Keywords
Sars-CoV-2, mass spectrometry, inflammation, biomarker, proteomics
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Sars-CoV-2, mass spectrometry, inflammation, biomarker, proteomics
Figure references have been corrected. Additional method information has been added. A brief mention is included of some of the pathways that the proteins in the multiplex assay are attributed to. An additional reference has been included and parts of the results section better clarified.
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See the authors' detailed response to the review by William J. griffiths
As more and more people are recovering from SARS-CoV-2 infection, one of the growing concerns is the increasing reports of the post viral fatigue symptoms or ‘long Covid’. This phenomenon is defined as not recovering for several weeks or months following the start of symptoms and whereby patients present with chronic and recurrent fatigue for weeks and even many months after a SARS-CoV-2 infection1,2. Understanding the effects and complications of ‘long Covid’, and then managing it, is the next challenge for public health services. Currently the UK is increasing its testing capacity for virus detection and antibody detection, but there still remains a gap in the understanding and diagnosis of long Covid.
Work has been performed to characterise the inflammatory response to SARS-CoV-2 infection in relation to disease severity3. There has been controversy as to whether severity is associated with a hyperinflammatory cytokine storm or failure of host protective immunity that results in unrestrained viral dissemination and organ injury. What has made addressing this question challenging has been the lack of diagnostic tools to evaluate immune function in Covid-19 infections. There are sets of simple but expensive immunoassay panels that are commercially available to look at known key inflammatory proteins such as cytokine panels; however, these only give information on known pathways and limit discovery of novel or less defined inflammatory responses. Targeted proteomics using mass spectrometry can also quantitate multiple diagnostic proteins without use of antibodies. Proteins can be easily added or removed from a panel thereby providing a custom tailored approach. This is ideal in addressing the need for evaluating less understood or defined immune response pathways. Novel assays for virus detection have been already developed using targeted mass spectrometry4,5 but there are no assays available yet to look at the symptoms for the diagnosis or understanding of ‘long Covid’.
From a previous study (unpublished reports) we developed a custom targeted mass spectrometry based assay panel that looks at up to 96 pro- and anti-inflammatory associated proteins (Figure 1a; see Table 1 on protocols.io6). Some of the pathways relevant to the proteins included in the multiplex include upstream regulation of cytokine and glucocorticoid expression; calpain activation; aging associated T-cell production and heat shock protein mediated immunostimulatory ‘danger signals’ for the innate and adaptive immune systems. Our hypothesis was long Covid symptoms could be related to a lingering ‘tail’ and an abnormal inflammatory response to an infection, by a type of virus the body has not seen before. We applied this assay to a cohort of samples taken from healthcare workers who had tested positive for SARS-CoV-2 infection by PCR and were either asymptomatic or had only a mild infection. Samples were taken at least 40–45 days post infection and demonstrated a positive antibody test. We compared these with serum from healthcare workers with a negative antibody test, no reported infection and no positive PCR test.
Samples were identified from the Health Research Authority approved project Co-Stars (Great Ormond Street Hospital NHS Trust COSTARS, IRAS 282713, ClinicalTrials.gov Identifier: NCT04380896, registered May 8th 2020) and all participants provided informed written consent.
A pilot group of 10 positive and 10 negative samples covering a broad age range was selected as proof of principle for this assay. The negative group was 60% female with an age-range of 21–57, median 38 years. The positive group was 69% female, with an age range 31–66 and median age of 44 years. Of the positive patients, seven were asymptomatic and six had loss of taste/smell or had abnormal taste/smell. None were admitted to hospital or reported other symptoms.
The detailed method for the multiplex assay is published and available at protocols.io6. Briefly, yeast enolase internal standard was added to serum samples. Proteins were precipitated and trypsin digested to peptides. Peptides were desalted, separated by reverse phase chromatography over a 16 min acetonitrile gradient and analysed on a Waters Aquity UPLC system coupled to a Xevo TQ-S mass spectrometer.
Raw data was acquired using MassLynx v 4.1 in multiple reaction monitoring mode. Raw files were processed using Skyline v 19. Protein-Peptide sequences were obtained from www.uniprot.org and settings optimised using custom synthesised peptides (Genscript USA). Peak intensity data were normalised to a spiked internal standard protein yeast enolase (Sigma, UK). Normalised data were exported to Microsoft Excel and analysed using SIMCA v 15 (Umetrics, Sweden) for multivariate analysis and Graphpad prism v 6 was used for statistical analysis.
A representative overlaid chromatogram of the proteins included in the multiplex assay is show in Figure 1a. Multivariate analysis of all inflammatory proteins measured in the control and SARS-CoV-2 positive patients is shown in Figure1b. The score plot that shows the first two principal components indicates a clear separation of the positive and negative samples indicating the serum immune profile from people infected with SARS-CoV-2 is still significantly affected even 40 days post-infection. Univariate analysis revealed six proteins (Figure 2) from the entire multiplex panel were significantly altered. The majority of these proteins are either anti-inflammatory or associated with the stress response. Two proteins originate from the mitochondria, peroxiredoxin 3 (PRDX3) and carbamoyl phosphate synthase (CPS1). PRDX3 is a known antioxidant. Its increase in serum of patients infected with SARS-CoV-2 is likely indicative of continued mitochondrial stress response. CPS1 is a major mitochondrial urea cycle enzyme in hepatocytes. Serum CPS1 originates from the bile duct and is usually rapidly cleared by peripheral blood mononuclear cells7. It is possible that basal levels of CPS1 in serum are reduced in patients infected by SARS-CoV-2 due to increased circulation and activity of peripheral blood mononuclear cells.
N-Myc downstream regulated gene 1 (NDRG1) is a cytosolic protein with many biological functions8. Its role in the immune response is undefined but deficiency of NDRG1 affects the differentiation process of macrophages9 and maturation of mast cells10. Collagen triple helix repeat containing 1 (CTHRC1) is anti-inflammatory and promotes wound healing by recruiting M2 macrophages and regulating the TGF-β and Notch pathways11. This increase of CTHRC1 indicates tissue damage has occurred even in moderately affected patients.
Cystatin C is a protease inhibitor and extracellular levels are used as a biomarker for disease prognosis in cancer, cardiovascular disease, and inflammatory lung disorders12. In mice serum cystatin C is controlled by the anti-inflammatory cytokine IL10 of which increasing levels suppress cystatin C expression12. A longitudinal study looking at immune mediators show IL10 levels are significantly elevated in only severe cases of SARS-CoV-2 infection at four weeks post infection and are not affected at four weeks in mild cases13. This would corroborate with what we observe for cystatin C as the mild patients have increased cystatin C that is not being suppressed by higher IL10 levels. We also observe a slight reduction in serum progranulin. Progranulin plays a fundamental role in the immune response which is better defined within its role in neurodegenerative disorders14 but the relevance of serum progranulin is not fully understood. It appears to have a pro-inflammatory role in adipocytes in diabetes15 and an anti-inflammatory protective role in the vascular endothelium against inflammatory reactions16.
Remarkably, even in patients who have suffered from an asymptomatic or mild SARS-CoV-2 infection, after 40 days post-infection they still exhibit a significantly raised group of biomarkers involved in inflammation and the stress response. This initial data using a custom designed inflammatory marker panel applied to mildly affected patients identifies potential drug targets, provides insight into the post infection inflammatory response. This approach using targeted proteomic technology has potential for application on further well-defined sample cohorts to understand what is abnormal about post infection inflammatory response in ‘long covid’ patients.
ProteomeXchange: Underlying mass spectrometry data on ProteomeXchange. Accession number PXD022159.
Underlying mass spectrometry data is also available on PanoramaWeb at https://panoramaweb.org/x1eZmn.url.
We wish to thank Annabelle Lea Mai Immunology department Great Ormond Street Hospital for help with samples and the Peto Foundation for their continuing support.
This work is partly funded by the NIHR GOSH BRC. The views expressed are those of the authors and not necessarily those of the NHS, the NIHR or the Department of Health.
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Competing Interests: No competing interests were disclosed.
Reviewer Expertise: Precision Medicine, Proteomics, Clinical Proteomics, Data Science, Biochemistry
Is the work clearly and accurately presented and does it cite the current literature?
Partly
Is the study design appropriate and is the work technically sound?
Yes
Are sufficient details of methods and analysis provided to allow replication by others?
Yes
If applicable, is the statistical analysis and its interpretation appropriate?
Yes
Are all the source data underlying the results available to ensure full reproducibility?
Yes
Are the conclusions drawn adequately supported by the results?
Yes
Competing Interests: No competing interests were disclosed.
Reviewer Expertise: Precision Medicine, Proteomics, Clinical Proteomics, Data Science, Biochemistry
Is the work clearly and accurately presented and does it cite the current literature?
Yes
Is the study design appropriate and is the work technically sound?
Yes
Are sufficient details of methods and analysis provided to allow replication by others?
Partly
If applicable, is the statistical analysis and its interpretation appropriate?
Yes
Are all the source data underlying the results available to ensure full reproducibility?
Yes
Are the conclusions drawn adequately supported by the results?
Yes
Competing Interests: No competing interests were disclosed.
Reviewer Expertise: Mass spectrometry
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