, TAT and MN conceptualized and established the HCW cohort. GJ, MH, KM, MF, KM, CM, TAT and JCM collected HCW samples. CT, GC, JCM, KM, WH, NP, CP, JMG, BOB, DMA, MKM, ÁM, TB, MN, AM, LS and RJB interpreted the data. All authors read and approved the final version of the manuscript.
Acknowledgements
We gratefully acknowledge all the HCW participants for donating their samples and data for these analyses, and the research teams involved in consenting, recruitment and sampling of the HCW participants.
The COVIDsortium is supported by funding donated by individuals, charitable Trusts, and corporations including Goldman Sachs, Citadel and Citadel Securities, The Guy Foundation, GW Pharmaceuticals, Kusuma Trust, and Jagclif Charitable Trust, and enabled by Barts Charity with support from University College London Hospitals (UCLH) Charity. Wider support is acknowledged on the COVIDsortium website. Institutional support from Barts Health National Health Service (NHS) Trust and Royal Free NHS Foundation Trust facilitated study processes, in partnership with University College London and Queen Mary University of London. This work was additionally supported by the Translational Mass Spectrometry Research Group and the Biomedical Research Center (BRC) at Great Ormond Street Hospital.
GC is supported by the British Heart Foundation (BHF, SP/20/2/34841), the BHF Accelerator Award (AA/18/6/34223) and by the National Institute of Health Research (NIHR) UCL Hospitals BRC. GC, JCM and BOB are supported by the Barts Charity HeartOME1000 Grant (MGU0427/G-001411). KM and WH are supported by the NIHR BRC at Great Ormond Street Hospital for Children NHS Foundation Trust and UCL. RJB and DMA are supported by MRC (MR/S019553/1, MR/R02622X/1, MR/V036939/1, MR/W020610/1), NIHR Imperial Biomedical Research Centre (BRC):ITMAT, Cystic Fibrosis Trust SRC (2019SRC015), NIHR EME Fast Track (NIHR134607), NIHR Long Covid (COV-LT2-0027), Innovate UK (SBRI 10008614) and Horizon 2020 Marie Skłodowska-Curie Innovative Training Network (ITN) European Training Network (No 860325). ÁM is supported by Rosetrees Trust, The John Black Charitable Foundation, and Medical College of St Bartholomew's Hospital Trust. JCM, CM and TAT are directly and indirectly supported by the UCLH and Barts NIHR BRCs and through the BHF Accelerator Award (AA/18/6/34223). TAT is funded by a BHF Intermediate Research Fellowship (FS/19/35/34374). MN is supported by the Wellcome Trust (207511/Z/17/Z) and by NIHR BRC Funding to UCL and UCLH. MKM is supported by UK Research and Innovation (UKRI)/NIHR UK-CIC, Wellcome Trust Investigator Award (214191/Z/18/Z) and Cancer Research UK Immunology grant (26603). ÁM, CM and JCM were supported by the UKRI/MRC Covid-19 Rapid response grant COV0331 MR/V027883/1.
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