WO2022018195A1 - Methods for prognosis and monitoring of critical form of coronavirus infection - Google Patents

Methods for prognosis and monitoring of critical form of coronavirus infection Download PDF

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Publication number
WO2022018195A1
WO2022018195A1 PCT/EP2021/070497 EP2021070497W WO2022018195A1 WO 2022018195 A1 WO2022018195 A1 WO 2022018195A1 EP 2021070497 W EP2021070497 W EP 2021070497W WO 2022018195 A1 WO2022018195 A1 WO 2022018195A1
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lox
cd66b
neutrophils
level
patients
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PCT/EP2021/070497
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French (fr)
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Christophe Combadiere
Behazine Combadiere
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INSERM (Institut National de la Santé et de la Recherche Médicale)
Centre National De La Recherche Scientifique (Cnrs)
Sorbonne Université,
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Publication of WO2022018195A1 publication Critical patent/WO2022018195A1/en

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0634—Cells from the blood or the immune system
    • C12N5/0642—Granulocytes, e.g. basopils, eosinophils, neutrophils, mast cells
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56966—Animal cells
    • G01N33/56972—White blood cells
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56983—Viruses
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/005—Assays involving biological materials from specific organisms or of a specific nature from viruses
    • G01N2333/08—RNA viruses
    • G01N2333/165—Coronaviridae, e.g. avian infectious bronchitis virus
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
    • G01N2333/70503—Immunoglobulin superfamily, e.g. VCAMs, PECAM, LFA-3
    • G01N2333/70535—Fc-receptors, e.g. CD16, CD32, CD64 (CD2314/705F)
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
    • G01N2333/70596—Molecules with a "CD"-designation not provided for elsewhere in G01N2333/705

Definitions

  • the present invention relates to methods and kits for prognostic and monitoring the severe / critical form of coronavirus infection. More specifically present invention relates to methods for prognosis of the critical form of coronavirus infection through detection of a specific population of neutrophils in a patient. The present invention also relates to a method of preventing or treating a coronavirus in a subject in need thereof
  • SARS-CoV-2 infection is characterized by a range of symptoms including fever, cough, fatigue and myalgia in the majority of cases and occasional headache and diarrhea (1, 3).
  • the inventors therefore set up a prognostic and monitoring method of the critical form of coronavirus infection that allows to directly reflect the immunological status of the patient.
  • a retrospective cohort of 201 patients with confirmed COVID- 19 pneumonia revealed that older age, neutrophilia, organ and coagulation dysfunction were the major risk factors associated with the development of ARDS and progression to death (9). ARDS and sepsis are frequent complications among deceased patients (10). In severe cases, bilateral lung involvement with ground-glass opacities is the most common chest computed tomography (CT) finding. More surprisingly, abnormal CT scans were also reported on asymptomatic COVID-19 patients (11). Immune transcriptome profiling of bronchoalveolar lavage fluids of COVID-19 patients also displayed high levels of pro-inflammatory cytokines (6).
  • NLR neutrophil-to-lymphocyte ratio
  • a first object of the present invention relates to an in vitro method for assessing a subject’s risk of having or developing severe or critical form of coronavirus infection , comprising the steps of i) determining in a sample obtained from the subject the level of neutrophil cells having cell surface expression of CD66b+CD10-CD16-CD64+CD123+ markers and/or CD66b+CD10-CD16-CD64+LOX-l+ markers, ii) comparing the level determined in step i) with a reference value and iii) concluding when the level of neutrophil having cell surface expression of CD66b+CD10-CD16-CD64+CD123+ markers and/or CD66b+CD10-CD16-CD64+LOXl+ markers determined at step i) is higher than the reference value is predictive of a high risk of having or developing severe or critical form of coronavirus infection.
  • An additional object of the invention relates to an in vitro method for monitoring a coronavirus infection comprising the steps of i) determining the level of a population of neutrophils having cell surface expression of CD66b+CD10-CD16-CD64+CD123+ markers and/or CD66b+CD10-CD16-CD64+LOXl+ markers in a sample obtained from the subject at a first specific time of the disease, ii) determining the level of a population of neutrophils having cell surface expression of CD66b+CD10-CD16-CD64+CD123+ markers and/or CD66b+CD10- CD16-CD64+LOX-1+ markers in a sample obtained from the subject at a second specific time of the disease, iii) comparing the level determined at step i) with the level determined at step ii) and iv) concluding that the disease has evolved in worse manner when the level determined at step ii) is higher than the level determined at step i).
  • An additional object of the invention relates to an in vitro method for monitoring the treatment of a of coronavirus infection comprising the steps of i) determining the level of a population of neutrophils having cell surface expression of CD66+CD10-CD16- CD64+CD123+ and/or CD66+CD10-CD16-CD64+LOX-1+ in a sample obtained from the subject before the treatment, ii) determining the level of a population of neutrophils having cell surface expression of CD66b+CD10-CD16-CD64+CD123+ markers and/or CD66b+CD10-CD16-CD64+LOX-l+ markers in a sample obtained from the subject after the treatment”, iii) comparing the level determined at step i) with the level determined at step ii) and iv) concluding that the treatment is efficient when the level determined at step ii) is lower than the level determined at step i).
  • the coronavirus infection is the severe or critical form of coronavirus infection.
  • Another object of the invention relates to an in vitro method for assessing a COVID patient’s risk of having or developing thrombosis comprising the steps of i) determining the level of neutrophil cells having cell surface expression of CD66b+CD10-CD16-CD64+LOX- 1+ markers in a sample obtained from the subject, ii) comparing the level determined at step i) with the with a reference value and iv) concluding that:
  • Another object of the invention relates to a LOX-1 (lectin-type oxidized LDL receptor 1) inhibitor for use in the prevention or the treatment of a coronavirus infection in a subject in need thereof.
  • LOX-1 lectin-type oxidized LDL receptor 1
  • the present invention relates to an in vitro method for assessing a subject’s risk of having or developing severe or critical form of coronavirus infection , comprising the steps of i) determining in a sample obtained from the subject the level of neutrophil cells having cell surface expression of CD66b+CD10-CD16-CD64+CD123+ markers and/or CD66b+CD10- CD16-CD64+LOX-1+ markers, ii) comparing the level determined in step i) with a reference value and iii) concluding when the level of neutrophil having cell surface expression of CD66b+CD 10-CD 16-CD64+CD 123+ markers and/or CD66b+CD10-CD16-CD64+LOX-l+ markers determined at step i) is higher than the reference value is predictive of a high risk of having or developing severe or critical form of coronavirus infection.
  • the present invention relates to an in vitro prognosis method of having or developing severe or critical form of coronavirus infection in a subject, comprising the steps of i) determining in a sample obtained from the subject the level of neutrophil cells having cell surface expression of CD66b+CD10-CD16-CD64+CD123+ markers and/or CD66b+CD10-CD16-CD64+LOX-l+ markers, ii) comparing the level determined in step i) with a reference value and iii) concluding when the level of neutrophil having cell surface expression of CD10-CD16-CD64+CD123+ markers and/or CD66b+CD10-CD16- CD64+LOX-1+ markers determined at step i) is higher than the reference value is predictive of having or developing severe or critical form of coronavirus infection
  • prognosis is a medical term for predicting the likely or expected development of a disease. Prognostic scoring is also used for disease outcome predictions.
  • the “prognosis” is associated with level of neutrophils having cell surface expression of CD66b+CD10-CD16-CD64+CD123+markers and/or the level of neutrophils having cell surface expression of CD66b+CD10-CD16- CD64+LOX-1+ markers which in turn may be a risk for developing critical form of coronavirus infection
  • subject refers to a mammalian, such as a rodent (e.g. a mouse or a rat), a feline, a canine or a primate.
  • rodent e.g. a mouse or a rat
  • feline e.g. a feline
  • canine e.g. a canine
  • primate e.g. a human subject
  • the subject according to the invention can be a healthy subject or a subject suffering from a given disease such as coronavirus infection.
  • coronavirus has its general meaning in the art and refers to any member or members of the Coronaviridae family.
  • Coronavirus is a virus whose genome is plus-stranded RNA of about 27 kb to about 33 kb in length depending on the particular virus.
  • the virion RNA has a cap at the 5’ end and a poly A tail at the 3’ end.
  • the length of the RNA makes coronaviruses the largest of the RNA virus genomes.
  • coronavirus RNAs encode: (1) an RNA-dependent RNA polymerase; (2) N-protein; (3) three envelope glycoproteins; plus (4) three non- structural proteins.
  • the coronavirus particle comprises at least the four canonical structural proteins E (envelope protein), M (membrane protein), N (nucleocapsid protein), and S (spike protein).
  • E envelope protein
  • M membrane protein
  • N membrane protein
  • S spike protein
  • the S protein is cleaved into 3 chains: Spike protein SI, Spike protein S2 and Spike protein S2'. Production of the replicase proteins is initiated by the translation of ORFla and ORFlab via a -1 ribosomal frame- shifting mechanism.
  • This mechanism produces two large viral polyproteins, ppla and pplab, that are further processed by two virally encoded cysteine proteases, the papain-like protease (PLpro) and a 3C-like protease (3CLpro), which is sometimes referred to as main protease (Mpro).
  • PLpro papain-like protease
  • 3CLpro 3C-like protease
  • Coronaviruses infect a variety of mammals and birds. They cause respiratory infections (common), enteric infections (mostly in infants >12 mo.), and possibly neurological syndromes. Coronaviruses are transmitted by aerosols of respiratory secretions.
  • Coronaviruses are exemplified by, but not limited to, human enteric coV (ATCC accession # VR-1475), human coV 229E (ATCC accession # VR-740), human coV OC43 (ATCC accession # VR-920), Middle East respiratory syndrome-related coronavirus (MERS-Cov) and Severe Acute Respiratory Syndrome (SARS)-coronavirus (Center for Disease Control), in particular SARS-CoVl and SARS-CoV2.
  • human enteric coV ATCC accession # VR-1475
  • human coV 229E ATCC accession # VR-740
  • human coV OC43 ATCC accession # VR-920
  • MERS-Cov Middle East respiratory syndrome-related coronavirus
  • SARS Severe Acute Respiratory Syndrome
  • the coronavirus can be a MERS-CoV, SARS-CoV, SARS- CoV-2 or any new future family members.
  • the method of the present invention is suitable for the treatment of Severe Acute Respiratory Syndrome (SARS) and any neurological manifestations (headaches, dizziness, nausea, seizures, stroke, cognitive or sensory disturbances, etc.) or cardiorespiratory manifestations (non-responsiveness to hypoxia, cardiac rhythm disturbances%) of brain viral infection, or disturbances of fluid balance, or anorexia/cachexia, or short or long-term neuroendocrine disturbances.
  • SARS Severe Acute Respiratory Syndrome
  • any neurological manifestations headaches, dizziness, nausea, seizures, stroke, cognitive or sensory disturbances, etc.
  • cardiorespiratory manifestations non-responsiveness to hypoxia, cardiac rhythm disturbances
  • brain viral infection or disturbances of fluid balance, or anorexia/cachexia, or short or long-term neuroendocrine disturbances.
  • SARS-CoV-2 refers to severe acute respiratory syndrome coronavirus 2 known by the provisional name 2019 novel coronavirus (2019-nCoV) is the cause of the respiratory coronavirus disease 2019 (COVID-19). Taxonomically, it is a strain of the Severe acute respiratory syndrome-related coronavirus (SARSr-CoV), a positive-sense single-stranded RNA virus. It is contagious in humans, and the World Health Organization (WHO) has designated the ongoing pandemic of COVID-19 a Public Health Emergency of International Concern. SARS-CoV-2 virion is approximately 50-200 nanometres in diameter.
  • SARS-CoV-2 has four structural proteins, known as the S (spike), E (envelope), M (membrane), and N (nucleocapsid) proteins; the N protein holds the RNA genome, and the S, E, and M proteins together create the viral envelope.
  • the spike protein which has been imaged at the atomic level using cryogenic electron microscopy is the protein responsible for allowing the virus to attach to the membrane of a host cell.
  • the subject of the present invention suffers from COVID- 19 and/or have been previously diagnosed with SARS-CoV-2.
  • SARS-CoV-2 infection refers to the transmission of this virus from an animal and/or human to another animal and/or human primarily via respiratory droplets from coughs and sneezes within a range of about 2 meters. Indirect contact via contaminated surfaces is another possible cause of infection.
  • the subject can be symptomatic or asymptomatic.
  • asymptomatic refers to a subject who experiences no detectable symptoms for the brain viral infection (e.g. coronavirus).
  • symptomatic refers to a subject who experiences detectable symptoms of a pathogen brain viral infection and particularly a coronavirus infection.
  • Symptoms of coronavirus infection include: neurological symptoms (headaches, dizziness, nausea, loss of consciousness, seizures, encephalitis stroke, cognitive or sensory disturbances%), as well as anosmia or ageusia; fatigue, cough, fever, difficulty to breathe or cardiorespiratory manifestations (non-responsiveness to hypoxia, cardiac rhythm disturbances%) of brain viral infection, or disturbances of fluid balance, or anorexia/cachexia, or short or long-term neuroendocrine disturbances
  • coronavirus infection refers to the progression of the disease to acute respiratory distress syndrome (ARDS), accountable for high mortality related to the damages of the alveolar lumen.
  • ARDS acute respiratory distress syndrome
  • sample refers to any biological sample of a subject and can include, by way of example and not limitation, bodily fluids and/or tissue extracts such as homogenates or solubilized tissue obtained from a subject. Tissue extracts are obtained routinely from tissue biopsy.
  • the biological sample is a body fluid sample (such blood or immune primary cell) or tissue biopsy of said subject.
  • the fluid sample is a blood sample.
  • blood sample means a whole blood sample obtained from a subject (e.g. an individual for which it is interesting to determine whether a population of neutrophils cells can be identified).
  • immune primary cell has its general meaning in the art and is intended to describe a population of white blood cells directly obtained from a subject.
  • immune primary cell is selected from the group consisting of PBMC, WBC, neutrophil.
  • PBMC peripheral blood mononuclear cells
  • PBMC sample according to the invention therefore contains lymphocytes (B cells, T cells, NK cells, NKT cells) and neutrophils.
  • lymphocytes B cells, T cells, NK cells, NKT cells
  • neutrophils typically, these cells can be extracted from whole blood using Ficoll, a hydrophilic polysaccharide that separates layers of blood, with the PBMC forming a cell ring under a layer of plasma.
  • PBMC can be extracted from whole blood using a hypotonic lysis buffer, which will preferentially lyse red blood cells. Such procedures are known to the expert in the art.
  • WBC White Blood Cells
  • All white blood cells are produced and derived from multipotent cells in the bone marrow known as hematopoietic stem cells.
  • Leukocytes are found throughout the body, including the blood and lymphatic system.
  • WBC or some cells among WBC can be extracted from whole blood by using i) immunomagnetic separation procedures, ii) percoll or ficoll density gradient centrifugation, iii) cell sorting using flow cytometer (FACS).
  • FACS flow cytometer
  • WBC can be extracted from whole blood using a hypotonic lysis buffer, which will preferentially lyse red blood cells. Such procedures are known to the expert in the art.
  • the fluid sample is a sample of purified neutrophil in suspension.
  • the sample of neutrophil is prepared by immunomagnetic separation methods preformed on a PBMC or WBC sample.
  • neutrophils cells are isolated by using antibodies for neutrophils -associated cell surface markers, such as CD66b (or CD66+/CRTH2-).
  • kits e.g. Direct Human Neutrophil Isolation Kit kits (Immunomagnetic negative selection from whole blood kit) using CD66b labelled antibodies (#19666 from Stem cells technologies) are available.
  • CD10 also known as cluster of differentiation 10 (or Neprilysin, membrane metallo-endopeptidase (MME), neutral endopeptidase (NEP), and common acute lymphoblastic leukemia antigen (CALLA)
  • MME membrane metallo-endopeptidase
  • NEP neutral endopeptidase
  • CALLA common acute lymphoblastic leukemia antigen
  • CD66b Cluster of Differentiation 66b also known as Carcinoembryonic antigen- related cell adhesion molecule 8 (CEACAM8) refers to a member of the carcinoembryonic antigen (CEA) gene family (CD66b / human gene (gene ID 1088)). Its main function is cell adhesion, cell migration, and pathogen binding. CD66b is expressed exclusively on neutrophils (granulocytes) and used as neutrophils marker (Eades-Perner AM et al. (1998) Blood. ;91(2):663-72).
  • CD 10 refers to a cell-surface marker in the diagnosis of human acute lymphocytic leukemia (ALL).
  • ALL human acute lymphocytic leukemia
  • CD 10 is present on leukemic cells of pre-B phenotype, which represent 85% of cases of ALL.
  • Hematopoetic progenitors expressing CD 10 are considered "common lymphoid progenitors", which means they can differentiate into T, B or natural killer cells.
  • CD10-“ means that the cell surface marker is not expressed on neutrophil (or not detected when contacted for instance with a labeled CD 10 antibody) .
  • CD 16 also known as FcyRIII, has its general meaning in the art and refers to a cluster of differentiation molecule found on the surface of natural killer cells, neutrophils, monocytes, and macrophages CD16 has been identified as Fc receptors FcyRIIIa (CD 16a / human gene (gene ID 2214)) and FcyRIIIb (CD 16b / human gene (gene ID 2214)), which participate in signal transduction.
  • Fc receptors FcyRIIIa CD 16a / human gene (gene ID 2214)
  • FcyRIIIb CD 16b / human gene (gene ID 2214)
  • CD 16 is a molecule of the immunoglobulin superfamily (IgSF) involved in antibody-dependent cellular cytotoxicity (ADCC). It can be used to isolate populations of specific immune cells through fluorescent-activated cell sorting (FACS) or magnetic-activated cell sorting, using antibodies directed towards CD 16.
  • FACS fluorescent-activated cell sorting
  • CD16-“ means that the cell surface marker is not expressed on neutrophil (or not detected when contacted for instance with a labeled CD 16 antibody) .
  • CD64 also knows as Cluster of Differentiation 64 or Fc- gamma receptor 1 (FcyRI) refers to a membrane glycoprotein known as an Fc receptor that binds monomeric IgG-type antibodies with high affinity (Hulett M, et al (1998). Mol Immunol. 35 (14-15): 989-96). After binding IgG, CD64 interacts with an accessory chain known as the common g chain (g chain), which possesses an IT AM motif that is necessary for triggering cellular activation (Nimmeijahn F, et al (2006). Immunity. 24 (1): 19-28).
  • FcyRI Fc- gamma receptor 1
  • CD64 is composed of a signal peptide that allows its transport to the surface of a cell, three extracellular immunoglobulin domains of the C2-type that it uses to bind antibody, a hydrophobic transmembrane domain, and a short cytoplasmic tail (Ernst L, et al (1998). Mol Immunol. 35 (14-15): 943-54).
  • CD64 is constitutively found on only macrophages and neutrophils, but treatment of polymorphonuclear leukocytes with cytokines like IFNy and G- CSF can induce CD64 expression on these cells.
  • FcyRIA CD64A / human gene (gene ID 2209)
  • FcyRIB CD64B/ human gene (gene ID 2210)
  • FcyRIC CD64C / human gene (gene ID 2211)
  • FcyRIA CD64A / human gene (gene ID 2209)
  • FcyRIB CD64B/ human gene (gene ID 2210)
  • FcyRIC CD64C / human gene (gene ID 2211)
  • CD 123 also knows as Cluster of Differentiation 64 or alpha-chain of the interleukin-3 receptor (IL-3RA) refers to receptor found on cells which helps transmit the signal of interleukin-3, a soluble cytokine important in the immune system.
  • the human gene coding for the receptor is located in the pseudoautosomal region of the X and Y chromosomes (human gene (gene ID 3553).
  • the receptor belongs to the type I cytokine receptor family and is a heterodimer with a unique alpha chain paired with the common beta (beta c or CD131) subunit.
  • CD123 found on pluripotent progenitor cells, induces tyrosine phosphorylation within the cell and promotes proliferation and differentiation within the hematopoietic cell lines. It can be found on basophils and pDCs as well as some cDCs among peripheral blood mononuclear cells. CD123 is expressed across acute myeloid leukemia (AML) subtypes, including leukemic stem cells.
  • AML acute myeloid leukemia
  • An experimental antibody-drug conjugate SGN-CD123A targets CD123 as a possible treatment for AML (www.businesswire. com/news/home/20170919005140/en/Seattle-Genetics-Initiates-Phase- 1 - Trial-SGN-CD123A /Sept 2016)
  • LOX-1 oxidized low-density lipoprotein receptor 1
  • Ox-LDL receptor 1 oxidized low-density lipoprotein receptor 1
  • LOX-1 is the main receptor for oxidized LDL on endothelial cells, macrophages, smooth muscle cells (Pirillo A, et al. Mediators of Inflammation. 2013: 1-12).
  • LOX-1 is a 50 kDa transmembrane glycoprotein which belongs to the C-type lectin superfamily. Its gene is regulated through the cyclic AMP signaling pathway. The protein binds, internalizes and degrades oxidized low-density lipoprotein.
  • LOX-1 expression on endothelial cells is low, but tumor necrosis factor alpha, oxidized LDL, blood vessel sheer stress, and other atherosclerotic stimuli substantially increase LOX-1 expression (Kakutani M, et al PNAS (2000). 97 (1): 360-364).
  • LOX-1 may be involved in the regulation of Fas-induced apoptosis. Oxidized LDL induces endothelial cell apoptosis through LOX-1 binding ((Pirillo A, et al. Mediators of Inflammation. 2013: 1-12).
  • ligands for LOX-1 include oxidized high- density lipoprotein, advanced gly cation end-products, platelets, and apoptotic cell.
  • the binding of platelets to LOX-1 causes a release of vasoconstrictive endothelin, which induces endothelial dysfunction (Kakutani M, et al PNAS (2000). 97 (1): 360-364).
  • LOX-1 human amino acid sequence (UniProtKB - P78380) is provided in SEQ ID NO:l (transcript variant 1/ NCBI Reference Sequence: NP_002534, Table 4).
  • SEQ ID NO:2 transcription variant 1NCBI Reference Sequence: NM_002543, Table 4.
  • variant sequences of the LOX-1 may be used in the context of the present invention (as biomarker or therapeutic target), those including but not limited to functional homologues, paralogues or orthologues, transcript variants of such sequences such as:
  • LOX-1 transcript variant 1 (NCBI Reference Sequence: NM_002543/ NP_002534). This variant (1) represents the longest transcript and encodes the longest isoform (1)
  • LOX-1 transcript variant 2 (NCBI Reference Sequence: NM_001172632/ NP OO 1166103). This variant (2) lacks an exon in the coding region, which results in a frameshift and an early stop codon, compared to variant 1. The encoded isoform (2) is shorter and has a distinct C-terminus, compared to isoform 1.
  • LOX-1 transcript variant 3 (NCBI Reference Sequence: NM_001172633.1/ NP OOl 166104) This variant (3) lacks an exon in the coding region, which results in a frameshift and an early stop codon, compared to variant 1.
  • the encoded isoform (3) is shorter and has a distinct C-terminus, compared to isoform 1.
  • Standard methods for detecting the expression of a specific surface marker such as CD64 or CD123 at cell surface are well known in the art.
  • the step consisting of detecting the surface expression of a surface marker (e.g. CD64, CD123 or LOX-1) or detecting the absence of the surface expression of a surface marker may consist in using at least one differential binding partner directed against the surface marker, wherein said cells are bound by said binding partners to said surface marker.
  • binding partner directed against the surface marker refers to any molecule (natural or not) that is able to bind the surface marker with high affinity.
  • the binding partners may be antibodies that may be polyclonal or monoclonal, preferably monoclonal antibodies. In another embodiment, the binding partners may be a set of aptamers.
  • Polyclonal antibodies of the invention or a fragment thereof can be raised according to known methods by administering the appropriate antigen or epitope to a host animal selected, e.g., from pigs, cows, horses, rabbits, goats, sheep, and mice, among others.
  • a host animal selected, e.g., from pigs, cows, horses, rabbits, goats, sheep, and mice, among others.
  • Various adjuvants known in the art can be used to enhance antibody production.
  • antibodies useful in practicing the invention can be polyclonal, monoclonal antibodies are preferred.
  • Monoclonal antibodies of the invention or a fragment thereof can be prepared and isolated using any technique that provides for the production of antibody molecules by continuous cell lines in culture.
  • Techniques for production and isolation include but are not limited to the hybridoma technique originally; the human B-cell hybridoma technique; and the EBV-hybridoma technique.
  • the binding partner of CD64 of the invention is the anti-human CD64 antibody available from Biolegend (CD64 (Fc gamma Receptor 1) Monoclonal Antibody (10.1), # 305029)
  • the binding partner of CD123 of the invention is the anti-human CD123 antibody available from Biolegend (CD 123 Monoclonal Antibody (6H6), 306006) or from Fluidigm (Anti-IL3RA/CD123 antibody (6H6) (#3151001).
  • the binding partner of CD66b of the invention is the anti-human CD66b antibody available from Biolegend (CD66b Monoclonal Antibody (G10F5), # 355005) or from Fluidigm (Anti-Human CD66b (80H3) (#3162023).
  • the binding partner of LOX-1 (or OLR1) of the invention is the antihuman LOX-1/OLR1 antibody available from Biolegend (anti human LOX1 antibody BV421 #358609) or from RD Systems (anti-human LOX-1/OLR1 (AF1798)
  • the binding partners of the invention such as antibodies or aptamers may be labelled with a detectable molecule or substance, such as preferentially a fluorescent molecule, or a radioactive molecule or any others labels known in the art.
  • Labels are known in the art that generally provide (either directly or indirectly) a signal.
  • the term "labelled" with regard to the antibody or aptamer is intended to encompass direct labelling of the antibody or aptamer by coupling (i.e., physically linking) a detectable substance, such as a fluorophore [e.g.
  • FITC fluorescein isothiocyanate
  • PE phycoerythrin
  • Cy5 Indocyanine
  • radioactive molecule or a non-radioactive heavy metals isotopes to the antibody or aptamer, as well as indirect labelling of the probe or antibody by reactivity with a detectable substance.
  • An antibody or aptamer of the invention may be labelled with a radioactive molecule by any method known in the art. More particularly, the antibodies are already conjugated to a fluorophore (e.g. FITC-conjugated and/or PE-conjugated).
  • the aforementioned assays may involve the binding of the binding partners (i.e. antibodies or aptamers) to a solid support.
  • the solid surface could a microtitration plate coated with the binding partner for the surface marker.
  • the solid surfaces may be beads, such as activated beads, magnetically responsive beads. Beads may be made of different materials, including but not limited to glass, plastic, polystyrene, and acrylic.
  • the beads are preferably fluorescently labelled. In a preferred embodiment, fluorescent beads are those contained in TruCount(TM) tubes, available from Becton Dickinson Biosciences, (San Jose, California).
  • methods of flow cytometry are preferred methods for detecting (presence or absence of) the surface expression of the surface markers (i.e. CD66b, CD 10, CD 16, CD64, CD 123 and LOX-1). Said methods are well known in the art. For example, fluorescence activated cell sorting (FACS) may be therefore used.
  • FACS fluorescence activated cell sorting
  • Cell sorting protocols using fluorescent labeled antibodies directed against the surface marker (or immunobeads coated with antibody) in combination with antibodies directed against CD66b, CD10, CD16, CD64, CD123 and LOX-1 coupled with distinct fluorochromes can allow direct sorting, using cell sorters with the adequate optic configuration.
  • Such methods comprise contacting a biological sample obtained from the subject to be tested under conditions allowing detection (presence or absence) of CD66b, CD 10, CD 16, CD64 and CD123 and/or CD66b, CD10, CD16, and LOX-1 surface markers.
  • the level of covid (critical form) biomarkers (“Biomarkerl23”: CD66b+CD10-CD16-CD64+CD123+ cells and/or “BiomarkerLoxl”: CD66b+CD10-CD16-LOX-l+ cells) may be measured by any known method in the art.
  • the high or low level of covid-associated neutrophil cell surface biomarkers (“Biomarkerl23”: CD66b+CD10-CD16-CD64+CD123+ cells and/or “BiomarkerLoxl”: CD66b+CD10-CD16-LOX-l+ cells) is intended by comparison to a control reference value.
  • Said reference control values may be determined in regard to the level of biomarker present in blood samples taken from one or more healthy subject(s) or to the cell surface biomarker in a control population.
  • the method according to the present invention comprises the step of comparing said level of covid- associated neutrophil biomarkers (“Biomarkerl23”: CD66b+CD 10-CD 16-CD64+CD 123+ cells and/or “BiomarkerLOX-l”: CD66b+CD10- CD16-LOX-1+ cells) to a control reference value wherein a high level of covid- associated neutrophil biomarkers (“Biomarkerl23”: CD66b+CD10-CD16-CD64+CD123+ cells and/or “BiomarkerLOX-l”: CD66b+CD10-CD16-LOX-l+cells) compared to said control reference value is predictive of a high risk of having a critical form of coronavirus infection and a low level of covid- associated neutrophil biomarkers (“Biomarkerl23”: CD66b+CD10-CD16- CD64+CD123+ cells and/or “BiomarkerLOX-l”: CD66b+CD10-CD16-LOX-l+l+
  • control reference is null (not detected), which means when the BiomarkerLOX-l is detected, subject have a high risk of having or developing a critical form of coronavirus infection.
  • the control reference value may depend on various parameters such as the method used to measure the level covid- associated neutrophil biomarker BiomarkerLOX-l (CD66b+CD10-CD16-LOX-l+ cells) or the gender of the subject.
  • a level of neutrophil CD66b+CD10-CD16-CD64+CD123+ using Flow Cytometry approach identify and quantify neutrophil population, a level of neutrophil CD66b+CD10-CD16-CD64+CD123+ superior to 1% is predictive of having or a high risk of having or developing a critical form of coronavirus infection and a level of neutrophil CD66b+CD10-CD16-CD64+CD123+ lower than 1% is predictive of not having or at a low risk of having a critical form of coronavirus infection.
  • a level of neutrophil CD66b+CD10-CD16-CD64+LOX-l+ is predictive of having or a high risk of having or developing a critical form of coronavirus infection and a level of neutrophil CD66b+CD10-CD16-CD64+CD123+ lower than 1% is predictive of not having or developing or at a low risk of having a critical form of coronavirus infection.
  • Control reference values are easily determinable by the one skilled in the art, by using the same techniques as for determining the level of cell surface biomarker or cell death in blood samples previously collected from the patient under testing.
  • a “reference value” can be a “threshold value” or a “cut-off value”. Typically, a “threshold value” or “cut-off value” can be determined experimentally, empirically, or theoretically.
  • a threshold value can also be arbitrarily selected based upon the existing experimental and/or clinical conditions, as would be recognized by a person of ordinary skilled in the art. The threshold value has to be determined in order to obtain the optimal sensitivity and specificity according to the function of the test and the benefit/risk balance (clinical consequences of false positive and false negative). Typically, the optimal sensitivity and specificity (and so the threshold value) can be determined using a Receiver Operating Characteristic (ROC) curve based on experimental data.
  • ROC Receiver Operating Characteristic
  • the person skilled in the art may compare the level of neutrophil biomarkers (“Biomarker 123”: CD66b+CD10- CD 16- CD64+CD123+ cells and/or “BiomarkerLOX-1”: CD66b+CD10-CD16-LOX-l+cells) with a defined threshold value.
  • the threshold value is derived from the neutrophil level (or ratio, or score) determined in a blood sample derived from one or more subjects who are responders (to the method according to the invention).
  • the threshold value may also be derived from neutrophil level (or ratio, or score) determined in a blood sample derived from one or more subjects or who are non-responders.
  • retrospective measurement of the activated neutrophil level (or ratio, or scores) in properly banked historical subject samples may be used in establishing these threshold values.
  • Reference values are easily determinable by the one skilled in the art, by using the same techniques as for determining the level of activated neutrophils in fluids samples previously collected from the patient under testing.
  • "Risk” in the context of the present invention relates to the probability that an event will occur over a specific time period, as in the conversion to critical form of coronavirus infection, and can mean a subject's "absolute” risk or “relative” risk. Absolute risk can be measured with reference to either actual observation post-measurement for the relevant time cohort, or with reference to index values developed from statistically valid historical cohorts that have been followed for the relevant time period.
  • Relative risk refers to the ratio of absolute risks of a subject compared either to the absolute risks of low risk cohorts or an average population risk, which can vary by how clinical risk factors are assessed. Odds ratios, the proportion of positive events to negative events for a given test result, are also commonly used (odds are according to the formula p/(l-p) where p is the probability of event and (1- p) is the probability of no event) to no conversion.
  • Alternative continuous measures which may be assessed in the context of the present invention, include time to critical form of coronavirus infection conversion risk reduction ratios.
  • Risk evaluation in the context of the present invention encompasses making a prediction of the probability, odds, or likelihood that an event or disease state may occur, the rate of occurrence of the event or conversion from one disease state to another, i.e., from a normal condition or asymptomatic form of Covid-19 or symptomic form of COVID to a critical form of coronavirus infection condition or to one at risk of developing a critical form of coronavirus infection.
  • Risk evaluation can also comprise prediction of future clinical parameters, traditional laboratory risk factor values, or other indices of critical form of coronavirus infection, such as cellular population determination in peripheral tissues, in serum or other fluid, either in absolute or relative terms in reference to a previously measured population.
  • the methods of the present invention may be used to make continuous or categorical measurements of the risk of conversion to critical form of coronavirus infection, thus diagnosing and defining the risk spectrum of a category of subjects defined as being at risk for a critical form of coronavirus infection.
  • the invention can be used to discriminate between normal and other subject cohorts at higher risk for critical form of coronavirus infection.
  • the present invention may be used so as to help to discriminate those having COVID from critical form of coronavirus infection.
  • the method of detection of the invention is consequently useful for the in vitro diagnosis of COVID from a biological sample.
  • the method of detection of the invention is consequently useful for the in vitro diagnosis of early stage covid from a biological sample.
  • BiomarkerLOX-1 After the identification of neutrophil subsets that harbour an immature phenotype (“Biomarkerl23”: CD66b+CD10-CD16-CD64+CD123+ cells, “BiomarkerLOX-1”:
  • CD66b+CD10-CD16-CD64+LOX-l+ cells inventors highlighted with both unsupervised and expert-gating strategies, that both LOX-1- and CD 123- expressing CD10-CD64+ neutrophil subsets (“immature neutrophils”) strongly correlated with SAPS II and SOFA severity scores, commonly used in clinical practice for sepsis prognosis. Accordingly, inventors provided evidence that this immature subset may serve as a severity biomarker in COVID-19 for prognosis and monitoring purpose.
  • Immature neutrophils refers to cells phenotypically and functionally immature. Immature neutrophils were described as cells expressing CD66b and CD64 and lacking the expression of CD10 and CD16 (Taylor OY Br. J. Haematol.: Elghetany MT. Blood Cells Mol Dis 2002;28:260-274; and Ng, L.G. et al. Nat Rev Immunol 19, 255- 265 (2019).).
  • “immature neutrophils” is a population of neutrophils having cell surface expression of CD66b+CD10-CD16-CD64+.
  • an additional object of the invention relates to an in vitro method for monitoring a coronavirus infection comprising the steps of i) determining the level of a population of neutrophils having cell surface expression of CD66b+CD10-CD16- CD64+CD123+ markers and/or CD66b+CD10-CD16-CD64+LOX-l+ markers in a sample obtained from the subject at a first specific time of the disease, ii) determining the level of a population of neutrophils having cell surface expression of CD66b+CD10- CD 16- CD64+CD123+ markers and/or CD66b+CD10-CD16-CD64+LOX-l+ markers in a sample obtained from the subject at a second specific time of the disease, iii) comparing the level determined at step i) with the level determined at step ii) and iv) concluding that the disease has evolved in worse manner when the level determined at step ii) is higher than the level determined at step i).
  • the coronavirus infection is the severe or critical form of coronavirus infection (i.e. COVID-19)
  • An additional object of the invention relates to an in vitro method for monitoring the treatment of a coronavirus infection comprising the steps of i) determining the level of a population of neutrophils having cell surface expression of CD66+CD10-CD16- CD64+CD123+ and/or CD66+CD10-CD64+LOX-1+ in a sample obtained from the subject before the treatment, ii) determining the level of a population of neutrophils having cell surface expression of CD66b+CD10-CD16-CD64+CD123+ markers and/or CD66b+CD10- CD16-CD64+LOX-1+ markers in a sample obtained from the subject after the treatment”, iii) comparing the level determined at step i) with the level determined at step ii) and iv) concluding that the treatment is efficient when the level determined at step ii) is lower than the level determined at step i).
  • the coronavirus infection is the severe or critical form of coronavirus infection (i.e. COVID-19)
  • the decrease can be e.g. at least 5%, or at least 10%, or at least 20%, more preferably at least 50% even more preferably at least 100%.
  • another object of the invention relates to an in vitro method for assessing a subject’s risk of having or developing thrombosis in a patient with coronavirus infection comprising the steps of i) determining the level of neutrophil cells having cell surface expression of CD66b+CD10-CD16-CD64+LOX-l+ markers in a sample obtained from the subject, ii) comparing the level determined at step i) with the with a reference value and iv) concluding that:
  • thrombosis has its general meaning in the art and is the process by which an unwanted blood clot forms in a blood vessel. It can occur in a vein or in an artery. Arterial thrombosis is the cause of almost all cases of myocardial infarction and the majority of strokes, collectively the most common cause of deaths in the developed world. Deep vein thrombosis and pulmonary embolism are referred to as venous thromboembolism, which is currently the third leading cause of cardiovascular-associated death.
  • thrombosis includes inter alia atrophic thrombosis, arterial thrombosis, cardiac thrombosis, coronary thrombosis, creeping thrombosis, mesenteric thrombosis, placental thrombosis, propagating thrombosis, traumatic thrombosis and venous thrombosis and venous thromboembolism.
  • LOX-1 deletion was previously evaluated in a murine model of polymicrobial sepsis, resulting in the reduction of IL-6 and TNFa levels in blood and lungs, enhancing bacterial clearance and preventing neutrophils activation (19). More recently, LOX-1 was identified as a marker on granulocytic myeloid-derived suppressor cells able to suppress T cell activity (18). However, LOX-1 is mostly acknowledged for its role in atherosclerosis. LOX-1 is a class E scavenger receptor contributing to the formation of atherosclerotic plaques by promoting endothelial cell activation, macrophage foam cell formation, and smooth muscle cells migration and proliferation (24). LOX-1 activation induces NFKB activation leading to pro-inflammatory cytokines release, endoplasmic reticulum stress, and reactive oxygen species (ROS) production which could damage the microenvironment (25, 26).
  • ROS reactive oxygen species
  • LOX-1 role on neutrophils remains elusive. LOX-1 is barely detected on neutrophils at homeostasis, while its expression increases on neutrophils from human cancer patients (18) and in murine sepsis (19, 27).
  • LOX-1 expression on neutrophils seems to be detrimental for patients as is associated with the secretion of several pro-inflammatory cytokines, such as IL-6, IL-Ib and TNFa, and with severity (as assessed by the SOFA score) and thrombosis.
  • cytokines such as IL-6, IL-Ib and TNFa
  • SOFA score severity
  • thrombosis as assessed by the SOFA score
  • the integrity of the lung is compromised by an exaggerated immune response leading to acute respiratory distress syndrome (10, 16).
  • Mechanisms contributing to microcirculation disorders in sepsis are capillary leakage, leukocytes adhesion and infiltration and intravascular coagulation, leading to thrombus formation.
  • oxLDL oxidized low-density lipoprotein
  • oxLDL oxidized low-density lipoprotein
  • the increase of LOX-1 expression especially by endothelial cells, leads to an increase of LDL uptake into vessel wall which activates the specific Oct-l/SIRT-1 thrombosis protective pathway (28).
  • SIRT1 is able to supress the NFkB-induced expression of tissue factor, also known as thromboplastin, a key initiator of the coagulation cascade involved in thrombus formation (29).
  • inventors show an increase of the incidence of vascular thrombotic events among individuals displaying a high frequency of immature LOX-1+ neutrophils. It remains to be seen whether thrombosis in COVID-19 patients results from functionally-diverted neutrophils expressing LOX-1 and/or from its expression on endothelial and smooth muscle cells.
  • LOX-1 The overexpression of LOX-1 might also be found in other cell types that might trigger the prothrombotic ERK1/2 pathway.
  • the polymorphic LOX-1 gene is also intensively associated with increased susceptibility to myocardial diseases. LOX-1 should be thus considered a potential target for therapeutic intervention.
  • LOX1 is expressed and dysregulated in the immature neutrophil cells of the COVID subject.
  • LOX-1 would have a potential role in coronavirus (e.g. SARS-CoV-2) pathogenesis.
  • the invention relates to a method of preventing or treating a coronavirus infection in a patient in need thereof comprising administering to the patient a therapeutically effective amount of a LOX-1 (lectin-type oxidized LDL receptor 1) inhibitor.
  • LOX-1 lectin-type oxidized LDL receptor 1
  • the LOX-1 inhibitor is administrated by intravenous administration or intranasal administration.
  • the invention relates to a LOX-1 (lectin-type oxidized LDL receptor 1) inhibitor for use in the prevention or the treatment of a coronavirus infection in a subject in need thereof.
  • LOX-1 lectin-type oxidized LDL receptor 1
  • the invention relates to a LOX-1 (lectin-type oxidized LDL receptor 1) inhibitor for use in the prevention or the treatment of a coronavirus infection in a subject in need thereof, wherein the level of a population of neutrophils CD66b+CD10- CD 16-CD64+CD 123+ and/or CD66b+CD10-CD16-CD64+LOX-l+ obtained from said patient, have been detected by one of the methods (prognostic or monitoring) of the invention
  • treating refers to reversing, alleviating, inhibiting the progress of coronavirus infection, preferably inhibiting the severe form of coronavirus infection.
  • prevention or “prophylactic treatment” of coronavirus infections may refer to the administration of the compounds of the present invention that prevent the symptoms of coronavirus infections, in particular the severe form of coronavirus infections.
  • the term “subject” denotes a mammal, such as a rodent, a feline, a canine, or a primate.
  • the subject is a human.
  • the subject is an elderly human.
  • the subject denotes a human with a pathogen viral infection.
  • the subject denotes a human with a coronavirus infection.
  • the subject is a human with co-morbidities and in the elderly (see for example Guan et al., 2020).
  • the term “subject” encompasses the term "patient”.
  • LOX-1 inhibitor refers to refers to a natural or synthetic compound that has a biological effect to inhibit the activity or the expression of LOX-1.
  • inhibitor refers to an agent that is capable of specifically binding and inhibiting signaling through a receptor to fully block, as does an inhibitor, or detectably inhibit a response mediated by the receptor.
  • LOX-1 inhibitor is a natural or synthetic compound which binds and inactivates fully or partially LOX-1 for initiating or participating to a pathway signaling (such as the ERK prothrombotic pathway) and further biological processes.
  • the LOX-1 inhibitor in particular prevents, decreases or suppresses the virus replication.
  • the virus replication decrease observed can be by at least about 1%, 2%, 5%, 10%, e.g. by 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90%, as compared to the replication observed in a referenced cell.
  • LOX-1 inhibitory activity may be assessed by various known methods.
  • a control LOX-1 can be exposed to no antibody or antigen binding molecule, an antibody or antigen binding molecule that specifically binds to another antigen, or an anti- LOX-1 antibody or antigen binding molecule known not to function as an inhibitor, for example as an inhibitor.
  • the LOX-1 inhibitor inhibits the LOX-1 actions that exacerbate the effects of viral invasion and pro-inflammatory cytokines release (cytokine burst) and/or thromboembolic events and would be an effective therapeutic option for coronavirus infection and its consequences.
  • biological activity of LOX-1 inducing cytokine burst (through the control of pro-inflammatory cytokines release) and/or inducing thromboembolic events (through the ERK prothrombotic pathway).
  • the inhibitor specifically binds to LOX-1 (protein or nucleic sequence (DNA or mRNA)) in a sufficient manner to inhibit the biological activity of LOX-1. Binding to LOX-1 and inhibition of the biological activity of LOX-1 may be determined by any competing assays well known in the art.
  • the assay may consist in determining the ability of the agent to be tested as a LOX-1 inhibitor to bind to LOX-1. The binding ability is reflected by the Kd measurement.
  • KD is intended to refer to the dissociation constant, which is obtained from the ratio of Kd to Ka (i.e. Kd/Ka) and is expressed as a molar concentration (M). KD values for binding biomolecules can be determined using methods well established in the art.
  • an inhibitor that "specifically binds to LOX-1" is intended to refer to an inhibitor that binds to human LOX-1 polypeptide with a KD of ImM or less, lOOnM or less, lOnM or less, or 3nM or less. Then a competitive assay may be settled to determine the ability of the agent to inhibit biological activity of LOX-1.
  • the functional assays may be envisaged such as evaluating the ability to: a) inhibit processes associated with pro- inflammatory cytokines release and/or b) inhibit processes associated thromboembolic events (through the ERK prothrombotic pathway.
  • LOX-1 inhibitor neutralizes, blocks, inhibits, abrogates, reduces or interferes with a biological activity of LOX-1.
  • LOX-1 activity or expression
  • processes associated with pro-inflammatory cytokines release and/or b) inhibit processes associated thromboembolic events (through the ERK prothrombotic pathway) may be performed with each inhibitor.
  • inhibiting pro-inflammatory cytokines release can be assessed by detecting inflammatory cytokines beta with specific antibody, ultrasensitive immunodetection (digital ELISA) as described in the Example section (see Table 3), and ERK prothrombotic pathway assay can be measured by Phospho-ERK Assays (as described in Garbison Kim E et al “Phospho-ERK Assays” Book Assay Guidance Manual (published May 2012) ,Sittampalam GS, Grossman A, Brimacombe K, et al., editors.Bethesda (MD): Eli Lilly & Company and the National Center for Advancing Translational Sciences)
  • a LOX-1 inhibitor according to the invention can be a molecule selected from a peptide, a peptide mimetic, a small organic molecule, an antibody, an aptamer, a phospholipid, a polynucleotide (inhibitor of LOX-1 gene expression) and a compound comprising such a molecule or a combination thereof.
  • a LOX-1 inhibitor for use in the context of the present invention may be selected but is not limited, from: a) Natural LOX-1 inhibitors such as
  • Tanshinone II-A a pharmacologically active derivative of danshen, which is an herbal drug, has been shown to inhibit LOX-1 and ox-LDL uptake by macrophages); (as described in Xu S, Liu Z, Huang Y, et al. Tanshinone II-A inhibits oxidized LDL- induced LOX-1 expressionin macrophages by reducing intracellular superoxide radical generation andNF-kB activation. Transl Res 2012;160:114-24);
  • Curcumin (diferuloylmethane), an active ingredient of turmeric , has been shown to exert an anti-inflammatory effect and to inhibit atherogenesis and post-ischemic myocardial fibrosis. Curcumin also reduces Ang II-mediated up-regulation of Ang II type 1 receptors and LOX-1, and it decreases oxidative stress in mouse cardiomyocytes by decreasing the expression of nuclear factor kappa B (as described in Kang BY, et al. “Curcumin reduces angiotensin II-mediated cardiomyocyte growth via LOX-1 inhibition. J Cardiovasc Pharmacol 2010;55:417-24)
  • Flavonoids from Hippophae rhamnoides (sea buckthorn) as described in Bao M, Lou Y. “Flavonoids from seabuckthorn protect endothelial cells (EA.hy926) from oxidized low-density lipoprotein induced injuries via regulation of LOX-1 and eNOS expression. J Cardiovasc Pharmacol 2006;48:834-41
  • Gingko biloba extract commonly used as a therapeutic agent for cardiovascular and neurological disorders, inhibits ox-LDL-mediated expression of intercellular adhesion molecule, vascular cell adhesion molecule, and E-selectin; decreases ROS generation; and attenuates platelet-induced LOX-1 expression in endothelial cells (as described in Ou HC, Lee WJ, Lee IT, et al. Ginkgo biloba extract attenuates oxLDL-induced oxidative functional damages in endothelial cells. J Appl Physiol 2009;106:1674-85.)
  • Tetramethylpyrazine an active ingredient of Ligusticum wallichii Franchat, described in Wang GF, Shi CG, Sun MZ, et al. “Tetramethylpyrazine attenuates atherosclerosis development and protects endothelial cells from ox-LDL.” Cardiovasc Drugs Ther 2013 ;27 : 199-210)
  • Resveratrol (3,5,4’-trihydroxy-transstilbene) a polyphenol phytoalexin present in a variety of plant species (White hellebore, Polygonum cuspidatum, grapes, peanuts, mulberries, red wine) as described in Li H, Xia N, Forstermann U. Cardiovascular effects and molecular targets of resveratrol. Nitric Oxide 2012;26:102-10)
  • Pterostilbene a natural dimethylated analog of resveratrol, as described in Zhang L, Zhou G, Song W, et al. Pterostilbene protects vascular endothelial cells against oxidized low-density lipoprotein-induced apoptosis in vitro and in vivo. Apoptosis 2012;17:25-36
  • 6-Shogaol the major bioactive compound present in Zingiber officinale which possesses the anti-atherosclerotic effect as described Wang YK, Hong YJ, Yao YH, et al. “6-Shogaol protects against oxidized LDL-induced endothelial injuries by inhibiting oxidized LDL-evoked LOX-1 signaling. Evid Based Complement Alternat Med 2013;2013:503521).
  • Ellagic acid a polyphenolic compound widely distributed in fruits and nuts, as described in Lee WJ, Ou HC, Hsu WC, et al. “Ellagic acid inhibits oxidized LDL- mediated LOX-1 expression, ROS generation, and inflammation in human endothelial cells”. J Vase Surg 2010;52:1290-300
  • PLAzPC a modified oxidized phospholipid, which binds to the tunnel binding site of the LOX-1 molecule and markedly inhibits interaction with ox-LDL and described in Falconi M, Ciccone S, D’Arrigo P, et al. Design of a novel LOX-1 receptor antagonist mimicking the natural substrate. Biochem Biophys Res Commun 2013;438:340-5.);
  • the LOX-1 inhibitor can also be a peptide or peptide molecule comprising amino acid residues.
  • amino acid residue refers to any natural/standard and non-natural/non-standard amino acid residue in (L) or (D) configuration, and includes alpha or alpha-di substituted amino acids. It refers to isoleucine, leucine, lysine, methionine, phenylalanine, threonine, tryptophan, valine, arginine, alanine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, proline, serine, tyrosine.
  • beta-alanine 3 -amino-propionic acid, 2,3-diamino propionic acid, alpha- aminoisobutyric acid (Aib), 4-amino-butyric acid, N-methylglycine (sarcosine), hydroxyproline, ornithine (e.g., L-ornithine), citrulline, t-butylalanine, t-butylglycine, N- methylisoleucine, phenylglycine, cyclohexylalanine, cyclopentylalanine, cyclobutylalanine, cyclopropylalanine, cyclohexylglycine, cyclopentylglycine, cyclobutylglycine, cyclopropylglycine, norleucine (Nle), norvaline, 2-napthylalanine, pyridylalanine, 3- benzothienyl alanine
  • Example of peptide used as a LOX-1 inhibitor for use in the context of the present invention can be selected from specific peptides identified by subtractive Phage Display that targets LOX-1 (in particular. Peptides 17, 32 and 40) as described in White SJ et al “Identification of Peptides That Target the Endothelial Cell–Specific LOX-1” Receptor”Hypertension.2001;37:449–455; Amaranth synthetic pure peptides as described in Montoya-Rodr ⁇ guez A.
  • the bond which replaces the amide bond conserves many or all of the properties of the amide bond, e.g. conformation, steric bulk, electrostatic character, potential for hydrogen bonding, etc.
  • Typical peptide bond replacements include esters, polyamines and derivatives thereof as well as substituted alkanes and alkenes, such as aminomethyl and ketomethylene.
  • the peptide may have one or more peptide linkages replaced by linkages such as -CH2NH-, -CH2S-, -CH2-CH2-, -CH ⁇ CH- (cis or trans), -CH(OH)CH2-, or -COCH2-, -N- NH-, -CH2NHNH-, or peptoid linkages in which the side chain is connected to the nitrogen atom instead of the carbon atom.
  • Such peptidomimetics may have greater chemical stability, enhanced biological/pharmacological properties (e.g., half-life, absorption, potency, efficiency, etc.) and/or reduced antigenicity relative its peptide equivalent.
  • the LOX-1 inhibitor can also be a small organic molecule.
  • small organic molecule refers to a molecule of a size comparable to those organic molecules generally used in pharmaceuticals. The term excludes biological macromolecules (e.g., proteins, nucleic acids, etc.). Preferred small organic molecules range in size up to about 5000 Da, more preferably up to 2000 Da, and most preferably up to about 1000 Da. Examples of small organic molecule are Molecule 4 and 5 described un Thakkar et al. and in patent application WO2017075418.
  • the LOX-1 inhibitor can also be an antibody or an antigen-binding molecule.
  • the antibody specifically recognize/bind LOX-1 (e.g. LOX-1 of SEQ ID NO:l) or an epitope thereof involved in the activation/stimulation of the ERK-pathway.
  • the antibody is a monoclonal antibody or single chain antibody.
  • Example of monoclonal antibody used as a LOX-1 inhibitor for use in the context of the present invention can be selected from the monocolal antibodies described in Iwamoto S et al. “Generation and characterization of chicken monoclonal antibodies against human LOX-1. MAbs 2009;1:357-63 (and US2012087926), monocolal antibodies developed by Novartis described in W02014205300, monocolal antibodies developed by Abgenics described in EP1418234.
  • Example of single chain antibody used as a LOX-1 inhibitor for use in the context of the present invention can be the anti -LOX-1 human single chain antibody by JILIN University described in CN107216390
  • antibody is used in the broadest sense, and covers monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, multispecific antibodies, chimeric antibodies and humanized antibodies, so long as they exhibit the desired biological activity (e.g., as indicated previously, a) inhibiting processes associated with pro- inflammatory cytokines release and/or b) inhibiting processes associated thromboembolic events (through the ERK prothrombotic pathway.).
  • Antibody fragments comprise a portion of a full-length antibody, generally an antigen binding or variable region thereof.
  • antibody fragments include Fab, Fab' , F(ab' )2, and Fv fragments, diabodies, linear antibodies, single-chain antibody molecules, single domain antibodies (e.g., from camelids), shark NAR single domain antibodies, and multispecific antibodies formed from antibody fragments.
  • Antibody fragments can also refer to binding moieties comprising CDRs or antigen binding domains including, but not limited to, V H regions (V H, V H-V H), anticalins, PepBodies, antibody-T-cell epitope fusions (Troybodies) or Peptibodies.
  • Antibodies according to the present invention can be of any class, such as IgG, IgA, IgDl IgEl IgMl or IgYl although IgG antibodies are typically preferred.
  • Antibodies can be of any mammalian or avian origin, including human, murine (mouse or rat), donkey, sheep, goat, rabbit, camel, horse, or chicken.
  • the antibodies can be modified by the covalent attachment of any type of molecule to the antibody.
  • the antibody derivatives include antibodies that have been modified, e.g., by glycosylation, acetylation, pegylation, phosphylation, amidation, derivatization by known protecting/blocking groups, proteolytic cleavage, linkage to a cellular ligand or other protein, or other modifications known in the art.
  • antibodies according to the present invention can be fused to marker sequences, such as a peptide tag to facilitate purification; a suitable tag is a hexahistidine tag.
  • the antibodies can also be conjugated to a diagnostic or therapeutic agent by methods known in the art. Techniques for preparing such conjugates are well known in the art. Other methods of preparing these monoclonal antibodies, as well as chimeric antibodies, humanized antibodies, and single-chain antibodies, are known in the art.
  • the LOX-1 inhibitor can also be an aptamer.
  • Aptamers are a class of molecule that represents an alternative to antibodies in term of molecular recognition.
  • Aptamers are oligonucleotide or oligopeptide sequences with the capacity to recognize virtually any class of target molecules with high affinity and specificity.
  • Such ligands may be isolated through Systematic Evolution of Ligands by Exponential enrichment (SELEX) of a random sequence library, as described in Tuerk C. and Gold L., 1990.
  • the random sequence library is obtainable by combinatorial chemical synthesis of DNA. In this library, each member is a linear oligomer, eventually chemically modified, of a unique sequence.
  • Peptide aptamers consists of a conformationally constrained antibody variable region displayed by a platform protein, such as E. coli Thioredoxin A that are selected from combinatorial libraries by two hybrid methods (Colas et al., 1996).
  • a phospholipid as a LOX-1 inhibitor.
  • LOX-1 inhibitor a modified phospholipid, PLAzPC, which binds to the tunnel binding site of the LOX-1 molecule and markedly inhibits interaction with ox-LDL.
  • phospholipid usable in the context of the invention is PLAzPC.
  • the LOX-1 inhibitor can also be a polynucleotide, typically an inhibitory nucleotide.
  • inhibitory nucleotide include short interfering RNA (siRNA), microRNA (miRNA), and synthetic hairpin RNA (shRNA), anti-sense nucleic acids, complementary DNA (cDNA) or guide RNA (gRNA usable in the context of a CRISPR/Cas system).
  • siRNA short interfering RNA
  • miRNA microRNA
  • shRNA synthetic hairpin RNA
  • anti-sense nucleic acids complementary DNA
  • gRNA guide RNA
  • gRNA guide RNA
  • gRNA targeting LOX-1 expression is used. Interference with the function and expression of endogenous genes by double-stranded RNA such as siRNA has been shown in various organisms.
  • siRNAs can include hairpin loops comprising self- complementary sequences or double stranded sequences.
  • siRNAs typically have fewer than 100 base pairs and can be, e.g., about 30 bps or shorter, and can be made by approaches known in the art, including the use of complementary DNA strands or synthetic approaches.
  • Such double-stranded RNA can be synthesized by in vitro transcription of single- stranded RNA read from both directions of a template and in vitro annealing of sense and antisense RNA strands.
  • Double-stranded RNA targeting LOX-1 can also be synthesized from a cDNA vector construct in which a LOX-1 gene (e.g., human LOX-1 gene) is cloned in opposing orientations separated by an inverted repeat. Following cell transfection, the RNA is transcribed and the complementary strands reanneal.
  • Double-stranded RNA targeting the LOX-1 gene can be introduced into a cell (e.g., a tumor cell) by transfection of an appropriate construct.
  • RNA interference mediated by siRNA, miRNA, or shRNA is mediated at the level of translation; in other words, these interfering RNA molecules prevent translation of the corresponding mRNA molecules and lead to their degradation. It is also possible that RNA interference may also operate at the level of transcription, blocking transcription of the regions of the genome corresponding to these interfering RNA molecules.
  • RNA molecules The structure and function of these interfering RNA molecules are well known in the art and are described, for example, in R. F. Gesteland et ah, eds, “The RNA World” (3rd, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 2006), pp. 535-565, incorporated herein by this reference.
  • cloning into vectors and transfection methods are also well known in the art and are described, for example, in J. Sambrook & D. R. Russell, “Molecular Cloning: A Laboratory Manual” (3rd, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, 2001), incorporated herein by this reference.
  • nucleic acid agents targeting LOX-1 can also be employed in the practice of the present invention, e.g., antisense nucleic acids.
  • Antisense nucleic acids are DNA or RNA molecules that are complementary to at least a portion of a specific target mRNA molecule. In the cell, the single stranded antisense molecule hybridizes to that mRNA, forming a double stranded molecule. The cell does not translate an mRNA in this double-stranded form. Therefore, antisense nucleic acids interfere with the translation of mRNA into protein, and, thus, with the expression of a gene that is transcribed into that mRNA.
  • Antisense methods have been used to inhibit the expression of many genes in vitro. See, e.g., Li D et ak, “Antisense to LOX-1 inhibits oxidized LDL- mediated upregulation of monocyte chemoattractant protein- 1 and monocyte adhesion to human coronary artery endothelial cells “Circulation. 2000 Jun 27; 101 (25):2889-95. doi: 10.1161; Amati F et al , “LOX-1 Inhibition in ApoE KO Mice Using a Schizophyllan-based Antisense Oligonucleotide Therapy,” Mol Ther Nucleic Acids. 2012 Dec; 1(12): e58;, incorporated herein by this reference.
  • LOX-1 polynucleotide sequences from human and many other animals in particular mammals have all been delineated in the art. Based on the known sequences, inhibitory nucleotides (e.g., siRNA, miRNA, or shRNA) targeting LOX-1 can be readily synthesized using methods well known in the art.
  • inhibitory nucleotides e.g., siRNA, miRNA, or shRNA
  • Exemplary siRNAs according to the invention could have up to 29 bps, 25 bps, 22 bps, 21 bps, 20 bps, 15 bps, 10 bps, 5 bps or any integral number of base pairs between these numbers.
  • Tools for designing optimal inhibitory siRNAs include that available from DNAengine Inc. (Seattle, Wash.) and Ambion, Inc. (Austin, Tex).
  • the guide RNA (gRNA) sequences direct a nuclease (i.e. CrispRCas9 protein) to induce a site-specific double strand break (DSB) in the genomic DNA in the target sequence.
  • a nuclease i.e. CrispRCas9 protein
  • DSB site-specific double strand break
  • the invention also relates to a method for treating coronavirus infection with a LOX-1 inhibitor in a subject wherein the level of a population of neutrophils CD66b+CD10-CD16- CD64+LOX-1+ obtained from said patient have been detected by one of method of the invention.
  • treating means reversing, alleviating, inhibiting the progress of, or preventing the disorder or condition to which such term applies, or reversing, alleviating, inhibiting the progress of, or preventing one or more symptoms of the disorder or condition to which such term applies.
  • a LOX-1 inhibitor according to the invention can be a molecule selected from a peptide, a peptide mimetic, a small organic molecule, an antibody, an aptamer, a phospholipid, a polynucleotide (inhibitor of LOX-1 gene expression) and a compound comprising such a molecule or a combination thereof.
  • Another object of the present invention is a method of treating coronavirus infection in a subject comprising the steps of: a) providing a sample containing neutrophil from a subject, b) detecting the level of a population of neutrophils CD66b+CD10-CD16- CD64+CD123+ and/or CD66b+CD10-CD16-CD64+LOX-l+ c) comparing the level determined at stet b) with a reference value and if level determined at stet b) is higher than the reference value, treating the subject with Lox-1 inhibitors.
  • anticoagulant is the current main treatment for the severe form of coronavirus infection or thrombosis associated with coronavirus infection.
  • the invention also relates to a method for treating coronavirus infection with anticoagulant in a subject wherein the level of a population of neutrophils CD66b+CD 10-CD 16-CD64+CD 123+ and/or CD66b+CD10-CD16-CD64+LOX-l+ obtained from said patient, have been detected by one of the methods of the invention.
  • Method of treating coronavirus infection in a subject comprising the steps of: a) providing a sample containing neutrophils from a subject, b) detecting the level of a population of neutrophils CD66b+CD10-CD16- CD64+CD123+ and/or CD66b+CD10-CD16-CD64+LOX-l+ c) comparing the level determined at stet b) with a reference value and if level determined at step b) is higher than the reference value, treating the subject with anticoagulant.
  • the coronavirus infection is the severe form of coronavirus infection and/or thrombosis associated with coronavirus infection
  • anticoagulant agents defined herein as agents that inhibit blood clot formation
  • agents that inhibit blood clot formation include, without limitation, specific inhibitors of thrombin, factor IXa, FXa, factor XIa, factor Xlla or factor Vila, heparin and derivatives, Vitamin K antagonists (VKA), Non- VKA Anticoagulant agents” and anti-tissue factor antibodies.
  • Non-VKA (Vitamin K Antagonists) anticoagulants includes non-VKA oral anticoagulants (NOAC) such as direct-oral anticoagulants (DOAC).
  • NOAC non-VKA oral anticoagulants
  • DOAC direct-oral anticoagulants
  • specific inhibitors of thrombin include hirudin, bivalirudin (Angiomax®), argatroban, and lepirudin (Refludan®).
  • heparin and derivatives include unfractionated heparin (UFH), low molecular weight heparin (LMWH), such as enoxaparin (Lovenox®), dalteparin (Fragmin®), tinzaparin (Innohep®), nadroparine (Fraxiparine® or Fraxodi®); and synthetic pentasaccharide, such as fondaparinux (Arixtra®).
  • DOAC anticoagulants include rivaroxaban (Xarelto®), apixaban (Eliquis®), edoxaban (Lixiana®), and dabigatran (Pradaxa®).
  • the anticoagulant is an inhibitor of FXa.
  • Vitamin K antagonists include, in a non-limitative manner, warfarin (Coumadin®), phenocoumarol, acenocoumarol (Sintrom®), clorindione, dicoumarol, diphenadione, ethyl biscoumacetate, phenprocoumon, phenindione, fluindione (Previscan®) and tioclomarol.
  • FIGURES are a diagrammatic representation of FIGURES.
  • Figure 1 Severe CO ID-19 patients displayed increased immature neutrophil subsets expressing CD123 or LOX-1.
  • viSNE analysis was performed on neutrophils from all samples with cells organized along t-SNE-1 and t-SNE-2 according to per-cell expression of CD15, CD10, CD64, LOX-1, CD123 and PD-L1.
  • Cell density for the concatenated file of each patient’s group (ICU vs Non-ICU) is shown on a black to yellow heat scale.
  • CD64 markers expression is presented on a rainbow heat scale in the t-SNE map of each group concatenated file.
  • Figure 2 Abundancy of LOX-l-expressing immature neutrophil correlate with clinical severity of COVID-19 patients.
  • Figure 3 Abundancy of LOX-l-expressing immature neutrophil correlate with thrombosis of COVID-19 patients.
  • Nonparametric Mann-Whitney test was used to compare differences in frequencies of neutrophil subsets between the two groups, with significance defined by a p-value ⁇ 0.05: * for p ⁇ 0.05; ** for p ⁇ 0.01; **** for p ⁇ 0.0001.
  • B Receiver operating characteristic (ROC) curve analysis was performed to assess the predictive value of LOX-1 with thrombosis.
  • Figure 4 Increased proportions of circulating immature neutrophils expressing either CD123 or LOX-1 in critical COVID-19 patients are associated with COVID-19 severity and thromboembolic complications.
  • A Abundance of CD10 CD64 + neutrophils expressing CD123, PD-L1, or LOX-1 in ICU and non-ICU patient groups.
  • B-C Box plots (min to max distribution) of the proportion of total ImNs and ImNs expressing CD123-, LOX- 1, or PD-L1 in patient groups with invasive mechanical ventilation (B) or with thromboembolic complications (C).
  • Nonparametric Mann-Whitney test was used to compare differences in cellular abundance of neutrophil subsets between groups, with significance defined by a p-value ⁇ 0.05: **p ⁇ 0.01; and ***p ⁇ 0.001.
  • FIG. 5 Subsets of immature neutrophils expressing LOX-1 infiltrate lung.
  • FIG. 6 Immature neutrophil subsets expressing either CD123, LOX-1, or PD- L1 are correlated with clinical severity, but only LOX-1+ subset proportion at entry is strongly associated with higher risk of thrombosis.
  • One-way ANOVA test was used to compare the three groups, with significance defined as follows: ***p ⁇ 0.001; and **** > ⁇ 0.0001.
  • Nonparametric Mann-Whitney test was used to compare differences between groups, with significance defined by a p-v alue ⁇ 0.05: * **p ⁇ 0.01; ***p ⁇ 0.001.
  • Nonparametric Mann- Whitney test was used to compare differences in cellular abundance of neutrophil subsets between groups, with significance defined by a p-v alue ⁇ 0.05: *p ⁇ 0.05; **p ⁇ 0.01.
  • Log-rank (Mantel-Cox) test was used to compare HR between groups, with significance defined by a p- value ⁇ 0.05: *p ⁇ 0.05; **p ⁇ 0.01; and ***p ⁇ 0.001.
  • FIG. 7 Immature neutrophil subsets and plasma levels of MPO and ELA are independent markers of COVID-19 severity.
  • the concentrations of MPO are expressed as pg/mL, ELA as 10-1 pg/mL, and MPO-DNA complexes as an arbitrary unit proportional to 10-5 of the ratio blank/sample of the absorbance measured at 450 nm.
  • One-way ANOVA test was used to compare the three groups, with significance defined as ***p ⁇ 0.001.
  • Nonparametric Mann-Whitney test was used to compare differences in cellular abundance of neutrophil subsets between groups, with significance defined by a p-value ⁇ 0.05: *p ⁇ 0.05; and **p ⁇ 0.01.
  • ACD Anticoagulant Citrate-Dextrose solution
  • Samples were diluted in brilliant violet buffer (BD biosciences) and incubated 20 min at room temperature in the dark.
  • the antibody panel (Table 3) included: CD15-BV786, CD14-BUV737, CD10-BUV395 (BD, Le Pont de Claix, France); CRTH2-FITC, CD 123 -PE, LOX-1-BV421, CD64-BV605 and LOX-1-BV711 (Biolegend, San Diego, USA).
  • BD FACS lysing (BD biosciences) solution IX was directly added to the cells to lyse red blood cells, incubated 20min, centrifuged, and washed with PBS. Leukocytes were resuspended in PBS before analysis with a BD LSR FORTES SA-X-20TM. FlowJoTM software 10.0 was used for analysis of marker expression on neutrophils. One hundred m ⁇ of whole blood were additionally stained for several patients with a FMO mix missing of antibodies targeting CD123, LOX-1, and LOX-1, in order to determine the threshold of expression of these markers.
  • Quanterix technology digital ELISA
  • the SimoaTM (single molecule array) HD-1 analyser (Quanterix, Lexington, MA, USA) was used for ultrasensitive immunodetection (digital ELISA) of IL-3, IL-17A, IL-18, GM-CSF and IFN-a, using singleplex bead-based assays (Table 3). Concentrations of IL-Ib, IFN-g, IL-6, IL-8, IL-22, TNF-a and IL-10 were determined using a multiplex planar array immunoassay on the Quanterix SP-XTM platform according to manufacturer’s instructions. Serum IFN-b levels were quantified with a highly sensitive ELISA kit (PBL Assay Science, Piscataway, NJ, USA).
  • IL-Ib (0.073-300 pg/mL), IFN-g (0.012-50 pg/mL), IL-6 (0.073-300 pg/mL), IL-8 (0.098-400 pg/mL), IL-22 (0.024-100 pg/mL), TNFa (0.098-400 pg/mL), IL-10 (0.024-100 pg/mL), IL-3 (0.686-500 pg/mL), IL- 17A (0.041-30 pg/mL), IL-18 (0.011-45 pg/mL), GM-CSF (0.041-30 pg/mL), IFN-a (0.028- 27.3 pg/mL) and IFN-b (1.2-150 pg/mL).
  • Treatment regimen at baseline was mostly anti hypertensive therapy (ACE inhibitors 26.3% and angiotensin II receptor blockers 15.8%). Severity at baseline was assessed by the SAPS II score for all patients (median 33, ranging from 25 to 78) and an additional SOFA score for ICU patients (median 8.5, ranging from 2 to 17). Twenty-eight patients were assessed with CT chest imaging, with ground-glass opacities and/or consolidation > 50% of the lung field among 50% of all patients, with up to 81.3% of the ICU patients.
  • SAPS II Simplified Acute Physiology Score
  • LOX-1+ neutrophil proportions are positively correlated with clinical severity and cytokine levels
  • COVID-19 patients from ICU were segregated into two groups based on severity at the time of admission.
  • Patients with low SOFA ( ⁇ 8) had significantly fewer CD123- and LOX-1 -expressing immature neutrophils than patients with high SOFA (>8), (p ⁇ 0.01 and p ⁇ 0.001 respectively) (Figure 2A).
  • the abundancy of LOX-1 -expressing immature neutrophils correlated positively with the SOFA score (Table 1), with inflammatory cytokines such as IL-Ib, IL-6, IL-8, TNFa and with the anti-inflammatory cytokine IL-10, whereas it correlated negatively with IFNa and the multipotent hematopoietic growth factor IL-3.
  • ROC curve analysis suggested that LOX-1 may be an accurate predictive marker of thrombosis for COVID-19 patients.
  • the antibody panel included CD15-BV786, CD14-BUV737, and CD10-BUV395 (BD, Le Pont-de-Claix, France); and CRTH2-FITC, CD123-PE, LOX-1-BV421, CD64-BV605, and PD-L1-BV711 (BioLegend, San Diego, CA, USA).
  • BD FACS lysing solution IX (BD Biosciences) was directly added to the cells to lyse red blood cells, which were incubated for 20 min, centrifuged, and washed with PBS.
  • Leukocytes were resuspended in PBS before analysis with a BD LSRFortessa X-20 (BD Biosciences).
  • FlowJo software 10.0 (FlowJo LLC, Ashland, OR, USA) was used for analysis of marker expression on neutrophils.
  • One hundred microliters of whole blood was stained with a fluorescence minus one (FMO) mix missing antibodies targeting CD 123, LOX-1, and PD-L1, in order to determine the threshold of expression of these markers.
  • FMO fluorescence minus one
  • BAL leukocyte phenotyping was performed similarly after filtration, two wash procedures of BAL cells, and staining with the same antibody mix. Acquired data were normalized and analyzed using the OMIQ platform (https://www.omiq.ai).
  • neutrophil subsets To identify neutrophil subsets and visualize all cells in a 2D map where position represents local phenotypic similarity, we used a dimensionality reduction tool: the opt-SNE implementation of t-SNE. Neutrophils (40 000 events) were randomly taken from the sample for the unsupervised analysis. Cells were also grouped in phenotypically homogeneous clusters using the FlowSOM algorithm.
  • MPO and neutrophil ELA were measured using Human Myeloperoxidase and Human Neutrophil Elastase/ELA2 DuoSet ELISA kits (R&D Systems, Minneapolis, MN, USA), with plasma or BAL diluted to 1:100 with PBS according to manufacturer’s instructions.
  • concentrations of MPO and ELA were expressed as picograms/milliliter or relative luminescence units.
  • Netosis was measured in the patient’s plasma by detecting MPO-DNA complexes using anti-human MPO primary antibody (clone 4A4; AbD Serotec, Marnes-la- Coquette, France) as the capture antibody and a peroxidase-labeled anti-DNA antibody (clone MCA-33; Roche, Mannheim, Germany) as the detection antibody. Plasma samples were diluted 1:4 in PBS.
  • Quanterix technology digital ELISA
  • the SimoaTM (single molecule array) HD-1 analyzer (Quanterix, Lexington, MA, USA) using singleplex bead-based assays was used for ultrasensitive immunodetection of IL- 3, IL-17A, IL-18, GM-CSF, and IFN-a.
  • Concentrations of IL-Ib, IFN-g, IL-6, IL-8, IL-22, TNFa, and IL-10 were determined using a multiplex planar array immunoassay on the Quanterix SP-X platform according to manufacturer’s instructions.
  • Serum IFN-b levels were quantified with a highly sensitive ELISA kit (PBL Assay Science, Piscataway, NJ, USA).
  • IL-Ib (0.073-300 pg/mL), IFN-g (0.012-50 pg/mL), IL-6 (0.073-300 pg/mL), IL-8 (0.098-400 pg/mL), IL-22 (0.024-100 pg/mL), TNFa (0.098-400 pg/mL), IL-10 (0.024-100 pg/mL), IL-3 (0.686-500 pg/mL), IL-17A (0.041-30 pg/mL), IL- 18 (0.011-45 pg/mL), GM-CSF (0.041-30 pg/mL), IFN-a (0.028-27.3 pg/mL), and IFN-b (1.2-150 pg/mL).
  • SAPS Simplified Acute Physiology Score
  • ImN subsets expressing either CD123, LOX-1, or PD-L1 may define specific profiles of severity associated with high levels of cytokines.
  • BALs neutrophil subsets in the bronchoalveolar lavages
  • Figure 5 neutrophil subsets in the bronchoalveolar lavages
  • BAL neutrophils were identified in the upper right quadrant of the map; blood neutrophils from COVID-19 patients were more central; and blood neutrophils from healthy donors (HD) were organized in the lower left quadrant of the map ( Figure 5A).
  • Automatic clustering using major (CD 15, CD 10, CD 16, CD64) and specific neutrophil markers split neutrophil signatures into positive and negative subpopulations for each marker (data not shown).
  • ImNs expressing LOX-1, PD-L1, or CD123 represented a few percent of COVID- 19 blood neutrophils (see also Figure 4A)
  • these subsets were much more present in patient BALs with ImNs expressing LOX-1 being the major subset, representing about 40% of total neutrophils.
  • a profusion of ImNs in COVID-19 BAL was associated with massive production of myeloperoxidase (MPO) and neutrophil elastase (ELA) ( Figure 5B), two antimicrobial and cytotoxic proteins known to be highly concentrated in the azurophilic granule of ImN.
  • MPO myeloperoxidase
  • ESA neutrophil elastase
  • ImNs preferentially those expressing LOX-1, infiltrate bronchoalveolar space in the lungs of COVID-19 patients, where they release their cytotoxic content, suggesting a potential role in disease severity.
  • Immature neutrophil subsets expressing CD123, LOX-1, or PD-L1 are correlated with clinical severity, but only the LOX-1+ subset proportion at entry is strongly associated with higher risk of thrombosis.
  • MPO, ELA, and MPO-DNA complexes representing NET formation were significantly associated with disease severity ( Figure 7A), whereas ELA-DNA complexes were not.
  • MPO and ELA levels at hospital admission were also significantly increased among COVID-19 patients who later died ( Figure 7B). MPO-DNA complex levels were not associated with survival.
  • the AUC was 0.89 for LOX-1 ImN abundance (p ⁇ 0.0001), indicating that LOX-1 expression on ImN in the blood at the time of hospital admission could accurately predict later thromboembolic events among COVID-19 patients during hospitalization.
  • a cutoff point of 0.5% abundance of the LOX-1 ImN subset was able to detect patients with thrombotic events with a sensitivity of 100% and patients without complications with a specificity of 53%.
  • a cutoff point of 2% reached a sensitivity of 82% and a specificity of 75%.
  • CD123 the alpha chain of the Interleukin-3 receptor
  • IL-3 cytokine the IL-3 cytokine
  • IL-3 promotes emergency myelopoiesis, exacerbating pro-inflammatory cytokines secretion and, consequently, systemic inflammation, organ dysfunction and death.
  • the authors further tested the prognostic value of IL-3 in two small cohorts of humans with sepsis and found that high plasma IL-3 levels were associated with high mortality even when adjusting for disease severity.
  • LOX-1 deletion was previously evaluated in a murine model of polymicrobial sepsis, resulting in the reduction of IL-6 and TNFa levels in blood and lungs, enhancing bacterial clearance and preventing neutrophils activation (19). More recently, LOX-1 was identified as a marker on granulocytic myeloid-derived suppressor cells able to suppress T cell activity (18). However, LOX-1 is mostly acknowledged for its role in atherosclerosis. LOX-1 is a class E scavenger receptor contributing to the formation of atherosclerotic plaques by promoting endothelial cell activation, macrophage foam cell formation, and smooth muscle cells migration and proliferation (24). LOX-1 activation induces NFKB activation leading to pro-inflammatory cytokines release, endoplasmic reticulum stress, and reactive oxygen species (ROS) production which could damage the microenvironment (25, 26).
  • ROS reactive oxygen species
  • LOX-1 role on neutrophils remains elusive. LOX-1 is barely detected on neutrophils at homeostasis, while its expression increases on neutrophils from human cancer patients (18) and in murine sepsis (19, 27).
  • LOX-1 expression on neutrophils seems to be detrimental for patients as it was associated with the secretion of several pro-inflammatory cytokines, such as IL-6, IL- 1b and TNFa, and with severity (as assessed by the SOFA score) and thrombosis.
  • cytokines such as IL-6, IL- 1b and TNFa
  • severity assessed by the SOFA score
  • thrombosis In severe cases of COVID-19, the integrity of the lung is compromised by an exaggerated immune response leading to acute respiratory distress syndrome (10, 16).
  • Mechanisms contributing to microcirculation disorders in sepsis are capillary leakage, leukocytes adhesion and infiltration and intravascular coagulation, leading to thrombus formation.
  • oxLDL oxidized low-density lipoprotein
  • oxLDL oxidized low-density lipoprotein
  • the increase of LOX-1 expression especially by endothelial cells, leads to an increase of LDL uptake into vessel wall which activates the specific Oct-l/SIRT-1 thrombosis protective pathway (28).
  • SIRT1 is able to supress the NFkB-induced expression of tissue factor, also known as thromboplastin, a key initiator of the coagulation cascade involved in thrombus formation (29).
  • LOX-1 The overexpression of LOX-1 might also be found in other cell types that might trigger the prothrombotic ERK1/2 pathway. Further investigations would be necessary, such as the titration of oxLDL in blood or the evaluation of the ERK1/2 pathway. In addition, some studies support the relationship between ACE/ACE2 axis and the expression of the pro- oxidative molecule LOX-1, which could increase the oxidative stress favoring prothrombotic state (30). SARS-CoV-2 virus requires binding to ACE2 and is particularly deleterious to patients with underlying cardiovascular disease (31). The polymorphic LOX-1 gene is also intensively associated with increased susceptibility to myocardial diseases. LOX-1 should be thus considered a potential target for therapeutic intervention.
  • the SimoaTM (single molecule array) HD-1 analyser was used for ultrasensitive multiplex immunodetection of cytokines as describe in methods section.
  • the potential association between serum cytokine levels or marker expressing neutrophils frequencies was evaluated by Spearman correlation (one-tailed), with significance defined by a p-value ⁇ 0.05:
  • n (%) cardiac, liver or kidney allograft, hematopoietic stem cell transplantation, or immunosuppressive agent for auto-immune disease o 28 patients were assessed
  • CT computed tomography
  • GGO ground-glass opacities
  • SAPS II Simplified Acute Physiology Score II
  • SOFA score Sequential organ failure assessment score
  • Lectin-type oxidized LDL receptor-1 distinguishes population of human polymorphonuclear myeloid-derived suppressor cells in cancer patients. Sci Immunol 2016;l:aaf8943-aaf8943.

Abstract

In the present invention, inventors investigate the representation of neutrophil subsets in severe and critical COVID-19 patients based on Intensive Care Units (ICU) and non-ICU admission. The results show that 80% of ICU patients develop strong myelemia with CD10- CD64+ immature neutrophils. Cellular profiling revealed two distinct neutrophil subsets expressing either the LOX‐1 or CD123 marker, both overrepresented in ICU patients compared to non-ICU patients. The proportion of LOX-1-expressing immature neutrophils positively correlated with clinical severity, with the cytokine storm (IL-1β, IL-6, IL-8, TNFα), and with intravascular coagulation. Importantly, high proportions of LOX-1+- immature neutrophils are associated with higher risk of severe thrombosis. The present invention relates to non-invasive, specific and rapid methods for prognostic and monitoring the severe / critical form of coronavirus infection. More specifically present invention relates to methods for prognosis and/or monitoring of the critical form of coronavirus infection through detection of a specific population of neutrophils in a covid patient. The present invention also relates to a method of preventing or treating a coronavirus infection in a subject in need thereof

Description

METHODS FOR PROGNOSIS AND MONITORING OF CRITICAL FORM OF
CORONA VIRUS INFECTION
FIELD OF THE INVENTION:
The present invention relates to methods and kits for prognostic and monitoring the severe / critical form of coronavirus infection. More specifically present invention relates to methods for prognosis of the critical form of coronavirus infection through detection of a specific population of neutrophils in a patient. The present invention also relates to a method of preventing or treating a coronavirus in a subject in need thereof
BACKGROUND OF THE INVENTION:
Since the first reports of an outbreak of a severe acute respiratory syndrome caused by coronavirus 2 (SARS-CoV-2) in China in December 2019 (1, 2), the coronavirus disease 2019 (COVID-19) has grown to be a global public health emergency, with cases of COVID-19 around the world reaching 8 385 440 cases and 450 686 deaths as of June 19th, 2020 (for up- to-date, https://www.who.int/emergencies/diseases/novel-coronavirus-2019). SARS-CoV-2 infection is characterized by a range of symptoms including fever, cough, fatigue and myalgia in the majority of cases and occasional headache and diarrhea (1, 3). Among reported cases, approximatively 80% present with mild condition, 13% serious condition, and 6% develop a critical state requiring intensive care, the latter associated with a fatality rate of 2 to 8% of reported cases (4). Some severe cases of COVID-19 progress to acute respiratory distress syndrome (ARDS), accountable for high mortality related to the damages of the alveolar lumen. Numerous patients with ARDS secondary to COVID-19 develop life-threatening thrombotic complications (5).
Previous coronaviruses-related infections have been characterized by the onset of a cytokine storm (6). It is therefore reasonable to postulate that the inflammatory response measured both at cellular and molecular levels could represent a strong prognostic signature of the disease. Molecular assays have been the gold standard to directly detect the presence of the virus as well as to characterize the infection onset. The cytokine storm remains as of today an uncontrollable inflammatory response leading to viral sepsis, acute respiratory distress syndrome, respiratory failure, shock, organ failure or death (7, 8). In addition to the life- threatening course of the disease, many critically ill patients develop complications with a high burden (e.g. thrombosis, ventilator-associated pneumonia, acute kidney injury...). Strong predictive markers are still missing for these complications.
Accordingly, there remains an unmet need in the art for specific and more rapid prognostic test for critical state of Covid patient, reflecting directly the dysfunction of immune process.
The inventors therefore set up a prognostic and monitoring method of the critical form of coronavirus infection that allows to directly reflect the immunological status of the patient.
A retrospective cohort of 201 patients with confirmed COVID- 19 pneumonia revealed that older age, neutrophilia, organ and coagulation dysfunction were the major risk factors associated with the development of ARDS and progression to death (9). ARDS and sepsis are frequent complications among deceased patients (10). In severe cases, bilateral lung involvement with ground-glass opacities is the most common chest computed tomography (CT) finding. More surprisingly, abnormal CT scans were also reported on asymptomatic COVID-19 patients (11). Immune transcriptome profiling of bronchoalveolar lavage fluids of COVID-19 patients also displayed high levels of pro-inflammatory cytokines (6). In addition, serum concentrations of both pro- and anti-inflammatory cytokines including IL-6, TNFa, and IL-10, were increased in the majority of severe cases and were markedly higher than those of moderate cases, suggesting that the cytokine storm might be associated with disease severity and leading the way to the development of potential immune-modulatory treatments (3, 12). The cytokine storm is associated with a massive influx of innate immune cells, namely neutrophils and monocytes, which could worsen lung injury. However, little is known about the innate immune features and the molecular mechanisms involved in COVID-19 severity.
Increasing clinical data indicated that the neutrophil-to-lymphocyte ratio (NLR) was a powerful predictive and prognostic indicator of severe COVID-19 (13-15). Lymphopenia, neutrophilia, and high NLR are associated with a more severe viral infection (13, 16).
Inventors previously identified two new CD10-CD64+ neutrophil subsets expressing either PD-L1 or CD123 that were specific to bacterial sepsis (17). In addition to these markers, previous work showed that LOX-1 is an important mediator of inflammation and neutrophils dysfunction in sepsis and cancers (18, 19). To test the hypothesis of a virally- driven neutrophil profile, we developed a multi -parametric neutrophil profiling strategy based on known neutrophil markers to distinguish COVID-19 phenotypes in critical and severe patients. SUMMARY OF THE INVENTION:
A first object of the present invention relates to an in vitro method for assessing a subject’s risk of having or developing severe or critical form of coronavirus infection , comprising the steps of i) determining in a sample obtained from the subject the level of neutrophil cells having cell surface expression of CD66b+CD10-CD16-CD64+CD123+ markers and/or CD66b+CD10-CD16-CD64+LOX-l+ markers, ii) comparing the level determined in step i) with a reference value and iii) concluding when the level of neutrophil having cell surface expression of CD66b+CD10-CD16-CD64+CD123+ markers and/or CD66b+CD10-CD16-CD64+LOXl+ markers determined at step i) is higher than the reference value is predictive of a high risk of having or developing severe or critical form of coronavirus infection.
An additional object of the invention relates to an in vitro method for monitoring a coronavirus infection comprising the steps of i) determining the level of a population of neutrophils having cell surface expression of CD66b+CD10-CD16-CD64+CD123+ markers and/or CD66b+CD10-CD16-CD64+LOXl+ markers in a sample obtained from the subject at a first specific time of the disease, ii) determining the level of a population of neutrophils having cell surface expression of CD66b+CD10-CD16-CD64+CD123+ markers and/or CD66b+CD10- CD16-CD64+LOX-1+ markers in a sample obtained from the subject at a second specific time of the disease, iii) comparing the level determined at step i) with the level determined at step ii) and iv) concluding that the disease has evolved in worse manner when the level determined at step ii) is higher than the level determined at step i).
An additional object of the invention relates to an in vitro method for monitoring the treatment of a of coronavirus infection comprising the steps of i) determining the level of a population of neutrophils having cell surface expression of CD66+CD10-CD16- CD64+CD123+ and/or CD66+CD10-CD16-CD64+LOX-1+ in a sample obtained from the subject before the treatment, ii) determining the level of a population of neutrophils having cell surface expression of CD66b+CD10-CD16-CD64+CD123+ markers and/or CD66b+CD10-CD16-CD64+LOX-l+ markers in a sample obtained from the subject after the treatment”, iii) comparing the level determined at step i) with the level determined at step ii) and iv) concluding that the treatment is efficient when the level determined at step ii) is lower than the level determined at step i).
In a particular embodiment regarding the method for monitoring (the disease or the treatment) the coronavirus infection is the severe or critical form of coronavirus infection. Another object of the invention relates to an in vitro method for assessing a COVID patient’s risk of having or developing thrombosis comprising the steps of i) determining the level of neutrophil cells having cell surface expression of CD66b+CD10-CD16-CD64+LOX- 1+ markers in a sample obtained from the subject, ii) comparing the level determined at step i) with the with a reference value and iv) concluding that:
- when the level of neutrophil having cell surface expression of CD66b+CD10-CD16- CD64+LOX-1+ markers determined at step i) is higher than the reference value, then said COVID patient is at high risk of having or developing thrombosis; or
- when the level of neutrophil having cell surface expression of CD66b+CD10-CD16- CD64+LOX-1+ markers determined at step i) is lower or equal than the reference value, then said COVID patient is at low risk of having or developing thrombosis or not having a thrombosis.
Another object of the invention relates to a LOX-1 (lectin-type oxidized LDL receptor 1) inhibitor for use in the prevention or the treatment of a coronavirus infection in a subject in need thereof.
DETAILED DESCRIPTION OF THE INVENTION:
In the present invention, inventors investigate the representation of neutrophil subsets in severe and critical COVID- 19 patients based on Intensive Care Units (ICU) and non-ICU admission. The results show that 80% of ICU patients develop strong myelemia with CD 10- CD64+ immature neutrophils. Cellular profiling revealed two distinct neutrophil subsets expressing either the LOX - 1 (lectin - like oxidized low - density lipoprotein receptor - 1) or the Interleukin-3 receptor alpha (CD123), both overrepresented in ICU patients compared to non-ICU patients. The proportion of LOX-1 -expressing immature neutrophils positively correlated with clinical severity, with the cytokine storm (IL-Ib, IL-6, IL-8, TNFa), and with intravascular coagulation. Importantly, high proportions of LOX-1 +-immature neutrophils are associated with higher risk of thrombosis. Together these data suggest that point of care enumeration of LOX-1 -immature neutrophils might help distinguish patients most likely to benefit from intensified anticoagulant therapy and surveillance of vascular complication. This minimal marker set may be used as prognosis tool in combination with clinical scores. These results thus set-up the basis for the development of a rapid functional specific test for critical form of COVID. Prognostic methods according to the invention
The present invention relates to an in vitro method for assessing a subject’s risk of having or developing severe or critical form of coronavirus infection , comprising the steps of i) determining in a sample obtained from the subject the level of neutrophil cells having cell surface expression of CD66b+CD10-CD16-CD64+CD123+ markers and/or CD66b+CD10- CD16-CD64+LOX-1+ markers, ii) comparing the level determined in step i) with a reference value and iii) concluding when the level of neutrophil having cell surface expression of CD66b+CD 10-CD 16-CD64+CD 123+ markers and/or CD66b+CD10-CD16-CD64+LOX-l+ markers determined at step i) is higher than the reference value is predictive of a high risk of having or developing severe or critical form of coronavirus infection.
In another term, the present invention relates to an in vitro prognosis method of having or developing severe or critical form of coronavirus infection in a subject, comprising the steps of i) determining in a sample obtained from the subject the level of neutrophil cells having cell surface expression of CD66b+CD10-CD16-CD64+CD123+ markers and/or CD66b+CD10-CD16-CD64+LOX-l+ markers, ii) comparing the level determined in step i) with a reference value and iii) concluding when the level of neutrophil having cell surface expression of CD10-CD16-CD64+CD123+ markers and/or CD66b+CD10-CD16- CD64+LOX-1+ markers determined at step i) is higher than the reference value is predictive of having or developing severe or critical form of coronavirus infection
The term “prognosis” is a medical term for predicting the likely or expected development of a disease. Prognostic scoring is also used for disease outcome predictions.
In the context of the present invention the “prognosis” is associated with level of neutrophils having cell surface expression of CD66b+CD10-CD16-CD64+CD123+markers and/or the level of neutrophils having cell surface expression of CD66b+CD10-CD16- CD64+LOX-1+ markers which in turn may be a risk for developing critical form of coronavirus infection
The term “subject” as used herein refers to a mammalian, such as a rodent (e.g. a mouse or a rat), a feline, a canine or a primate. In a preferred embodiment, said subject is a human subject. The subject according to the invention can be a healthy subject or a subject suffering from a given disease such as coronavirus infection.
As used herein, the term “coronavirus” has its general meaning in the art and refers to any member or members of the Coronaviridae family. Coronavirus is a virus whose genome is plus-stranded RNA of about 27 kb to about 33 kb in length depending on the particular virus. The virion RNA has a cap at the 5’ end and a poly A tail at the 3’ end. The length of the RNA makes coronaviruses the largest of the RNA virus genomes. In particular, coronavirus RNAs encode: (1) an RNA-dependent RNA polymerase; (2) N-protein; (3) three envelope glycoproteins; plus (4) three non- structural proteins. In particular, the coronavirus particle comprises at least the four canonical structural proteins E (envelope protein), M (membrane protein), N (nucleocapsid protein), and S (spike protein). The S protein is cleaved into 3 chains: Spike protein SI, Spike protein S2 and Spike protein S2'. Production of the replicase proteins is initiated by the translation of ORFla and ORFlab via a -1 ribosomal frame- shifting mechanism. This mechanism produces two large viral polyproteins, ppla and pplab, that are further processed by two virally encoded cysteine proteases, the papain-like protease (PLpro) and a 3C-like protease (3CLpro), which is sometimes referred to as main protease (Mpro). Coronaviruses infect a variety of mammals and birds. They cause respiratory infections (common), enteric infections (mostly in infants >12 mo.), and possibly neurological syndromes. Coronaviruses are transmitted by aerosols of respiratory secretions. Coronaviruses are exemplified by, but not limited to, human enteric coV (ATCC accession # VR-1475), human coV 229E (ATCC accession # VR-740), human coV OC43 (ATCC accession # VR-920), Middle East respiratory syndrome-related coronavirus (MERS-Cov) and Severe Acute Respiratory Syndrome (SARS)-coronavirus (Center for Disease Control), in particular SARS-CoVl and SARS-CoV2.
According to the invention, the coronavirus can be a MERS-CoV, SARS-CoV, SARS- CoV-2 or any new future family members. In particular, the method of the present invention is suitable for the treatment of Severe Acute Respiratory Syndrome (SARS) and any neurological manifestations (headaches, dizziness, nausea, seizures, stroke, cognitive or sensory disturbances, etc.) or cardiorespiratory manifestations (non-responsiveness to hypoxia, cardiac rhythm disturbances...) of brain viral infection, or disturbances of fluid balance, or anorexia/cachexia, or short or long-term neuroendocrine disturbances.
As used herein, the term “SARS-CoV-2” refers to severe acute respiratory syndrome coronavirus 2 known by the provisional name 2019 novel coronavirus (2019-nCoV) is the cause of the respiratory coronavirus disease 2019 (COVID-19). Taxonomically, it is a strain of the Severe acute respiratory syndrome-related coronavirus (SARSr-CoV), a positive-sense single-stranded RNA virus. It is contagious in humans, and the World Health Organization (WHO) has designated the ongoing pandemic of COVID-19 a Public Health Emergency of International Concern. SARS-CoV-2 virion is approximately 50-200 nanometres in diameter. Like other coronaviruses, SARS-CoV-2 has four structural proteins, known as the S (spike), E (envelope), M (membrane), and N (nucleocapsid) proteins; the N protein holds the RNA genome, and the S, E, and M proteins together create the viral envelope. The spike protein, which has been imaged at the atomic level using cryogenic electron microscopy is the protein responsible for allowing the virus to attach to the membrane of a host cell.
In particular embodiments, the subject of the present invention suffers from COVID- 19 and/or have been previously diagnosed with SARS-CoV-2.
As used herein, “SARS-CoV-2 infection” refers to the transmission of this virus from an animal and/or human to another animal and/or human primarily via respiratory droplets from coughs and sneezes within a range of about 2 meters. Indirect contact via contaminated surfaces is another possible cause of infection.
In some embodiments, the subject can be symptomatic or asymptomatic. As used herein, the term "asymptomatic" refers to a subject who experiences no detectable symptoms for the brain viral infection (e.g. coronavirus). As used herein, the term "symptomatic" refers to a subject who experiences detectable symptoms of a pathogen brain viral infection and particularly a coronavirus infection. Symptoms of coronavirus infection include: neurological symptoms (headaches, dizziness, nausea, loss of consciousness, seizures, encephalitis stroke, cognitive or sensory disturbances...), as well as anosmia or ageusia; fatigue, cough, fever, difficulty to breathe or cardiorespiratory manifestations (non-responsiveness to hypoxia, cardiac rhythm disturbances...) of brain viral infection, or disturbances of fluid balance, or anorexia/cachexia, or short or long-term neuroendocrine disturbances
The term “severe or critical form of coronavirus infection” refers to the progression of the disease to acute respiratory distress syndrome (ARDS), accountable for high mortality related to the damages of the alveolar lumen. Numerous patients with ARDS secondary to COVID-19 develop life-threatening thrombotic complications (5). More precisely severe form of COVID-19 can lead to critical illness, with acute respiratory distress (ARDS) and multiorgan failure as its primary complications, eventually followed by intravascular coagulopathy.
As used herein, the term “sample “ or "biological sample" as used herein refers to any biological sample of a subject and can include, by way of example and not limitation, bodily fluids and/or tissue extracts such as homogenates or solubilized tissue obtained from a subject. Tissue extracts are obtained routinely from tissue biopsy. In a particular embodiment regarding the prognostic method of the critical form of the coronavirus according to the invention, the biological sample is a body fluid sample (such blood or immune primary cell) or tissue biopsy of said subject. In preferred embodiments, the fluid sample is a blood sample. The term “blood sample” means a whole blood sample obtained from a subject (e.g. an individual for which it is interesting to determine whether a population of neutrophils cells can be identified).
The term “immune primary cell” has its general meaning in the art and is intended to describe a population of white blood cells directly obtained from a subject.
In the context of the present invention immune primary cell is selected from the group consisting of PBMC, WBC, neutrophil.
The term “PBMC” or “peripheral blood mononuclear cells” or “unfractionated PBMC”, as used herein, refers to whole PBMC, i.e. to a population of white blood cells having a round nucleus, which has not been enriched for a given sub-population (which contain neutrophils, T cells, B cells, natural killer (NK) cells, NK T cells and DC precursors). A PBMC sample according to the invention therefore contains lymphocytes (B cells, T cells, NK cells, NKT cells) and neutrophils. Typically, these cells can be extracted from whole blood using Ficoll, a hydrophilic polysaccharide that separates layers of blood, with the PBMC forming a cell ring under a layer of plasma. Additionally, PBMC can be extracted from whole blood using a hypotonic lysis buffer, which will preferentially lyse red blood cells. Such procedures are known to the expert in the art.
The term “WBC” or “White Blood Cells”, as used herein, also refers to leukocytes population, are the cells of the immune system. All white blood cells are produced and derived from multipotent cells in the bone marrow known as hematopoietic stem cells. Leukocytes are found throughout the body, including the blood and lymphatic system. Typically, WBC or some cells among WBC can be extracted from whole blood by using i) immunomagnetic separation procedures, ii) percoll or ficoll density gradient centrifugation, iii) cell sorting using flow cytometer (FACS). Additionally, WBC can be extracted from whole blood using a hypotonic lysis buffer, which will preferentially lyse red blood cells. Such procedures are known to the expert in the art.
In some embodiments, the fluid sample is a sample of purified neutrophil in suspension. Typically, the sample of neutrophil is prepared by immunomagnetic separation methods preformed on a PBMC or WBC sample. For example, neutrophils cells are isolated by using antibodies for neutrophils -associated cell surface markers, such as CD66b (or CD66+/CRTH2-). Commercial kits, e.g. Direct Human Neutrophil Isolation Kit kits (Immunomagnetic negative selection from whole blood kit) using CD66b labelled antibodies (#19666 from Stem cells technologies) are available. As used herein, the term "CD10", also known as cluster of differentiation 10 (or Neprilysin, membrane metallo-endopeptidase (MME), neutral endopeptidase (NEP), and common acute lymphoblastic leukemia antigen (CALLA)) has its general meaning in the art refers to an enzyme that in humans is encoded by the MME gene (gene ID 4311).
CD66b (Cluster of Differentiation 66b) also known as Carcinoembryonic antigen- related cell adhesion molecule 8 (CEACAM8) refers to a member of the carcinoembryonic antigen (CEA) gene family (CD66b / human gene (gene ID 1088)). Its main function is cell adhesion, cell migration, and pathogen binding. CD66b is expressed exclusively on neutrophils (granulocytes) and used as neutrophils marker (Eades-Perner AM et al. (1998) Blood. ;91(2):663-72).
CD 10 refers to a cell-surface marker in the diagnosis of human acute lymphocytic leukemia (ALL). CD 10 is present on leukemic cells of pre-B phenotype, which represent 85% of cases of ALL. Hematopoetic progenitors expressing CD 10 are considered "common lymphoid progenitors", which means they can differentiate into T, B or natural killer cells.
In the context of the method of the invention “CD10-“ means that the cell surface marker is not expressed on neutrophil (or not detected when contacted for instance with a labeled CD 10 antibody) .
As used herein, the term "CD 16" also known as FcyRIII, has its general meaning in the art and refers to a cluster of differentiation molecule found on the surface of natural killer cells, neutrophils, monocytes, and macrophages CD16 has been identified as Fc receptors FcyRIIIa (CD 16a / human gene (gene ID 2214)) and FcyRIIIb (CD 16b / human gene (gene ID 2214)), which participate in signal transduction. The most well-researched membrane receptor implicated in triggering lysis by NK cells, CD 16 is a molecule of the immunoglobulin superfamily (IgSF) involved in antibody-dependent cellular cytotoxicity (ADCC). It can be used to isolate populations of specific immune cells through fluorescent-activated cell sorting (FACS) or magnetic-activated cell sorting, using antibodies directed towards CD 16.
In the context of the method of the invention “CD16-“ means that the cell surface marker is not expressed on neutrophil (or not detected when contacted for instance with a labeled CD 16 antibody) ..
As used herein, the term "CD64" also knows as Cluster of Differentiation 64 or Fc- gamma receptor 1 (FcyRI) refers to a membrane glycoprotein known as an Fc receptor that binds monomeric IgG-type antibodies with high affinity (Hulett M, et al (1998). Mol Immunol. 35 (14-15): 989-96). After binding IgG, CD64 interacts with an accessory chain known as the common g chain (g chain), which possesses an IT AM motif that is necessary for triggering cellular activation (Nimmeijahn F, et al (2006). Immunity. 24 (1): 19-28). Structurally CD64 is composed of a signal peptide that allows its transport to the surface of a cell, three extracellular immunoglobulin domains of the C2-type that it uses to bind antibody, a hydrophobic transmembrane domain, and a short cytoplasmic tail (Ernst L, et al (1998). Mol Immunol. 35 (14-15): 943-54). CD64 is constitutively found on only macrophages and neutrophils, but treatment of polymorphonuclear leukocytes with cytokines like IFNy and G- CSF can induce CD64 expression on these cells. There are three distinct (but highly similar) genes in humans for CD64 called FcyRIA (CD64A / human gene (gene ID 2209)), FcyRIB (CD64B/ human gene (gene ID 2210)), and FcyRIC (CD64C / human gene (gene ID 2211)) that are located on chromosome 1 (Ernst L, et al (1992). J Biol Chem. 267 (22): 15692-700). These three genes produce six different mRNA transcripts; two from CD64A, three from CD64B, and one from CD64C; by alternate splicing of the genes (Ernst L, et al (1998). Mol Immunol. 35 (14-15): 943-54).
As used herein, the term "CD 123" also knows as Cluster of Differentiation 64 or alpha-chain of the interleukin-3 receptor (IL-3RA) refers to receptor found on cells which helps transmit the signal of interleukin-3, a soluble cytokine important in the immune system. The human gene coding for the receptor is located in the pseudoautosomal region of the X and Y chromosomes (human gene (gene ID 3553). The receptor belongs to the type I cytokine receptor family and is a heterodimer with a unique alpha chain paired with the common beta (beta c or CD131) subunit. CD123 found on pluripotent progenitor cells, induces tyrosine phosphorylation within the cell and promotes proliferation and differentiation within the hematopoietic cell lines. It can be found on basophils and pDCs as well as some cDCs among peripheral blood mononuclear cells. CD123 is expressed across acute myeloid leukemia (AML) subtypes, including leukemic stem cells. An experimental antibody-drug conjugate SGN-CD123A targets CD123 as a possible treatment for AML (www.businesswire. com/news/home/20160919005140/en/Seattle-Genetics-Initiates-Phase- 1 - Trial-SGN-CD123A /Sept 2016)
As use herein, the term “lectin-type oxidized LDL receptor 1” or “LOX-1” also known as “Oxidized low-density lipoprotein receptor 1” (or “Ox-LDL receptor 1”) is a protein that in humans is encoded by the OLR1 gene, (human gene: Gene ID: 4973).
LOX-1 is the main receptor for oxidized LDL on endothelial cells, macrophages, smooth muscle cells (Pirillo A, et al. Mediators of Inflammation. 2013: 1-12). LOX-1 is a 50 kDa transmembrane glycoprotein which belongs to the C-type lectin superfamily. Its gene is regulated through the cyclic AMP signaling pathway. The protein binds, internalizes and degrades oxidized low-density lipoprotein. Normally, LOX-1 expression on endothelial cells is low, but tumor necrosis factor alpha, oxidized LDL, blood vessel sheer stress, and other atherosclerotic stimuli substantially increase LOX-1 expression (Kakutani M, et al PNAS (2000). 97 (1): 360-364). LOX-1 may be involved in the regulation of Fas-induced apoptosis. Oxidized LDL induces endothelial cell apoptosis through LOX-1 binding ((Pirillo A, et al. Mediators of Inflammation. 2013: 1-12). Other ligands for LOX-1 include oxidized high- density lipoprotein, advanced gly cation end-products, platelets, and apoptotic cell. The binding of platelets to LOX-1 causes a release of vasoconstrictive endothelin, which induces endothelial dysfunction (Kakutani M, et al PNAS (2000). 97 (1): 360-364). Finally LOX-1 may play a role as a scavenger receptor and mutations of OLR1 gene have been associated with atherosclerosis, risk of myocardial infarction (Entrez Gene: OLR1 oxidized low density lipoprotein (lectin-like) receptor 1": www.ncbi.nlm.nih.gov/gene =4973)
One example of LOX-1 human amino acid sequence (UniProtKB - P78380) is provided in SEQ ID NO:l (transcript variant 1/ NCBI Reference Sequence: NP_002534, Table 4). One example of nucleotide sequence encoding wild-type LOX-1 is provided in SEQ ID NO:2 (transcript variant 1NCBI Reference Sequence: NM_002543, Table 4).
Of course variant sequences of the LOX-1 may be used in the context of the present invention (as biomarker or therapeutic target), those including but not limited to functional homologues, paralogues or orthologues, transcript variants of such sequences such as:
LOX-1 transcript variant 1: (NCBI Reference Sequence: NM_002543/ NP_002534). This variant (1) represents the longest transcript and encodes the longest isoform (1)
LOX-1 transcript variant 2: (NCBI Reference Sequence: NM_001172632/ NP OO 1166103). This variant (2) lacks an exon in the coding region, which results in a frameshift and an early stop codon, compared to variant 1. The encoded isoform (2) is shorter and has a distinct C-terminus, compared to isoform 1.
LOX-1 transcript variant 3: (NCBI Reference Sequence: NM_001172633.1/ NP OOl 166104) This variant (3) lacks an exon in the coding region, which results in a frameshift and an early stop codon, compared to variant 1. The encoded isoform (3) is shorter and has a distinct C-terminus, compared to isoform 1.
Standard methods for detecting the expression of a specific surface marker such as CD64 or CD123 at cell surface (e.g. neutrophil surface) are well known in the art. Typically, the step consisting of detecting the surface expression of a surface marker (e.g. CD64, CD123 or LOX-1) or detecting the absence of the surface expression of a surface marker (e.g. CD10 or CD 16) may consist in using at least one differential binding partner directed against the surface marker, wherein said cells are bound by said binding partners to said surface marker.
As used herein, the term “binding partner directed against the surface marker” refers to any molecule (natural or not) that is able to bind the surface marker with high affinity. The binding partners may be antibodies that may be polyclonal or monoclonal, preferably monoclonal antibodies. In another embodiment, the binding partners may be a set of aptamers.
Polyclonal antibodies of the invention or a fragment thereof can be raised according to known methods by administering the appropriate antigen or epitope to a host animal selected, e.g., from pigs, cows, horses, rabbits, goats, sheep, and mice, among others. Various adjuvants known in the art can be used to enhance antibody production. Although antibodies useful in practicing the invention can be polyclonal, monoclonal antibodies are preferred.
Monoclonal antibodies of the invention or a fragment thereof can be prepared and isolated using any technique that provides for the production of antibody molecules by continuous cell lines in culture. Techniques for production and isolation include but are not limited to the hybridoma technique originally; the human B-cell hybridoma technique; and the EBV-hybridoma technique.
For example, the binding partner of CD64 of the invention is the anti-human CD64 antibody available from Biolegend (CD64 (Fc gamma Receptor 1) Monoclonal Antibody (10.1), # 305029)
For example, the binding partner of CD123 of the invention is the anti-human CD123 antibody available from Biolegend (CD 123 Monoclonal Antibody (6H6), 306006) or from Fluidigm (Anti-IL3RA/CD123 antibody (6H6) (#3151001).
For example, the binding partner of CD66b of the invention is the anti-human CD66b antibody available from Biolegend (CD66b Monoclonal Antibody (G10F5), # 355005) or from Fluidigm (Anti-Human CD66b (80H3) (#3162023).
For example, the binding partner of LOX-1 (or OLR1) of the invention is the antihuman LOX-1/OLR1 antibody available from Biolegend (anti human LOX1 antibody BV421 #358609) or from RD Systems (anti-human LOX-1/OLR1 (AF1798)
The binding partners of the invention such as antibodies or aptamers may be labelled with a detectable molecule or substance, such as preferentially a fluorescent molecule, or a radioactive molecule or any others labels known in the art. Labels are known in the art that generally provide (either directly or indirectly) a signal. As used herein, the term "labelled", with regard to the antibody or aptamer, is intended to encompass direct labelling of the antibody or aptamer by coupling (i.e., physically linking) a detectable substance, such as a fluorophore [e.g. fluorescein isothiocyanate (FITC) or phycoerythrin (PE) or Indocyanine (Cy5)]) or radioactive molecule or a non-radioactive heavy metals isotopes to the antibody or aptamer, as well as indirect labelling of the probe or antibody by reactivity with a detectable substance. An antibody or aptamer of the invention may be labelled with a radioactive molecule by any method known in the art. More particularly, the antibodies are already conjugated to a fluorophore (e.g. FITC-conjugated and/or PE-conjugated).
The aforementioned assays may involve the binding of the binding partners (i.e. antibodies or aptamers) to a solid support. The solid surface could a microtitration plate coated with the binding partner for the surface marker. Alternatively, the solid surfaces may be beads, such as activated beads, magnetically responsive beads. Beads may be made of different materials, including but not limited to glass, plastic, polystyrene, and acrylic. In addition, the beads are preferably fluorescently labelled. In a preferred embodiment, fluorescent beads are those contained in TruCount(TM) tubes, available from Becton Dickinson Biosciences, (San Jose, California). According to the invention, methods of flow cytometry are preferred methods for detecting (presence or absence of) the surface expression of the surface markers (i.e. CD66b, CD 10, CD 16, CD64, CD 123 and LOX-1). Said methods are well known in the art. For example, fluorescence activated cell sorting (FACS) may be therefore used. Cell sorting protocols using fluorescent labeled antibodies directed against the surface marker (or immunobeads coated with antibody) in combination with antibodies directed against CD66b, CD10, CD16, CD64, CD123 and LOX-1 coupled with distinct fluorochromes (or immunobeads coated with anti-CD66b, anti CD 10 antibodies, anti CD 16 antibodies, anti CD64 antibodies, anti CD 123 antibodies and anti -LOX-1 antibodies) can allow direct sorting, using cell sorters with the adequate optic configuration.
Such methods comprise contacting a biological sample obtained from the subject to be tested under conditions allowing detection (presence or absence) of CD66b, CD 10, CD 16, CD64 and CD123 and/or CD66b, CD10, CD16, and LOX-1 surface markers. Once the sample from the subject is prepared, the level of covid (critical form) biomarkers (“Biomarkerl23”: CD66b+CD10-CD16-CD64+CD123+ cells and/or “BiomarkerLoxl”: CD66b+CD10-CD16-LOX-l+ cells) may be measured by any known method in the art. Typically, the high or low level of covid-associated neutrophil cell surface biomarkers (“Biomarkerl23”: CD66b+CD10-CD16-CD64+CD123+ cells and/or “BiomarkerLoxl”: CD66b+CD10-CD16-LOX-l+ cells) is intended by comparison to a control reference value.
Said reference control values may be determined in regard to the level of biomarker present in blood samples taken from one or more healthy subject(s) or to the cell surface biomarker in a control population.
In one embodiment, the method according to the present invention comprises the step of comparing said level of covid- associated neutrophil biomarkers (“Biomarkerl23”: CD66b+CD 10-CD 16-CD64+CD 123+ cells and/or “BiomarkerLOX-l”: CD66b+CD10- CD16-LOX-1+ cells) to a control reference value wherein a high level of covid- associated neutrophil biomarkers (“Biomarkerl23”: CD66b+CD10-CD16-CD64+CD123+ cells and/or “BiomarkerLOX-l”: CD66b+CD10-CD16-LOX-l+cells) compared to said control reference value is predictive of a high risk of having a critical form of coronavirus infection and a low level of covid- associated neutrophil biomarkers (“Biomarkerl23”: CD66b+CD10-CD16- CD64+CD123+ cells and/or “BiomarkerLOX-l”: CD66b+CD10-CD16-LOX-l+ cells) compared to said control reference value is predictive of a low risk of having or developing a critical form of coronavirus infection.
In one embodiment, for covid- associated neutrophil biomarker “BiomarkerLOX-l” (CD66b+CD10-CD16-LOX-l+ cells) the control reference is null (not detected), which means when the BiomarkerLOX-l is detected, subject have a high risk of having or developing a critical form of coronavirus infection.
The control reference value may depend on various parameters such as the method used to measure the level covid- associated neutrophil biomarker BiomarkerLOX-l (CD66b+CD10-CD16-LOX-l+ cells) or the gender of the subject.
Typically regarding the reference value using “Biomarkerl23” (CD66b+CD10-CD16- CD64+CD123+ cells), as indicated in the Example section (figure 1), for a level of neutrophil CD66b+CD10-CD16-CD64+CD123+ using Flow Cytometry approach identify and quantify neutrophil population, a level of neutrophil CD66b+CD10-CD16-CD64+CD123+ superior to 1% is predictive of having or a high risk of having or developing a critical form of coronavirus infection and a level of neutrophil CD66b+CD10-CD16-CD64+CD123+ lower than 1% is predictive of not having or at a low risk of having a critical form of coronavirus infection.
Typically regarding the reference value using “BiomarkerLOX-l” (CD66b+CD10- CD16-LOX-1+ cells), as indicated in the Example section, for a level of neutrophil CD66b+CD10-CD16-CD64+LOX-l+ using Flow Cytometry approach identify and quantify neutrophil specific population, a level of neutrophil CD66b+CD10-CD16-CD64+LOX-l+ superior to 1%, is predictive of having or a high risk of having or developing a critical form of coronavirus infection and a level of neutrophil CD66b+CD10-CD16-CD64+CD123+ lower than 1% is predictive of not having or developing or at a low risk of having a critical form of coronavirus infection.
Control reference values are easily determinable by the one skilled in the art, by using the same techniques as for determining the level of cell surface biomarker or cell death in blood samples previously collected from the patient under testing.
A “reference value” can be a “threshold value” or a “cut-off value”. Typically, a "threshold value" or "cut-off value" can be determined experimentally, empirically, or theoretically. A threshold value can also be arbitrarily selected based upon the existing experimental and/or clinical conditions, as would be recognized by a person of ordinary skilled in the art. The threshold value has to be determined in order to obtain the optimal sensitivity and specificity according to the function of the test and the benefit/risk balance (clinical consequences of false positive and false negative). Typically, the optimal sensitivity and specificity (and so the threshold value) can be determined using a Receiver Operating Characteristic (ROC) curve based on experimental data. Preferably, the person skilled in the art may compare the level of neutrophil biomarkers (“Biomarker 123”: CD66b+CD10- CD 16- CD64+CD123+ cells and/or “BiomarkerLOX-1”: CD66b+CD10-CD16-LOX-l+cells) with a defined threshold value. In one embodiment of the present invention, the threshold value is derived from the neutrophil level (or ratio, or score) determined in a blood sample derived from one or more subjects who are responders (to the method according to the invention). In one embodiment of the present invention, the threshold value may also be derived from neutrophil level (or ratio, or score) determined in a blood sample derived from one or more subjects or who are non-responders. Furthermore, retrospective measurement of the activated neutrophil level (or ratio, or scores) in properly banked historical subject samples may be used in establishing these threshold values.
In particular embodiment, when using LOX-1 neutrophil subsets proportions, inventors (for risk of thrombosis) shows that the reference value is 5% (see figure 3B).
Reference values are easily determinable by the one skilled in the art, by using the same techniques as for determining the level of activated neutrophils in fluids samples previously collected from the patient under testing. "Risk" in the context of the present invention, relates to the probability that an event will occur over a specific time period, as in the conversion to critical form of coronavirus infection, and can mean a subject's "absolute" risk or "relative" risk. Absolute risk can be measured with reference to either actual observation post-measurement for the relevant time cohort, or with reference to index values developed from statistically valid historical cohorts that have been followed for the relevant time period. Relative risk refers to the ratio of absolute risks of a subject compared either to the absolute risks of low risk cohorts or an average population risk, which can vary by how clinical risk factors are assessed. Odds ratios, the proportion of positive events to negative events for a given test result, are also commonly used (odds are according to the formula p/(l-p) where p is the probability of event and (1- p) is the probability of no event) to no conversion. Alternative continuous measures, which may be assessed in the context of the present invention, include time to critical form of coronavirus infection conversion risk reduction ratios.
"Risk evaluation," or "evaluation of risk" in the context of the present invention encompasses making a prediction of the probability, odds, or likelihood that an event or disease state may occur, the rate of occurrence of the event or conversion from one disease state to another, i.e., from a normal condition or asymptomatic form of Covid-19 or symptomic form of COVID to a critical form of coronavirus infection condition or to one at risk of developing a critical form of coronavirus infection. Risk evaluation can also comprise prediction of future clinical parameters, traditional laboratory risk factor values, or other indices of critical form of coronavirus infection, such as cellular population determination in peripheral tissues, in serum or other fluid, either in absolute or relative terms in reference to a previously measured population. The methods of the present invention may be used to make continuous or categorical measurements of the risk of conversion to critical form of coronavirus infection, thus diagnosing and defining the risk spectrum of a category of subjects defined as being at risk for a critical form of coronavirus infection. In the categorical scenario, the invention can be used to discriminate between normal and other subject cohorts at higher risk for critical form of coronavirus infection. In other embodiments, the present invention may be used so as to help to discriminate those having COVID from critical form of coronavirus infection.
Accordingly, the method of detection of the invention is consequently useful for the in vitro diagnosis of COVID from a biological sample. In particular, the method of detection of the invention is consequently useful for the in vitro diagnosis of early stage covid from a biological sample. Monitoring methods and Management
After the identification of neutrophil subsets that harbour an immature phenotype (“Biomarkerl23”: CD66b+CD10-CD16-CD64+CD123+ cells, “BiomarkerLOX-1”:
CD66b+CD10-CD16-CD64+LOX-l+ cells), inventors highlighted with both unsupervised and expert-gating strategies, that both LOX-1- and CD 123- expressing CD10-CD64+ neutrophil subsets (“immature neutrophils”) strongly correlated with SAPS II and SOFA severity scores, commonly used in clinical practice for sepsis prognosis. Accordingly, inventors provided evidence that this immature subset may serve as a severity biomarker in COVID-19 for prognosis and monitoring purpose.
As used herein, the term “Immature neutrophils” refers to cells phenotypically and functionally immature. Immature neutrophils were described as cells expressing CD66b and CD64 and lacking the expression of CD10 and CD16 (Taylor OY Br. J. Haematol.: Elghetany MT. Blood Cells Mol Dis 2002;28:260-274; and Ng, L.G. et al. Nat Rev Immunol 19, 255- 265 (2019).).
Accordingly, “immature neutrophils” according to the invention, is a population of neutrophils having cell surface expression of CD66b+CD10-CD16-CD64+.
Accordingly, an additional object of the invention relates to an in vitro method for monitoring a coronavirus infection comprising the steps of i) determining the level of a population of neutrophils having cell surface expression of CD66b+CD10-CD16- CD64+CD123+ markers and/or CD66b+CD10-CD16-CD64+LOX-l+ markers in a sample obtained from the subject at a first specific time of the disease, ii) determining the level of a population of neutrophils having cell surface expression of CD66b+CD10- CD 16- CD64+CD123+ markers and/or CD66b+CD10-CD16-CD64+LOX-l+ markers in a sample obtained from the subject at a second specific time of the disease, iii) comparing the level determined at step i) with the level determined at step ii) and iv) concluding that the disease has evolved in worse manner when the level determined at step ii) is higher than the level determined at step i).
In particular embodiment, the coronavirus infection is the severe or critical form of coronavirus infection (i.e. COVID-19)
An additional object of the invention relates to an in vitro method for monitoring the treatment of a coronavirus infection comprising the steps of i) determining the level of a population of neutrophils having cell surface expression of CD66+CD10-CD16- CD64+CD123+ and/or CD66+CD10-CD64+LOX-1+ in a sample obtained from the subject before the treatment, ii) determining the level of a population of neutrophils having cell surface expression of CD66b+CD10-CD16-CD64+CD123+ markers and/or CD66b+CD10- CD16-CD64+LOX-1+ markers in a sample obtained from the subject after the treatment”, iii) comparing the level determined at step i) with the level determined at step ii) and iv) concluding that the treatment is efficient when the level determined at step ii) is lower than the level determined at step i).
In particular embodiment, the coronavirus infection is the severe or critical form of coronavirus infection (i.e. COVID-19)
The decrease can be e.g. at least 5%, or at least 10%, or at least 20%, more preferably at least 50% even more preferably at least 100%.
Prognosis methods for thrombosis event
As demonstrated by the inventors, most COVID patients with a high proportion LOX- 1 neutrophil subset developed thromboembolic events. The ROC curve analysis (figure 3B) shows that Biomarker LOX-1” (CD66b+CD10-CD64+LOX-l+ cells) may be a strong accurate predictive marker of thrombosis event for COVID-19 patients (AUC :95% for p < 0.0001)
Accordingly, another object of the invention relates to an in vitro method for assessing a subject’s risk of having or developing thrombosis in a patient with coronavirus infection comprising the steps of i) determining the level of neutrophil cells having cell surface expression of CD66b+CD10-CD16-CD64+LOX-l+ markers in a sample obtained from the subject, ii) comparing the level determined at step i) with the with a reference value and iv) concluding that:
- when the level of neutrophil having cell surface expression of CD66b+CD10-CD16- CD64+LOX-1+ markers determined at step i) is higher than the reference value, then said patient is at risk of having or developing thrombosis; or
- when the level of neutrophil having cell surface expression of CD10-CD16- CD64+CD123+ markers and/or CD66b+CD10-CD16-CD64+LOX-l+ markers determined at step i) is lower or equal than the reference value, then said patient is at low risk of having or developing or not having a thrombosis.
As used herein, the term “thrombosis” has its general meaning in the art and is the process by which an unwanted blood clot forms in a blood vessel. It can occur in a vein or in an artery. Arterial thrombosis is the cause of almost all cases of myocardial infarction and the majority of strokes, collectively the most common cause of deaths in the developed world. Deep vein thrombosis and pulmonary embolism are referred to as venous thromboembolism, which is currently the third leading cause of cardiovascular-associated death.
Thus the term "thrombosis" includes inter alia atrophic thrombosis, arterial thrombosis, cardiac thrombosis, coronary thrombosis, creeping thrombosis, mesenteric thrombosis, placental thrombosis, propagating thrombosis, traumatic thrombosis and venous thrombosis and venous thromboembolism.
Reports that COVID-19 is associated with venous and arterial thrombosis and pulmonary embolism and increased rates of thrombosis in cannulae and extracorporeal circuits for renal replacement or membrane oxygenation, have drawn attention to the coagulant effects of the disease.
Case reports and small case series of thromboembolic complications in patients with COVID-19 have included pulmonary embolism (Poggiali E. et al. Eur J Case Rep Intern Med. 2020; 7(5): 001646; Yuanliang Xie et al. Radiology: Cardiothoracic Imaging Volume 2: Number 2 — 2020), femoral vein thrombosis ((Poggiali E. et al. Eur J Case Rep Intern Med. 2020; 7(5): 001646), phlegmasia caerulea dolens (Morales MH et al. Am J Emerg Med. 2020 May 15 doi: 10.1016), cerebral venous sinus thrombosis (Poillon G et al J Neuroradiol. 2020 May 11 doi: 10.1016/; Hughes C et al. Eur J Case Rep Intern Med. 2020; 7(5): 001691), aortic thrombosis (Le Berre A. et al. Diagn Interv Imaging. 2020 May; 101(5): 321-322.; Lushina N. et al, Radiology. 2020 Apr 23: 2016238), aorto-iliac thrombosis (Bellosta R et al. J Vase Surg. 2020 Apr 29 doi: 10.1016) humeral artery thrombosis (Bellosta R et al. J Vase Surg. 2020 Apr 29 doi: 10.1016), acro-ischaemic presentations (Zhang Y. et al. Chin J Hematol, 2020,41(04): 302-307. DOI: 10.3760), and strokes in young patients (Oxley TJ et al. N Engl J Med. 2020 Apr 28: NEJMc2009787). Thrombotic events can occur during COVID- 19; they may be a presenting feature (Poggiali E. et al. Eur J Case Rep Intern Med. 2020; 7(5): 001646; 11 Oxley TJ et al. N Engl J Med. 2020 Apr 28: NEJMc2009787); and they may occur during convalescence (Tveita A et al. Tidsskr Nor Legeforen 2020(13 May 2020) doi: 10.4045/tidsskr.20.0366)
Therapeutic method
The impact of LOX-1 deletion was previously evaluated in a murine model of polymicrobial sepsis, resulting in the reduction of IL-6 and TNFa levels in blood and lungs, enhancing bacterial clearance and preventing neutrophils activation (19). More recently, LOX-1 was identified as a marker on granulocytic myeloid-derived suppressor cells able to suppress T cell activity (18). However, LOX-1 is mostly acknowledged for its role in atherosclerosis. LOX-1 is a class E scavenger receptor contributing to the formation of atherosclerotic plaques by promoting endothelial cell activation, macrophage foam cell formation, and smooth muscle cells migration and proliferation (24). LOX-1 activation induces NFKB activation leading to pro-inflammatory cytokines release, endoplasmic reticulum stress, and reactive oxygen species (ROS) production which could damage the microenvironment (25, 26).
However, LOX-1 role on neutrophils remains elusive. LOX-1 is barely detected on neutrophils at homeostasis, while its expression increases on neutrophils from human cancer patients (18) and in murine sepsis (19, 27).
In the present invention, inventors show that LOX-1 expression on neutrophils seems to be detrimental for patients as is associated with the secretion of several pro-inflammatory cytokines, such as IL-6, IL-Ib and TNFa, and with severity (as assessed by the SOFA score) and thrombosis. In severe cases of COVID-19, the integrity of the lung is compromised by an exaggerated immune response leading to acute respiratory distress syndrome (10, 16). Mechanisms contributing to microcirculation disorders in sepsis are capillary leakage, leukocytes adhesion and infiltration and intravascular coagulation, leading to thrombus formation. Over the course of systemic inflammatory diseases such as sepsis, the microenvironment is highly oxidative, leading notably to an increase of oxidized low-density lipoprotein (oxLDL) in plasma, which triggers LOX-1 overexpression through a positive feedback loop. In physiological conditions, the increase of LOX-1 expression, especially by endothelial cells, leads to an increase of LDL uptake into vessel wall which activates the specific Oct-l/SIRT-1 thrombosis protective pathway (28). The activation of SIRT1 is able to supress the NFkB-induced expression of tissue factor, also known as thromboplastin, a key initiator of the coagulation cascade involved in thrombus formation (29).
In the present invention, inventors show an increase of the incidence of vascular thrombotic events among individuals displaying a high frequency of immature LOX-1+ neutrophils. It remains to be seen whether thrombosis in COVID-19 patients results from functionally-diverted neutrophils expressing LOX-1 and/or from its expression on endothelial and smooth muscle cells.
Additionally, inventors show a slight correlation between LOX-1 expressing immature neutrophils and the concentration of D-Dimers (spearman test, r=0.42 p= 0.023). D-dimers are used to determine the risk of venous thromboembolism. However, in this study, the predictive score of D-dimers for thromboembolic events was not significant (ROC test, AUC=0.64, p=ns) compared to LOX-1 expressing neutrophil abundancy in the blood (AUC=0.977, p < 0.0001). These results suggest that the high LOX-1 expression by immature neutrophils might be sufficient to predict thromboembolic events among critically-ill COVID-19 patients. The overexpression of LOX-1 might also be found in other cell types that might trigger the prothrombotic ERK1/2 pathway. In addition, some studies support the relationship between ACE/ACE2 axis and the expression of the pro-oxidative molecule LOX-1, which could increase the oxidative stress favoring prothrombotic state (30). SARS-CoV-2 virus requires binding to ACE2 and is particularly deleterious to patients with underlying cardiovascular disease (31). The polymorphic LOX-1 gene is also intensively associated with increased susceptibility to myocardial diseases. LOX-1 should be thus considered a potential target for therapeutic intervention.
The inventors show that LOX1 is expressed and dysregulated in the immature neutrophil cells of the COVID subject. LOX-1 would have a potential role in coronavirus (e.g. SARS-CoV-2) pathogenesis.
Accordingly, in an additional aspect the invention relates to a method of preventing or treating a coronavirus infection in a patient in need thereof comprising administering to the patient a therapeutically effective amount of a LOX-1 (lectin-type oxidized LDL receptor 1) inhibitor. In some embodiments, the LOX-1 inhibitor is administrated by intravenous administration or intranasal administration.
In a particular embodiment, the invention relates to a LOX-1 (lectin-type oxidized LDL receptor 1) inhibitor for use in the prevention or the treatment of a coronavirus infection in a subject in need thereof.
In a particular embodiment, the invention relates to a LOX-1 (lectin-type oxidized LDL receptor 1) inhibitor for use in the prevention or the treatment of a coronavirus infection in a subject in need thereof, wherein the level of a population of neutrophils CD66b+CD10- CD 16-CD64+CD 123+ and/or CD66b+CD10-CD16-CD64+LOX-l+ obtained from said patient, have been detected by one of the methods (prognostic or monitoring) of the invention
In its broadest meaning, the term "treating" or "treatment" refers to reversing, alleviating, inhibiting the progress of coronavirus infection, preferably inhibiting the severe form of coronavirus infection. In particular, "prevention" or "prophylactic treatment" of coronavirus infections may refer to the administration of the compounds of the present invention that prevent the symptoms of coronavirus infections, in particular the severe form of coronavirus infections.
According to the invention, the term “subject” denotes a mammal, such as a rodent, a feline, a canine, or a primate. In some embodiments, the subject is a human. In some embodiments, the subject is an elderly human. Particularly, the subject denotes a human with a pathogen viral infection. Particularly, the subject denotes a human with a coronavirus infection. In a particular embodiment the subject is a human with co-morbidities and in the elderly (see for example Guan et al., 2020). As used herein, the term “subject” encompasses the term "patient”.
As used herein, the term “LOX-1 inhibitor” refers to refers to a natural or synthetic compound that has a biological effect to inhibit the activity or the expression of LOX-1.
The term “inhibitor” as used herein, refers to an agent that is capable of specifically binding and inhibiting signaling through a receptor to fully block, as does an inhibitor, or detectably inhibit a response mediated by the receptor. For example, as used herein the term “LOX-1 inhibitor” is a natural or synthetic compound which binds and inactivates fully or partially LOX-1 for initiating or participating to a pathway signaling (such as the ERK prothrombotic pathway) and further biological processes. In the context of the invention the LOX-1 inhibitor in particular prevents, decreases or suppresses the virus replication. The virus replication decrease observed can be by at least about 1%, 2%, 5%, 10%, e.g. by 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90%, as compared to the replication observed in a referenced cell.
LOX-1 inhibitory activity may be assessed by various known methods. A control LOX-1 can be exposed to no antibody or antigen binding molecule, an antibody or antigen binding molecule that specifically binds to another antigen, or an anti- LOX-1 antibody or antigen binding molecule known not to function as an inhibitor, for example as an inhibitor.
In some embodiment, the LOX-1 inhibitor inhibits the LOX-1 actions that exacerbate the effects of viral invasion and pro-inflammatory cytokines release (cytokine burst) and/or thromboembolic events and would be an effective therapeutic option for coronavirus infection and its consequences.
By "biological activity" of LOX-1 is meant inducing cytokine burst (through the control of pro-inflammatory cytokines release) and/or inducing thromboembolic events (through the ERK prothrombotic pathway).
Tests for determining the capacity of a compound to be a LOX-1 inhibitor are well known to the person skilled in the art. In a preferred embodiment, the inhibitor specifically binds to LOX-1 (protein or nucleic sequence (DNA or mRNA)) in a sufficient manner to inhibit the biological activity of LOX-1. Binding to LOX-1 and inhibition of the biological activity of LOX-1 may be determined by any competing assays well known in the art. For example, the assay may consist in determining the ability of the agent to be tested as a LOX-1 inhibitor to bind to LOX-1. The binding ability is reflected by the Kd measurement. The term "KD", as used herein, is intended to refer to the dissociation constant, which is obtained from the ratio of Kd to Ka (i.e. Kd/Ka) and is expressed as a molar concentration (M). KD values for binding biomolecules can be determined using methods well established in the art. In specific embodiments, an inhibitor that "specifically binds to LOX-1" is intended to refer to an inhibitor that binds to human LOX-1 polypeptide with a KD of ImM or less, lOOnM or less, lOnM or less, or 3nM or less. Then a competitive assay may be settled to determine the ability of the agent to inhibit biological activity of LOX-1. The functional assays may be envisaged such as evaluating the ability to: a) inhibit processes associated with pro- inflammatory cytokines release and/or b) inhibit processes associated thromboembolic events (through the ERK prothrombotic pathway.
The skilled in the art can easily determine whether a LOX-1 inhibitor neutralizes, blocks, inhibits, abrogates, reduces or interferes with a biological activity of LOX-1. To check whether the LOX-1 inhibitor binds to LOX-1 and/or is able to inhibit LOX-1 activity (or expression) such as processes associated with pro-inflammatory cytokines release and/or b) inhibit processes associated thromboembolic events (through the ERK prothrombotic pathway) may be performed with each inhibitor. For instance, inhibiting pro-inflammatory cytokines release can be assessed by detecting inflammatory cytokines beta with specific antibody, ultrasensitive immunodetection (digital ELISA) as described in the Example section (see Table 3), and ERK prothrombotic pathway assay can be measured by Phospho-ERK Assays (as described in Garbison Kim E et al “Phospho-ERK Assays” Book Assay Guidance Manual (published May 2012) ,Sittampalam GS, Grossman A, Brimacombe K, et al., editors.Bethesda (MD): Eli Lilly & Company and the National Center for Advancing Translational Sciences)
In a particular embodiment, a LOX-1 inhibitor according to the invention can be a molecule selected from a peptide, a peptide mimetic, a small organic molecule, an antibody, an aptamer, a phospholipid, a polynucleotide (inhibitor of LOX-1 gene expression) and a compound comprising such a molecule or a combination thereof.
A LOX-1 inhibitor for use in the context of the present invention may be selected but is not limited, from: a) Natural LOX-1 inhibitors such as
• Tanshinone II-A a pharmacologically active derivative of danshen, which is an herbal drug, has been shown to inhibit LOX-1 and ox-LDL uptake by macrophages); (as described in Xu S, Liu Z, Huang Y, et al. Tanshinone II-A inhibits oxidized LDL- induced LOX-1 expressionin macrophages by reducing intracellular superoxide radical generation andNF-kB activation. Transl Res 2012;160:114-24);
• Curcumin ((diferuloylmethane), an active ingredient of turmeric , has been shown to exert an anti-inflammatory effect and to inhibit atherogenesis and post-ischemic myocardial fibrosis. Curcumin also reduces Ang II-mediated up-regulation of Ang II type 1 receptors and LOX-1, and it decreases oxidative stress in mouse cardiomyocytes by decreasing the expression of nuclear factor kappa B (as described in Kang BY, et al. “Curcumin reduces angiotensin II-mediated cardiomyocyte growth via LOX-1 inhibition. J Cardiovasc Pharmacol 2010;55:417-24)
• Flavonoids from Hippophae rhamnoides (sea buckthorn) as described in Bao M, Lou Y. “Flavonoids from seabuckthorn protect endothelial cells (EA.hy926) from oxidized low-density lipoprotein induced injuries via regulation of LOX-1 and eNOS expression. J Cardiovasc Pharmacol 2006;48:834-41
• Gingko biloba extract, commonly used as a therapeutic agent for cardiovascular and neurological disorders, inhibits ox-LDL-mediated expression of intercellular adhesion molecule, vascular cell adhesion molecule, and E-selectin; decreases ROS generation; and attenuates platelet-induced LOX-1 expression in endothelial cells (as described in Ou HC, Lee WJ, Lee IT, et al. Ginkgo biloba extract attenuates oxLDL-induced oxidative functional damages in endothelial cells. J Appl Physiol 2009;106:1674-85.)
• the compound Tetramethylpyrazine an active ingredient of Ligusticum wallichii Franchat, described in Wang GF, Shi CG, Sun MZ, et al. “Tetramethylpyrazine attenuates atherosclerosis development and protects endothelial cells from ox-LDL.” Cardiovasc Drugs Ther 2013 ;27 : 199-210)
• Resveratrol (3,5,4’-trihydroxy-transstilbene) a polyphenol phytoalexin present in a variety of plant species (White hellebore, Polygonum cuspidatum, grapes, peanuts, mulberries, red wine) as described in Li H, Xia N, Forstermann U. Cardiovascular effects and molecular targets of resveratrol. Nitric Oxide 2012;26:102-10)
• Pterostilbene a natural dimethylated analog of resveratrol, as described in Zhang L, Zhou G, Song W, et al. Pterostilbene protects vascular endothelial cells against oxidized low-density lipoprotein-induced apoptosis in vitro and in vivo. Apoptosis 2012;17:25-36
• 6-Shogaol the major bioactive compound present in Zingiber officinale which possesses the anti-atherosclerotic effect as described Wang YK, Hong YJ, Yao YH, et al. “6-Shogaol protects against oxidized LDL-induced endothelial injuries by inhibiting oxidized LDL-evoked LOX-1 signaling. Evid Based Complement Alternat Med 2013;2013:503521).
• Berberine, a compound isolated from Rhizoma coptidis as described in Huang Z, Dong
F, Li S, et al. “Berberine-induced inhibition of adipocyte enhancer-binding protein 1 attenuates oxidized low-density lipoprotein accumulation and foam cell formation in phorbol 12-myristate 13 -acetate-induced macrophages. Eur J Pharmacol
2012;690:164-9.
• Selaginellin a compound extracted from Saussurea pulvinata as described in Zhang WF, Xu YY, Xu KP, et al. “Inhibitory effect of selaginellin on high glucose-induced apoptosis in differentiated PC12 cells: role of NADPH oxidase and LOX-1”. Eur J Pharmacol 2012;694:60-8.
• Ellagic acid a polyphenolic compound widely distributed in fruits and nuts, as described in Lee WJ, Ou HC, Hsu WC, et al. “Ellagic acid inhibits oxidized LDL- mediated LOX-1 expression, ROS generation, and inflammation in human endothelial cells”. J Vase Surg 2010;52:1290-300
• Bergamot peel the nonvolatile fraction (NVF) and the antioxidant component of bergamot essential oil (BEO), as described in Mollace V, Ragusa S, Sacco I, et al. “The protective effect of bergamot oil extract on lecitinelike oxyLDL receptor- 1 expression in balloon injury-related neointima formation”. J Cardiovasc Pharmacol Ther 2008;13:120-9. b) synthetic molecule such as
• PLAzPC a modified oxidized phospholipid, which binds to the tunnel binding site of the LOX-1 molecule and markedly inhibits interaction with ox-LDL and described in Falconi M, Ciccone S, D’Arrigo P, et al. Design of a novel LOX-1 receptor antagonist mimicking the natural substrate. Biochem Biophys Res Commun 2013;438:340-5.);
• small-molecule inhibitors of Lox-1 (Molecules 1 to Molecules 5), targeting the hydrophobic binding tunnel of LOX-1 and described in Thakkar S, Wang X, Khaidakov M, et al. “Structure-based design targeted at LOX-1, a receptor for oxidized low-density lipoprotein. Sci Rep 2015;5: 16740. Using structure-based drug- design techniques, Thakkar et al. evaluated the effect of 5 different molecules on LOX-1 inhibition. Of the 5 molecules tested, 2 (termed Mol 4 and Mol 5) were shown to decrease uptake of ox-LDL by human umbilical vein endothelial cells. In addition, treatment with these small molecules led to decreased expression of LOX-1 mRNA and reduced downstream mitogen-activated protein kinase and adhesion molecule expression. Specifically, Mol 5 was the most potent, with almost an 80% reduction in ox-LDL uptake when endothelial cells were pre-treated with this agent at 200 nM concentration. There was also no evidence of experimental cytotoxicity with these agents.
The Molecule 4 and 5 described in Thakkar et al. 2015 the most potent LOX-1 inhibitors (and derived compounds) can also be found in patent application WO2017075418 (Inhibitors of Oxidized Low-Density Lipoprotein Receptor 1 and methods of use thereof)
• monoclonal antibodies specific for human LOX-1 described in Iwamoto S et al. “Generation and characterization of chicken monoclonal antibodies against human LOX-l.MAbs 2009;1:357-63. Iwamoto et al. generated a total of 53 monoclonal antibodies specific for human LOX-1 by a phage display technique, by using chickens immunized with recombinant human LOX-1. Using these antibodies, investigators constructed chimeric chicken-human antibodies with characteristics similar to those of the original antibodies. Use of these chimeric antibodies has been shown to decrease ox-LDL uptake.
The monoclonal antibodies described in Iwamoto et al. (2009) as LOX-1 inhibitors (and chimeric antibodies) can also be found in patent application W02010147171, US2012087926
• Peptide molecule
As indicated previously the LOX-1 inhibitor can also be a peptide or peptide molecule comprising amino acid residues. As used herein the term “amino acid residue” refers to any natural/standard and non-natural/non-standard amino acid residue in (L) or (D) configuration, and includes alpha or alpha-di substituted amino acids. It refers to isoleucine, leucine, lysine, methionine, phenylalanine, threonine, tryptophan, valine, arginine, alanine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, proline, serine, tyrosine. It also includes beta-alanine, 3 -amino-propionic acid, 2,3-diamino propionic acid, alpha- aminoisobutyric acid (Aib), 4-amino-butyric acid, N-methylglycine (sarcosine), hydroxyproline, ornithine (e.g., L-ornithine), citrulline, t-butylalanine, t-butylglycine, N- methylisoleucine, phenylglycine, cyclohexylalanine, cyclopentylalanine, cyclobutylalanine, cyclopropylalanine, cyclohexylglycine, cyclopentylglycine, cyclobutylglycine, cyclopropylglycine, norleucine (Nle), norvaline, 2-napthylalanine, pyridylalanine, 3- benzothienyl alanine, 4-chlorophenylalanine, 2-fluorophenylalanine, 3-fluorophenylalanine, 4-fluorophenylalanine, penicillamine, 1,2,3,4-tetrahydro-isoquinoline-3-carboxylix acid, beta- 2-thienylalanine, methionine sulfoxide, L-homoarginine (hArg), N-acetyl lysine, 2-amino butyric acid, 2-amino butyric acid, 2,4,-diaminobutyric acid (D- or L-), p- aminophenylalanine, N-methylvaline, selenocysteine, homocysteine, homoserine (HoSer), cysteic acid, epsilon-amino hexanoic acid, delta-amino valeric acid, or 2,3-diaminobutyric acid (D- or L-), etc. These amino acids are well known in the art of biochemistry/peptide chemistry. Example of peptide used as a LOX-1 inhibitor for use in the context of the present invention can be selected from specific peptides identified by subtractive Phage Display that targets LOX-1 (in particular. Peptides 17, 32 and 40) as described in White SJ et al “Identification of Peptides That Target the Endothelial Cell–Specific LOX-1” Receptor”Hypertension.2001;37:449–455; Amaranth synthetic pure peptides as described in Montoya-Rodríguez A. et al “Pure peptides from amaranth (Amaranthus hypochondriacus) proteins inhibit LOX-1 receptor and cellular markers associated with atherosclerosis development in vitro”. Food Research International Volume 77, Part 2, November 2015, Pages 204-214; Compounds of the present invention which include peptides may comprise replacement of at least one of the peptide bonds with an isosteric modification. Compounds of the present invention which include peptides may be peptidomimetics. A peptidomimetic is typically characterised by retaining the polarity, three dimensional size and functionality (bioactivity) of its peptide equivalent, but wherein one or more of the peptide bonds/linkages have been replaced, often by proteolytically more stable linkages. Generally, the bond which replaces the amide bond (amide bond surrogate) conserves many or all of the properties of the amide bond, e.g. conformation, steric bulk, electrostatic character, potential for hydrogen bonding, etc. Typical peptide bond replacements include esters, polyamines and derivatives thereof as well as substituted alkanes and alkenes, such as aminomethyl and ketomethylene. For example, the peptide may have one or more peptide linkages replaced by linkages such as -CH2NH-, -CH2S-, -CH2-CH2-, -CH═CH- (cis or trans), -CH(OH)CH2-, or -COCH2-, -N- NH-, -CH2NHNH-, or peptoid linkages in which the side chain is connected to the nitrogen atom instead of the carbon atom. Such peptidomimetics may have greater chemical stability, enhanced biological/pharmacological properties (e.g., half-life, absorption, potency, efficiency, etc.) and/or reduced antigenicity relative its peptide equivalent. • Small organic molecule
The LOX-1 inhibitor can also be a small organic molecule. The term “small organic molecule” refers to a molecule of a size comparable to those organic molecules generally used in pharmaceuticals. The term excludes biological macromolecules (e.g., proteins, nucleic acids, etc.). Preferred small organic molecules range in size up to about 5000 Da, more preferably up to 2000 Da, and most preferably up to about 1000 Da. Examples of small organic molecule are Molecule 4 and 5 described un Thakkar et al. and in patent application WO2017075418.
• Antibody or an antigen-binding molecule
The LOX-1 inhibitor can also be an antibody or an antigen-binding molecule. In an embodiment, the antibody specifically recognize/bind LOX-1 (e.g. LOX-1 of SEQ ID NO:l) or an epitope thereof involved in the activation/stimulation of the ERK-pathway. In another preferred embodiment, the antibody is a monoclonal antibody or single chain antibody.
Example of monoclonal antibody used as a LOX-1 inhibitor for use in the context of the present invention can be selected from the monocolal antibodies described in Iwamoto S et al. “Generation and characterization of chicken monoclonal antibodies against human LOX-1. MAbs 2009;1:357-63 (and US2012087926), monocolal antibodies developed by Novartis described in W02014205300, monocolal antibodies developed by Abgenics described in EP1418234.
Example of single chain antibody used as a LOX-1 inhibitor for use in the context of the present invention can be the anti -LOX-1 human single chain antibody by JILIN University described in CN107216390
The term “antibody” is used in the broadest sense, and covers monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, multispecific antibodies, chimeric antibodies and humanized antibodies, so long as they exhibit the desired biological activity (e.g., as indicated previously, a) inhibiting processes associated with pro- inflammatory cytokines release and/or b) inhibiting processes associated thromboembolic events (through the ERK prothrombotic pathway.). Antibody fragments comprise a portion of a full-length antibody, generally an antigen binding or variable region thereof. Examples of antibody fragments include Fab, Fab' , F(ab' )2, and Fv fragments, diabodies, linear antibodies, single-chain antibody molecules, single domain antibodies (e.g., from camelids), shark NAR single domain antibodies, and multispecific antibodies formed from antibody fragments. Antibody fragments can also refer to binding moieties comprising CDRs or antigen binding domains including, but not limited to, V H regions (V H, V H-V H), anticalins, PepBodies, antibody-T-cell epitope fusions (Troybodies) or Peptibodies. Antibodies according to the present invention can be of any class, such as IgG, IgA, IgDl IgEl IgMl or IgYl although IgG antibodies are typically preferred. Antibodies can be of any mammalian or avian origin, including human, murine (mouse or rat), donkey, sheep, goat, rabbit, camel, horse, or chicken. The antibodies can be modified by the covalent attachment of any type of molecule to the antibody. For example, but not by way of limitation, the antibody derivatives include antibodies that have been modified, e.g., by glycosylation, acetylation, pegylation, phosphylation, amidation, derivatization by known protecting/blocking groups, proteolytic cleavage, linkage to a cellular ligand or other protein, or other modifications known in the art.
In general, techniques for preparing antibodies (including polyclonal antibodies, monoclonal antibodies and hybridomas) and for detecting antigens using antibodies are well known in the art, see e.g., Harlow & Lane, Using Antibodies, A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1998; Kohler & Milstein, Nature 256:495-497 (1975); Kozbor et ah, Immunology Today 4:72 (1983); and Cole et ah, pp. 77- 96 in Monoclonal Antibodies and Cancer Therapy, 1985. Additionally, antibodies according to the present invention can be fused to marker sequences, such as a peptide tag to facilitate purification; a suitable tag is a hexahistidine tag. The antibodies can also be conjugated to a diagnostic or therapeutic agent by methods known in the art. Techniques for preparing such conjugates are well known in the art. Other methods of preparing these monoclonal antibodies, as well as chimeric antibodies, humanized antibodies, and single-chain antibodies, are known in the art.
• Aptamer
The LOX-1 inhibitor can also be an aptamer. Aptamers are a class of molecule that represents an alternative to antibodies in term of molecular recognition. Aptamers are oligonucleotide or oligopeptide sequences with the capacity to recognize virtually any class of target molecules with high affinity and specificity. Such ligands may be isolated through Systematic Evolution of Ligands by Exponential enrichment (SELEX) of a random sequence library, as described in Tuerk C. and Gold L., 1990. The random sequence library is obtainable by combinatorial chemical synthesis of DNA. In this library, each member is a linear oligomer, eventually chemically modified, of a unique sequence. Possible modifications, uses and advantages of this class of molecules have been reviewed in Jayasena S.D., 1999. Peptide aptamers consists of a conformationally constrained antibody variable region displayed by a platform protein, such as E. coli Thioredoxin A that are selected from combinatorial libraries by two hybrid methods (Colas et al., 1996).
• Phospholipid
Also within the scope of the invention is a phospholipid as a LOX-1 inhibitor. As above mention Falconi et al. (Biochem Biophys Res Commun 2013) developed a modified phospholipid, PLAzPC, which binds to the tunnel binding site of the LOX-1 molecule and markedly inhibits interaction with ox-LDL.
An example of phospholipid usable in the context of the invention is PLAzPC.
• polynucleotide
The LOX-1 inhibitor can also be a polynucleotide, typically an inhibitory nucleotide. (Inhibitor of LOX-1 gene expression) These include short interfering RNA (siRNA), microRNA (miRNA), and synthetic hairpin RNA (shRNA), anti-sense nucleic acids, complementary DNA (cDNA) or guide RNA (gRNA usable in the context of a CRISPR/Cas system). In some embodiments, a siRNA targeting LOX-1 expression is used. Interference with the function and expression of endogenous genes by double-stranded RNA such as siRNA has been shown in various organisms. See, e.g., Zhao Y et al, “Co-delivery of LOX-1 siRNA and statin to endothelial cells and macrophages in the atherosclerotic lesions by a dual-targeting core-shell nanoplatform: A dual cell therapy to regress plaques,” Journal of Controlled Release Volume 283, 10 August 2018, p.241-260; Aijuman A et al “LOX-1: A potential target for therapy in atherosclerosis; an in vitro study “Int J Biochem Cell Biol. 2017 Oct;91(Pt A):65-80. doi: 10.1016. siRNAs can include hairpin loops comprising self- complementary sequences or double stranded sequences. siRNAs typically have fewer than 100 base pairs and can be, e.g., about 30 bps or shorter, and can be made by approaches known in the art, including the use of complementary DNA strands or synthetic approaches. Such double-stranded RNA can be synthesized by in vitro transcription of single- stranded RNA read from both directions of a template and in vitro annealing of sense and antisense RNA strands. Double-stranded RNA targeting LOX-1 can also be synthesized from a cDNA vector construct in which a LOX-1 gene (e.g., human LOX-1 gene) is cloned in opposing orientations separated by an inverted repeat. Following cell transfection, the RNA is transcribed and the complementary strands reanneal. Double-stranded RNA targeting the LOX-1 gene can be introduced into a cell (e.g., a tumor cell) by transfection of an appropriate construct.
Typically, RNA interference mediated by siRNA, miRNA, or shRNA is mediated at the level of translation; in other words, these interfering RNA molecules prevent translation of the corresponding mRNA molecules and lead to their degradation. It is also possible that RNA interference may also operate at the level of transcription, blocking transcription of the regions of the genome corresponding to these interfering RNA molecules.
The structure and function of these interfering RNA molecules are well known in the art and are described, for example, in R. F. Gesteland et ah, eds, “The RNA World” (3rd, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 2006), pp. 535-565, incorporated herein by this reference. For these approaches, cloning into vectors and transfection methods are also well known in the art and are described, for example, in J. Sambrook & D. R. Russell, “Molecular Cloning: A Laboratory Manual” (3rd, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, 2001), incorporated herein by this reference.
In addition to double stranded RNAs, other nucleic acid agents targeting LOX-1 can also be employed in the practice of the present invention, e.g., antisense nucleic acids. Antisense nucleic acids are DNA or RNA molecules that are complementary to at least a portion of a specific target mRNA molecule. In the cell, the single stranded antisense molecule hybridizes to that mRNA, forming a double stranded molecule. The cell does not translate an mRNA in this double-stranded form. Therefore, antisense nucleic acids interfere with the translation of mRNA into protein, and, thus, with the expression of a gene that is transcribed into that mRNA. Antisense methods have been used to inhibit the expression of many genes in vitro. See, e.g., Li D et ak, “Antisense to LOX-1 inhibits oxidized LDL- mediated upregulation of monocyte chemoattractant protein- 1 and monocyte adhesion to human coronary artery endothelial cells “Circulation. 2000 Jun 27; 101 (25):2889-95. doi: 10.1161; Amati F et al , “LOX-1 Inhibition in ApoE KO Mice Using a Schizophyllan-based Antisense Oligonucleotide Therapy,” Mol Ther Nucleic Acids. 2012 Dec; 1(12): e58;, incorporated herein by this reference. LOX-1 polynucleotide sequences from human and many other animals in particular mammals have all been delineated in the art. Based on the known sequences, inhibitory nucleotides (e.g., siRNA, miRNA, or shRNA) targeting LOX-1 can be readily synthesized using methods well known in the art.
Exemplary siRNAs according to the invention could have up to 29 bps, 25 bps, 22 bps, 21 bps, 20 bps, 15 bps, 10 bps, 5 bps or any integral number of base pairs between these numbers. Tools for designing optimal inhibitory siRNAs include that available from DNAengine Inc. (Seattle, Wash.) and Ambion, Inc. (Austin, Tex).
The guide RNA (gRNA) sequences direct a nuclease (i.e. CrispRCas9 protein) to induce a site-specific double strand break (DSB) in the genomic DNA in the target sequence.
Example of commercial gRNAs against LOX-1 are available. Therapeutic Method of a specific population
The invention also relates to a method for treating coronavirus infection with a LOX-1 inhibitor in a subject wherein the level of a population of neutrophils CD66b+CD10-CD16- CD64+LOX-1+ obtained from said patient have been detected by one of method of the invention.
In the context of the invention, the term "treating" or "treatment", as used herein, means reversing, alleviating, inhibiting the progress of, or preventing the disorder or condition to which such term applies, or reversing, alleviating, inhibiting the progress of, or preventing one or more symptoms of the disorder or condition to which such term applies.
In a particular embodiment, a LOX-1 inhibitor according to the invention can be a molecule selected from a peptide, a peptide mimetic, a small organic molecule, an antibody, an aptamer, a phospholipid, a polynucleotide (inhibitor of LOX-1 gene expression) and a compound comprising such a molecule or a combination thereof.
Another object of the present invention is a method of treating coronavirus infection in a subject comprising the steps of: a) providing a sample containing neutrophil from a subject, b) detecting the level of a population of neutrophils CD66b+CD10-CD16- CD64+CD123+ and/or CD66b+CD10-CD16-CD64+LOX-l+ c) comparing the level determined at stet b) with a reference value and if level determined at stet b) is higher than the reference value, treating the subject with Lox-1 inhibitors.
As mentioned, anticoagulant is the current main treatment for the severe form of coronavirus infection or thrombosis associated with coronavirus infection.
Accordingly, the invention also relates to a method for treating coronavirus infection with anticoagulant in a subject wherein the level of a population of neutrophils CD66b+CD 10-CD 16-CD64+CD 123+ and/or CD66b+CD10-CD16-CD64+LOX-l+ obtained from said patient, have been detected by one of the methods of the invention.
Method of treating coronavirus infection in a subject comprising the steps of: a) providing a sample containing neutrophils from a subject, b) detecting the level of a population of neutrophils CD66b+CD10-CD16- CD64+CD123+ and/or CD66b+CD10-CD16-CD64+LOX-l+ c) comparing the level determined at stet b) with a reference value and if level determined at step b) is higher than the reference value, treating the subject with anticoagulant.
In particular embodiments, the coronavirus infection is the severe form of coronavirus infection and/or thrombosis associated with coronavirus infection
Example of anticoagulant agents (defined herein as agents that inhibit blood clot formation) include, without limitation, specific inhibitors of thrombin, factor IXa, FXa, factor XIa, factor Xlla or factor Vila, heparin and derivatives, Vitamin K antagonists (VKA), Non- VKA Anticoagulant agents" and anti-tissue factor antibodies.
Non-VKA (Vitamin K Antagonists) anticoagulants includes non-VKA oral anticoagulants (NOAC) such as direct-oral anticoagulants (DOAC). Examples of specific inhibitors of thrombin include hirudin, bivalirudin (Angiomax®), argatroban, and lepirudin (Refludan®). Examples of heparin and derivatives include unfractionated heparin (UFH), low molecular weight heparin (LMWH), such as enoxaparin (Lovenox®), dalteparin (Fragmin®), tinzaparin (Innohep®), nadroparine (Fraxiparine® or Fraxodi®); and synthetic pentasaccharide, such as fondaparinux (Arixtra®). Examples of DOAC anticoagulants include rivaroxaban (Xarelto®), apixaban (Eliquis®), edoxaban (Lixiana®), and dabigatran (Pradaxa®). In one preferred embodiment, the anticoagulant is an inhibitor of FXa. “Vitamin K antagonists (VKA)” include, in a non-limitative manner, warfarin (Coumadin®), phenocoumarol, acenocoumarol (Sintrom®), clorindione, dicoumarol, diphenadione, ethyl biscoumacetate, phenprocoumon, phenindione, fluindione (Previscan®) and tioclomarol.
The invention will be further illustrated by the following figures and examples. However, these examples and figures should not be interpreted in any way as limiting the scope of the present invention.
FIGURES:
Figure 1: Severe CO ID-19 patients displayed increased immature neutrophil subsets expressing CD123 or LOX-1. (A) viSNE analysis was performed on neutrophils from all samples with cells organized along t-SNE-1 and t-SNE-2 according to per-cell expression of CD15, CD10, CD64, LOX-1, CD123 and PD-L1. Cell density for the concatenated file of each patient’s group (ICU vs Non-ICU) is shown on a black to yellow heat scale. Neutrophils’ CD 10, CD64 markers expression is presented on a rainbow heat scale in the t-SNE map of each group concatenated file. (B) Box plots representation (min to max distribution) of CD10-CD64+ neutrophil subset abundancy among total neutrophils of each group samples. (C) Representative expression of LOX-1 and CD123 on CD10-CD64+ neutrophils. (D) Abundancy of CD10-CD64+ neutrophil expressing CD123 or LOX-1 in ICU and non-ICU patients’ groups identify the median and min to max distribution. Nonparametric Mann-Whitney test was used to compare differences in cellular abundance of neutrophil subsets between groups, with significance defined by a p-value < 0.05: * for p < 0.05; ** for p < 0.01; *** for p < 0.001. (E) Principal component analysis (PCA) using LOX- 1+, CD123+ CD10-CD64+ neutrophil abundancy and SAPS II variables on sample sizes: ICLH24 (black circles), non-ICU=14 (empty circles). Percent contribution (contrib) of each variable is indicated.
Figure 2: Abundancy of LOX-l-expressing immature neutrophil correlate with clinical severity of COVID-19 patients. (A) Box plots representation (min to max distribution) of the proportion of immature neutrophil expressing CD 123- or LOX-1 into patients’ groups with high (n=13) or low SOFA score (n=ll) among all ICU patients. Non parametric two-tailed Mann-Whitney test was used to compare differences in cellular abundance of neutrophil subsets between high and low SOFA score: CD123+ neutrophils (**p<0.0022), LOX-1+ neutrophils (****p<0.0001). (B) Principal component analysis (PCA) using serum cytokines and SOFA score variables on ICU patients sample sizes: high SOFA score (n=l 1), low SOFA score (n=10) (SOFA<8= black circles, SOFA>8 = empty circles).
Figure 3: Abundancy of LOX-l-expressing immature neutrophil correlate with thrombosis of COVID-19 patients. (A) Box plots representation (min to max distribution) of the proportion of immature neutrophils expressing either CD123- or LOX-1 in blood of patients with (yes) or without (no) thrombosis. Sample sizes: thrombosis n=8, no thrombosis n=24. Nonparametric Mann-Whitney test was used to compare differences in frequencies of neutrophil subsets between the two groups, with significance defined by a p-value < 0.05: * for p < 0.05; ** for p < 0.01; **** for p < 0.0001. (B) Receiver operating characteristic (ROC) curve analysis was performed to assess the predictive value of LOX-1 with thrombosis.
Figure 4: Increased proportions of circulating immature neutrophils expressing either CD123 or LOX-1 in critical COVID-19 patients are associated with COVID-19 severity and thromboembolic complications. (A) Abundance of CD10 CD64+ neutrophils expressing CD123, PD-L1, or LOX-1 in ICU and non-ICU patient groups. (B-C) Box plots (min to max distribution) of the proportion of total ImNs and ImNs expressing CD123-, LOX- 1, or PD-L1 in patient groups with invasive mechanical ventilation (B) or with thromboembolic complications (C). Nonparametric Mann-Whitney test was used to compare differences in cellular abundance of neutrophil subsets between groups, with significance defined by a p-value < 0.05: **p < 0.01; and ***p < 0.001.
Figure 5: Subsets of immature neutrophils expressing LOX-1 infiltrate lung. (A) Opt-SNE analysis was performed on 40 000 randomly chosen neutrophils from blood and bronchoalveolar (BAL) samples (n = 16). Cells were organized along t-SNE-1 and t-SNE-2 according to per-cell expression of CD10, CD15, CD16, CD64, LOX-1, CD123, and PD-L1. Cell density for the concatenated file of each group; blood samples from healthy donor (HD) blood; n = 8); blood and BAL samples from COVID-19 patients (COVID-19 BAL; n = 16). Cell density is shown on a black-to-yellow heat scale. (B) Box and whisker plots with min and max of myeloperoxidase (MPO) and neutrophil elastase (EL A) in BAL of COVID-19 patients (n = 12) and of control patients (CRTL; n = 12).
Figure 6: Immature neutrophil subsets expressing either CD123, LOX-1, or PD- L1 are correlated with clinical severity, but only LOX-1+ subset proportion at entry is strongly associated with higher risk of thrombosis. (A) Box plots (min to max distribution) of the proportion of ImNs expressing CD123-, LOX-1, or PD-L1 in severity patient groups in mild {n = 18), severe (// = 19), or critical (// = 51) clinical condition. One-way ANOVA test was used to compare the three groups, with significance defined as follows: ***p < 0.001; and **** > < 0.0001. (B) Box plots (min to max distribution) of the abundance of CD10-CD64+ neutrophil subsets among discharged (// = 74) and deceased (// = 14) patients. Nonparametric Mann-Whitney test was used to compare differences between groups, with significance defined by a p-v alue < 0.05: * **p < 0.01; ***p < 0.001. (C) Box plots (min to max distribution) of the abundance of CD10-CD64+ neutrophil subsets among patients without (i n = 75) or with (// = 12) thromboembolic complications. Nonparametric Mann- Whitney test was used to compare differences in cellular abundance of neutrophil subsets between groups, with significance defined by a p-v alue < 0.05: *p < 0.05; **p < 0.01. (D) Forest plots comparing hazard ratio (log-rank test) for survival, invasive mechanical ventilation (IMV), and thrombotic events (thrombosis) in 118 patients according to median frequency of ImN subsets (reference group with ImN subset frequencies below the median) and comorbidities (reference group with no comorbidities or age below 61). Log-rank (Mantel-Cox) test was used to compare HR between groups, with significance defined by a p- value < 0.05: *p < 0.05; **p < 0.01; and ***p < 0.001.
Figure 7: Immature neutrophil subsets and plasma levels of MPO and ELA are independent markers of COVID-19 severity. (A) Box plots (min to max distribution) of the plasma levels of MPO, ELA, and MPO-DNA complexes (NET) in severity patient groups in mild (n = 23), severe (n = 22), or critical (n = 63) clinical condition. The concentrations of MPO are expressed as pg/mL, ELA as 10-1 pg/mL, and MPO-DNA complexes as an arbitrary unit proportional to 10-5 of the ratio blank/sample of the absorbance measured at 450 nm. One-way ANOVA test was used to compare the three groups, with significance defined as ***p < 0.001. (B) Box plots (min to max distribution) of the plasma levels of MPO, ELA, and NET among discharged (n = 90) and deceased (n = 17) patients. Nonparametric Mann-Whitney test was used to compare differences in cellular abundance of neutrophil subsets between groups, with significance defined by a p-value < 0.05: *p < 0.05; and **p < 0.01. (C) Box plots (min to max distribution) of the plasma levels of MPO, ELA, and NET among patients without (n = 94) or with (n = 12) thromboembolic complications. Nonparametric Mann-Whitney test was used to compare differences in cellular abundance of neutrophil subsets between groups, with significance defined by a p-value < 0.05: ns= not significant. (D) Principal component analysis (PCA) using granule protein plasma levels (MPO, ELA, and NET), immature phenotypic markers (LOX-1+, PD-L1+, CD123+ ImNs) and severity variables: critical patients, n = 63; mild + severe patients, n = 45. (E) Receiver operating characteristic (ROC) curve analysis performed on discovery and validation studies to assess the predictive value of LOX-1 with thrombosis (n = 118).
EXAMPLE 1:
Methods:
Study participants
Fresh blood samples from 38 consecutive adult patients with COVID-19 referred to the Department of Internal Medicine 2, Department of Infectious Diseases and Intensive Care Units (ICU), Pitie-Salpetriere Hospital, Paris were included in the study between March 18, 2020 and April 29, 2020. The diagnosis of COVID-19 relied on SARS-CoV-2 carriage in the nasopharyngeal swab, as confirmed by real-time reverse transcription-PCR analysis. Demographic and clinical characteristics are detailed in Table 2. All flow cytometric analyses were performed on fresh whole blood cells collected at the admission to the hospital and ICU. Sera were collected for cytokine measurement.
Study approval
The study was conducted in accordance with the Declaration of Helsinki and the International Conference on Harmonization Good Clinical Practice guidelines and approved by the relevant regulatory and independent ethics committees. All patients gave oral informed consent. The study was registered and approved by local ethical committee of Sorbonne- Universite/assistance-publique hopitaux de Paris for standard hospitalized patients (N°2020- CER2020-21) and ICU patients (N° CER-2020-31).
Flow cytometry
One hundred mΐ of fresh whole blood collected on Anticoagulant Citrate-Dextrose solution (ACD) for patients in intensive care or on EDTA for patients in standard hospitalization were stained with a mix of monoclonal antibodies. Samples were diluted in brilliant violet buffer (BD biosciences) and incubated 20 min at room temperature in the dark. The antibody panel (Table 3) included: CD15-BV786, CD14-BUV737, CD10-BUV395 (BD, Le Pont de Claix, France); CRTH2-FITC, CD 123 -PE, LOX-1-BV421, CD64-BV605 and LOX-1-BV711 (Biolegend, San Diego, USA). One ml of BD FACS lysing (BD biosciences) solution IX was directly added to the cells to lyse red blood cells, incubated 20min, centrifuged, and washed with PBS. Leukocytes were resuspended in PBS before analysis with a BD LSR FORTES SA-X-20™. FlowJo™ software 10.0 was used for analysis of marker expression on neutrophils. One hundred mΐ of whole blood were additionally stained for several patients with a FMO mix missing of antibodies targeting CD123, LOX-1, and LOX-1, in order to determine the threshold of expression of these markers.
Data presentation and statistical analysis
Statistical analyses of the immunological data and graphic representations were performed with Prism 8.0 (GraphPad Software Inc.). Two-tailed Student’s t-test were used for group comparisons, and one-way and two-way ANOVA tests with Bonferoni for multiple comparison tests. The potential association between serum cytokine levels or marker expressing neutrophils frequencies was evaluated using Spearman correlation (one-tailed), with significance defined by a p-value < 0.05: * for p < 0.05; ** for p < 0.01; *** for p < 0.001; **** for p < 0.0001. Principal Component Analyses were performed with R software 3.3.1.
Quanterix technology (digital ELISA)
The Simoa™ (single molecule array) HD-1 analyser (Quanterix, Lexington, MA, USA) was used for ultrasensitive immunodetection (digital ELISA) of IL-3, IL-17A, IL-18, GM-CSF and IFN-a, using singleplex bead-based assays (Table 3). Concentrations of IL-Ib, IFN-g, IL-6, IL-8, IL-22, TNF-a and IL-10 were determined using a multiplex planar array immunoassay on the Quanterix SP-X™ platform according to manufacturer’s instructions. Serum IFN-b levels were quantified with a highly sensitive ELISA kit (PBL Assay Science, Piscataway, NJ, USA). The concentrations of cytokines in unknown samples were interpolated from a standard curve performed with two replicates of each level of recombinant calibrator proteins, representing the dynamic range of the assay: IL-Ib (0.073-300 pg/mL), IFN-g (0.012-50 pg/mL), IL-6 (0.073-300 pg/mL), IL-8 (0.098-400 pg/mL), IL-22 (0.024-100 pg/mL), TNFa (0.098-400 pg/mL), IL-10 (0.024-100 pg/mL), IL-3 (0.686-500 pg/mL), IL- 17A (0.041-30 pg/mL), IL-18 (0.011-45 pg/mL), GM-CSF (0.041-30 pg/mL), IFN-a (0.028- 27.3 pg/mL) and IFN-b (1.2-150 pg/mL).
Computational data analysis of neutrophils viSNE analysis was performed with Cytobank software (Table 3) using CD10, CD64, PD-L1, CD123 and LOX-1 markers. We selected a total of 100,000 CD15+ granulocytes (data not shox), divided equally between 24 ICU patients and 13 non-ICU patients, using one single concatenated file for each condition.
Results
Demographics and baseline characteristics of ICU and non-ICU COVID-19 patients
Thirty-eight COVID-19 patients admitted to either ICU departments or non-ICU departments were included. SARS-CoV-2 infection was confirmed on nasopharyngeal swab by positive RT-PCR in accordance with WHO interim guidance. Clinical and biological characteristics of the 38 patients are shown in Table 2. Median age was 57 years (range 25-79 years), with 65.8% of them being males. Analysis was performed on median average 8 days after the onset of symptoms (median was 8 days for the ICU patients, 13 days for the non-ICU patients). The most common past medical comorbidities were hypertension (50%), type 2 diabetes (34.2%) and obesity (36.8%). Treatment regimen at baseline was mostly anti hypertensive therapy (ACE inhibitors 26.3% and angiotensin II receptor blockers 15.8%). Severity at baseline was assessed by the SAPS II score for all patients (median 33, ranging from 25 to 78) and an additional SOFA score for ICU patients (median 8.5, ranging from 2 to 17). Twenty-eight patients were assessed with CT chest imaging, with ground-glass opacities and/or consolidation > 50% of the lung field among 50% of all patients, with up to 81.3% of the ICU patients. Laboratory findings showed a decreased median lymphocyte count at 0.94X109/L, an increased median neutrophil count at 7.87xl09/L, an increased median lactate dehydrogenase at 475.5 U/L and an increased median D-dimer level at 2450 ng/mL. During hospitalization, 8 patients received hydroxychloroquine (42.1%), while all patients received antibiotics. Oxygen therapy was administered to 100% of patients; ICU patients were ventilated with invasive mechanical ventilation for 87.5% of them, while 54.2% received extracorporeal membrane oxygenation. Acute respiratory distress syndrome occurred among 55.3% of all patients (87.5% of ICU patients) and acute kidney injury among 31.2% of all patients. Among the 38 patients, 2 patients were diagnosed with pulmonary embolism (5.3%) and 10 patients (all ICU) (26.3%) were diagnosed with venous thromboembolism. 76.3% of all patients were discharged as of June 8, 2020, while 10.5% remained in hospital and 13.2% had died, the latter all being ICU patients.
Increased proportions of circulating immature neutrophils expressing either CD123 or LOX-1 in severe COVID-19 patients.
In order to identify neutrophil surface markers that may help to predict the severity of the infection at hospital admission, we designed an observational study including 38 individuals and analyzed their neutrophil phenotypes, comparing them among patients admitted to ICU (n=24) or not (n=14) within the first day following their admission (Table 2). COVID-19 patients from ICU displayed more severe clinical and biological signs than non- ICU patients, with an elevated Simplified Acute Physiology Score (SAPS II); (ICU35.5, n=24 and non-ICU:25.5, n=14; p=0.056), higher serum lactate dehydrogenase (ICU:504, n=24 and non-ICU:324, n=14; p=0.005), and higher D-dimers (ICU2760, n=23 and non- ICU:1860, n=12; p=0.25). However, they did not differ while comparing neutrophils counts (ICU8.75, n=24 and non-ICU:5.185, n=14; p=0.06) and level of lymphopenia (ICU0.925, n=24 and non-ICU: 1.185, n=14; p=0.41). Patients characteristics confirmed previously published data with notably a high prevalence of obese patients in COVID-19 patients. Main differences between ICU and non-ICU patients reflected case severity with a high proportion of patient with lung lesions (as observed by ground-glass opacities on chest CT) requiring mechanical ventilation and resulting in high in-hospital mortality.
Whole blood immunostaining was performed, within 3h after blood drawing, using a previously published panel designed to give a precise evaluation of immature circulating neutrophils (17). Neutrophils were automatically identified and visualized using Visualization of t-Distributed Stochastic Neighbor Embedding (viSNE implementation of t-SNE) in order to define an imprint for each sample group (Figure 1A). Using an unsupervised classification, neutrophils from ICU patients were organized in the upper left quadrant of the map, whereas the non-ICU patients’ neutrophils were in the upper right quadrant.
This analysis allowed precise delimitation of two main subsets of neutrophils based on the expression of CD 10 and CD64 markers; the ICU-abundant upper left area was composed of neutrophils with mid-to-low expression of CD 10 and high expression of CD64, and the non-ICU-abundant upper right area was composed of high-to-mid expression of CD 10 and high-to-mid expression of CD64. We next determined whether the identified neutrophil signature would be confirmed using conventional analysis applicable by experts. Neutrophils were identified on the CD 15 neutrophil marker and excluding prototypical markers of eosinophil and monocyte, respectively CRTH2 and CD14 (data not shown). Expert-gating strategy confirmed the high abundance of CD10 CD64+ neutrophils among ICU patients compared non-ICU patients (Figure IB). Then, we compared the expression of CD123, LOX-1 and LOX-1 surface molecules, formerly known as dysregulated in sepsis (17). All three of them were barely co-expressed on neutrophils and lead to the identification of three distinct neutrophil subpopulations (Figure 1C and data not shown). Subsets of neutrophils expressing either CD 123 or LOX-1 were more abundant in ICU than in non-ICU patients (Figure ID) unlike those expressing LOX-1 (data not shown). Principal component analysis (Figure IE) revealed that both the expression of CD123 and LOX-1 on immature neutrophils contributed independently to patients’ severity as appreciated by SAPS II regardless of age, obesity and other potential confounding factors. These data suggested that immature neutrophil subsets expressing CD123 or LOX-1 may contribute to the severity of the disease.
LOX-1+ neutrophil proportions are positively correlated with clinical severity and cytokine levels
COVID-19 patients from ICU were segregated into two groups based on severity at the time of admission. Patients with low SOFA (<8) had significantly fewer CD123- and LOX-1 -expressing immature neutrophils than patients with high SOFA (>8), (p<0.01 and p<0.001 respectively) (Figure 2A). The abundancy of LOX-1 -expressing immature neutrophils correlated positively with the SOFA score (Table 1), with inflammatory cytokines such as IL-Ib, IL-6, IL-8, TNFa and with the anti-inflammatory cytokine IL-10, whereas it correlated negatively with IFNa and the multipotent hematopoietic growth factor IL-3. In contrast, expression of CD123 on immature neutrophils did not correlate with any of these cytokines but with IL-17, IL-18, IL-22, while negatively correlating with PTMb (Table 1). In addition, we observed that the expression of LOX-1 significantly correlated with serum D- dimer concentrations. Principal Component Analysis confirmed that the ICU patients’ severity was associated with the expression of CD123 and LOX-1 on immature neutrophils and distinct patterns of cytokines (Figure 2B). These results lead to the identification of three profiles: 1) patients with high LOX-1+ neutrophils proportions and high IL-Ib, IL-6, IL-8, TNFa serum levels, 2) patients with CD123 expression and IL-18, IL-22, IFNy secretion, and 3) a bulk of the patients with lower severity associated with high type-1 interferons levels. These data suggested that immature neutrophil subsets expressing either CD 123 or LOX-1 may define a specific profile of severity associated with high levels of pro-inflammatory cytokines.
Increased LOX-1+ neutrophil proportions at entry are associated with higher thrombosis risk
Because high values of D-dimer have been associated with disseminated intravascular coagulation in COVID-19 patients (1), we aimed to seek a correlation between the overexpression of LOX-1 and thromboembolic events. The occurrence of thrombosis (Figure 3A) was associated with higher levels of CD123- and LOX-1 -expressing immature neutrophils (p<0.01 and p<0.0001 respectively). In order to assess the predictive value of LOX-1 on the occurrence of thrombosis, we performed a Receiver Operating Characteristic (ROC) curve analysis (Figure 3B), with data from patients who survived more that 10 days after inclusion (n=34). The area under the curve (AUC) was 0.95 for LOX-1 immature neutrophil abundancy (p < 0.0001) (Figure 3B) compared with CD123 (AUC=0.81, p=0.003), the SAPS II score (AUC=0.73, p=0.03), D-Dimers level (AUC=0.63, non significant), and age (AUC=0.61, non-significant). Thus, ROC curve analysis suggested that LOX-1 may be an accurate predictive marker of thrombosis for COVID-19 patients.
EXAMPLE 2:
Methods:
Study participants
Fresh blood samples from 38 (pilot study, Mar- Apr 2020) and 118 (validation study, Sep-Nov 2020) consecutive adult patients with COVID-19 referred to the Department of Internal Medicine 2, Department of Infectious Diseases and Intensive Care Units of Pitie- Salpetriere University Hospital, Paris, were included. The diagnosis of COVID-19 relied on SARS-CoV-2 carriage on the nasopharyngeal swab, as confirmed by real-time reverse transcription PCR analysis. In addition, blood samples were collected from eight healthy donors obtained from the French blood donation center. When ventilator-associated pneumonia was suspected, patients underwent fiber-optic bronchoscopy and BAL (n = 16) to sample distal respiratory secretions. Leukocyte phenotyping was performed on BAL when possible. Demographic and clinical characteristics are detailed in Tables 2 and 5.
Study approval
The studies were conducted in accordance with the Declaration of Helsinki and the International Conference on Harmonization (ICH) Good Clinical Practice (GCP) Guideline and approved by the relevant regulatory and independent ethics committees. In accordance with current French law, informed written consent was obtained from patients or relatives. The studies were registered and approved by the local ethical committee of Sorbonne Universite/Assistance Publique - Hopitaux de Paris for standard hospitalized patients (N°2020-CER2020-21 ) and ICU patients (N° CER-2020-31).
Flow cytometry
One hundred microliters of fresh whole blood collected at admission to the hospital — on anticoagulant citrate-dextrose solution (ACD) for patients in the ICU or on EDTA for patients in standard hospitalization — was stained with a mix of monoclonal antibodies. Samples were diluted in Brilliant Violet buffer (BD Biosciences, Le Pont-de-Claix, France) and incubated 20 min at room temperature in the dark. The antibody panel (data not shown) included CD15-BV786, CD14-BUV737, and CD10-BUV395 (BD, Le Pont-de-Claix, France); and CRTH2-FITC, CD123-PE, LOX-1-BV421, CD64-BV605, and PD-L1-BV711 (BioLegend, San Diego, CA, USA). One milliliter of BD FACS lysing solution IX (BD Biosciences) was directly added to the cells to lyse red blood cells, which were incubated for 20 min, centrifuged, and washed with PBS. Leukocytes were resuspended in PBS before analysis with a BD LSRFortessa X-20 (BD Biosciences). FlowJo software 10.0 (FlowJo LLC, Ashland, OR, USA) was used for analysis of marker expression on neutrophils. One hundred microliters of whole blood was stained with a fluorescence minus one (FMO) mix missing antibodies targeting CD 123, LOX-1, and PD-L1, in order to determine the threshold of expression of these markers. BAL leukocyte phenotyping was performed similarly after filtration, two wash procedures of BAL cells, and staining with the same antibody mix. Acquired data were normalized and analyzed using the OMIQ platform (https://www.omiq.ai). To identify neutrophil subsets and visualize all cells in a 2D map where position represents local phenotypic similarity, we used a dimensionality reduction tool: the opt-SNE implementation of t-SNE. Neutrophils (40 000 events) were randomly taken from the sample for the unsupervised analysis. Cells were also grouped in phenotypically homogeneous clusters using the FlowSOM algorithm.
ELISA for antimicrobial proteins
MPO and neutrophil ELA were measured using Human Myeloperoxidase and Human Neutrophil Elastase/ELA2 DuoSet ELISA kits (R&D Systems, Minneapolis, MN, USA), with plasma or BAL diluted to 1:100 with PBS according to manufacturer’s instructions. The concentrations of MPO and ELA were expressed as picograms/milliliter or relative luminescence units. Netosis was measured in the patient’s plasma by detecting MPO-DNA complexes using anti-human MPO primary antibody (clone 4A4; AbD Serotec, Marnes-la- Coquette, France) as the capture antibody and a peroxidase-labeled anti-DNA antibody (clone MCA-33; Roche, Mannheim, Germany) as the detection antibody. Plasma samples were diluted 1:4 in PBS.
Quanterix technology (digital ELISA)
The SimoaTM (single molecule array) HD-1 analyzer (Quanterix, Lexington, MA, USA) using singleplex bead-based assays was used for ultrasensitive immunodetection of IL- 3, IL-17A, IL-18, GM-CSF, and IFN-a. Concentrations of IL-Ib, IFN-g, IL-6, IL-8, IL-22, TNFa, and IL-10 were determined using a multiplex planar array immunoassay on the Quanterix SP-X platform according to manufacturer’s instructions. Serum IFN-b levels were quantified with a highly sensitive ELISA kit (PBL Assay Science, Piscataway, NJ, USA). The concentrations of cytokines in unknown samples were interpolated from a standard curve created with two replicates of each level of recombinant calibrator proteins representing the dynamic range of the assay: IL-Ib (0.073-300 pg/mL), IFN-g (0.012-50 pg/mL), IL-6 (0.073-300 pg/mL), IL-8 (0.098-400 pg/mL), IL-22 (0.024-100 pg/mL), TNFa (0.098-400 pg/mL), IL-10 (0.024-100 pg/mL), IL-3 (0.686-500 pg/mL), IL-17A (0.041-30 pg/mL), IL- 18 (0.011-45 pg/mL), GM-CSF (0.041-30 pg/mL), IFN-a (0.028-27.3 pg/mL), and IFN-b (1.2-150 pg/mL).
Data presentation and statistical analysis
Statistical analyses of the immunological data and graphic representations were performed with Prism 9 (GraphPad Software Inc.). A two-tailed Student’s t-test was used for group comparisons, and one-way and two-way ANOVA tests with a Bonferroni adjustment was used for multiple comparison tests. The association between variables was evaluated using Spearman’s correlation (one-tailed), with significance defined by a p-value of <0.05. Survival curves were compared with a log-rank (Mantel-Cox) test and were considered statistically significant with a p of <0.05. HR values with 95% Cl were computed. ROC curves were created using Prism 9. PC As were performed with R-software 3.3.1.
Results
Increased proportions of circulating immature neutrophils expressing either CD123 or LOX-1 in critical COVID-19 patients are associated with COVID-19 severity and thromboembolic complications.
We designed a first observational study with 38 individuals and analyzed their neutrophil phenotypes, comparing them with those of patients admitted to the ICU (n = 24) or not (n = 14), within the first day following their admission to the ICU or hospitalization units (see Table 2). COVID-19 patients from ICUs displayed more severe clinical and biological signs than non-ICU patients, with an elevated Simplified Acute Physiology Score (SAPS) II (ICU: 35.5, n = 24 and non-ICU: 25.5, n = 14; p-value = 0.05), higher serum lactate dehydrogenase (ICU: 504, n = 24 and non-ICU: 324, n = 14; p-value = 0.005), and higher D- dimers (ICU: 2760, n = 23 and non-ICU: 1860, n = 12; p-value = 0.25). However, they did not differ in neutrophil counts or levels of lymphopenia (Table 2). Patients’ characteristics confirmed previously published data, with a notably high prevalence of obese COVID-19 patients. The main differences between ICU and non-ICU patients reflected case severity, with a high proportion of patients with lung lesions (observed as ground-glass opacities (GGOs) on chest CT scans) requiring invasive mechanical ventilation (IMV) and resulting in high in-hospital mortality.
Within 3 h of drawing blood, we performed whole blood immunostaining using a previously published panel designed to precisely evaluate immature circulating neutrophils (17). Neutrophils were automatically identified and visualized using a dimensionality reduction tool (opt-SNE for optimal implementation of t-distributed stochastic neighbor embedding (t-SNE)) to define an imprint for each sample group (Figure 1A). Under unsupervised classification, neutrophils from ICU patients were organized in the upper left quadrant of the map, whereas those from the non-ICU patients ended up in the upper right quadrant. This analysis allowed delimitation of two main subsets of neutrophils based on the expression of CD 10 and CD64 markers: the ICU-abundant upper left area was composed of neutrophils with mid-to-low expression of CD 10 and high expression of CD64, and the non- ICU-abundant upper right area was composed of high-to-mid expression of CD 10 and high- to-mid expression of CD64. We next determined whether the identified neutrophil signature would be confirmed by conventional analysis undertaken by experts. Neutrophils were identified with the CD 15 neutrophil marker, while excluding prototypical markers of eosinophils and monocytes, respectively CRTH2 and CD14 (data not shown). An expert gating strategy confirmed the high abundance of CD10-CD64+ ImNs among ICU patients compared with non-ICU patients (Figure IB). Correlations between ImN markers and COVID severity were independent of age, obesity, and other potential confounding factors (data not shown). We next compared the expression of CD 123, LOX-1, and PD-L1 surface molecules, formerly known to be dysregulated in sepsis (17). All three of them were barely co-expressed on neutrophils (Figure 1C), which led to the identification of three distinct ImN subpopulations. Subsets of neutrophils expressing either CD123 or LOX-1 were more abundant in ICU than in non-ICU patients (Figure 4A), unlike those expressing PD-L1, for which overabundance did not reach statistical significance (p = 0.1). Principal component analysis (PCA) revealed that CD123-, LOX-1-, or PD-L1 -expressing ImNs contributed independently to the disease severity of patients as evaluated by SAPS II (data not shown). In addition, PCA confirmed that the disease severity in ICU patients was associated with the expression of CD123, LOX-1, and PD-L1 on ImNs and distinct patterns of cytokines (Figure 2B and Table 1). We identified three profiles: (a) patients with high LOX-1 -expressing ImN proportions and high IL-Ib, IL-6, IL-8, and TNFa serum levels; (b) patients with CD123 and PD-L1 expression and IL-18, IL-22, and IFNy secretion; and (c) patients with a lower severity score associated with high type-1 interferon levels. Thus, ImN subsets expressing either CD123, LOX-1, or PD-L1 may define specific profiles of severity associated with high levels of cytokines.
Because the mortality in COVID-19 cases is associated with the virally driven cytokine storm, especially in patients with comorbidities (1), we sought to correlate ImN subsets with severe symptoms such as ARDS (patients receiving intermittent mandatory ventilation (IMV)) or thrombosis (Figures 4B and 4C, respectively). The requirement for IMV or the occurrence of thromboembolic events was associated with higher proportions of ImN expressing CD123 or LOX-1, unlike those expressing PD-L1 (p = 0.1). No differences in the proportions of ImN subsets could be detected between discharged and deceased patients (data not shown). These data suggested that increased proportions of circulating ImNs expressing either CD 123 or LOX-1 in critical COVID-19 patients are associated with COVID-19 severity and thromboembolic complications.
Subsets of immature neutrophils expressing LOX-1 infiltrate lung.
A pulmonary immune environment during critical COVID-19 infection is one of the major features of disease complications. We thus sought neutrophil subsets in the bronchoalveolar lavages (BALs) when available and compared them to blood samples from patients and healthy donors (Figure 5). With a opt-SNE algorithm, BAL neutrophils were identified in the upper right quadrant of the map; blood neutrophils from COVID-19 patients were more central; and blood neutrophils from healthy donors (HD) were organized in the lower left quadrant of the map (Figure 5A). Automatic clustering using major (CD 15, CD 10, CD 16, CD64) and specific (CD 123, LOX-1, PD-L1) neutrophil markers split neutrophil signatures into positive and negative subpopulations for each marker (data not shown). This unsupervised analysis allowed the identification of nine clusters, representing three main subsets of neutrophils (data not shown): (a) the mature neutrophils (MatNs) with high expression of CD 15 and CD 10 and low expression of CD64; (b) the ImNs with high expression of CD 15 and CD64 and low expression of CD 10; and (c) the activated neutrophils (ActN) with high expression of CD15, CD10, and CD64. Expression of CD123, PD-L1, and LOX-1 was spotted on both ActN and ImN subsets. MatNs were abundant in healthy donor blood, and both ActNs and ImNs were abundant in the blood of COVID-19 patients (data not shown). If ImNs expressing LOX-1, PD-L1, or CD123 represented a few percent of COVID- 19 blood neutrophils (see also Figure 4A), these subsets were much more present in patient BALs with ImNs expressing LOX-1 being the major subset, representing about 40% of total neutrophils. A profusion of ImNs in COVID-19 BAL was associated with massive production of myeloperoxidase (MPO) and neutrophil elastase (ELA) (Figure 5B), two antimicrobial and cytotoxic proteins known to be highly concentrated in the azurophilic granule of ImN.
These data revealed that ImNs, preferentially those expressing LOX-1, infiltrate bronchoalveolar space in the lungs of COVID-19 patients, where they release their cytotoxic content, suggesting a potential role in disease severity.
Immature neutrophil subsets expressing CD123, LOX-1, or PD-L1 are correlated with clinical severity, but only the LOX-1+ subset proportion at entry is strongly associated with higher risk of thrombosis.
COVID-19 patients from the validation study were segregated into three groups based on severity of disease at the time of admission: 18 in mild, 19 in severe, and 51 in critical condition (Table 5 and data not shown). The proportion of CD123-, LOX-1-, and PD-L1- expressing ImNs correlated positively with severity (Figure 6A). Interestingly, abundances of all three ImN subsets were associated with patient death (Figure 6B) and with patients requiring IMV (data not shown), but only the LOX-1 -expressing ImN subset was associated with thromboembolic events (Figure 6C), confirming our results in the pilot study (see Figure 4C). We segregated our COVID-19 patients into two groups based on the median proportion of each ImN subset and compared their relative risk of ARDS, thrombosis, and death using a Cox proportional hazards model with other variables (age, gender, hypertension, obesity, and diabetes) (Figure 6D). Patients with a high abundance of ImN subsets were at higher risk of ARDS requiring IMV and of death. Hypertension and diabetes were risk factors of survival but not for ARDS. Again, only patients with a high abundance of LOX-1- expressing ImNs were at higher risk of thromboembolic complications (HR, 5.99; 95% Cl, 2.02-17.81; p = 0.007).
These data from the validation study confirmed that ImN subsets expressing either CD 123, PD-L1, or LOX-1 were associated with COVID-19 severity, but only LOX-1 expression remained associated with thromboembolic complications. Immature neutrophil subsets and plasma levels of MPO and ELA are independent markers of COVID-19 severity.
Because ImNs that recently emigrated from the bone marrow are enriched in granule antimicrobial, cytotoxic, and NET-forming proteins, we measured plasma levels of MPO, ELA, and MPO-DNA complexes representing NET formation in three groups of patients on the basis of severity at the time of admission: 23 in mild, 22 in severe, and 63 in critical condition. MPO and ELA plasma levels were significantly associated with disease severity (Figure 7A), whereas ELA-DNA complexes were not. In addition, MPO and ELA levels at hospital admission were also significantly increased among COVID-19 patients who later died (Figure 7B). MPO-DNA complex levels were not associated with survival. There was no association between MPO, ELA, or MPO-DNA complexes and thromboembolic events (Figure 7C). PCA (Figure 7D) that combined abundances of ImN subsets and plasma levels of neutrophil microbicidal proteins revealed at least two independent patient profiles at risk of severity: those with high proportions of ImN subsets and those with high plasma levels of MPO, ELA, and NET. To further evaluate the ability of LOX-1 neutrophil markers to segregate patients with thrombosis, we plotted a receiver operating characteristic (ROC) curve (Figure 7E). The ROC analysis of these abundances indicated the optimal threshold yielding the best separation of the two groups of patients with optimal sensitivity and specificity. The AUC was 0.89 for LOX-1 ImN abundance (p < 0.0001), indicating that LOX-1 expression on ImN in the blood at the time of hospital admission could accurately predict later thromboembolic events among COVID-19 patients during hospitalization. A cutoff point of 0.5% abundance of the LOX-1 ImN subset was able to detect patients with thrombotic events with a sensitivity of 100% and patients without complications with a specificity of 53%. A cutoff point of 2% reached a sensitivity of 82% and a specificity of 75%.
Discussion:
Stratification of patients using biomarkers remains an unmet need in COVID-19 patients care. Here, we sought to identify innate immune cellular signatures that may help predict the outcome of COVID-19 patients with severe symptoms. Conventional whole blood flow cytometry identified classical hallmarks of severe infections, such as neutrophilia and myelemia, but also revealed two novel neutrophils subsets able to discriminate patients requiring ICU or not. Both LOX-1- and CD123- expressing CD10 CD64+ neutrophil subsets strongly correlated with SAPS II and SOFA severity scores, commonly used in clinical practice for sepsis prognosis. They were also associated with distinct cytokines profiles. Most patients with a high proportion LOX-1 neutrophil subset developed thromboembolic events.
Our data indicated that patients’ severity could be predicted based on the proportion of immature CD10 CD64+ neutrophils using both unsupervised and expert-gating strategies. This neutrophil subset has been described as an immature subset (20) unlike the CD64+-activated neutrophils which still express the neutral endopeptidase (CD 10) and the low-affinity immuno-globulin-Fc fragment III (CD16). Numerous studies have shown an association between circulating immature neutrophils and bacterial sepsis (21). Here, we provided evidence that this immature subset may serve as a severity biomarker in COVID-19.
We recently reported that the proportion of CD 123 -expressing immature neutrophils correlated with bacterial sepsis severity (17). Here, we showed that the expression of CD123 on CD10 CD64+ neutrophils was related to a higher SOFA score among critically-ill COVID- 19 patients. Indeed, both CD123 (the alpha chain of the Interleukin-3 receptor) and its cognate ligand, the IL-3 cytokine, were suggested to play an important role in sepsis. Recent studies demonstrated that a high IL-3 plasma levels were associated with lung inflammation, lung injury and high mortality rates in an animal model, but also in humans (Weber 2015, Tong 2020). In addition, these studies showed that IL3 neutralization and anti-CD 123 treatment improved mice outcome by decreasing inflammation, and decreased mortality rates (Weber et ak, 2015). IL-3 promotes emergency myelopoiesis, exacerbating pro-inflammatory cytokines secretion and, consequently, systemic inflammation, organ dysfunction and death. The authors further tested the prognostic value of IL-3 in two small cohorts of humans with sepsis and found that high plasma IL-3 levels were associated with high mortality even when adjusting for disease severity. However, we did not observe similar results in COVID-19, as we report an inverse correlation between IL-3 levels and SOFA score. Because IL-3 is mainly produced by activated T-lymphocytes, severe lymphopenia observed in critically-ill COVID- 19 patients may limit the IL-3 T cell production.
The association between CD123 expression on immature neutrophils and high serum levels of IL-17, IL-22 and IFNy was, to the best of our knowledge, never reported and may reveal a yet unidentified link between innate and adaptive immune responses. These findings open the way to new therapeutic opportunities aiming to control the excessive inflammation induced by SARS-CoV-2 infection. The evaluation of CD 123 expression on CD10 CD64+ immature neutrophils could also be a helpful predictor of COVID-19 severity.
The impact of LOX-1 deletion was previously evaluated in a murine model of polymicrobial sepsis, resulting in the reduction of IL-6 and TNFa levels in blood and lungs, enhancing bacterial clearance and preventing neutrophils activation (19). More recently, LOX-1 was identified as a marker on granulocytic myeloid-derived suppressor cells able to suppress T cell activity (18). However, LOX-1 is mostly acknowledged for its role in atherosclerosis. LOX-1 is a class E scavenger receptor contributing to the formation of atherosclerotic plaques by promoting endothelial cell activation, macrophage foam cell formation, and smooth muscle cells migration and proliferation (24). LOX-1 activation induces NFKB activation leading to pro-inflammatory cytokines release, endoplasmic reticulum stress, and reactive oxygen species (ROS) production which could damage the microenvironment (25, 26).
However, LOX-1 role on neutrophils remains elusive. LOX-1 is barely detected on neutrophils at homeostasis, while its expression increases on neutrophils from human cancer patients (18) and in murine sepsis (19, 27).
In this study, LOX-1 expression on neutrophils seems to be detrimental for patients as it was associated with the secretion of several pro-inflammatory cytokines, such as IL-6, IL- 1b and TNFa, and with severity (as assessed by the SOFA score) and thrombosis. In severe cases of COVID-19, the integrity of the lung is compromised by an exaggerated immune response leading to acute respiratory distress syndrome (10, 16). Mechanisms contributing to microcirculation disorders in sepsis are capillary leakage, leukocytes adhesion and infiltration and intravascular coagulation, leading to thrombus formation. Over the course of systemic inflammatory diseases such as sepsis, the microenvironment is highly oxidative, leading notably to an increase of oxidized low-density lipoprotein (oxLDL) in plasma, which triggers LOX-1 overexpression through a positive feedback loop. In physiological conditions, the increase of LOX-1 expression, especially by endothelial cells, leads to an increase of LDL uptake into vessel wall which activates the specific Oct-l/SIRT-1 thrombosis protective pathway (28). The activation of SIRT1 is able to supress the NFkB-induced expression of tissue factor, also known as thromboplastin, a key initiator of the coagulation cascade involved in thrombus formation (29). In this study, we observed an increase of the incidence of vascular thrombotic events among individuals displaying a high frequency of immature LOX-l+ neutrophils. It remains to be seen whether thrombosis in COVID-19 patients results from functionally-diverted neutrophils expressing LOX-1 and/or from its expression on endothelial and smooth muscle cells.
Additionally, we observed a slight correlation between LOX-1 expressing immature neutrophils and the concentration of D-Dimers (spearman test, r=0.42 p= 0.023). D-dimers are used to determine the risk of venous thromboembolism. However, in our study, the predictive score of D-dimers for thromboembolic events was not significant (ROC test, AUC=0.64, p=ns) compared to LOX-1 expressing neutrophil abundancy in the blood (AUC=0.977, p < 0.0001). These results suggest that the high LOX-1 expression by immature neutrophils might be sufficient to predict thromboembolic events among critically-ill COVID-19 patients. The overexpression of LOX-1 might also be found in other cell types that might trigger the prothrombotic ERK1/2 pathway. Further investigations would be necessary, such as the titration of oxLDL in blood or the evaluation of the ERK1/2 pathway. In addition, some studies support the relationship between ACE/ACE2 axis and the expression of the pro- oxidative molecule LOX-1, which could increase the oxidative stress favoring prothrombotic state (30). SARS-CoV-2 virus requires binding to ACE2 and is particularly deleterious to patients with underlying cardiovascular disease (31). The polymorphic LOX-1 gene is also intensively associated with increased susceptibility to myocardial diseases. LOX-1 should be thus considered a potential target for therapeutic intervention.
In conclusion, we outline two potential biomarkers of COVID-19 severity measurable among immature neutrophils, the CD123 and LOX-1 surface markers. These markers are significantly correlated with disease severity in general, and more particularly to thromboembolic events.
Figure imgf000052_0001
The SimoaTM (single molecule array) HD-1 analyser was used for ultrasensitive multiplex immunodetection of cytokines as describe in methods section. The potential association between serum cytokine levels or marker expressing neutrophils frequencies was evaluated by Spearman correlation (one-tailed), with significance defined by a p-value < 0.05:
* for p < 0.05; ** for p < 0.01; *** for p < 0.001; **** for p < 0.0001.
Figure imgf000053_0001
median (quartiles) [normal range: 1.5 - 4] - 1.34) - 2) - 1.81) Lactate dehydrogenase, U/L, 475.5 (234 504 (375 - 324 (234 - median (range) [normal range: 135-215] - 2030) 1087) 2030)
Ddimer, ng/mL, median (range) 2450 (540 2760 (540 1860 (540 - 20000) 20000) 20000)
Figure imgf000054_0001
Values are expressed as n (%), unless stated otherwise. including cardiac, liver or kidney allograft, hematopoietic stem cell transplantation, or immunosuppressive agent for auto-immune disease o 28 patients were assessed
† As of June 8th, 2020
CT, computed tomography; GGO, ground-glass opacities; SAPS II, Simplified Acute Physiology Score II; SOFA score, Sequential organ failure assessment score
Figure imgf000055_0001
Table 4: Useful nucleotide and amino acid sequences for practicing the invention
Figure imgf000056_0001
Figure imgf000057_0001
Figure imgf000057_0002
Figure imgf000058_0001
Figure imgf000059_0001
>75% 6(5.1) 6(8.7) 0(0)
Figure imgf000060_0001
Arterial 0 (0) 0 (0) 0(0)
Figure imgf000061_0001
REFERENCES:
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Claims

CLAIMS:
1. An in vitro method for assessing a subject’s risk of having or developing severe or critical form of coronavirus infection, comprising the steps of i) determining in a sample obtained from the subject the level of neutrophil cells having cell surface expression of CD66b+CD10-CD16- CD64+CD123+ markers and/or
CD66b+CD10-CD16-CD64+LOX-l+ markers, ii) comparing the level determined in step i) with a reference value and iii) concluding when the level of neutrophil having cell surface expression of CD10-CD16-CD64+CD123+ markers and/or CD66b+CD10-CD64+LOXl+ markers determined at step i) is higher than the reference value is predictive of a high risk of having or developing severe or critical form of coronavirus infection.
2. The in vitro method according to claim 1, wherein the sample is a blood sample or immune primary cells.
3. The in vitro method according to claim 2, wherein the immune primary cells are selected from the group consisting of PBMC, WBC or neutrophils.
4. An in vitro method for monitoring a coronavirus infection comprising the steps of i) determining the level of a population of neutrophils having cell surface expression of CD66b+CD10-CD16-CD64+CD123+ markers and/or CD66b+CD10-CD64+LOXl+ markers in a sample obtained from the subject at a first specific time of the disease, ii) determining the level of a population of neutrophils having cell surface expression of CD66b+CD10-CD16- CD64+CD123+ markers and/or CD66b+CD10-CD16-CD64+LOX-l+ markers in a sample obtained from the subject at a second specific time of the disease, iii) comparing the level determined at step i) with the level determined at step ii) and iv) concluding that the disease has evolved in worse manner when the level determined at step ii) is higher than the level determined at step i).
5. An in vitro method for monitoring the treatment of coronavirus infection comprising the steps of i) determining the level of a population of neutrophils having cell surface expression of CD66+CD10-CD16-CD64+CD123+ and/or CD66+CD10-CD64+LOX-1+ in a sample obtained from the subject before the treatment, ii) determining the level of a population of neutrophils having cell surface expression of CD66b+CD10-CD16-CD64+CD123+ markers and/or CD66b+CD10-CD16-CD64+LOX-l+ markers in a sample obtained from the subject after the treatment”, iii) comparing the level determined at step i) with the level determined at step ii) and iv) concluding that the treatment is efficient when the level determined at step ii) is lower than the level determined at step i).
6. An in vitro method for assessing a covid patient’s risk of having or developing thrombosis comprising the steps of i) determining the level of neutrophil cells having cell surface expression of CD66b+CD10-CD16-CD64+LOX-l+ markers in a sample obtained from the subject, ii) comparing the level determined at step i) with the with a reference value and iv) concluding that:
- when the level of neutrophil having cell surface expression of CD66b+CD10-
CD16-CD64+LOX-1+ markers determined at step i) is higher than the reference value, then said covid patient is at high risk of having or developing thrombosis; or
- when the level of neutrophil having cell surface expression of CD66b+CD10-
CD16-CD64+LOX-1+ markers determined at step i) is lower or equal than the reference value, then said covid patient is at low risk of having or developing thrombosis or not having a thrombosis.
7. The in vitro method according to any one of claim 4 to 6, wherein the sample is a blood sample or immune primary cells.
8. The in vitro method according to any one of claim 4 to 6, wherein the immune primary cells selected from the group consisting of PBMC, WBC or neutrophils.
9. The in vitro method according to any one of claim 1 and 4 to 6, wherein coronavirus infection is the Middle East respiratory syndrome-related coronavirus (MERS-CoV), the Severe Acute Respiratory (SARS-CoV) or the Severe Acute Respiratory 2 (SARS-CoV-2) infection.
10. A LOX-1 (lectin-type oxidized LDL receptor 1) inhibitor for use in the prevention or the treatment of a coronavirus infection in a subject in need thereof.
11. The LOX-1 inhibitor for use according to claim 10, wherein coronavirus infection is the Middle East respiratory syndrome-related coronavirus (MERS- CoV), the Severe Acute Respiratory (SARS-CoV) or the Severe Acute Respiratory 2 (SARS-CoV-2) infection.
12. The LOX-1 inhibitor for use according to any one of claim 10 or 11 wherein the inhibitors is selected from a peptide, a peptide mimetic, a small organic molecule, an antibody, an aptamer, a phospholipid, and a polynucleotide.
13. The LOX-1 inhibitor for use according to any one of claim 10 to 12, wherein the level of a population of neutrophils CD66b+CD10-CD16-CD64+CD123+ and/or CD66b+CD10-CD16-CD64+LOX-l+ obtained from said patient, have been detected by one of the methods of claim 1 or claim 4 to 6.
14. A method for treating coronavirus infection with anticoagulant in a subject wherein the level of a population of neutrophils CD66b+CD10-CD16- CD64+CD123+ and/or CD66b+CD10-CD16-CD64+LOX-l+ obtained from said patient, have been detected by one of the methods of claim 1 or claim 4 to 6.
15. A method of treating coronavirus infection in a subject comprising the steps of: a) providing a sample containing neutrophil from a subject, b) detecting the level of a population of neutrophils CD66b+CD10-CD16- CD64+CD123+ and/or CD66b+CD10-CD16-CD64+LOX-l+ c) comparing the level determined at stet b) with a reference value and if level determined at step b) is higher than the reference value, treating the subject with anticoagulant.
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