WO2022084300A1 - Methods for diagnosis and monitoring form of coronavirus infection - Google Patents

Abstract

During COVID-19, dysregulated immune response is the key factor leading to unfavorable outcome. Depending on the pathogen-associated-molecular-pattern, the NLRP3 inflammasome can play a crucial role during innate immunity activation. To determine Caspase-1 activation levels in multiple blood myeloid cells, the inventors used the FAM- FLICA® Caspase-1 Assay together with CD45, CD14, CD66b and CD16 cell markers in the whole blood of patients. Caspase-1 activation in CD66b+CD16dim cells was lower in severe and critical presentation of COVID-19 as well as the number of cell having cell surface expression of CD14dim CD16+ markers. Accordingly, the present invention relates to method for assessing a subject's risk of having or developing a mild, severe or critical coronavirus infection, comprising the steps of i) determining in a sample obtained from the subject the level of caspase-1 activation in myeloid cells having cell surface expression of CD66b+ CD16dim markers and/or the number of cell having cell surface expression of CD14dim CD16+ markers, ii) comparing the level and/or the number determined in step i) with a reference value and iii) concluding when the level of caspase-1 activation in myeloid cells having cell surface expression of CD66b+ CD16dim markers and/or the number of cell having cell surface expression of CD14dim CD16+ markers determined at step i) is lower than the reference value is predictive of a high risk of having or developing severe or critical form of coronavirus infection.

Description

METHODS FOR DIAGNOSIS AND MONITORING FORM OF

CORONAVIRUS INFECTION

FIELD OF THE INVENTION:

The present invention relates to methods and kits for prognostic and monitoring the form of coronavirus infection. More specifically present invention relates to methods for prognosis form of coronavirus infection through detection of a specific population of myeloid cell in a patient.

BACKGROUND OF THE INVENTION:

SARS-CoV-2 is a novel human coronavirus which emerged in December 2019 in Wuhan, China (1). The virus is responsible for a contagious respiratory illness named COVID- 19 (COronaVIrus Disease-19) that can evolve in a life-threatening Severe Acute Respiratory Syndrome (SARS) in some cases (2). However, some patients infected by SARS-CoV-2 suffer from mild COVID-19 conditions, reporting only slight cough and low-grade fever, and even cases of asymptomatic carriers have been reported (2). As for most viral infections, it is very likely that the outcome of the infection is mainly governed by the interplay between virus and host antiviral immunity (3, 4).

Innate immunity is the first line of defense against pathogen invasion in naive patients. It plays an essential role in restricting viral replication and activating adaptive immunity during the first stages of infection. Innate immune defects have been involved in susceptibility to infection while activating mutations can cause auto-inflammatory diseases (5). Both innate and adaptive immunity work as a continuum that starts by an efficient detection of the pathogen by the innate immune system (6). The innate immune detection system of viruses relies on Pattern Recognition Receptors (PRRs). PRRs are conserved proteins able to sense Pathogen- Associated-Molecular-Patterns (PAMPS) that are specific to microbes (7). Viral nucleic acids as well as viral proteins have been shown to interplay with PRR (8). Among the PPR involved in virus detection, Toll-like receptors (TLRs), Nod-like receptors (NLRs), RIG-I-like receptors (RLRs) as well as the cGAS-STING pathway are critical for the antiviral response. Indeed, TLRs, RLRs and cGAS-STING stimulation triggers the activation of the NF-kB transcription factor as well as Interferon Responsive Factors (IRFs) leading to the production of proinflammatory cytokines and type I interferons; while inflammasomes control the maturation of the Interleukine-ip (IL-ip) and IL- 18 cytokines (9). Among inflammasomes, the NLRP3 inflammasome is the most extensively studied. The NLRP3 inflammasome is activated by microbial or endogenous triggers respectively PAMPs and Damage Associated Molecular Patterns (DAMPs). Among these triggers, Nigericin is a bacterial pore forming toxin widely used as a specific activator of the NLRP3 inflammasome. The stimulation of NLRP3 by Nigericin results in the assembly of the inflammasome through the recruitment of the adaptor protein ASC (apoptosis-associated speck-like protein containing CARD) and the recruitment of the Caspase- 1 that has a proteolytic activity and allows the maturation of pro-IL-ip into active IL-ip.

Recent studies suggest that the death of COVID-19 patients with no medical history can be attributed to a cytokine storm that is similar to what is observed during sepsis with excessive plasma IL-6 and IL-ip levels (10-12). Recent reports have suggested a potential role of NLRP3 inflammasome during the COVID-19 cytokine storm but clinical evidence of the NLRP3 inflammasome involvement during COVID-19 is still lacking (13-18). Furthermore, clinical trials have been designed to dampen either the NLRP3 inflammasome or the IL-ip cytokine dependent inflammation, but predictive biological markers allowing the determination of the therapeutic window for these treatments are lacking.

Accordingly, there remains an unmet need in the art for specific and more rapid prognostic test for state of Covid patient. To address this point, the inventors designed an assay to monitor the NLRP3 -triggered Caspase- 1 activation in the whole blood of CO VID-19 patients. Here, the inventors used this assay to determine the innate immune status of patients and identify myeloid cells activation profiles that could be used as a marker to stratify patients during COVID-19.

SUMMARY OF THE INVENTION:

The present invention relates to methods and kits for prognostic and monitoring the form of coronavirus infection. More specifically present invention relates to methods for prognosis form of coronavirus infection through detection of a specific population of myeloid cell in a patient. In particular, the invention is claimed by the claims.

DETAILED DESCRIPTION OF THE INVENTION:

During COVID-19, dysregulated immune response is the key factor leading to unfavorable outcome. Depending on the pathogen-associated-molecular-pattem, the NLRP3 inflammasome can play a crucial role during innate immunity activation. To date, studies describing NLRP3 response to SARS-CoV-2 infection in patients are lacking. Patients with mild to critical COVID-19 were prospectively included. To determine Caspase-1 activation levels in multiple blood myeloid cells, the inventors used the FAM- FLICA® Caspase-1 Assay together with CD45, CD14, CD66b and CD16 cell markers in the whole blood of patients. Basal activation and response to Nigericin stimulation were analyzed by flow cytometry and compared between the different severity of illness during infection and after recovery. Blood samples from healthy donors were used for comparison and characterization purposes. Circulating myeloid cells from 15 COVID-19 patients and 11 healthy donors were analyzed. CD14dimCD16+ monocytes were measured with a higher level of Caspase-1 activation during SARS-CoV-2 infection. Caspase-1 activation in CD66b+CD16dim cells was lower in severe and critical presentation of COVID-19. CD66b+CD16dim cells of critical patients showed a 3-fold decreased of basal activation of Caspase-1 relative to controls and mild COVID-19 patients. In CD66b+CD16dim cells the Nigericin-triggered Caspase- 1 activation potential was decreased specifically in severe and critical patients. In patients who recovered from COVID-19, Nigericin-triggered Caspase-1 activation potential in CD66b+CD16dim cells was restored. Those results reveal that both basal and triggered NLRP3 inflammasome activation differs among myeloid cells. This assay could be a useful tool to categorize disease severity and help in therapeutic decision involving immunomodulatory drugs.

Prognostic methods according to the invention

The present invention relates to a method for assessing a subject’s risk of having or developing a mild, severe or critical coronavirus infection, comprising the steps of i) determining in a sample obtained from the subject the level of caspase- 1 activation in myeloid cells having cell surface expression of CD66b+ CD16dim markers and/or the number of cell having cell surface expression of CD14dim CD16+ markers, ii) comparing the level and/or the number determined in step i) with a reference value and iii) concluding when the level of caspase-1 activation in myeloid cells having cell surface expression of CD66b+ CD16dim markers and/or the number of cell having cell surface expression of CD14dim CD16+ markers determined at step i) is lower than the reference value is predictive of a high risk of having or developing severe or critical form of coronavirus infection.

In particular, the present invention relates to a method for assessing a subject’s risk of having or developing a mild, severe or critical coronavirus infection, comprising the steps of i) determining in a sample obtained from the subject the level of caspase- 1 activation in myeloid cells having cell surface expression of CD66b+ CD16dim markers, ii) comparing the level determined in step i) with a reference value and iii) concluding when the level of caspase- 1 activation in myeloid cells having cell surface expression of CD66b+ CD16dim markers determined at step i) is lower than the reference value is predictive of a high risk of having or developing severe or critical form of coronavirus infection.

In particular, the present invention relates to a method for assessing a subject’s risk of having or developing a mild, severe or critical coronavirus infection, comprising the steps of i) determining in a sample obtained from the subject the number of cell having cell surface expression of CD14dim CD 16+ markers, ii) comparing the number determined in step i) with a reference value and iii) concluding when the number of cell having cell surface expression of CD14dim CD16+ markers determined at step i) is lower 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 a prognosis method of having or developing coronavirus infection in a subject, comprising the steps of i) determining in a sample obtained from the subject the level of caspase-1 activation in myeloid cells having cell surface expression of CD66b+ CD16dim markers and/or the number of cell having cell surface expression of CD14dim CD 16+ markers, ii) comparing the level and/or the number determined in step i) with a reference value and iii) concluding when the level of caspase- 1 activation in myeloid cells having cell surface expression of CD66b+ CD16dim markers and/or the number of cell having cell surface expression of CD14dim CD16+ markers determined at step i) is lower than the reference value is predictive of having or developing severe or critical form of coronavirus infection.

In particular, the present invention relates to a prognosis method of having or developing coronavirus infection in a subject, comprising the steps of i) determining in a sample obtained from the subject the level of caspase-1 activation in myeloid cells having cell surface expression of CD66b+ CD16dim markers, ii) comparing the level determined in step i) with a reference value and iii) concluding when the level of caspase-1 activation in myeloid cells having cell surface expression of CD66b+ CD16dim markers determined at step i) is lower than the reference value is predictive of having or developing severe or critical form of coronavirus infection.

In particular, the present invention relates to a prognosis method of having or developing coronavirus infection in a subject, comprising the steps of i) determining in a sample obtained from the subject the number of cell having cell surface expression of CD14dim CD 16+ markers, ii) comparing the number determined in step i) with a reference value and iii) concluding when the number of cell having cell surface expression of CD14dim CD 16+ markers determined at step i) is lower than the reference value is predictive of having or developing severe or critical form of coronavirus infection.

As used herein, the term “NLRP3” refers to Nucleotide-binding oligomerization domain-like receptor including a pyrin domain 3. Nucleotide-binding oligomerization domainlike receptors ("NLRs") include a family of intracellular receptors that detects pathogen- associated molecular patterns ("PAMPs") and endogenous signal danger molecules. NLRPs represent a subfamily of NLRs that include a Pyrin domain and are constituted by proteins such as NLRP1, NLRP3, NLR.P4, NLRP6, NLRP7, and NLRP12. NLRPs are involved in the formation of multiprotein complexes termed inflammasomes. The NLRP3 inflammasome forms a molecular platform inside macrophages and microglial cells, catalyzing the activation of the protease Caspase-1. Caspase-1 is responsible for converting the potent pro-inflammatory cytokine interleukin-1 beta (IL- 1 P) from an inactive to an active secreted form.

In some embodiment, the level of caspase-1 activation in myeloid cells having cell surface expression of CD66b+ CD16dim markers is measured.

In some embodiment, the number of cell having cell surface expression of CD14dim CD 16+ markers (non classical monocytes) is measured.

As used herein, the term “nigericin” has its general meaning in the art and refers to a bacterial pore forming toxin widely used as a specific activator of the NLRP3 inflammasome. The stimulation of NLRP3 by Nigericin results in the assembly of the inflammasome through the recruitment of the adaptor protein ASC (apoptosis-associated speck-like protein containing CARD) and the recruitment of the Caspase- 1 that has a proteolytic activity and allows the maturation of pro-IL-ip into active IL-ip.

As used herein, the term “prognosis” refers to 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 the number of cell having cell surface expression of CD14dim CD 16+ markers (non classical monocytes) and/or the level of caspase-1 activation in myeloid cells (e.g. granulocytes) having cell surface expression of CD66b+CD16dim markers which in turn may be a risk for developing severe and critical form of coronavirus infection.

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 a human infant. In some embodiments, the subject is a human child. In some embodiments, the subject is a human adult. In some embodiments, the subject is an elderly human. In some embodiments, the subject is a premature human infant. In some embodiments, the subject is a pregnant women. In some embodiments, the subject is a fetus. 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 “subject” encompasses the term "patient”.

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 singlestranded RNA virus. It is contagious in humans, and the World Health Organization (WHO) has designated the ongoing pandemic of CO VID-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 some embodiments, the subject of the present invention suffers from COVID-19.

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 viral infection (e.g. coronavirus). As used herein, the term "symptomatic" refers to a subject who experiences detectable symptoms of a pathogen 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 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. In some embodiment, the level of caspase-1 activation in multiple blood myeloid cells is determined by the FAM-FLICA® Caspase-1 Assay together with CD45, CD14, CD66b and CD 16 cell markers in the whole blood of patients.

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 pronostic method of the 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 myeloid cells can be identified).

As used herein, the term “the myeloid lineage” as its general meaning in the art and relates to the lineage of white blood cells: that is meaning monocytes (macrophages, dendritic cells and osteoclasts) and granulocytes (neutrophils, basophils and eosinophils), including of course all of their immature bone marrow precursors.

In some embodiment, the myeloid cells of the present invention are selected from the group consisting of granulocytes or monocytes.

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.

As used herein, the term “immune primary cell” has its general meaning in the art and is intended to describe a population of white blood cells (WBC) directly obtained from a subject.

In some embodiment, immune primary cell is selected from the group consisting of peripheral blood mononuclear cells (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.

As used herein, the term “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-Pemer AM et al. (1998) Blood. ;91(2):663-72).

In the context of the method of the invention “CD66b-” means that the cell surface marker is not expressed on myeloid cell. In the context of the method of the invention “CD66b+” means that the cell surface marker is expressed on myeloid cell.

As used herein, the term "CD16" 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, granulocytes, monocytes, and macrophages CD 16 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, CD16 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 myeloid cell. In the context of the method of the invention “CD16+” means that the cell surface marker is expressed on myeloid cell. In the context of the method of the invention “CD16dim” means that the cell surface marker is dim on myeloid cell.

In some embodiment, the level of caspase-1 activation in myeloid cells having cell surface expression of CD45+CD14+ markers is measured. In a particular embodiment, the level of caspase- 1 activation in monocytes having cell surface expression of CD45+CD14+ markers is measured. In some embodiment, caspase-1 activation in three populations of monocytes are measured: classical monocytes (CD45+CD14highCD16-), intermediate monocytes (CD45+CD14highCD16+) and non-classical monocytes (CD45+CD14dimCD16+).

As used herein the term “CD45” has its general meaning in the art and refers to the protein tyrosine phosphatase (PTP) encoded by the PTPRC gene, which is specifically expressed in hematopoietic cells ¹⁰. CD45 regulates receptor signalling by direct interaction with components of the receptor complexes or by activating and dephosphorylating various Src family kinases (SFK) i.e. Lek 12. But it can inhibit cytokine receptor signalling by inhibiting JAK kinases or by dephosphorylating the activating residues of Src ¹². Typically it is possible to distinguish two isoforms of CD45: CD45RA and CD45RO. In the context of the method of the invention “CD45-” means that the cell surface marker is not expressed on myeloid cell. In the context of the method of the invention “CD45+” means that the cell surface marker is expressed on myeloid cell.

As used herein, the term “CD14” has its general meaning in the art and refers to the a glycolipid-anchored membrane glycoprotein expressed on cells of the myelomonocyte lineage including monocytes, macrophages, and some granulocytes. CD14 is a key molecule in the activation of innate immune cells and exists as a membrane-anchored or soluble form. In the context of the method of the invention “CD14-” or “CD14dim” means that the cell surface marker is not expressed on myeloid cell. In the context of the method of the invention “CD14+” or “CD14high” means that the cell surface marker is expressed on myeloid cell.

Standard methods for detecting the expression of a specific surface marker such as CD66b or CD16 at cell surface (e.g. granulocyte surface) or CD14 or CD45 or CD16 at cell surface (e.g. monocytes surface) are well known in the art. Typically, the step consisting of detecting the surface expression or the absence of the surface expression of a surface marker (e;g. CD66b 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 CD66b of the invention is the anti-CD66b antibody available from Biolegend (CD66b Monoclonal Antibody (G10F5), # 355005) or from Fluidigm (Anti-Human CD66b (80H3) (#3162023) or from Miltenyi Biotec (CD66b-PEVIo770 (clone REA306)).

For example, the binding partner of CD16 of the invention is the anti-CD16 antibody available from Biolegend (CD16 (IgG receptor III (FcyRIII)) Monoclonal Antibody (#302001) or from Fluidigm (Anti-Human CD16 (3G8) (# 3145008B) or from Miltenyi Biotec (CD16-PE (clone REA423)).

For example, the binding partner of CD45 of the invention is the anti-CD45 antibody available from Miltenyi Biotec (CD45-VIoGreen (clone REA747)).

For example, the binding partner of CD14 of the invention is the anti-CD14 antibody available from Miltenyi Biotec CD14-APC-VIo770 (clone REA599).

In another embodiment, the binding partners may be aptamers. 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. The random sequence library is obtainable by combinatorial chemical synthesis of DNA or RNA. In this library, each member is a linear oligomer, eventually chemically modified, of a unique sequence. Peptide aptamers consist of conformationally constrained antibody variable regions displayed by a platform protein, such as E. coli Thioredoxin A that are selected from combinatorial libraries by two hybrid methods.

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 (Cy 5)]) 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 (ie. 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, CD16, CD45 and CD14). 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, CD 16, CD45 and CD 14 coupled with distinct fluorochromes (or immunobeads coated with anti-CD66b, anti CD 16 antibodies, anti CD45 antibodies and anti CD 14 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 and/or CD 16 and/or CD45 and CD14 surface markers. Once the sample from the subject is prepared, the level of covid (severe and critical form) biomarkers CD66b+CD16dim or CD14dimCD16+ may be measured by any known method in the art.

Typically, the high or low level of caspase- 1 activation in myeloid cell surface biomarkers biomarkers CD66b+CD16dim CD14dimCD16+ is intended by comparison to a control reference value. Typically, the high or low level of the number of cell having cell surface expression of CD14dim CD16+ markers 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 caspase- 1 activation in myeloid cell biomarkers CD66b+CD16dim to a control reference value wherein a low level of caspase- 1 activation in myeloid cell biomarkers CD66b+CD16dim compared to said control reference value is predictive of a high risk of having a critical form of coronavirus infection and a high level of caspase-1 activation in myeloid cell biomarkers CD66b+CD16dim 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, the method according to the present invention comprises the step of comparing said number of cell having cell surface expression of CD14dim CD 16+ markers to a control reference value wherein a low level of the number of cell having cell surface expression of CD14dim CD16+ markers compared to said control reference value is predictive of a high risk of having a critical form of coronavirus infection and a high level of the number of cell having cell surface expression of CD14dim CD 16+ markers compared to said control reference value is predictive of a low risk of having or developing a critical form of coronavirus infection.

In some embodiment, if the percentage of FAM-FLIC A positive CD66b+-CD16dim cells is inferior to 20% (control reference) after Nigericin stimulation is predictive of having or a high risk of having or developing a severe form of coronavirus infection.

In some embodiment, if the percentage of FAM-FLIC A positive CD66b+-CD16dim cells is inferior to 5% (control reference) after Nigericin stimulation is predictive of having or a high risk of having or developing 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 caspase- 1 activation in myeloid cell biomarkers (CD66b+CD16dim) and/or the number of cell having cell surface expression of CD14dim CD16+ markers with a defined threshold value. In one embodiment of the present invention, the threshold value is derived from the myeloid cell 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 myeloid cell 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 myeloid cell 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 myeloid cell 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 symptomatic form of COVID-19 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 diagnosis of CO VID-19 from a biological sample. In particular, the method of detection of the invention is consequently useful for the diagnosis of early stage COVID-19 from a biological sample.

Monitorins method

An additional object of the invention relates to a method for monitoring a coronavirus infection comprising the steps of i) determining the level of caspase-1 activation in a population of myeloid cells having cell surface expression of CD66b+CD16dim markers and/or the number of cell having cell surface expression of CD14dim CD 16+ markers in a sample obtained from the subject at a first specific time of the disease, ii) determining the level of caspase-1 activation in a population of myeloid cells having cell surface expression of CD66b+CD16dim markers and/or the number of cell having cell surface expression of CD14dim CD16+ markers in a sample obtained from the subject at a second specific time of the disease, iii) comparing the level and/or the number 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 and/or the number determined at step ii) is lower than the level determined at step i).

In particular, the invention relates to a method for monitoring a coronavirus infection comprising the steps of i) determining the level of caspase-1 activation in a population of myeloid cells having cell surface expression of CD66b+CD16dim markers in a sample obtained from the subject at a first specific time of the disease, ii) determining the level of caspase-1 activation in a population of myeloid cells having cell surface expression of CD66b+CD16dim markers in a sample obtained from the subject at a second specific time of the disease, iii) comparing the level and/or the number 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 lower than the level determined at step i).

In particular, the invention relates to a method for monitoring a coronavirus infection comprising the steps of i) determining the number of cell having cell surface expression of CD14dim CD 16+ markers in a sample obtained from the subject at a first specific time of the disease, ii) determining the number of cell having cell surface expression of CD14dim CD16+ markers in a sample obtained from the subject at a second specific time of the disease, iii) comparing the number determined at step i) with the number determined at step ii) and iv) concluding that the disease has evolved in worse

An additional object of the invention relates to a method for monitoring the treatment of a coronavirus infection comprising the steps of i) determining the level of caspase- 1 activation in a population of myeloid cells having cell surface expression of CD66b+CD16dim markers and/or the number of cell having cell surface expression of CD14dim CD16+ markers in a sample obtained from the subject before the treatment, ii) determining the level of caspase- 1 activation in a population of myeloid cells having cell surface expression of CD66b+ CD16dim markers and/or the number of cell having cell surface expression of CD14dim CD16+ markers in a sample obtained from the subject after the treatment, iii) comparing the level and/or the number determined at step i) with the level and/or the number determined at step ii) and iv) concluding that the treatment is efficient when the level and/or the number determined at step ii) is higher than the level determined at step i).

In particular, the invention relates to a method for monitoring the treatment of a coronavirus infection comprising the steps of i) determining the level of caspase-1 activation in a population of myeloid cells having cell surface expression of CD66b+CD16dim markers in a sample obtained from the subject before the treatment, ii) determining the level of caspase- 1 activation in a population of myeloid cells having cell surface expression of CD66b+ CD16dim 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 higher than the level determined at step i).

In particular, the invention relates to a method for monitoring the treatment of a coronavirus infection comprising the steps of i) determining the number of cell having cell surface expression of CD14dim CD 16+ markers in a sample obtained from the subject before the treatment, ii) determining the number of cell having cell surface expression of CD14dim CD 16+ markers in a sample obtained from the subject after the treatment, iii) comparing the number determined at step i) with the number determined at step ii) and iv) concluding that the treatment is efficient when the number determined at step ii) is higher than the number determined at step i).

In a particular embodiment regarding the method for monitoring (the disease or the treatment) the coronavirus infection is determined as severe or critical form of coronavirus infection.

Therapeutic method

The present invention relates to a method for monitoring the treatment efficacy of coronavirus infection in a subject in need thereof comprising a step of i) determining in a sample obtained from the subject the level of caspase-1 activation in myeloid cells having cell surface expression of CD66b+CD16dim markers and/or the number of cell having cell surface expression of CD14dim CD 16+ markers, ii) comparing the level and/or the number determined in step i) with a reference value and iii) when the level of caspase-1 activation in myeloid cells having cell surface expression of CD66b+CD16dim markers and/or the number of cell having cell surface expression of CD14dim CD16+ markers determined at step i) is lower than the reference value, administering said subject with a therapeutically effective amount of the appropriate treatment.

In particular, the present invention relates to a method for monitoring the treatment efficacy of coronavirus infection in a subject in need thereof comprising a step of i) determining in a sample obtained from the subject the level of caspase-1 activation in myeloid cells having cell surface expression of CD66b+CD16dim markers, ii) comparing the level determined in step i) with a reference value and iii) when the level of caspase-1 activation in myeloid cells having cell surface expression of CD66b+CD16dim markers determined at step i) is lower than the reference value, administering said subject with a therapeutically effective amount of the appropriate treatment.

The present invention relates to a method for monitoring the treatment efficacy of coronavirus infection in a subject in need thereof comprising a step of i) determining in a sample obtained from the subject the number of cell having cell surface expression of CD14dim CD 16+ markers, ii) comparing the number determined in step i) with a reference value and iii) when the number of cell having cell surface expression of CD14dim CD 16+ markers determined at step i) is lower than the reference value, administering said subject with a therapeutically effective amount of the appropriate treatment. In some embodiment, the appropriate treatment for the severe form of coronavirus infection is an immunomodulatory drug such as corticoids, i.e. Dexamethasone or an inflammasome inhibitor, i.e. an inhibitor of NLRP3 inflammasome.

The present invention relates to a method for treating coronavirus infection with NLRP3 inhibitor in a subject in need thereof wherein the level of caspase- 1 activation in a population of myeloid cells having cell surface expression of CD66b+CD16dim markers and/or the number of cell having cell surface expression of CD14dim CD 16+ markers obtained from said patient, have been detected by one of the methods of the present invention.

In some embodiment, the present invention also relates to NLRP3 inhibitor for use in the prevention or the treatment of a coronavirus infection in a subject in need thereof.

As used herein, the term “an inhibitor of NLRP3” refers to a natural or synthetic compound that has a biological effect to inhibit the activity or the expression of NLRP3. More particularly, such inhibitor inhibits the recruitment of the associated protein Nek7 and/or the adapter protein the apoptosis-associated speck-like (ASC) pro-caspase-1 leading to caspase-1 production and subsequent IL-ip maturation and release.

In a particular embodiment, the inhibitor of NLRP3 is a peptide, peptidomimetic, small organic molecule, antibody, aptamers, siRNA or antisense oligonucleotide. The term “peptidomimetic” refers to a small protein-like chain designed to mimic a peptide. In a particular embodiment, the inhibitor of NLRP3 is 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.

In a particular embodiment, the inhibitor of NLRP3 is 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.

In a particular embodiment, the inhibitor of NLRP3 is MCC950. MCC950 blocks the release of IL-lbeta induced by NLRP3 activators, such as ATP, MSU and Nigericin, by preventing oligomerization of the inflammasome adaptor protein ASC (apoptosis-associated speck-like protein containing CARD). MCC950 is well known in the art and has the cas number 210826-40-7 and chemical formula: C20H24N2O5S. In a particular embodiment, the inhibitor of NLRP3 is described in the following patent applications: WO2017/129897; WO2013/007763; WO2016/12322; W02017/031161; WO20 17/017469; WO2017/184746; WO2019/025467; WO2019/034693.

In a particular embodiment, the inhibitor of NLRP3 is selected from the group consisting of but not limited to: a sufonylurea drug such as glyburide, including functionally equivalent derivatives thereof, for example, glyburide precursors or derivatives that lack the cyclohexylurea moiety, or functionally equivalent precursors or derivatives that contain the sulfonyl and benamido groups. Examples include 5-chloro-2-methoxy-N-[2-(4- sulfamoylphenyl)-ethyl]- benzamide and l-[(4-methylbenzene)-sulfonyl]-lH-l,3-benzodiazol- 2-amine. Functionally equivalent precursors or derivatives of glyburide include precursors or derivatives that retain the activity of glyburide, at least in part, to inhibit or reduce the activity of NLRP3 inflammasome, e.g. retain at least about 25% of the activity of glyburide, preferably about 50% of glyburide activity, for example, at least about 70%, 80%, or 90% if glyburide activity.

In some embodiments, the inhibitor of NLRP3 is an antibody. As used herein, the term “antibody” is used in the broadest sense and specifically covers monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g. bispecific antibodies) formed from at least two intact antibodies, and antibody fragments so long as they exhibit the desired biological activity. The term includes antibody fragments that comprise an antigen binding domain such as Fab', Fab, F(ab')2, single domain antibodies (DABs), TandAbs dimer, Fv, scFv (single chain Fv), dsFv, ds-scFv, Fd, linear antibodies, minibodies, diabodies, bispecific antibody fragments, bibody, tribody (scFv-Fab fusions, bispecific or trispecific, respectively); sc-diabody; kappa(lamda) bodies (scFv-CL fusions); BiTE (Bispecific T-cell Engager, scFv-scFv tandems to attract T cells); DVD-Ig (dual variable domain antibody, bispecific format); SIP (small immunoprotein, a kind of minibody); SMIP ("small modular immunopharmaceutical" scFv-Fc dimer; DART (ds-stabilized diabody "Dual Affinity ReTargeting"); small antibody mimetics comprising one or more CDRs and the like. The techniques for preparing and using various antibody -based constructs and fragments are well known in the art (see Kabat et al., 1991, specifically incorporated herein by reference). Diabodies, in particular, are further described in EP 404, 097 and WO 93/1 1 161; whereas linear antibodies are further described in Zapata et al. (1995). Antibodies can be fragmented using conventional techniques. For example, F(ab')2 fragments can be generated by treating the antibody with pepsin. The resulting F(ab')2 fragment can be treated to reduce disulfide bridges to produce Fab' fragments. Papain digestion can lead to the formation of Fab fragments. Fab, Fab' and F(ab')2, scFv, Fv, dsFv, Fd, dAbs, TandAbs, ds-scFv, dimers, minibodies, diabodies, bispecific antibody fragments and other fragments can also be synthesized by recombinant techniques or can be chemically synthesized. Techniques for producing antibody fragments are well known and described in the art. For example, each of Beckman et al., 2006; Holliger & Hudson, 2005; Le Gall et al., 2004; Reff & Heard, 2001 ; Reiter et al., 1996; and Young et al., 1995 further describe and enable the production of effective antibody fragments. In some embodiments, the antibody is a “chimeric” antibody as described in U.S. Pat. No. 4,816,567. In some embodiments, the antibody is a humanized antibody, such as described U.S. Pat. Nos. 6,982,321 and 7,087,409. In some embodiments, the antibody is a human antibody. A “human antibody” such as described in US 6,075,181 and 6,150,584. In some embodiments, the antibody is a single domain antibody such as described in EP 0368 684, WO 06/030220 and WO 06/003388.

In a particular embodiment, the inhibitor is a monoclonal antibody. Monoclonal antibodies 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, the human B-cell hybridoma technique and the EBV-hybridoma technique.

In a particular, the inhibitor is an intrabody having specificity for NLRP3. As used herein, the term "intrabody" generally refer to an intracellular antibody or antibody fragment. Antibodies, in particular single chain variable antibody fragments (scFv), can be modified for intracellular localization. Such modification may entail for example, the fusion to a stable intracellular protein, such as, e.g., maltose binding protein, or the addition of intracellular trafficking/localization peptide sequences, such as, e.g., the endoplasmic reticulum retention. In some embodiments, the intrabody is a single domain antibody. In some embodiments, the antibody according to the invention is a single domain antibody. The term “single domain antibody” (sdAb) or "VHH" refers to the single heavy chain variable domain of antibodies of the type that can be found in Camelid mammals which are naturally devoid of light chains. Such VHH are also called “nanobody®”. According to the invention, sdAb can particularly be llama sdAb.

In some embodiments, the NLRP3 inhibitor is an inhibitor of NLRP3 expression. An "inhibitor of expression" refers to a natural or synthetic compound that has a biological effect to inhibit the expression of a gene.

In a particular embodiment of the invention, the inhibitor of gene expression is a siRNA, an antisense oligonucleotide or a ribozyme. For example, anti-sense oligonucleotides, including anti-sense RNA molecules and antisense DNA molecules, would act to directly block the translation of NLRP3 mRNA by binding thereto and thus preventing protein translation or increasing mRNA degradation, thus decreasing the level of NLRP3, and thus activity, in a cell.

For example, antisense oligonucleotides of at least about 15 bases and complementary to unique regions of the mRNA transcript sequence encoding NLRP3 can be synthesized, e.g., by conventional phosphodiester techniques. Methods for using antisense techniques for specifically inhibiting gene expression of genes whose sequence is known are well known in the art (e.g. see U.S. Pat. Nos. 6,566, 135; 6,566,131; 6,365,354; 6,410,323; 6,107,091; 6,046,321; and 5,981,732). Small inhibitory RNAs (siRNAs) can also function as inhibitors of expression for use in the present invention. NLRP3 gene expression can be reduced by contacting a subject or cell with a small double stranded R A (dsPvNA), or a vector or construct causing the production of a small double stranded R A, such that NLRP3 gene expression is specifically inhibited (i.e. RNA interference or RNAi). Antisense oligonucleotides, siRNAs, shRNAs and ribozymes of the invention may be delivered in vivo alone or in association with a vector. In its broadest sense, a "vector" is any vehicle capable of facilitating the transfer of the antisense oligonucleotide, siRNA, shRNA or ribozyme nucleic acid to the cells and typically cells expressing NLRP3. Typically, the vector transports the nucleic acid to cells with reduced degradation relative to the extent of degradation that would result in the absence of the vector. In general, the vectors useful in the invention include, but are not limited to, plasmids, phagemids, viruses, other vehicles derived from viral or bacterial sources that have been manipulated by the insertion or incorporation of the antisense oligonucleotide, siRNA, shRNA or ribozyme nucleic acid sequences. Viral vectors are a preferred type of vector and include, but are not limited to nucleic acid sequences from the following viruses: retrovirus, such as moloney murine leukemia virus, harvey murine sarcoma virus, murine mammary tumor virus, and rous sarcoma virus; adenovirus, adeno-associated virus; SV40-type viruses; polyoma viruses; Epstein-Barr viruses; papilloma viruses; herpes virus; vaccinia virus; polio virus; and RNA virus such as a retrovirus. One can readily employ other vectors not named but known to the art. In some embodiments, the inhibitor consists in a vector that comprises the CRISPR/cas 9 protein and the appropriate RNA guide for disrupting the expression level of the gene encoding for NLRP3. In some embodiments, the endonuclease is CRISPR-Cpfl which is the more recently characterized CRISPR from Provotella and Francisella 1 (Cpfl) in Zetsche et al. (“Cpfl is a Single RNA-guided Endonuclease of a Class 2 CRISPR-Cas System (2015); Cell; 163, 1-13). As used herein, the terms “treating” or “treatment” refer to both prophylactic or preventive treatment as well as curative or disease modifying treatment, including treatment of subject at risk of contracting the disease or suspected to have contracted the disease as well as subject who are ill or have been diagnosed as suffering from a disease or medical condition, and includes suppression of clinical relapse. The treatment may be administered to a subject having a medical disorder or who ultimately may acquire the disorder, in order to prevent, cure, delay the onset of, reduce the severity of, or ameliorate one or more symptoms of a disorder or recurring disorder, or in order to prolong the survival of a subject beyond that expected in the absence of such treatment. By "therapeutic regimen" is meant the pattern of treatment of an illness, e.g., the pattern of dosing used during therapy. A therapeutic regimen may include an induction regimen and a maintenance regimen. The phrase "induction regimen" or "induction period" refers to a therapeutic regimen (or the portion of a therapeutic regimen) that is used for the initial treatment of a disease. The general goal of an induction regimen is to provide a high level of drug to a subject during the initial period of a treatment regimen. An induction regimen may employ (in part or in whole) a "loading regimen", which may include administering a greater dose of the drug than a physician would employ during a maintenance regimen, administering a drug more frequently than a physician would administer the drug during a maintenance regimen, or both. The phrase "maintenance regimen" or "maintenance period" refers to a therapeutic regimen (or the portion of a therapeutic regimen) that is used for the maintenance of a subject during treatment of an illness, e.g., to keep the subject in remission for long periods of time (months or years). A maintenance regimen may employ continuous therapy (e.g., administering a drug at a regular intervals, e.g., weekly, monthly, yearly, etc.) or intermittent therapy (e.g., interrupted treatment, intermittent treatment, treatment at relapse, or treatment upon achievement of a particular predetermined criteria [e.g., pain, disease manifestation, etc.]).

As used herein the terms "administering" or "administration" refer to the act of injecting or otherwise physically delivering a substance as it exists outside the body (e.g., NLRP3 inhibitor) into the subject, such as by oral, mucosal, intradermal, intravenous, subcutaneous, intramuscular delivery and/or any other method of physical delivery described herein or known in the art. When a disease, or a symptom thereof, is being treated, administration of the substance typically occurs after the onset of the disease or symptoms thereof. When a disease or symptoms thereof, are being prevented, administration of the substance typically occurs before the onset of the disease or symptoms thereof. More particularly, the PAK-1 and/or PAK-2 inhibitors are formulated for an oral administration is performed to the subject. In a further embodiment, intravenous administration is performed to the subject. In a particular embodiment, the NLRP3 inhibitor is according to the invention is formulated for a nasal administration.

Any therapeutic agent of the invention may be combined with pharmaceutically acceptable excipients, and optionally sustained-release matrices, such as biodegradable polymers, to form pharmaceutical compositions.

"Pharmaceutically" or "pharmaceutically acceptable" refers to molecular entities and compositions that do not produce an adverse, allergic or other untoward reaction when administered to a mammal, especially a human, as appropriate. A pharmaceutically acceptable carrier or excipient refers to a non-toxic solid, semi-solid or liquid filler, diluent, encapsulating material or formulation auxiliary of any type.

The form of the pharmaceutical compositions, the route of administration, the dosage and the regimen naturally depend upon the condition to be treated, the severity of the illness, the age, weight, and sex of the patient, etc.

The pharmaceutical compositions of the invention can be formulated for a topical, oral, intranasal, parenteral, intraocular, intravenous, intramuscular or subcutaneous administration and the like. Pharmaceutical compositions of the present invention may comprise a further therapeutic active agent.

By a "therapeutically effective amount" of the appropriate treatment (e.g NLRP3 inhibitor) of the present invention as above described is meant a sufficient amount of the appropriate treatment for treating coronavirus infection s at a reasonable benefit/risk ratio applicable to any medical treatment. It will be understood, however, that the total daily usage of the appropriate treatment (e.g NLRP3 inhibitor) and compositions of the present invention will be decided by the attending physician within the scope of sound medical judgment. The specific therapeutically effective dose level for any particular subject will depend upon a variety of factors including the disorder being treated and the severity of the disorder; activity of the specific appropriate treatment (e.g NLRP3 inhibitor) employed; the specific composition employed, the age, body weight, general health, sex and diet of the subject; the time of administration, route of administration, and rate of excretion of the specific appropriate treatment (e.g NLRP3 inhibitor) employed; the duration of the treatment; drugs used in combination or coincidental with the specific appropriate treatment (e.g NLRP3 inhibitor) employed; and like factors well known in the medical arts. For example, it is well within the skill of the art to start doses of the appropriate treatment (e.g NLRP3 inhibitor) at levels lower than those required to achieve the desired therapeutic effect and to gradually increase the dosage until the desired effect is achieved. However, the daily dosage of the products may be varied over a wide range from 0.01 to 1,000 mg per adult per day. Typically, the compositions contain 0.01, 0.05, 0.1, 0.5, 1.0, 2.5, 5.0, 10.0, 15.0, 25.0, 50.0, 100, 250 and 500 mg of the appropriate treatment (e.g NLRP3 inhibitor) of the present invention for the symptomatic adjustment of the dosage to the subject to be treated. A medicament typically contains from about 0.01 mg to about 500 mg of the appropriate treatment (e.g NLRP3 inhibitor) of the present invention, preferably from 1 mg to about 100 mg of the appropriate treatment (e.g NLRP3 inhibitors) of the present invention. An effective amount of the drug is ordinarily supplied at a dosage level from 0.0002 mg/kg to about 20 mg/kg of body weight per day, especially from about 0.001 mg/kg to 7 mg/kg of body weight per day.

The NLRP3 inhibitor as described above is also combined with a classical treatment.

As used herein, the term “classical treatment” refers to antiviral treatment, antibiotics, anti-parasitic treatment, immunosuppressive corticosteroids or non-steroidal therapies; immunotherapy: recombinant human IL-1B receptor antagonist; neutralizing monoclonal anti- IL-ip antibody; PAK-l/PAK-2 inhibitors; or caspase-1 inhibitor.

Accordingly, in a second aspect, the invention relates to i) a NLRP3 inhibitor and ii) a classical treatment used as a combined preparation for treating SARS-CoV-2 infection in a subject in need thereof.

In a particular embodiment, the combined preparation according to the invention, wherein the SARS-CoV-2 is COVID-19.

As used herein, the term “combination” is intended to refer to all forms of administration that provide a first drug together with a further (second, third. . .) drug. The drugs may be administered simultaneous, separate or sequential and in any order. According to the invention, the drug is administered to the subject using any suitable method that enables the drug to reach the lungs. In some embodiments, the drug administered to the subject systemically (i.e. via systemic administration). Thus, in some embodiments, the drug is administered to the subject such that it enters the circulatory system and is distributed throughout the body. In some embodiments, the drug is administered to the subject by local administration, for example by local administration to the lungs.

As used herein, the terms “combined treatment”, “combined therapy” or “therapy combination” refer to a treatment that uses more than one medication. The combined therapy may be dual therapy or bi-therapy.

As used herein, the term “administration simultaneously” refers to administration of 2 active ingredients by the same route and at the same time or at substantially the same time. The term “administration separately” refers to an administration of 2 active ingredients at the same time or at substantially the same time by different routes. The term “administration sequentially” refers to an administration of 2 active ingredients at different times, the administration route being identical or different.

In a particular embodiment, the invention relates to i) immunosuppressive drug ie Corticoids or a NLRP3 inhibitor and ii) antiviral treatment used as a combined preparation for treating SARS-CoV-2 infection in a subject in need thereof.

As used herein, the term “antiviral treatment” to an inhibitor which inhibits the viral entry, viral internalization, viral replication and/or viral release. Such inhibitor prevents or reduces the occurrence of viral resistance, and the like. Typically, the antiviral treatment includes but not limited to interferons (e.g., interferon-alpha, pegylated interferon-alpha), ribavirin, anti-HCV, (monoclonal or polyclonal) antibodies, RNA polymerase inhibitors, protease inhibitors, IRES inhibitors, helicase inhibitors, antisense compounds, ribozymes, neuraminidase inhibitor, nucleoside analogues of guanine, nucleoside analogue of thymidine, nucleoside reverse transcriptase inhibitors (NRTI), nucleotide reverse transcriptase inhibitor (NtRTI), protease inhibitors and any combination thereof.

Typically, the antiviral treatment is selected from the group consisting of but not limited to Abacavir, Acyclovir (Aciclovir), Adefovir, Amantadine, Ampligen, Amprenavir (Agenerase), Arbidol, Atazanavir, Atripla, Balavir, Baloxavir marboxil (Xofluza), Biktarvy, Boceprevir (Victrelis), Cidofovir, Cobicistat (Tybost), Combivir, Daclatasvir (Daklinza), Darunavir, Delavirdine, Descovy, Didanosine, Docosanol Dolutegravir, Doravirine (Pifeltro), Ecoliever, Edoxudine, Efavirenz, Elvitegravir, Emtricitabine, Enfuvirtide, Entecavir, Etravirine (Intel ence), Famciclovir, Fomivirsen, Fosamprenavir, Foscamet, Fosfonet, Fusion inhibitor, Ganciclovir (Cytovene), Ibacitabine, Ibalizumab (Trogarzo), Idoxuridine, Imiquimod, Imunovir, Indinavir, Inosine, Integrase inhibitor, Interferon type I, Interferon type II, Interferon type III, Interferon, Lamivudine, Letermovir (Prevymis), Lopinavir, Loviride, Maraviroc, Methisazone, Moroxydine, Nelfinavir, Nevirapine, Nexavir, Nitazoxanide, Norvir, Nucleoside analogues, Oseltamivir (Tamiflu), Peginterferon alfa-2a, Peginterferon alfa-2b, Penciclovir, Peramivir (Rapivab), Pleconaril, Podophyllotoxin, Protease inhibitor, Pyramidine, Raltegravir, Remdesivir, Reverse transcriptase inhibitor Ribavirin, Rilpivirine (Edurant), Rimantadine, Ritonavir, Saquinavir, Simeprevir (Olysio), Sofosbuvir, Stavudine, Synergistic enhancer, Telaprevir, Telbivudine (Tyzeka), Tenofovir alafenamide, Tenofovir disoproxil, Tenofovir, Tipranavir, Trifluridine, Trizivir, Tromantadine, Truvada, Valaciclovir (Valtrex), Valganciclovir, Vicriviroc, Vidarabine, Viramidine, Zalcitabine, Zanamivir (Relenza) and/or Zidovudine.

In a particular embodiment, the invention relates to i) immunosuppressive drug ie Corticoids or a NLRP3 inhibitor and ii) an antibiotic used as a combined preparation for treating SARS-CoV-2 infection in a subject in need thereof.

As used herein, the term “antibiotic” refers to an antimicrobial substance active against bacteria and is the most important type of antibacterial agent for fighting bacterial infections. Each antibiotic belongs to an antibiotic class which is a grouping of different drugs that have similar chemical and pharmacologic properties. Their chemical structures may look comparable, and drugs within the same class may kill the same or related bacteria. Typically, the antibiotic class includes but not limited to Penicillins, Tetracyclines, Cephalosporins, Quinolones, Lincomycins, Macrolides, Sulfonamides, Glycopeptides, Aminoglycosides, and Carbapenems.

Typically, the antibiotic is selected from the group consisting of but not limited to amoxicillin, amoxicillin and clavulanate, ampicillin, dicloxacillin, oxacillin, penicillin V potassium, demeclocycline, doxycycline, eravacycline, minocycline, omadacycline, tetracycline, cefaclor, cefdinir, cefotaxime, ceftazidime, ceftriaxone, cefuroxime, ciprofloxacin, levofloxacin, moxifloxacin, clindamycin, lincomycin, azithromycin, clarithromycin, erythromycin, sulfamethoxazole and trimethoprim, sulfasalazine, dalbavancin, oritavancin, telavancin, vancomycin, gentamicin, tobramycin, amikacin, imipenem and cilastatin, meropenem, doripenem and ertapenem.

In a particular embodiment, the invention relates to i) immunosuppressive drug ie Corticoids or a NLRP3 inhibitor and ii) an anti-parasitic treatment used as a combined preparation for treating SARS-CoV-2 infection in a subject in need thereof.

As used herein, the term “anti-parasitic treatment” refers to a treatment of parasitic diseases, such as those caused by helminths, amoeba, ectoparasites, parasitic fungi, and protozoa. Antiparasitics target the parasitic agents of the infections by destroying them or inhibiting their growth.

In a particular embodiment, the anti-parasitic drug is selected from the group consisting of but not limited to: chloroquine, amodiaquine, mefloquine, halofantrine, artemether, artesunate, arteminol, sulfadoxine, pyrimethamine, proguanil, atovaquone, quinine, abamectine, albendazole, diethylcarbamazine, mebendazole, niclosamide, ivermectin, suramine, thiabendazole, levamisole, praziquantel, triclabendazole, flubendazole, metronidazole, tinidazole, secnidazole, tenonitrozole, pyrimethamine, amphotericin B, pentamidine, miltefosine, nifurtimox, benznidazole, amphotericin B, ketoconazole, econazole, griseofulvin, miconazole orfluconazole.

In a particular embodiment, the anti-parasitic drug is an anti-malaria drug. Typically, the anti-parasitic drug is chloroquine.

In a particular embodiment, the i) immunosuppressive drug ie Corticoids or a NLRP3 inhibitor and ii) chloroquine used as a combined preparation for treating SARS-CoV-2 infection in a subject.

Accordingly, in a particular embodiment, the invention relates to i) immunosuppressive drug ie Corticoids or a NLRP3 inhibitor ii) an anti-parasitic treatment used as a combined preparation for treating SARS-CoV-2 infection in a subject in need thereof, wherein the NLRP3 inhibitor is MCC950 and the anti-parasitic drug is chloroquine.

In a particular embodiment, the invention relates to i) a NLRP3 inhibitor and ii) immunosuppressive corticosteroids used as a combined preparation for treating SARS-CoV-2 infection in a subject in need thereof.

As used herein, the term “corticosteroid” is well known in the art and refers to class of steroid hormones that are produced in the adrenal cortex as well as the synthetic analogues of these hormones. Two types of classes of corticosteroid exist in the art: glucocorticoids and mineralocorticoids. The corticosteroid for use in the invention is selected from the group consisting of: Flugestone (flurogestone); FluoromethoIone; Medrysone; Prebediolone acetate; chlormadinone acetate, cyproterone acetate, medrogestone, medroxyprogesterone acetate, megestrol acetate, and segesterone acetate; Chloroprednisone; Cloprednol; Difluprednate; Fludrocortisone; Fluocinolone; Fluperolone; Fluprednisolone; Loteprednol; Methylprednisolone; Prednicarbate; Prednisolone; Prednisone; Tixocortol; Triamcinolone; Alclometasone; Beclometasone; Betamethasone; Clobetasol; Clobetasone; Clocortolone; Desoximetasone; Dexamethasone; Diflorasone; Difluocortolone; Fluclorolone; Flumetasone; Fluocortin; Fluocortolone; Fluprednidene; Fluticasone; Fluticasone furoate; Halometasone; Meprednisone; Mometasone; Mometasone furoate; Paramethasone; Prednylidene; Rimexolone; Ulobetasol (halobetasol); Amcinonide; Budesonide; Ciclesonide; Deflazacort; Desonide; Formocortal (fluoroformylone); Fluclorolone acetonide (flucloronide); Fludroxycortide (flurandr enol one, flurandrenolide); Flunisolide; Fluocinolone acetonide; Fluocinonide; Halcinonide; Triamcinolone acetonide; Cortivazol; RU-28362.

In a particular embodiment, the invention relates to i) immunosuppressive drug ie Corticoids or a NLRP3 inhibitor and ii) non-steroidal drug used as a combined preparation for treating SARS-CoV-2 infection in a subject in need thereof. As used herein, the term “nonsteroidal drug” refers to a class of drugs which decrease inflammation. The nonsteroidal drug for use in the invention is selected from the group consisting of: Aspirin (acetylsalicylic acid); Diflunisal (Dolobid); Salicylic acid and other salicylates Salsalate (Disalcid); Ibuprofen; Dexibuprofen ; Naproxen ; Fenoprofen ; Ketoprofen ; Dexketoprofen ; Flurbiprofen ; Oxaprozin; Loxoprofen; Indomethacin; Tolmetin; Sulindac; Etodolac; Ketorolac; Diclofenac; Aceclofenac; Nabumetone; Piroxicam; Meloxicam; Tenoxicam; Droxicam; Lornoxicam; Phenylbutazone; Mefenamic acid; Meclofenamic acid; Flufenamic acid; Tolfenamic acid; Celecoxib; Clonixin.

In a particular embodiment, the invention relates to i) immunosuppressive drug ie Corticoids or a NLRP3 inhibitor and ii) an immunotherapy treatment used as a combined preparation for treating SARS-CoV-2 infection in a subject in need thereof.

As used herein, the term “immunotherapy” has its general meaning in the art and refers to the treatment that consists in administering an immunogenic agent i.e. an agent capable of inducing, enhancing, suppressing or otherwise modifying an immune response.

In a further embodiment, the invention relates to i) immunosuppressive drug ie Corticoids or a NLRP3 inhibitor and ii) a neutralizing monoclonal anti-IL-ip antibody used as a combined preparation for treating SARS-CoV-2 infection in a subject in need thereof.

As used herein, the term “a neutralizing monoclonal anti-IL-ip antibody” refers to an antibody that blocks or reduces at least one activity of a polypeptide comprising the epitope to which the antibody specifically binds. The neutralizing antibody reduces IL-ip biological activity in in cellulo and/or in vivo tests. In the context of the invention, the neutralizing monoclonal anti-IL-ip antibody is canakinumab (trade name Haris, developed by Novartis).

In a particular embodiment, the invention relates i) a NLRP3 inhibitor and ii) a caspase- 1 inhibitor used as a combined preparation for treating SARS-CoV-2 infection in a subject in need thereof.

In another embodiment, the inhibitor is a Caspase-1 inhibitor. The Caspase-1 inhibitor may be a direct inhibitor of Caspase- 1 enzymatic activity, or may be an indirect inhibitor that inhibits initiation of inflammasome assembly or inflammasome signal propagation. Caspase- 1 inhibitors for use in the present invention may be antioxidants, including reactive oxygen species (ROS) inhibitors. Examples of such Caspase-1 inhibitors include, but are not limited to, flavonoids including flavones such as apigenin, luteolin, and diosmin; flavonols such as myricetin, fisetin and quercetin; flavanols and polymers thereof such as catechin, gallocatechin, epicatechin, epigallocatechin, epigallocatechin-3- gallate and theaflavin; isoflavone phytoestrogens; and stilbenoids such as resveratrol. Also included are phenolic acids and their esters such as gallic acid and salicyclic acid; terpenoids or isoprenoids such as andrographolide and parthenolide; vitamins such as vitamins A, C and E; vitamin cofactors such as co-enzyme Q10, manganese and iodide, other organic antioxidants such as citric acid, oxalic acid, phytic acid and alpha-lipoic acid, and Rhus verniciflua stokes extract. The Caspase-1 inhibitor may be a combination of these compounds, for example, a combination of a-lipoic acid, co-enzyme Q10 and vitamin E, or a combination of a Caspase 1 inhibitor(s) with another inflammasome inhibitor such as glyburide or a functionally equivalent precursor or derivative thereof. The Caspase- 1 inhibitor may be a small molecule inhibitor, as one of skill in the art will appreciate. Non-limiting examples include cyanopropanate-containing molecules such as (S)-3-((S)-l- ((S)-2-(4-amino-3-chlorobenzamido)-3,3-dimethylbutanoyl)pyrrolidine-2-carboxamido)-3- cyano- propanoic acid, as well as other small molecule caspase- 1 inhibitors such as (S)-1-((S)- 2-{[l-(4-amino- 3 -chloro-phenyl)-methanoyl] -amino } -3 ,3 -dimethyl-butanoyl)-pyrrolidine- 2-carboxylic acid ((2R,3 S)- 2-ethoxy-5-oxo-tetrahydro-furan-3-yl)-amide. Such inhibitors may be chemically synthesized.

Pharmaceutical composition:

The NLRP3 inhibitor for use according to the invention alone and/or combined with NLRP3 inhibitor and classical treatment as described above may be combined with pharmaceutically acceptable excipients, and optionally sustained-release matrices, such as biodegradable polymers, to form pharmaceutical compositions.

Accordingly, in a further aspect, the invention relates to a pharmaceutical composition comprising a NLRP3 inhibitor for treating SARS-CoV-2 infection in a subject in need thereof.

In a particular embodiment, the pharmaceutical composition according the invention, wherein the NLRP3 inhibitor is MCC950.

In a particular embodiment, the pharmaceutical composition according the invention comprising i) a NLRP3 inhibitor and ii) a classical treatment.

In a particular embodiment, the pharmaceutical composition according the invention comprising i) a NLRP3 inhibitor and ii) an antiviral treatment.

In a particular embodiment, the pharmaceutical composition according the invention comprising i) a NLRP3 inhibitor and ii) an antibiotic.

In a particular embodiment, the pharmaceutical composition according the invention comprising i) a NLRP3 inhibitor and ii) an anti-parasitic drug.

In a particular embodiment, the pharmaceutical composition according the invention comprising i) a NLRP3 inhibitor and ii) immunosuppressive corticosteroids. In a particular embodiment, the pharmaceutical composition according the invention comprising i) a NLRP3 inhibitor and ii) non-steroidal drug.

In a particular embodiment, the pharmaceutical composition according the invention comprising i) a NLRP3 inhibitor and ii) a caspase- 1 inhibitor.

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: Caspase-1 activation level in myeloid cells in the blood of COVID-19 patients. Whole peripheral blood cells of healthy donors or COVID-19 patients with mild to critical symptoms were stained for active Caspase-1 (detected using the FAM-FLICA probe) and for CD45, CD14, CD16 and CD66b markers. Cells were immunophenotyped by flow cytometry. Leukocytes were defined as CD45 positive and were analyzed for monocytes and granulocytes surface markers. (A-C) Monocytes were defined as CD 14 positive and subpopulations were gated using CD14 and CD16 markers. The indicated monocytes subsets were analyzed for the MFI of FAM-FLICA corresponding to the activation of Caspase-1. (D- E) Granulocytes were defined as CD66b positive and the different subsets were gated using CD66b and CD 16 markers. The indicated granulocytes subsets were analyzed for the FAM- FLICA MFI. P value : ** < 0.01

Figure 2: Non-classical monocyte disappearance and increased Nigericin-triggered Caspase-1 activation in non-classical monocytes are associated with COVID-19 severity. Whole peripheral blood cells of healthy donors or COVID-19 patients were analyzed by flow cytometry using CD45, CD14 and CD16 markers. Whole peripheral blood was treated with vehicle (control) or Nigericin (5 pM) for 30min and monocytes subsets were analyzed for Nigericin-induced fold of FAM-FLICA MFI compared to control (A, C, E). Leucocytes were defined as CD45+ (B, D, F) and frequency of monocytes subsets among leukocytes were analyzed : CD14dim CD 16+ non-classical monocytes (B) CD14high CD 16- classical monocytes (D) and CD14high CD16+ intermediate monocytes (F). P value : ** < 0.01, *** < 0.001

Figure 3: CD66b+ CD16high granulocytes display an increased Nigericin- triggered Caspase-1 activation in severe COVID-19 while CD66b+ CD16dim granulocytes of severe COVID-19 loss their capacity to respond to the NLRP3 stimulation. Whole peripheral blood cells of healthy donors or COVID-19 patients were analyzed by flow cytometry using CD45, CD66b and CD 16 markers. (A-D) Whole peripheral blood was treated with vehicle (control) or Nigericin (5 pM) for 30min and granulocytes subsets were analyzed for Nigericin-induced fold of FAM-FLICA MFI compared to control (A,B). (C,D) Leucocytes were defined as CD45+ and frequency of granulocytes subsets among leukocytes were analyzed. P value : * < 0.05, ** < 0.01, **** <0.0001.

Figure 4: Myeloid cell response to NLRP3 inflammasome stimulation in recovered COVID-19 patients. Peripheral blood cells of recovered COVID-19 patients were collected 30 to 50 days after the first analysis. Whole peripheral blood cells of recovered COVID-19 patients were analyzed by flow cytometry using CD45, CD16 and CD66b markers. Whole peripheral blood was treated with vehicle (control) or Nigericin (5 pM) for 30min and CD66b+ CD16dim granulocytes were analyzed for FAM-FLICA MFI (Caspase-1 activation).

Figure 5: Prognosis score: % Non classical monocytes X Fold FAM-FLICA CD66b+CD16dim granulocytes.

EXAMPLE:

Material & Methods

Study design and Ethics

This prospective study was performed in the infectious diseases department and one intensive care unit (ICU) of the University Hospital of Nice, France, and in the intensive care unit of Cannes Hospital, France between May and June 2020. A French ethics committee (Comite de Protection des Personnes NORD OUEST-1) approved the study (national registration number: 2020-00959-30). The study was declared on ClinicalTrials.gov under the identifier: NCT04385017. The study design is summarized below:

All adult patients managed for COVID-19 in both institutions were eligible. COVID-19 diagnosis was confirmed by positive SARS-CoV-2 RT-PCR on nasopharyngeal swab specimen. The exclusion criteria included pregnancy, breast feeding, bone marrow aplasia, HIV-infection with a CD4 T-cells count < 200/pL. Eligible participants provided written informed consent. When required during ICU management the written informed consent was provided by the surrogate decision maker and confirmed later by the patient himself. The following characteristics of patients were collected: gender, age, comorbidities; acquired, druginduced or congenital immunosuppression; oxygen supply or mechanical ventilation, complete blood cell count for hospitalized patients. COVID-19 disease severity was classified according to WHO guidelines (WHO, 2020). Blood samples from healthy donors were used for comparison and characterization purposes. Informed consent was provided according to the Declaration of Helsinki following the recommendations of an independent scientific review board. The project has been validated by The Etablissement Francais du Sang, the French national agency for blood collection (13-PP-l l / CCTIRS N°14.266).

Statistical analysis

The statistical analysis of flow cytometry data was performed with a Mann-Whitney nonparametric test using the GraphPad Prism software. Patient characteristics were analyzed using a Fisher’s exact test using the GraphPad Prism software.

Ex vivo stimulation of whole blood and flow cytometry

Whole blood samples were collected into sodium citrate collection tubes and analyzed 24h after collection. The peripheral blood was diluted 1 : 1 with RPMI medium and treated with 5 pM Nigericin (Invivogen) or vehicle for 30 min at 37 °C, under agitation (500 rpm on Eppendorf ThermoMixer). Caspase-1 activation was detected using the FAM-FLICA® Caspase-1 Assay kit (Immunochemistry) according to the manufacturer’s instructions. Briefly, cells were incubated with the FAM-FLICA probe for 30 min at 37 °C before to be washed: 1 mL of RPMI was added to dilute the non-bounded probe. Cell surface markers were stained for 10 min in the dark at room temperature using the following recombinant antibodies (1/100) (Miltenyi Biotec): CD45-VioGreen (clone REA747), CD14-APC-Vio770 (clone REA599), CD66b-PEVio770 (clone REA306), CD16-PE (clone REA423), CD15-APC (clone VIMC6), CD 10 APCVio770 (clone REA877) Siglec-8 PE-Vio615 (Clone REA1045). Red blood cells were lysed using the BD Pharm Lyse buffer (BD Biosciences) according to the manufacturer’s instructions. Cells were fixed with 4% PFA for 10 min. Cells were analyzed using MACSQuant 10 flow cytometer from Miltenyi. Data were analyzed with the FlowJo and GraphPad Prism softwares. After single cells were gated and debris excluded, peripheral blood mononuclear cells (PBMCs) were identified as CD45-positive cells. Monocytes and granulocytes were then gated respectively as CD14 and CD66b positive cells.

Results

Patients recruitment

44 COVID-19 patients and 24 age matched healthy donors were included during the study period. The main clinical characteristics of those patients COVID-19 patients were recruited upon SARS-CoV-2 positive RT-PCR and healthy donors were negative for SARS- CoV-2 serological assays. Patients were classified in 4 groups (mild, moderate, severe and critical) in accordance with the WHO guidelines (WHO, 2020). None had any acquired, baseline drug-induced or congenital immunosuppression. Blood from 24 healthy donors with a mean age of 62.3 years old underwent the same assay at the same time in parallel to COVID- 19 patients.

Steady state Caspase-1 activity in circulating myeloid cells

The FAM-FLICA probe (FAMYVAD-FMK) was previously shown to be a powerful tool to monitor inflammatory Caspase- 1 activation in monocytes during bacterial infection (Martinez-Garcia et al., 2019). To determine Caspase-1 activation levels in multiple blood myeloid cells of COVID-19 patients, we used the FAM-FLICA probe together with specific extracellular immune cell markers (CD45, CD14, CD66b and CD16) in the blood of patients and analyses were performed by flow cytometry (Figure 1). Peripheral blood cells of healthy donors or CO VID-19 patients were analyzed for the expression of monocytes and granulocytes surface markers and FAM-FLICA. To monitor Caspase- 1 activity in monocytes from healthy donors or COVID-19 patients, we first gated the CD45+CD14+ monocyte population that was subsequently subdivided into 3 subpopulations: classical monocytes (CD45+CD14highCD16-), intermediate monocytes (CD45+CD14highCD16+) and non-classical monocytes (CD45+CD14dimCD16+) (Data not shown). At steady state, we did not observe statistical difference in the level of Caspase-1 activation in any of these monocytes subsets in CO VID-19 patients compared to healthy donors (Figures 1A-C).

We next focused our analysis on two sub-populations of granulocytes: CD66b+ CD16high and CD66b+ CD16dim (Data not shown). CD66b+ CD16high cells of mild to moderate COVID-19 patients as well as severe to critical COVID-19 patients showed a decreased Caspase-1 activation compared to healthy controls (Figure ID). We measured a lower Caspase- 1 activation in CD66b+ CD16dim granulocytes in the severe and critical forms of COVID-19 that was not observed in mild cases (Figure IE). CD66b+ CD16dim granulocytes of critical patients showed a 2-fold decreased of basal Caspase- 1 activation relative to healthy controls (Figure IE). Thus, our assay revealed specific regulations of Caspase- 1 activation in different myeloid cell populations depending on the clinical severity of COVID-19 patients.

Nigericin-triggered Caspase-1 activation in circulating myeloid cells

We further investigated the activation potential of the NLRP3 inflammasome in COVID-19 patients. To this aim, we incubated 100 pL of blood samples with the NLRP3 trigger Nigericin and the Caspase-1 activation was monitored in myeloid innate immune cells of healthy donors and COVID-19 patients. We first investigated the Nigericin-triggered NLRP3 activation in monocytes (Figure 2). We observed an increased activation specifically in CD14dim CD16+ non-classical monocytes isolated from severe to critical COVID-19 patients (Figure 2A). Interestingly, this effect was inversely correlated with the decreased number of these cells in severe to critical COVID-19 patients (Figure 2B). In contrast, the Nigericin-triggered NLRP3 activation in intermediate and classical monocytes was found similar to healthy donors and their number remained unchanged (Figures 2C-2F). These data reveal differences in the activation level of monocytes and/or the priming state of the NLRP3 inflammasome not only between healthy controls versus COVID-19 patients but also depending on the subpopulation and the severity of the disease. Next, we investigated the Nigericin-triggered NLRP3 activation in granulocytes (Figure 3). In contrast to CD66b+ CD16high granulocytes in which we observed an increased Caspase- 1 activation, we measured an impaired response to the NLRP3 inflammasome trigger in CD66b+ CD16dim cells that corelated with the severity of symptoms (Figures 3A-3D). The proportion of CD66b+ CD16high granulocytes was found to be increased in correlation with the severity whereas the number of CD66b+ CD16dim granulocytes stayed similar in CO VID- 19 patients and in heathy donors. Importantly, CD66b+ CD16dim cells were found to exhibit a higher response to Nigericin treatment in both healthy donors and mild COVID-19 patients (Figure 3B and data not shown). Interestingly, CD66b+ CD16dim granulocytes from healthy donors displayed a 4-fold increase in the Nigericin-triggered Caspase- 1 activation compared to untreated (Figure 3B). In contrast, we observed that the CD66b+ CD16dim cells response to Nigericin was lost in severe and critical COVID-19 patients (Figure 3B and data not shown).

Caspase-1 activation in circulating myeloid cells after recovery

Next, we wondered whether the impaired response to Nigericin in CD66b+ CD16dim cells was due to a preexisting susceptibility that could be the cause of the symptom’s severity or rather a consequence of the infection. To address this question, after recovery, we reanalyzed the blood of patients after a mean time of 39 days following inclusion using the same settings (Figure 4). Our data revealed that recovered patients had a restored Nigericin-triggered Caspase-1 activation potential of their CD66b+ CD16dim cells compared to the same assay performed when they were hospitalized with symptoms (Figure 4). Although one patient (number 12) still presented a low Nigericin-triggered Caspase-1 activation (Data not shown), both severe and critical patients tested had recovered the capacity to respond to Nigericin treatment (Figure 4 and data not shown). Identification of immature neutrophils as severity marker of COVID-19 patients

We further attempted to characterize the CD66b+ CD16dim cells impaired in the NLRP3 inflammasome response in the most severe forms of COVID-19. We observed that these cells showed a differential CD45 expression levels suggesting the presence of two different populations with a respective proportion depending on the severity of the CO VID-19 (Data not shown). Indeed, we found that CD45 is highly expressed in CD66b+ CD16dim cells of both healthy donors and mild cases of CO VID-19 whereas in severe and critical CO VID-19 patients we observed a low CD45 expression (Data not shown). CD66b+CD16dim cell could be either eosinophils or immature neutrophils depending on their CD45 expression pattern. To discriminate between these populations, we introduced in our immunophenotyping panel the CD 15, Siglec-8 and CD 10 markers (Data not shown). Siglec-8 was used to identify eosinophils and CD 15 and CD 10 as markers of mature neutrophils (Grieshaber-Bouyer andNigrovic, 2019). Corelating with the CD66b+CD16dimCD45high cells profile, we observed majority of Siglec- 8 expressing cells in healthy donors and their proportion decreased in Siglec-8 expressing cells in the severe forms of COVID-19 (Data not shown). In the same severe patients, we observed an increased number of CD66b+CD16dimCD 15+CD 10- immature neutrophils in accordance with the CD66b+CD16dimCD45dim profile that we found increased in the severe forms (Data not shown).

Importantly, both eosinophils and immature neutrophils were found to have an impaired inflammasome activation in severe and critical forms (Data not shown). Critical patients tha recovered from COVID-19 showed a restored CD45 profile with a marked disappearance o CD66b+CD16dimCD45dim cells (Data not shown). Interestingly, the patient 12 that we found with a low recovery rate in the Nigericin triggered Caspase- 1 response still had a profile with numerous CD66b+CD16dimCD45dim cells (Data not shown).

In conclusion, we described an assay that allows either the monitoring of the basal Caspase- 1 activation or the activation of the NLRP3 inflammasome triggered by Nigericin in blood myeloid cells obtained from healthy donors and COVID-19 patients. This assay allowed us to determine non-classical monocytes as major NLRP3 responsive myeloid cells specifically in the severe forms of COVID-19. Our results showed that the CD66b+ CD16dim cells of CO VID-19 patients were decreased both in the basal level of Caspase- 1 activation as well as in the Nigericin-triggered Caspase-1 activation in severe to critical patients. We show that patients who recovered from COVID-19 had a restored Nigericin-triggered Caspase- 1 activation potential in CD66b+ CD16dim cells. Finally, we identify specifically in the CD66b+ CD16dim cells of severe to critical COVID-19 patients a large proportion of CD66b+CD16dimCD 15+CD 10- cells indicating the emergence of immature neutrophils in thes patients. These cells presented a strong defect of the NLRP3 inflammasome activation in response to Nigericin.

Moreover, a prognosis score (% Non classical monocytes X Fold FAM-FLICA CD66b+CD16dim granulocytes) was determined (Figure 5).

Discussion

The involvement of inflammasomes controlling the IL-ip maturation during CO VID- 19 cytokine storm is under extensive investigation, and drugs inhibiting inflammasomes are expected to dampen this detrimental inflammation. Strategies targeting directly the inflammasome components or the IL-ip signaling pathways are currently evaluated in clinical trials (Jamilloux et al., 2020). Among them, the use of the IL-1R antagonist anakinra in COVID- 19 patients has been reported to reduce both mortality and ICU admission providing a first evidence of the importance of this pathway during COVID-19 cytokine storm (Cavalli et al., 2020). Here, by using a probe that labels active Caspase-1, we investigated whether myeloid cells in the blood of COVID-19 patients had modulated Caspase-1 activation, a hallmark of inflammation, and whether this response is related to the severity of COVID-19 symptoms.

Our study revealed that both basal and triggered inflammasome activation differ among myeloid cell populations. We identified non-classical monocytes as a population of monocytes with a COVID-19 severity signature. Indeed, the Nigericin-triggered NLRP3 inflammasome activation of non-classical monocytes was increased in the severe forms. These data indicated non-classical monocytes as the major NLRP3 inflammasome responsive/primed cells in COVID-19 patients and suggest that their decreased proportion in the severe forms may be a consequence of pyroptotic cell death occurring downstream of Caspase- 1 activation. In contrast, we measured a lower basal Caspase-1 activation in granulocytes of COVID-19 patients. As a major result of our study, we observed that the Nigericin-triggered Caspase-1 activation of CD66b+ CD16dim granulocytes inversely correlated with the severity of the symptoms of COVID-19 patients. Here, our data indicated that in severe and critical patients, CD66b+ CD16dim granulocytes are not able to respond to the NLRP3 inflammasome stimulation. This result suggests that CD66b+ CD16dim granulocytes cells could be either exhausted or paralyzed. Interestingly, a paralysis of the NLRP3 inflammasome was previously observed in patients during sepsis (Martinez-Garcia et al., 2019). Another possibility to explain this absence of responsiveness is that severe or critical CO VID-19 patients exhibited a more immature subset of neutrophils associated with an altered response. Such a situation was previously observed during sepsis where CD66b+ CD16dim neutrophils are released from the bone marrow and display less immune functionality (Pillay et al., 2010). Interestingly, we identify in severe and critical COVID-19 patients the emergence of CD66b+CD16dimCD 15+CD 10- immature neutrophils favoring this hypothesis and reinforcing the parallel between the cytokine storm observed in the severe forms of COVID-19 and during sepsis. Recent reports have indicated the increased number of immature neutrophils in the severe forms of COVID-19 patients (Schulte-Schrepping et al., 2020; Silvin et al., 2020; Vitte et al., 2020). Complementing these studies, we here provide evidence for an impaired function of these cells in correlation with the severity of the COVID-19. Strikingly, in patients who recovered from COVID-19, we found that CD66b+ CD16dim granulocytes had restored a normal response to Nigericin treatment. These data shows that the tested COVID-19 patients did not constitutively exhibit a NLRP3 inflammasome impairment, but that this reduced response is rather a consequence of the SARS- CoV-2 infection. This observation is in agreement with the occurrence of the NLRP3 inflammasome transient paralysis observed during sepsis (Martinez-Garcia et al.. 2019). Additionally, this set of data suggests that bone marrow stem cells, responsible for neutrophil generation, are not altered in recovered COVID-19 patients.

By monitoring Caspase- 1 activation directly in myeloid cells of COVID-19 patients, we provide a first evidence of the involvement of myeloid cells, Caspase- 1 and the NLRP3 inflammasome complex during COVID-19 disease. We believe that our results will serve as a springboard for the future development of a clinical test to monitor the innate immune status in SARS-CoV-2 infected patients. Such an assay could be used either for personalized therapy or for drug screening to treat COVID-19 patients.

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Claims

CLAIMS:

1. A method for assessing a subject’s risk of having or developing a mild, severe or critical coronavirus infection, comprising the steps of i) determining in a sample obtained from the subject the level of caspase-1 activation in myeloid cells having cell surface expression of CD66b+ CD16dim markers and/or the number of cell having cell surface expression of CD14dim CD16+ markers, ii) comparing the level and/or the number determined in step i) with a reference value and iii) concluding when the level of caspase-1 activation in myeloid cells having cell surface expression of CD66b+ CD16dim markers and/or the number of cell having cell surface expression of CD14dim CD 16+ markers determined at step i) is lower than the reference value is predictive of a high risk of having or developing severe or critical form of coronavirus infection.

2. A method for monitoring the treatment efficacy of coronavirus infection in a subject in need thereof comprising a step of i) determining in a sample obtained from the subject the level of caspase-1 activation in myeloid cells having cell surface expression of CD66b+CD16dim markers and/or the number of cell having cell surface expression of CD14dim CD16+ markers, ii) comparing the level and/or the number determined in step i) with a reference value and iii) when the level of caspase-1 activation in myeloid cells having cell surface expression of CD66b+CD16dim markers and/or the number of cell having cell surface expression of CD14dim CD 16+ markers determined at step i) is lower than the reference value, administering said subject with a therapeutically effective amount of the appropriate treatment.

3. The method according to claim 1 or 2, wherein the sample is a blood sample.

4. The method according to claim 1 or 2, wherein the myeloid cells are selected from the group consisting of granulocytes or monocytes.

5. The method according to claim 1 or 2, 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.

6. The method according to claim 1 or 2, wherein the level of caspase-1 activation in multiple blood myeloid cells is determined by the FAM-FLICA® Caspase-1 Assay.

7. The method according to claim 2, wherein the appropriate treatment is an immunomodulatory drug or an inflammasome inhibitor.

8. The method according to claim 7, wherein the immunomodulatory drug is. Dexamethasone.

9. The method according to claim 7, wherein the inflammasome inhibitor is an inhibitor of NLRP3 inflammasome.