IDENTIFICATION OF NEW BIOMARKERS FOR SEVERITY, DISEASE PROGRESSION AND RESPONSE TO TREATMENT FOR

COVID-19

Proposal’s significance.

The project aims to analyse the blood samples collected at different time points (longitudinal) from patients infected with COVID-19/SARS-COV2 for more than 85 inflammatory markers to identify a new set of biomarkers for:

1) Rapid evaluation of the severity of the diseases (level of inflammation and immune activation);

2) Estimate the rate of progression of the disease (getting worse or improving);3) Response to potential treatments i.e. hydroxychloroquine, Vitamin C, zinc, etc… (from non-

responders to responder with rate of improvement).

PROPOSAL

BACKGROUNDNeuroinflammation and “cytokine storm” triggered by SARS-CoV-2Several cytokines include IFN-γ, TNF-α, IL-6, IL-10, IL-2, IL-1, and others could play critical roles in the severe form of SARS-CoV-2 infection. suggesting that The magnitude of the cytokine storm is associated with severity of disease. IL-6 concentrations have been shown to be 3 times higher in patients with severe disease and maximal IL-6 highly predictive of respiratory failure1. These data also suggest that monitoring these inflammatory markers over time could be a way to identify patients early on at risk for developing severe disease2.

The kynurenine pathway (KP) is the principal route of L-tryptophan catabolism leading to the formation of the essential pyridine nucleotide, nicotinamide adenine dinucleotide (NAD+), and important neuroactive metabolites, including the excitotoxin and proinflammatory, quinolinic acid (QUIN), the TH17/Treg balance modulator, 3-hydroxyanthranilic acid (3-HAA), and the immunosuppressive agent, L-Kynurenine (KYN). Interestingly, DAMPS, IFN-γ, TNF-α, that are increased during the cytokine storm, are known to be potent activator of IDO1.1

KP dysregulation play a key role in limiting antiviral immune response KP metabolites could be involved at several levels in SARS-COV2 infection: 1) Immunomodulation (KYN, 3HAA); 2) inflammation (QUIN). The KP plays an important role in anti-microbial defence and immune regulation. The IDO1 - aryl hydrocarbon receptor (AhR) axis acts by both depleting tryptophan to starve the invaders and by contributing to the state of immunosuppression with microorganisms.Pathogens using IDO1-mediated tryptophan deprivation include bacteria, parasites, and viruses. Viral infections highjack the host immune response to create a state of disease tolerance using kynurenine catabolites (KYN, 3HAA) include human immunodeficiency virus (HIV), hepatitis B virus (HBV), hepatitis C virus (HCV), herpes, cytomegalovirus (CMV) and likely SARS-COV2. To date, no study has look at the involvement of the KP in SARS-COV2 infection.

RESEARCH PLAN

The originality the present project is that it will concurrently assess a large number (85x) of inflammatory and immunological biomarkers in a longitudinal study. Then using biostatistical analyses we will test any potential combinatory correlations between sets of markers and COVID-19 infection severity, progression, and response to treatment.

COVID-19 Samples from the Sentinel Travellers and Research Preparedness Platform for Emerging Infectious Disease (SETREP-ID): Longitudinal samples will be collected from COVID-19 patients at diagnosis and collected at 0, 3, 6, 10, 14, 18, 24 and 30 days. We will also obtain the full clinical assessment for all the patients. SETREP-ID has already recruited more than 16 COVID-19 patient samples into their biobank and will have sufficient longitudinal samples to perform the proposed project.We will use five groups of samples:

1) 30 healthy participants (Controls)2) 30 patients with minor or moderate symptoms3) 30 patients with severe symptoms4) 30 patients with severe symptoms + treatment (Ivermectin; lopinavir/ritonavir, hydroxychloroquine)5) 10 patients with “cold symptoms” or influenza infection but COVID-19 negative.

N=30 is based upon power calculations to detect differences of at least 5%. Control participants will be age-matched, and often a close relative of the patient. Serum, urine and hair samples will be used for this project.

METHODS.

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Samples from the same patients will be tested at 9 different time points (100 patients and 30 controls with 9 time points = 1040 tests).

Quantification of the KP metabolites: Nine different KP metabolites including quinolinic acid with quantified using ultra-High-Pressure Liquid Chromatography (uHPLC) and gas chromatography-mass spectrometry (GC-MS) in blood (plasma) samples from GBM patients and age-matching/sex controls.

Inflammatory status will be assessed using a Human Cytokine/chemokines 71-plex Discovery PCR arrays performed by Eve Technology, Canada. https://www.evetechnologies.com/product/human-cytokine-chemokine-65-plex-panel-hd71-copy/

Both of these methods are optimized and routinely used in our laboratory. They have been both used in our publication describing the MS prognostic biomarker3. See https://www.ncbi.nlm.nih.gov/pubmed/28155867

Quantification of CRP, NAD+, GSH levels using commercial ELISA kits

Biostatistical analysis. Dr Ayse Bilgin who has the Head of the Biostatistics Department at Macquarie university will analyse the results from this study will help us to help with biomedical parameters. We aim to establish the correlation between levels of kynurenine pathway metabolites together with all the other inflammatory markers and severity of COVID-19 infection and response to treatment.

Biological samples. We need only 1mL of plasma or serum longitudinal samples collected at four different time points (longitudinal) from the patients infected with COVID-19. We have already Ethic approval for this project from the HREC at Macquarie University. (#5201300333).

EXPECTED PROJECT OUTCOMES The point of the study is the integration of these 85 molecules to create nuanced biomarker fingerprints that can accurately and predictively capture the complexities of this disease in a way that will provide real advances for clinical diagnosis and management and for new research directions in therapies.Note: Our biomarker set has already been validated with a new anti-inflammatory drug in clinical trial in Australia and USA (due to confidentiality agreement – cannot mention the company /drug)

FEASIBILITY

EXPERTISE: Prof Guillemin. For the last two decades, Prof Guillemin’s team has been focusing on the biological functions of the amino acid tryptophan in brain diseases. Prof Guillemin is the World leader in this field; he is the President of the international Society for Tryptophan research (ISTRY) and the Editor in Chief of the scientific journal International Journal on tryptophan Research (IJTR). He has also been the President of the international Neurotoxicity Society (2012-2015) showing his strongly expertise in neuroinflammation, neurotoxicity and neuroprotection. His team together with an large international network of collaborators has demonstrated the importance of small molecules deriving from tryptophan catabolism in the brain in several neurodegenerative diseases and neuropsychiatric disorders including suicide4-6 and depression7,8. In 2017, our group has successfully completed a similar study for multiple sclerosis (MS), identifying the first set of 3

blood biomarkers able to differentiate between the three subtypes of MS9 and also assess response to treatment in patients and finally assess new drug efficacy (three drug companies are using our biomarker set). See http://www.msra.org.au/world-first-blood-test-aide-prognosis-and-treatment-ms. We aim to use a similar approach for this project on SARS-COV2.

BUDGET

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References:1 Alijotas-Reig, J. et al. Immunomodulatory therapy for the management of severe COVID-

19. Beyond the anti-viral therapy: A comprehensive review. Autoimmunity reviews, 102569, doi:10.1016/j.autrev.2020.102569 (2020).

2 Jamilloux, Y. et al. Should we stimulate or suppress immune responses in COVID-19? Cytokine and anti-cytokine interventions. Autoimmunity reviews, 102567 (2020).

3 Lim, C. K. et al. Kynurenine pathway metabolomics predicts and provide mechanistic insight into multiple sclerosis progression. Scientific reports 7, in press, doi:DOI: 10.1038 (2017).

4 Erhardt, S. et al. Connecting inflammation with glutamate agonism in suicidality. Neuropsychopharmacology 38, 743-752, doi:10.1038/npp.2012.248 (2013).

5 Brundin, L. et al. An enzyme in the kynurenine pathway that governs vulnerability to suicidal behavior by regulating excitotoxicity and neuroinflammation. Translational psychiatry 6, e865, doi:10.1038/tp.2016.133 (2016).

6 Bay-Richter, C. et al. A role for inflammatory metabolites as modulators of the glutamate N-methyl-D-aspartate receptor in depression and suicidality. Brain Behav Immun, doi:10.1016/j.bbi.2014.07.012 (2014).

7 Steiner, J. et al. Severe depression is associated with increased microglial quinolinic acid in subregions of the anterior cingulate gyrus: Evidence for an immune-modulated glutamatergic neurotransmission? Journal of Neuroinflammation 8, 1-9 (2011).

8 Zepf, F. D., Stewart, R. M., Guillemin, G. & Ruas, J. L. Inflammation, immunology, stress and depression: a role for kynurenine metabolism in physical exercise and skeletal muscle. Acta neuropsychiatrica, 1-2, doi:10.1017/neu.2015.69 (2016).

9 Lim, C. K. et al. Kynurenine pathway metabolomics predicts and provides mechanistic insight into multiple sclerosis progression. Scientific reports 7, 41473, doi:10.1038/srep41473 (2017).

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