Determination of the risk of death of a patient infected by a respiratory virus by measuring the expression level of the adgre3 gene

Abstract

The invention relates to an in vitro or ex vivo determination method for determining the risk of death of a patient infected by a respiratory virus, said method comprising the steps of measuring, in a biological sample from said patient, the expression level of the ADGRE3 gene, and of comparing the expression level thus measured or a value derived from this quantity with a predetermined reference value. The method thus makes it possible to conclude that there is an increased risk of death of the patient when an underexpression of the ADGRE3 gene is statistically demonstrated from the biological sample. Advantageously, the measurement of the expression level of ADGRE3 can be supplemented by the measurement of one or more additional genes such as C3AR1, CD177, OAS2, CIITA, IL-10, IL1R2, CD74, TDRD9 and combinations thereof. The invention also relates to kits for measuring the expression of ADGRE3 and optionally of one or more additional genes.

Description

Description Title: DETERMINATION OF THE RISK OF DEATH OF A SUBJECT INFECTED BY A RESPIRATORY VIRUS BY MEASURING THE LEVEL OF EXPRESSION OF THE ADGRE3 GENE Technical field

[0001] The present invention relates to the technical field of in vitro diagnostic methods and kits. In particular, the invention relates to methods and kits for determining the presence of an increased risk of death in a subject infected with a respiratory virus, in particular with a respiratory virus such as SARS-CoV-2 or one of its variants. Prior art

[0002] The coronavirus disease (COVID-19) pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has so far infected more than 550 million patients worldwide and caused more than 6.3 million deaths.

[0003] Disease severity varies greatly among patients, and the majority of patients are asymptomatic or present with minimal symptoms such as fever, cough, and / or shortness of breath. Nevertheless, 5–10% of patients require intensive care due to the rapid progression (9–12 days) to severe or critical illness with the development of, for example, acute respiratory distress syndrome (ARDS) and / or severe hypoxemia, acute lung injury (ALI), multiple organ failure, and even death (C. Huang et al., 2020).

[0004] The immune response plays a major role in the pathophysiology of COVID-19 and the most severe phenotype upon admission to intensive care of infected patients is characterized by a complex immune profile that evolves over time (EZ Ong et al., 2020).

[0005] However, in severe COVID-19 patients, the immune response can be defined by impaired inflammatory and immune responses with marked lymphopenia, elevated neutrophil counts and of monocytes, decreased monocyte HLA-DR expression, moderate plasma cytokine storm, inadequate type I interferon signaling response, and downregulation of interferon-stimulated genes (ISGs) (F. Venet et al., Crit Care, 2021). These alterations can lead to microthrombosis and tissue damage, ultimately resulting in ARDS, multiple organ failure, and death (Hadjadj J ét al., 2020).

[0006] During the pandemic, numerous exploratory studies have been conducted to decipher the immune processes involved in COVID-19. Overall, these studies used various mixed flow approaches (spectral flow, multicolor flow, time-of-flight mass spectrometry), functional assays, but also multiplex measurement of soluble mediators. The results were mainly analyzed by multi-data / -omics approaches. Although they provide crucial information on the pathophysiology of COVID-19, these approaches are mainly based on clinical research tools that are not usable in clinical routine at the bedside or in the central laboratory to characterize the immune profile and therefore the possible risks of death, due to a significant number of limitations: significant implementation time, lack of standardization, low reproducibility between cohorts, substantial cost.

[0007] Therefore, there is a need to develop alternative approaches to enable, in a short time and at the patient's bedside or in a central laboratory, the effective prediction or identification of the mortality risk of patients infected with respiratory viruses such as those responsible for COVID-19. In this sense, the measurement of biomarker(s), particularly transcriptomics, is an avenue under constant exploration.

[0008] In this context, it has notably been established that the longitudinal trajectories of 11 circulating immunity-based biomarkers could be associated with patient mortality when they were increased (10) or decreased (1), thus providing initial evidence that immunity-based biomarkers could provide early warning of the outcome of patients with COVID-19 (Abers et al., 2021).

[0009] Gene expression profiles have also been described to predict the outcome of patients with COVID-19 (Guardela B et al., 2021).

[0010] Thus, the CD177 biomarker has been described as potentially associated with the severity and risk of death of patients with COVID-19. Particularly, the stability of CD177 protein levels in serum in patients with severe COVID-19 during the course of the disease is described as a sign of a worse prognosis, which may lead to death (Levy Y et al., 2021).

[0011] Nevertheless, and in view of the global prevalence, it is still necessary to identify new biomarkers in order to complete the clinician's arsenal with other alternatives making it possible to effectively predict or identify the risk of death in subjects infected by respiratory viruses, in particular those responsible for COVID-19, in order to be able to adapt the management, preferably early, through guided therapies and thus improve their chances of survival. Summary

[0012] A first subject of the invention relates to an in vitro or ex vivo method for determining the risk of death in a subject infected with a respiratory virus, said method comprising a step of measuring in a biological sample of said subject the level of expression of the ADGRE3 gene, followed by a step of comparing the level of expression of the ADGRE3 gene thus measured, in particular at the mRNA level, or a value derived from said level, to a predetermined reference value.

[0013] Based on the result of the comparison, a conclusion regarding the increased risk of death in the said subject is made since a decrease in the expression of the ADGRE3 gene is identified.

[0014] Advantageously, the reference value corresponds to the average of the level of expression of ADGRE3 obtained from biological samples from a population of subjects not presenting any infection by a respiratory virus or to the average of the level of expression of ADGRE3 obtained from biological samples from a population of subjects infected by a respiratory virus and of which They are known to have survived after infection, particularly within 28 days of admission to a healthcare facility or within 37 days post-infection.

[0015] In a preferred embodiment, the respiratory virus is SARS-CoV-2 or one of its variants.

[0016] Advantageously, the performance of the determination of the risk of death can be improved by measuring the level of expression of additional genes. Thus, according to a particular embodiment, the method also comprises a step of measuring in the biological sample of said subject the level of expression of at least one additional gene chosen from C3AR1, CD177, OAS2, CIITA, IL-10, IL1 R2, CD74, TDRD9 and their combinations, preferably chosen from C3AR1, CD177, OAS2, CIITA, IL-10, IL1 R2 and their combinations, and more preferably, chosen from C3AR1, OAS2, CIITA, IL-10 and their combinations.

[0017] The measured expression of the additional gene(s) is then compared to a reference value of the respective expression level of said genes and it may be concluded that there is an increased risk of death in said subject when the comparison of the expression level of the ADGRE3 gene, in particular at the mRNA level, with a predetermined threshold value shows that there is a decrease in the expression level or under-expression, and when the comparison of the level of mRNA transcripts of the additional genes below with a reference value of their respective expression level shows that there is: - an increase in the level of expression or overexpression of C3AR1, and / or - an increase in the level of expression or overexpression of CD177, and / or - an increase in the level of expression or overexpression of OAS2, and / or - a decrease in the level of expression or underexpression of CIITA, and / or - an increase in the level of expression or overexpression of IL-10, and / or - an increase in the level of expression or overexpression of IL1 R2, and / or - a decrease in the level of expression or under-expression of CD74, and / or - an increase in the level of expression or overexpression of TDRD9.

[0018] According to a preferred embodiment, the biological sample is a blood sample, preferably a whole blood sample.

[0019] According to another preferred embodiment, the expression of ADGRE3, and optionally that of additional genes, is measured at the messenger RNA (mRNA) level.

[0020] According to a preferred embodiment, the expression is measured by a molecular detection method, such as, for example, amplification, sequencing or hybridization. Preferably, the expression is measured by amplification via RT-PCR, in particular RT-qPCR.

[0021] According to a preferred embodiment, the measured expression of ADGRE3, and optionally that of the additional genes, is normalized relative to the expression of one or more housekeeping genes. Preferably, the expression is normalized relative to the housekeeping genes selected from DECR1, HPRT1, PPIB, GAPDH, ACTB and combinations thereof.

[0022] Another subject of the invention relates to a kit for the in vitro or ex vivo measurement of the expression of ADGRE3 in a biological sample comprising means for determining the level of expression of ADGRE3 in said sample. Preferably, the determination means are chosen from amplification primers or probes.

[0023] Advantageously, the kit may comprise a positive control sample calibrated to contain the amount of ADGRE3 that corresponds to the amount or concentration representative of the level of expression measured in a pool of samples from subjects not presenting infection by a respiratory virus or from subjects infected by a respiratory virus and known to have survived, and / or a negative control sample calibrated to contain the amount of ADGRE3 that corresponds to the average amount measured in a pool of samples from subjects who did not survive following infection by a respiratory virus.

[0024] The kit according to the invention may also comprise means for determining the level of expression of at least one additional gene selected from C3AR1, CD177, OAS2, CIITA, IL-10, IL1 R2, CD74, TDRD9 and their combinations, preferably selected from C3AR1, CD177, OAS2, CIITA, IL-10, IL1 R2 and their combinations, and more preferably selected from C3AR1, OAS2, CIITA, IL-10 and combinations thereof. Preferably, said determination means are chosen from amplification primers or probes.

[0025] Finally, another subject of the invention relates to the use of the kit according to the invention for determining the risk of death, preferably the risk of death within 28 days following admission to a healthcare establishment or within 37 days post infection, of a subject infected with a respiratory virus such as SARS-CoV-2 or one of its variants. Description of the embodiments

[0026] Certain terms and expressions used in the context of the invention are detailed below.

[0027] A first subject of the invention relates to an in vitro or ex vivo method for determining the risk of death in a subject infected with a respiratory virus, said method comprising the following steps of: a) measuring in a biological sample of said subject the level of expression of the ADGRE3 gene, b) comparing the level of expression of the ADGRE3 gene measured in step a) or a value derived from this quantity, with a predetermined reference value.

[0028] Surprisingly, it was found that measuring ADGRE3 gene expression could determine or identify a risk of death in a subject infected with a respiratory virus. Thus, and in a context where personalized medicine is becoming increasingly important, subjects at increased risk of mortality could benefit from individualized care. Moreover, considering the global prevalence of respiratory viruses such as SARS-CoV-2 and associated diseases, particularly their severe forms, it is essential to make available the most comprehensive arsenal possible to quickly and effectively determine the risk of death presented by patients infected with these viruses.

[0029] The method which is the subject of the invention has the advantage of being able to easily assess the increased risk of death of a subject, for example a patient admitted to a healthcare establishment such as an intensive care unit or emergency room, by having an easily measurable biomarker and the measurement of which can be made directly in the healthcare facility hosting it or in a local laboratory. In addition, the measurement of the ADGRE3 biomarker, like that of the additional biomarkers of the invention, is entirely suitable for being carried out by automated analysis machines or by so-called rapid tests.

[0030] The term "biomarker" or "marker" means an objectively measurable biological characteristic that represents an indicator of normal or pathological biological processes or of pharmacological response to a therapeutic intervention. For the purposes of this description, biomarkers are genes and their level of expression is detectable in particular at the level of transcripts, in particular mRNA transcripts.

[0031] The ADGRE3 gene (also known as EMR3) is located on chromosome 19 from position 14,619,117 to position 14,674,844 (GRCh38 / hg38), or 55728 bp. This gene encodes a member of the class B seven-domain transmembrane receptor (TM7) family, which is expressed primarily by cells of the immune system. Members of this family are characterized by an extended extracellular region with a variable number of N-terminal epidermal growth factor (EGF)-like domains coupled to a TM7 domain via a mucin-like spacer domain. This gene is closely linked to the gene encoding mucin-like hormone receptor 2 containing an EGF-like molecule on chromosome 19. Alternative splicing of ADGRE3 results in multiple transcript variants encoding different isoforms.

[0032] The nucleotide sequence of the ADGRE3 gene is known to those skilled in the art and is available from the NCBI under the reference NC_000019.10 (assembly GRCh38.p14).

[0033] The term "risk of death in a subject" refers to the risk that the subject has of succumbing in the days following infection with a respiratory virus or in the days following admission to a health facility following the infection. An increased risk of death is particularly expressed when the subject has a statistically significant risk of dying in the days following infection, generally compared to infected subjects who are known to have survived or to uninfected subjects.

[0034] Advantageously, the method according to the invention makes it possible to determine the increased risk of death in a subject in the 37 days following the day on which the infection is confirmed, also called post-infection days, for example via a test for detecting a respiratory virus.

[0035] Thus, the risk of death corresponds to the risk of death of the subject within 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36 or 37 days following infection, also called post-infection days. Preferably, the risk of death corresponds to the risk of death of the subject within 16 days, 23 days, 30 days, 31 days, 32 days, 33 days, 34 days, 35 days, 36 days, or 37 days post-infection of the subject. Preferably, the risk of death corresponds to the risk of death within 37 days postinfection.

[0036] According to a particular embodiment, the risk of death may also correspond to the number of days following admission to a healthcare facility, also called post-admission days, it being understood that at the time of admission the subjects have already been infected with a respiratory virus for generally several days. Thus, according to this embodiment, the risk of death corresponds to the risk of death of the subject within 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 15, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27 or 28 days after admission to the healthcare facility of the subject. Preferably, the risk of death corresponds to the risk of the subject dying within 7 days, 14 days, 21 days, 22 days, 23 days, 24 days, 25 days, 26 days, 27 days, or 28 days after the subject's admission. More preferably, the risk of death corresponds to the risk of dying within 28 days after admission.

[0037] For the purposes of this description, a healthcare facility means a hospital or clinic, preferably an emergency department, an intensive care unit, an intensive care unit (ICU) or a continuing care unit, or a medical facility for the elderly, for example an EHPAD.

[0038] The term “subject” means a human being and preferably, the subject is a patient. The patient is a person who has come into contact with a healthcare professional, including a doctor, a medical facility or a healthcare institution.

[0039] According to a particular embodiment, the subject is a patient within a healthcare establishment, preferably within a hospital, more preferably within the emergency department, the intensive care department, in an intensive care unit (ICU) or in a continuing care unit, and in particular a patient in an ICU.

[0040] A "respiratory virus" means a virus that infects the respiratory tract and / or lungs. Such viruses are typically found in samples collected from a subject's nose, throat, and / or mouth, including nasal or nasopharyngeal samples (which require a sample deeper in the nose), oropharyngeal samples (which require a sample from the back of the throat), or saliva.

[0041] The expression "subject infected with a respiratory virus" means a subject whose test for detecting the presence of a respiratory virus gives a positive result. These subjects, in particular those developing the most severe forms of the disease, are subjects for whom it is all the more relevant to implement the method according to the invention measuring the level of expression of ADGRE3.

[0042] Conversely, a subject not infected by a respiratory virus is a subject whose test for the detection of the presence of a respiratory virus is negative.

[0043] Examples of respiratory viruses include seasonal coronaviruses, the SARS-CoV-2 virus (“Severe Acute Respiratory Syndrome Coronavirus-2”), regardless of its variants, the influenza virus, the respiratory syncytial virus (RSV), rhinoviruses, metapneumoviruses, parainfluenza viruses and adenoviruses. To date, the known variants of the SARS-CoV-2 virus include, in particular, its so-called English, Brazilian, South African, American, Indian or Omicron variants. Knowledge of these variants and their names are evolving and the invention is applicable to each of them (https: / / www.who.int / en / activities / tracking-SARS-CoV-2-variants / ).

[0044] The discovery of the "English variant" of SARS-CoV-2 dates back to September 20, 2020, in Kent (Southeast England). On December 14, the United Kingdom reported the circulation of this variant to the World Health Organization (WHO). Initially named VUI 202012 / 01 (for Variant Under Investigation, year 2020, month 12, variant 01), it was quickly renamed, on December 18, 2020, VOC 202012 / 01 (for Variant Of Concern, literally "variant of concern"), then Alpha variant. It is part of the B.1.1.7 lineage in the phylogenetic tree and contains the 69-70 deletion, still designated AH69 / V70. The Brazilian P.1 variant (Gamma variant) is a descendant of the B.1.1.28 lineage. This Brazilian P.1 variant contains numerous mutations, in particular the E484K, K417T and N501Y mutations. The Japanese variant which derives from a lineage present in Brazil (B.1.1.28) contains a very high number of genetic changes. It has twelve amino acid mutations in the spike protein, including N501Y, E484K, and K417T. The South African variant (Beta variant) is called 501 Y. V2 and belongs to the B.1 lineage.351 also contains various mutations, including three, K417N, E484K and N501Y, located in the RBD domain of the spike protein, the receptor binding domain. The Indian variant, called the Delta variant, which belongs to the B.1.617.2 lineage and contains the S417N and S484K mutations. Finally, there is also the Omicron variant identified in November 2021 and which belongs to the B.1.1.529 lineage.

[0045] According to a particular embodiment, the respiratory virus is a coronavirus, in particular SARS-CoV-2 or one of its variants such as for example the Alpha, Beta, Gamma, Delta or Omicron variants.

[0046] The expressions “respiratory virus detection test”, “respiratory virus diagnostic test” or “test for detecting the presence of a respiratory virus infection” are synonymous and refer to any test known to those skilled in the art that makes it possible to provide such a conclusion, in particular tests for detecting the DNA or RNA of said respiratory virus, such as PCR tests, antigenic tests or even self-tests.

[0047] By "biological sample" we refer here to any sample originating from a subject, and which may be of different natures, such as blood or its derivatives, sputum, urine, stools, skin, cerebrospinal fluid, bronchoalveolar lavage fluid, abdominal cavity puncture fluid, saliva, gastric secretions, semen, seminal fluid, tears, spinal cord, trigeminal nerve ganglion, adipose tissue, lymphoid tissue, tissue placental, gastrointestinal tract tissue, genital tract tissue, or central nervous system tissue.

[0048] In particular, the biological sample may be a biological fluid, such as a blood sample or a blood-derived sample, which may in particular be chosen from whole blood (as collected venously, i.e. containing white and red cells, platelets and plasma), plasma, serum, as well as all types of cells extracted from blood, such as for example peripheral blood mononuclear cells (or PBMC, containing B lymphocytes, T lymphocytes, NK cells, dendritic cells and monocytes), B cell subpopulations, purified monocytes, or neutrophils.

[0049] According to a preferred embodiment, the biological sample used in the method according to the invention is a blood sample, preferably a whole blood sample.

[0050] For the purposes of this description, the biological sample of a subject, namely that of an infected subject whose risk of death is to be determined, corresponds to the biological sample to be tested, or test sample, as opposed to the reference sample used as a comparison.

[0051] For the purposes of this description, the expression "reference value" or "predetermined reference value" is synonymous with the expressions "control value" or "threshold value", and serves as a point of comparison to determine whether the level of expression of a target gene is decreased or increased.

[0052] According to the method which is the subject of the invention, the risk of death in a subject infected by a respiratory virus is in particular determined through the implementation of a step of measuring the level of expression of the ADGRE3 gene.

[0053] Measuring the level of expression of a gene is well known to those skilled in the art and consists in particular of quantifying at least one expression product of the gene. The expression product of a gene, within the meaning of the present invention, may be any biological molecule resulting from the expression of said gene. More particularly, the expression product of the gene may be a transcript.

[0054] The term "transcript" refers to RNA, and in particular messenger RNA (mRNA), resulting from gene transcription. More precisely, transcripts are the RNA produced by the transcription of a gene followed by post-transcriptional modifications of the pre-RNA forms.

[0055] According to a preferred embodiment, the measurement of the expression level of ADGRE3, and possibly that of one or more additional genes as listed below, is carried out at the RNA level, in particular at the messenger level (mRNA), in a biological sample from a subject infected with a respiratory virus. According to this embodiment, the measurement of the expression level of the ADGRE3 gene therefore concerns the determination of the mRNA level of said gene.

[0056] The transcripts of the ADGRE3 gene are known to those skilled in the art. Examples include transcripts with the following references in the NCBI database: XM_011528374.3 (2298nt), NM_001289159.2 (1971 nt), NM_001289158.2 (2193 nt), XM_047439546.1 (2151 nt) and NM_032571.5 (2349 nt).

[0057] As mentioned above, the measurement of the expression level of a gene is well known to those skilled in the art. In the case of a transcript such as, for example, mRNA, the measurement can be carried out by a direct method, according to any method known to those skilled in the art for determining the presence of said transcript in the biological sample, or by indirect detection of the transcript after transformation of the latter into DNA, or after amplification of said transcript or after amplification of the DNA obtained after transformation of said transcript into DNA. Many methods exist for the detection of nucleic acids and are well known to those skilled in the art (see, for example, Kricka et al., Clinical Chemistry, 1999, No. 45(4), p.453-458; Relier GH et al., DNA Probes, 2nd Ed., Stockton Press, 1993, sections 5 and 6, p.173-249).

[0058] For example, gene expression can be determined as follows: (1) extraction of total RNA from a blood sample or PBMCs and carrying out a reverse transcription step in order to obtain the different complementary DNAs of the different messenger RNAs initially present in the sample or PBMCs (or cDNA), (2) specific amplification of cDNAs. In this case, the specific reagent used includes at least one gene-specific amplification primer. This step can be carried out by a PCR-type amplification reaction or by any other suitable amplification technique, (3) determination of gene expression by quantifying cDNAs.

[0059] Gene expression can be measured in particular by Reverse Transcription-Polymerase Chain Reaction or RT-PCR, preferably by quantitative RT-PCR or RT-qPCR (for example using FilmArray® technology or Fluidigm's Biomark™ platform), by sequencing (preferably by high-throughput sequencing) or by hybridization techniques (for example with hybridization microarrays or by techniques of the NanoString® nCounter® type). All these methods are also well known to those skilled in the art and it is not necessary to detail them here.

[0060] According to a particular embodiment, the measurement of the expression of ADGRE3 is carried out by a molecular detection method, in particular by RT-PCR, by sequencing or by hybridization. Preferably, the measurement of the expression is carried out by RT-PCR, and in particular by RT-qPCR.

[0061] According to a particular embodiment, the level of expression of the ADGRE3 gene is measured by quantitative RT-qPCR detection of the mRNA transcripts of said gene. A person skilled in the art is in fact able to determine the primers necessary for the amplification of at least one transcript of the ADGRE3 gene, and possibly additional genes, to determine the level(s) of expression.

[0062] Measuring the expression level allows to determine the quantity of one or more ADGRE3 transcripts in the biological sample or also to give a derived value.

[0063] Thus, according to a particular embodiment, the level of expression of the ADGRE3 gene is a value derived from the quantity of its transcripts, in particular mRNA. For example, a value derived from the quantity of transcripts may for example be the absolute concentration, calculated using a calibration curve obtained from successive dilutions of an amplicon solution of known concentration. The derived value may also correspond to the value of the standardized and calibrated quantity, such as the CNRQ (Calibrated Normalized Relative Quantity, (Hellemans et al (2007), Genome biology 8(2): R19), which integrates the values ​​of a reference sample (or a calibrator) and one or more housekeeping genes (also called reference genes). Examples of housekeeping genes include DECR1, HPRT1, PPIB, RPLPO, PPIA, GLYR1, RANBP3, 18S, B2M, TBP, GAPDH and ACTB.

[0064] According to a particular embodiment, the expression of ADGRE3 is normalized relative to the expression of one or more housekeeping genes (or reference genes) according to methods known to those skilled in the art. Thus, the expression is normalized using one or more of the following housekeeping genes: DECR1 (chromosomal location: chr8, 90001352-90053633), HPRT1 (chromosomal location: chrX, 134452842-134520513) and PPIB (chromosomal location: chr15:64155812-64163205), RPLPO (chromosomal location: chr12, 120196699-120201111), PPIA (chromosomal location: chr7, 44795960-44803117), GLYR1 (chromosomal location: chr16, 4803203-4847288), RANBP3 (chromosomal location: chr19, 5916139-5978140), B2M (chromosomal location: chr15, 44711492-44718145), TBP (chromosomal location: chr6, 170554369- 170572859), GAPDH (chromosomal location: chr12, 6534517-6538371 ) and ACTB (chromosomal location: chr14 , 5527148-5530601 ). Chromosomal locations are given according to GRCh38 / hg38. Preferably, the expression is normalized using one or more of the housekeeping genes selected from: DECR1, HPRT1, PPIB, GAPDH, ACTB and combinations thereof, more preferably, selected from DECR1, HPRT1, PPIB and combinations thereof.

[0065] In such a case, the reference level used is also standardized beforehand, in the same way. The standardization, whether for the reference level or for the transcript level of the biological sample to be tested, is carried out before the comparison, in particular before calculating a ratio between the transcript level of said sample to be tested and the reference level. When a threshold value different from a reference level is used to issue a conclusion, this standardization may be taken into account when choosing the threshold value.

[0066] In the case where the level of transcripts of ADGRE3 is normalized relative to the level of transcripts of one or more housekeeping genes, of course, this implies that the method according to the invention includes the determination of the level of transcripts of the housekeeping gene(s) used for the normalization.

[0067] Generally speaking, in the method according to the invention, whatever its embodiments, the level of expression of ADGRE3, preferably the normalized expression, in the biological sample of the subject is compared to a reference value or to the expression of the same gene, preferably the normalized expression, obtained in a reference biological sample.

[0068] According to a particular embodiment, the level of expression of the ADGRE3 gene is determined from a biological sample of a subject and it can be concluded that there is an increased risk of death in said subject when the comparison of the level of expression of said ADGRE3 gene with a predetermined reference value shows that there is a significant difference with said reference value corresponding to said gene. More precisely, said difference corresponds to a level of transcripts, in particular mRNA, for the biological sample to be tested which is lower than that corresponding to the reference value. In other words, it can be concluded that there is an increased risk of death in said subject when an under-expression of the ADGRE3 gene is demonstrated.

[0069] By "underexpression" is meant a significant decrease in the level of expression compared to a reference value. A person skilled in the art is able to determine the statistical test to be used to determine this reference value with which the level of expression of ADGRE3 is to be compared. The exemplary embodiments present one of the possible methods.

[0070] Advantageously, according to a first variant, said reference value corresponds to a reference expression level of the ADGRE3 gene which is the level of transcripts of said gene obtained from a biological sample of a subject not presenting any infection by a respiratory virus. According to this variant, the reference value can also correspond to the average of the expression level of transcripts of ADGRE3 obtained from biological samples from a population of subjects not presenting any infection by a respiratory virus.

[0071] According to a second variant, the reference value corresponds to a reference expression level of the ADGRE3 gene which is the level of transcripts of said gene obtained from a biological sample of a subject infected with a respiratory virus but known to have survived after infection, in particular within 37 days following infection. According to this variant, the reference value may also correspond to the average of the expression level of ADGRE3 transcripts obtained from biological samples from a population of subjects infected with a respiratory virus but known to have survived after infection, in particular within 37 days following infection.

[0072] Preferably, the difference between the level of transcripts of the ADGRE3 gene determined in the test biological sample and the reference level of said gene (reference value) corresponds to the fact that the level of transcripts of said gene determined in the test sample is significantly reduced, in particular by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80% or at least 90% compared to the reference expression level, or reference value, of said ADGRE3 gene.

[0073] The reduction considered relevant to provide a conclusion will depend on the reference value considered, and in particular on the reference level used as a reference value, and may be adapted by a person skilled in the art. In particular, if the reference value corresponds to the reference level of the ADGRE3 gene, which is the level of transcripts of said gene in a subject not presenting any infection by a respiratory virus, in a population of subjects not presenting any infection by a respiratory virus, or in a subject or a population of subjects presenting a respiratory virus infection but having survived within 37 days following the infection, it may be sufficient for the level of ADGRE3 transcripts determined in the test biological sample to be simply lower than said reference threshold value.

[0074] According to the invention, the method makes it possible to conclude that there is an increased risk of death of the subject when an under-expression of ADGRE3 is highlighted in the biological sample to be tested.

[0075] The method according to the invention can thus comprise the following steps: - determine the level of expression of the ADGRE3 gene by measuring the quantity of at least one ADGRE3 transcript in a biological sample from the subject, - comparing the quantity of said transcript determined for said biological sample or a value derived from this quantity, to a reference value predetermined from a reference sample, and - to draw a conclusion about the presence of an increased risk of death when the result of the comparison shows a decrease in the expression of the ADGRE3 gene.

[0076] According to this embodiment, the reference sample may be, for example, a sample from a subject or a mixture of samples from several subjects, said subjects being uninfected by a respiratory virus. The reference value may in particular correspond to an average value of the level of expression of ADGRE3 measured from several samples each from different subjects uninfected by a respiratory virus or infected by a respiratory virus but known to have survived, in particular within 28 days following admission to a healthcare facility or within 37 days post-infection.

[0077] According to a preferred variant of this embodiment, the reference sample is of the same nature as the biological sample to be tested or at least of a compatible nature to constitute a reference for determining the level of expression of ADGRE3.

[0078] A "comparison" or verification of a "difference" between two values, or levels of values, may be made by any known technique. The comparison or verification of a "difference" may involve the calculation of a ratio or a difference.

[0079] Within the scope of the invention, the issuing of a conclusion regarding the risk of death in the subject from whom the test biological sample originates may also be carried out by any automated technique, carried out by a computer or assisted by a computer.

[0080] The method as described above, in all its embodiments, may also comprise, in addition to the step of measuring the expression of the ADGRE3 gene, a step of measuring the expression of one or more additional genes.

[0081] Thus, according to this particular embodiment, the additional gene(s) are chosen from C3AR1, CD177, OAS2, CIITA, IL-10, IL1 R2, CD74, TDRD9 and combinations thereof. According to this embodiment, the method also comprises a step of comparing the expression level of the additional gene(s) with a reference value of the respective expression level of said additional genes. The reference values ​​of the additional genes are as defined previously with ADGRE3.

[0082] The chromosomal locations of the additional genes are given in Table 1 below:

[0083] [Table 1]

[0084] Thus, it may be concluded that there is an increased risk of death in said patient when the comparison of the level of expression of the ADGRE3 gene, in particular the mRNA transcripts of said gene, with a predetermined reference value shows that there is a decrease in the level of expression or under-expression, and when the comparison of the level of expression of the additional genes below, in particular the mRNA transcripts of said genes, with a reference value of their respective level of expression, shows that there is: - an increase in the level of expression or overexpression of C3AR1, and / or - an increase in the level of expression or overexpression of CD177, and / or - an increase in the level of expression or overexpression of OAS2, and / or - a decrease in the level of expression or underexpression of CIITA, and / or - an increase in the level of expression or overexpression of IL-10, and / or - an increase in the level of expression or overexpression of IL1 R2, and / or - a decrease in the level of expression or under-expression of CD74, and / or - an increase in the level of expression or overexpression of TDRD9.

[0085] According to a variant of this embodiment, the method comprises, with regard to the measurement of gene expression, in addition to the step of measuring the expression of the ADGRE3 gene, only a step of measuring the expression of one or more additional genes chosen from C3AR1, CD177, OAS2, CIITA, IL-10, IL1 R2, and combinations thereof, preferably from C3AR1, CD177, CIITA, IL-10, IL1 R2 and combinations thereof.

[0086] According to another variant of this particular embodiment, the method comprises concerning the measurement of gene expression, in addition to the step of measuring of the expression of the ADGRE3 gene, only a step of measuring the expression of the additional IL-10 gene.

[0087] According to another variant of this embodiment, the method comprises, with regard to the measurement of gene expression, in addition to the step of measuring the expression of the ADGRE3 gene, only a step of measuring the expression of the additional gene IL-10 and a step of measuring the expression of at least one other additional gene chosen from C3AR1, CD177, OAS2, CIITA, IL1 R2, CD74, TDRD9 and their combinations, preferably chosen from C3AR1, CD177, OAS2, CIITA and their combinations, and more preferably, chosen from C3AR1, CIITA, and their combinations.

[0088] According to another variant of this particular embodiment, the method comprises, with regard to the measurement of gene expression, in addition to the step of measuring the expression of the ADGRE3 gene, only a step of measuring the expression of the additional CIITA gene.

[0089] According to another variant of this particular embodiment, the method comprises, with regard to the measurement of gene expression, in addition to the step of measuring the expression of the ADGRE3 gene, only a step of measuring the expression of the additional gene CIITA and a step of measuring the expression of at least one other additional gene chosen from C3AR1, CD177, OAS2, IL-10, IL1 R2, CD74, TDRD9 and their combinations, preferably chosen from C3AR1, CD177, OAS2, IL-10 and their combinations, and more preferably, chosen from C3AR1, IL-10 and their combinations.

[0090] According to another variant of this particular embodiment, the method comprises, with regard to the measurement of gene expression, in addition to the step of measuring the expression of the ADGRE3 gene, only a step of measuring the expression of the additional CD74 gene.

[0091] According to another variant of this particular embodiment, the method comprises, with regard to the measurement of gene expression, in addition to the step of measuring the expression of the ADGRE3 gene, only a step of measuring the expression of the additional gene CD74 and a step of measuring the expression of at least one other additional gene chosen from C3AR1, CD177, OAS2, IL-10, IL1 R2, CIITA, TDRD9 and combinations thereof, preferably among C3AR1, CD177, OAS2, IL-10, IL1 R2, CIITA and combinations thereof, and more preferably, among C3AR1, IL-10, CIITA and combinations thereof.

[0092] According to another variant of this embodiment, the method comprises, with regard to the measurement of gene expression, in addition to the step of measuring the expression of the ADGRE3 gene, only a step of measuring the expression of the additional OAS2 gene.

[0093] According to another variant of this particular embodiment, the method comprises, with regard to the measurement of gene expression, in addition to the step of measuring the expression of the ADGRE3 gene, only a step of measuring the expression of the additional gene OAS2 and a step of measuring the expression of at least one other additional gene chosen from C3AR1, CD177, CD74, IL-10, IL1 R2, CIITA, TDRD9 and their combinations, preferably chosen from C3AR1, CD177, IL-10, IL1 R2, CIITA and their combinations, and more preferably chosen from C3AR1, IL-10, CIITA and their combinations.

[0094] According to another variant of this embodiment, the method comprises, with regard to the measurement of gene expression, in addition to the step of measuring the expression of the ADGRE3 gene, only a step of measuring the expression of the additional C3AR1 gene.

[0095] According to another variant of this particular embodiment, the method comprises, with regard to the measurement of gene expression, in addition to the step of measuring the expression of the ADGRE3 gene, only a step of measuring the expression of the additional gene C3AR1 and a step of measuring the expression of at least one other additional gene chosen from OAS2, CD177, CD74, IL-10, IL1 R2, CIITA, TDRD9 and their combinations, and preferably chosen from OAS2, CD177, IL-10, IL1 R2, CIITA and their combinations, and more preferably chosen from IL-10, CIITA and their combinations.

[0096] According to another variant of this embodiment, the method comprises, with regard to the measurement of gene expression, in addition to the step of measuring the expression of the ADGRE3 gene, only a step of measuring the expression of the additional IL1 R2 gene.

[0097] According to another variant of this particular embodiment, the method comprises, with regard to the measurement of gene expression, in addition to the step of measuring the expression of the ADGRE3 gene, only a step of measuring the expression of the additional gene IL1 R2 and a step of measuring the expression of at least one other additional gene chosen from C3AR1, CD177, CD74, IL-10, OAS2, CIITA, TDRD9 and their combinations, preferably chosen from C3AR1, CD177, IL-10, OAS2, CIITA and their combinations, and more preferably chosen from C3AR1, IL-10, CIITA and their combinations.

[0098] According to another variant of this embodiment, the method comprises, with regard to the measurement of gene expression, in addition to the step of measuring the expression of the ADGRE3 gene, only a step of measuring the expression of the additional TDRD9 gene.

[0099] According to another variant of this embodiment, the method comprises, with regard to the measurement of gene expression, in addition to the step of measuring the expression of the ADGRE3 gene, only a step of measuring the expression of the additional gene TDRD9 and a step of measuring the expression of at least one other additional gene chosen from C3AR1, CD177, OAS2, IL-10, IL1 R2, CIITA and their combinations, preferably from C3AR1, CD177, OAS2, IL-10, IL1 R2, CIITA and their combinations, and more preferably, from C3AR1, OAS2, IL-10, CIITA and their combinations.

[0100] According to another variant of this embodiment, the method comprises, in addition to the step of measuring the expression of the ADGRE3 gene, a step of measuring the expression of the following 8 additional genes: C3AR1, CD177, OAS2, CIITA, IL-10, IL1 R2, CD74 and TDRD9.

[0101] According to a particularly preferred embodiment variant, the method according to the invention comprises, together with the measurement of the expression of the ADGRE3 gene, the measurement of the expression of the additional genes presented in Table 2:

[0102] [Table 2] C3AR1, 0AS2 CIITA, IL-10 Combinations of 2 additional genes CIITA, OAS2 IL-10, OAS2

[0103] For all the embodiment variants described above, the method comprises a step of concluding as to the increased risk of death on the basis of the measurement of the expression levels of said genes, and of the comparison of the levels thus measured (increase or decrease) with reference values ​​determined as described above.

[0104] Another subject of the invention relates to a kit for the in vitro or ex vivo measurement of the level of expression of the ADGRE3 gene in a biological sample of a subject, said kit comprising at least one means for determining the level of expression of ADGRE3 in said biological sample.

[0105] The means for determining the level of expression of the ADGRE3 gene are known to those skilled in the art and may be specific tools or reagents for measuring said expression in a biological sample. These may, for example, be primers or probes.

[0106] The specific or preferred embodiments described in connection with the methods according to the invention apply, of course, to the kits and uses which are the subject of the invention.

[0107] The term "primer" or "amplification primer" means a nucleotide fragment which may consist of 5 to 100 nucleotides, preferably 15 to 30 nucleotides, and having a hybridization specificity with a target nucleotide sequence, under conditions determined for the initiation of an enzymatic polymerization, for example in an enzymatic amplification reaction of the target nucleotide sequence. Generally, "primer pairs" are used, consisting of two primers. When it is desired to carry out the amplification of several different biomarkers (e.g. genes), several different primer pairs are preferably used, each preferably having a capacity to hybridize specifically with a different biomarker.

[0108] The person skilled in the art is thus able, from the sequence of the gene and in particular the corresponding transcripts, to determine the primers and probes necessary for the amplification, in particular for the amplification of one or more mRNA transcripts of said gene.

[0109] The term "probe" or "hybridization probe" means a nucleotide fragment typically consisting of 5 to 100 nucleotides, preferably 15 to 90 nucleotides, even more preferably 15 to 35 nucleotides, having a hybridization specificity under determined conditions to form a hybridization complex with a target nucleotide sequence. The probe also comprises a reporter (such as a fluorophore, an enzyme or any other detection system), which will allow the detection of the target nucleotide sequence. In the present invention, the target nucleotide sequence may be a nucleotide sequence included in a messenger RNA (mRNA) or a nucleotide sequence included in a complementary DNA (cDNA) obtained by reverse transcription of said mRNA. When it is desired to target several biomarkers (egdifferent genes), several different probes are preferably used, each preferably having the capacity to hybridize specifically with a different biomarker.

[0110] "Hybridization" means the process in which, under appropriate conditions, two nucleotide fragments, such as for example a hybridization probe and a target nucleotide fragment, having sufficiently complementary sequences, are capable of forming a double strand with stable and specific hydrogen bonds. A nucleotide fragment "capable of hybridizing" with a polynucleotide is a fragment capable of hybridizing with said polynucleotide under hybridization conditions, which can be determined in each case in a known manner. The hybridization conditions are determined by the stringency, i.e. the strictness of the operating conditions. The higher the stringency, the more specific the hybridization is. Stringency is defined in particular as a function of the base composition of a probe / target duplex, as well as by the degree of mismatch between two nucleic acids. Stringency can also be a function of the reaction parameters, such as the concentration and type of ionic species present in the hybridization solution, the nature and concentration of denaturing agents and / or the hybridization temperature. The stringency of the conditions under which a hybridization reaction must be carried out will depend mainly on the hybridization probes used.All of these data are well known and the appropriate conditions can be determined by those skilled in the art.

[0111] In general, depending on the length of the hybridization probes used, the temperature for the hybridization reaction is between about 20 and 70°C, in particular between 35 and 65°C in a saline solution at a concentration of about 0.5 to 1 M. A step of detecting the hybridization reaction is then carried out.

[0112] According to a particular embodiment, at least one reference value is stored on a computer-readable medium, for example a barcode, and / or can be used in the form of a computer-executable code configured to compare the level of ADGRE3 transcripts determined using the determination means, or data obtained from said level of transcripts of the ADGRE3 gene, to said reference value.

[0113] According to this embodiment, the reference value may correspond to the transcript level, preferably the mRNA level, of ADGRE3 in a subject having no infection with a respiratory virus or having an infection with a respiratory virus but having survived within 37 days following the infection. The reference value may also correspond to the average of the transcript level, preferably the mRNA level, of ADGRE3 in a population of subjects having no infection with a respiratory virus or having an infection with a respiratory virus but having survived within 37 days following the infection. infection with a respiratory virus but having survived within 37 days of infection.

[0114] According to another embodiment, the kit further comprises at least one reference level for the marker gene ADGRE3, stored on a computer-readable medium, for example a barcode and / or used in the form of a computer-executable code configured to compare the level of transcripts of ADGRE3 determined by the means of determining said reference level, with said reference level which is, preferably, the level of transcripts of ADGRE3, in a subject not infected with a respiratory virus, or in a population of such subjects.

[0115] According to another embodiment, the kit further comprises a positive control sample which is a sample calibrated to contain the amount of ADGRE3 which corresponds to the amount or concentration representative of the level of expression measured in a pool of samples from subjects known not to have a respiratory virus infection, and / or a negative control sample which is a sample calibrated to contain the amount of ADGRE3 which corresponds to the average amount measured in a pool of samples from subjects known not to have survived, in particular within 37 days, following a respiratory virus infection.

[0116] According to another embodiment, the kit further comprises a positive control sample which is a sample calibrated to contain the amount of ADGRE3 which corresponds to the amount or concentration representative of the level of expression measured in a pool of samples from subjects infected with a respiratory virus and known to have survived after 37 days following infection, and / or a negative control sample which is a sample calibrated to contain the amount of ADGRE3 which corresponds to the average amount measured in a pool of samples from subjects known not to have survived, in particular within 37 days, following infection with a respiratory virus.

[0117] The kit, in all its embodiments, may also comprise at least one additional means for determining the level of expression of one or more additional genes selected from C3AR1, CD177, OAS2, CIITA, IL-10, IL1 R2, CD74, TDRD9 and combinations thereof.

[0118] According to this embodiment, the kit may comprise, in the same manner as defined previously, a reference value of the level of expression of the additional genes and / or control samples (positive or negative) of said additional genes. The means for determining the level of expression may be specific tools or reagents as defined previously, for example primers or probes.

[0119] According to a preferred embodiment, the kit may also comprise, in addition to a means for determining the level of expression of ADGRE3, at least one other additional means for determining the level of expression, from a biological sample of a subject, of one or more additional genes chosen from C3AR1, CD177, OAS2, CIITA, IL-10, IL1 R2 and combinations thereof, preferably chosen from C3AR1, OAS2, CIITA, IL-10 and combinations thereof.

[0120] According to this embodiment, the kit may also comprise a reference value of the level of expression of the additional genes and / or control samples (positive or negative) of said additional genes.

[0121] Another object concerns the use of an in vitro or ex vivo measurement kit as defined above to determine the risk of death in a subject infected with a respiratory virus.

[0122] The invention also relates to in vitro or in vivo determination methods for determining the risk of death in a subject infected with a respiratory virus as defined by the present description, which further comprise a step of treating the infection of said respiratory virus.

[0123] In particular, treatment may be initiated if it is concluded that there is a risk of death in the said subject infected by a respiratory virus.

[0124] Treatment may involve administering a suitable antiviral medication. Examples of antiviral treatments include Iopinavir®, Ritonavir®, and recombinant interferons, including interferon beta, alpha, and lambda. Numerous therapeutic treatments for respiratory viruses, including those responsible for COVID-19, are currently being tested (Canedo-Marroquîn, G. et al., 2020).

[0125] According to a particular embodiment, the treatment step consists of administering one or more monoclonal antibodies to said subject. Examples of such antibodies are casirivimab, imdevimab, regdanvimab, tixagevimab or cilgavimab. Preferred treatments may consist of the combination of casirivimab and imdevimab (Ronapreve), or the combination of tixagevimab and cilgavimab.

[0126] Another subject matter relates to a method comprising the following steps: - obtain a biological sample of blood, preferably whole blood, from a subject infected with a respiratory virus which is SARS-CoV-2, - bringing said biological sample into contact with reagents specific for the expression products of the genes ADGRE3, C3AR1, CD177, OAS2, CIITA, IL-10, IL1 R2, CD74, TDRD9, preferably ADGRE3, C3AR1, CD177, OAS2, CIITA, IL-10, IL1 R2, and more preferably ADGRE3, C3AR1, CIITA, and IL-10, - measure the expression of said target genes.

[0127] The reagents specific for the expression products are selected from amplification primers, hybridization probes or antibodies, and are as defined previously.

[0128] According to a particular embodiment, the method may comprise a step of administering a suitable antiviral drug as defined previously, in particular when the expression of the target genes indicates that the subject presents a risk of death within 37 days.

[0129] Another subject matter relates to a method for determining whether a patient infected with a respiratory virus is at increased risk of death comprising the following steps: - obtain a biological sample, preferably whole blood, from a patient infected with a respiratory virus, - measure the level of expression of the ADGRE3 gene in said biological sample, - compare the level of expression of the ADGRE3 gene with a reference value obtained from patients infected with a respiratory virus and who survived, in which, if the level of expression of the ADGRE3 gene is lower than the reference value, it is determined that the patient has an increased risk of death.

[0130] According to a particular embodiment, the method also comprises measuring the level of expression of one or more additional genes as defined above and comparing the measured levels with respective reference values ​​of each additional gene also obtained from patients infected with a respiratory virus and who survived. In this case, the determination of the increased risk of death is made

[0131] Finally, another subject relates to a method comprising the quantitative measurement, in particular by RT-qPCR, of the mRNA of the ADGRE3 gene, and possibly of one or more additional genes as defined previously, in a biological sample of blood from a subject infected with a respiratory virus such as SARS-CoV-2.

[0132] The present invention is illustrated in a non-limiting manner from the following examples. Examples Materials and methods 1. Description of patient cohorts

[0133] The RICO (REA-IMMUNO-COVID) cohort is an ongoing prospective observational clinical study. In this ancillary study, 309 patients were recruited between August 2020 and August 2021 in five intensive care units of affiliated university hospitals (Hospices Civils de Lyon, France).

[0134] All patients had a lung infection due to SARS-CoV- 2. The results of this cohort have been published previously (F. Venet et al., 2021). Briefly, the inclusion criteria were as follows: (1) male or female > 18 years, (2) hospitalization in intensive care units (ICU) for SARS-CoV-2 respiratory infection, (3) first hospitalization in ICU, (4) positive diagnosis of SARS-CoV-2 infection made by PCR or another approved method in at least one respiratory sample, (5) blood sample collection within the first 24 hours after ICU admission (D-0) feasible and (6) patient or close relative having been informed of the terms of the study and not having objected to their participation.

[0135] Exclusion criteria were pregnancy, institutionalized patients, and inability to obtain informed consent.

[0136] From this cohort, samples were collected and analyzed on D-0. Patients had a mean age of 65 years [InterQuartile Range (IQR), 57-72] and a disparate distribution of men and women (68 / 32).

[0137] Healthy volunteer cohort: Blood samples from 49 healthy volunteers were independently obtained from the Etablissement Français du Sang (EFS, Lyon, France). Healthy donors were aged 40 years [IQR, 27-54] with a heterogeneous male / female distribution. All samples were collected in April 2020 and November 2021. 2. Transcriptome analysis

[0138] Blood samples were collected in PAXgene® tubes (ref. 762165, PreAnalytiX GmbH Hombrechtikon Switzerland) on D0 following the manufacturer's recommendations. Briefly, samples were left at room temperature for 2 h with the reagents present in the tubes before being transferred to -20°C for at least 24 h, then stored at -80°C.

[0139] Samples were tested using a FilmArray® pouch optimized to detect genes involved in host response, including mRNAs of genes such as ADGRE3, by nested PCR. The pouches are analyzed using the FilmArray®Torch instrument (BioFire, USA) following the manufacturer's recommendations. Expression level results are obtained automatically, in less than one hour, before being compiled for analysis.

[0140] Normalized expression values ​​of the markers (relative to the reference genes DECR1, HPRT1 and PPIB) were calculated and used for the analyses. 3. Measurements of immunological markers

[0141] The number of CD3+ T lymphocytes was determined on an automated volumetric flow cytometer (Aquios CL, Beckman Coulter).

[0142] Standardized values ​​(Antibody / Cells or Ac / C) of HLA-DR expression by monocytes (mHLA-DR) were obtained with a flow cytometer (Navios, Beckman Coulter) with HLA-DR Quantribite reagents (Becton Dickinson) as previously described (F. Venet et al., 2021). 4. Statistical analyses

[0143] The RICO cohort was randomly divided to obtain two datasets balanced according to 3 parameters: age, sex and mortality. Thus, a first dataset of 216 patients was used for machine learning and an independent test dataset of 93 patients was used for performance validation. For the description of the datasets, qualitative data were reported as numbers or frequency and quantitative data were reported as median [IQR interval],

[0144] Clinical characteristics were compared using the nonparametric Mann-Whitney-Wilcoxon test for continuous variables, and Fisher's exact test or chi-square test (where appropriate) for categorical variables. The significance level was set at 5% for two-sided tests. Statistical analyses were performed using R software, version 3.6.2. Data were centered and reduced to perform unsupervised principal component analysis via the FactoMineR package (version 2.4).

[0145] Genes significantly associated with 28-day mortality in a univariate logistic regression model were used to build multivariate models for predicting 28-day survival. The trained models were logistic regression models with L1 (Lasso), L2 (Ridge), and mixed (ElasticNet) regularization, Partial Least Squares-Discriminant (PLS) analysis, and Support Vector Machines with linear kernels (linear SVM) using the CARET package (version 6.0-84).

[0146] To compensate for the unbalanced distribution of mortality in the datasets, the synthetic minority oversampling technique (SMOTE) was applied for hyperparameter tuning (NV Chawla et al., 2002).

[0147] The hyperparameters of the models were chosen due to the low incidence of the outcome of interest within the cohort (AUPRC) among the cross-validation tests (k-fold=5, number of repetitions=10) in the RICO cohort training (B. Ozenne et al., 2015), with sensitivity, specificity, F1 score and Positive Predictive Values ​​(PPV) being the parameters of interest.

[0148] In summary, among the 5 machine learning algorithms evaluated, the selected hyper parameters are: Lasso (a=1, Δ=0.031), Ridge (a=0, Δ=0.556), Elastic net (a=0.35, Δ=0.37), PLS (ncomp=1), and Linear SVM (C=0.367).

[0149] The AUPRC and its 95% bootstrap confidence interval were obtained with the PRROC (version 1.3.1) and boot (version 1.3-28) packages. The number of boostrap resamples was set to N=1000. The importance of relative variables in the Linear SVM model was calculated using the FIRM method from the vip package (version 0.3.2)(B Greenwell et al., 2018).

[0150] The area under the ROC curve (AUROC), 95% bootstrap confidence interval, and diagnostic performance (sensitivity, specificity, positive and negative predictive values, and F1 score) at the optimal thresholds for the 9 mRNA panel and individual parameters were obtained by considering the respective Youden values ​​of the cutpointr package (version 1.1.1) defined on the training dataset and then applied to the values ​​of the test dataset. The F1 score (harmonic mean of recall and precision) was used as a measure of model accuracy due to data imbalance. 5. Ethics

[0151] The RICO study protocol was approved by the Ethics Committee (Comité de Protection des Personnes Ile de France 1 - N°IRB / IORG #: IORG0009918) under agreement number 2020-A01079-30. This clinical study was registered on ClinicalTrials.gov (NCT04392401). Results 1. Clinical characteristics of patients on admission to the intensive care unit

[0152] Patient characteristics are presented in Table 3 below. A total of 309 patients were hospitalized in 5 hospitals in Lyon (FRANCE) between August 2020 and August 2021 and included in the cohort.

[0153] Briefly and as previously mentioned, 70% of patients were male. Patients were admitted to the intensive care unit with a median of 9 days after the presentation of the first symptoms [IQR, 6-11], and presented notably a median body mass index (BMI) (kg / m3) of 29.1 [IQR, 26.1-33.2], In terms of disease severity, patients presented a decreased PaO2 / FiO2 (mmHg) with a median of 97.5 [IQR: 74.3-146.5], a high SOFA score [median: 2.0; IQR: 1.0-5.0] and a SAPS II score [median: 30.0; IQR: 23.5-39.0], In addition, at admission, 17.2% of patients required invasive mechanical ventilation.

[0154] All patients were on corticosteroid treatment before or at admission (6 mg / day of dexamethasone).

[0155] Overall, patients spent a median hospital stay of 18 days [IQR, 11.0-31.8], including 8 days [IQR, 4.0-17.0] in intensive care. One-third of the cohort developed secondary infections, most of which were pneumonias (87 / 99), 16 of which were fungal.

[0156] Finally, among the 309 patients, 52 (17%) died at the end of the 28th day after admission.

[0157] [Table 3] Comorbidities Diabetes 213 (69%) 131 (73.2%) 18 (48.7%) 0.001 55 (70.5%) 9 (60.0%) 0.534 none - n (%) Diabetes: with deterioration - n 15 (4.9%) 6 (3.4%) 6 (16.2%) 3 (3.9%) 0 (0.0%) (%) Diabetes: without deterioration 81 (26.2%) 42 (23.4%) 13 ( 35.1%) 20 (25.6%) 6 (40.0%) organic - n (%) Charlson score - 1.0 1.0 2.0 .0-2.0] [0.0-1.0] [1.0-4.0] <0.001 0.5 1.0 points [0 [0.0-1.0] [1.0-2.0] 0.043 Clinical severity on admission Time from first symptoms to ICU admission 9.0 9.0 7.5 [6.0-11.0] [7.0-12.0] [5 0.013 9.0 6.0 0.041 .0-9.8] [6.8-10.3] [5.5-9.0] Days SOFA score - 2.0 2.0 4.0 .0-5.0] [2.0-6.0] 0. 2.0 3.0 points [1.0-5.0] [0 008 [0.3-3.0] [1.5-7.5] 0.068 SAPS II score - 30.0 30.0 39.0 points [23.5-39.0] [23.0-38.8] [33.0-47.0] <0.001 27.5 32.0 [21.0-34.0] [26.8-41.3] 0.086 PaO2 / FIO2- 97.5 95.0 82.0 98.0 104.5 mmHg [74.3-146.5] [77.5-146.0] [70.5-147.8] 0.376 [89.0- [93.8- 0.844 149.0] 128.3] 7.45 7.46 7.44 [7.42-7.49] [7.42-7.49] [7.40-7.49] 0.591 7.46 7.47 pH [7.43-7.49] [7.40-7.49] 0.769 Lactate - mmol / L 1.65 1.70 1.90 1.50 1.40 [1.30-2.00] [1.37-2.02] [1.40-2.20] 0.326 [1.30-1.90] [1.30-1.80] 0.785 Organ support Invasive mechanical ventilation at day 0 53 (17.2%) 29 (16.2%) 10 (27%) 0.186 9 (11.5%) 5 (33.3%) 0.046 n (%) Vasoactive drugs - n 35 (11.4%) 19 (10.7%) 8 (21.6%) 0.120 6 (7.7%) 2 (13.3%) 0.611 (%) Renal replacement therapy 31 (10.0%) 13 (7.3%) 11 (29.7%) <0.001 4 (5.1%) 3 (20.0%) 0.080 - n (%) Follow up MV duration - 14.0 17.0 12.0 22.5 12.0 days [7.0-27.3] [7.0-34.0] [7.0-20.0] 0.110 [11.3-30.8] [6.5-15.5] 0.030 Days in Units 8.0 8.0 12.0 8.0 11.0 of Care 0.017 0.871 [4.0-17.0] [3.0-16.0] [8.0-19.0] [5.0-16.8] [5.5-15.5] Intensive 20.5 18.0 18.0 15.0 14.0 Days in hospital 0.024 [13.0- 0.014 [11.0-31.8] [10.0-36.5] [9.0-21.0] [7.5-18.5] 34.8] Mortality at 28 15 52 (16.8%) 0 ( 0%) 37 (100%) <0.001 0 ( 0%) <0.001 days - n (%) (100%) Mortality at 90 15 66 (21.9%) 12 (6.8%) 37 (100%) <0.001 2 (2.7%) <0.001 days - n (%) (100%) Infections acquired in 19 (54.3%) 0.018 (20 1 .0 5 %) (66 1 .7 0 %) ICU - n (%) <0 001 ICU-acquired pneumonia - 18 / 19 (94.7%) 0.366 (12 / 15 9 / 100 504 n (% IAI) 80.0%) (90.0%) Immunological parameters at admission

[0158] Medians and interquartile ranges [Q1-Q3] are shown for continuous variables, while numbers and percentages are presented for categorical variables. Patients with COVID-19 were separated into two groups based on their survival status at 28 days after admission. Sequential Organ Failure (SOFA) and Simplified Acute Physiology II (SAPS II) scores were calculated during the first 24 hours after admission. Acute respiratory distress on admission was based on the Berlin definition. Data were compared using the non-parametric Mann-Whitney test for continuous variables or the Fisher exact test for categorical variables. 2. Association between biomarker expression and patient mortality at 28 days post admission to a healthcare facility

[0159] Using univariate logistic regression analysis on a dataset of 216 patients, the ADGRE3 gene was identified as significantly associated with 28-day mortality in patients infected with respiratory viruses. In particular, the results show a significant underexpression of ADGRE3 in non-surviving patients compared to surviving patients. The results are presented in Table 4 below.

[0160] [Table 4] ORIQR [95% Cl] IQR p value ADGRE3 0.66 [0.45-0.93] 1.17 0.021 mHLA-DR 0.97 [0.67-1.30] 6246 0.856 [antibodies / cells] L J CD3 T cells o.56 [0.31 -0.90] 258.5 0.031 [cells / pL] L J

[0161] 216 patients were included in the training set, 179 survived to day 28, and 37 died. The association between 28-day survival status and ADGRE3 gene or classical immune parameters was performed by implementing univariate logistic regression models. To allow comparison between models, the odds ratios calculated for ADGRE3 and each immune parameter were standardized to an increment from the first to the third quartile (odd ratios inter quartile range, ORIQR). P values ​​< 0.05 are highlighted in bold.

[0162] Among the cellular parameters, the measurement of mHLA-DR is not significantly associated with patient mortality at 28 days, thus demonstrating the full interest of the ADGRE3 biomarker.

[0163] The ADGRE3 biomarker was then used in the 5 learning models in combination with one or more additional biomarkers to validate signatures predictive of the risk of death of patients at 28 days post-admission.

[0164] The performance results are presented in the tables below:

[0165] Combination of ADGRE3 and IL-10

[0166] [Table 5]

[0167] Combination of ADGRE3 and CIITA

[0168] [Table 6]

[0169] Combination of ADGRE3 and C3AR1

[0170] [Table 7]

[0171] Combination of ADGRE3 and OAS2

[0172] [Table 8]

[0173] Combination of ADGRE3, IL-10 and CD74

[0174] [Table 9]

[0175] Combination of ADGRE3, C3AR1 and CIITA

[0176] [Table 10]

[0177] Combination ADGRE3, C3AR1, IL-10

[0178] [Table 11]

[0179] Combination of ADGRE3, C3AR1 and OAS2

[0180] [Table 12]

[0181] Combination of ADGRE3, CIITA and IL-10

[0182] [Table 13]

[0183] Combination of ADGRE3, CIITA and OAS2

[0184] [Table 14]

[0185] Combination of ADGRE3, OAS2 and IL-10

[0186] [Table 15]

[0187] Combination of ADGRE3, IL-10, CD74 and CIITA

[0188] [Table 16]

[0189] Combination of ADGRE3, C3AR1, CIITA and IL-10

[0190] [Table 17]

[0191] Combination of ADGRE3, C3AR1, CIITA and OAS2

[0192] [Table 18]

[0193] Combination of ADGRE3, C3AR1, OAS2 and IL-10

[0194] [Table 19]

[0195] Combination of ADGRE3, OAS2, CIITA and IL-10

[0196] [Table 20]

[0197] Combination of ADGRE3, C3AR1, CIITA, IL-10 and OAS2

[0198] [Table 21]

[0199] Combination of ADGRE3, C3AR1, CD177, IL10, CIITA, IL1R2 and OAS2

[0200] [Table 22]

[0201] Finally, the models were applied to a signature containing the biomarker ADGRE3 and the eight additional biomarkers C3AR1, CD177, IL10, CIITA, IL1 R2, OAS2, CD74 and TDRD9:

[0202] [Table 23] UROCfraining AUPRCtrainin AUROCtest AUPRCtest [95% CI] [95% CI] [95% CI] [95% CI] 0.715 0.361 0.721 0.380 Elastic Net [0.575-0.844] [0.243-0.524 0.493-0.938] [0.171-0.662] 0.737 0.406 0.751 0.326 Ridge [0.612-0.859] [0.257-0.584 0.575-0.927] [0.164-0.558] 0.754 0.402 0.748 0.346 Lasso [0.630-0.874] [0.256-0.576 0.554-0.932] [0.168-0.620] 0.732 0.406 0.744 0.312 PLS [0.605-0.853] [0.256-0.579 0.567-0.924] [0.156-0.566] 0.744 0.431 0.764 0.431 svmLin [0.600-0.881] [0.278-0.610 0.536-0.960] [0.214-0.720]

[0203] The overall performance thus demonstrates that the measurement of the level of expression of ADGRE3, particularly in combination with the measurement of the level expression of one or more specific additional genes can determine whether a patient infected with a respiratory virus such as SARS-CoV-2 is at increased risk of death.

[0204] Preferably, the most advantageous biomarker combinations for predicting the increased risk of death in a subject infected with a respiratory virus are those for which the majority of the learning models make it possible to obtain an AUROC of at least 0.7. In addition, since the mortality in the test data set is 17%, it is particularly advantageous to also target the combinations for which the majority of the models make it possible to obtain an AUROC of at least 0.3, preferably at least 0.32. BIBLIOGRAPHICAL REFERENCES

[0205] C. Huang, et al., “Clinical features of patients infected with 2019 novel coronavirus in Wuhan, China” Lancet, 2020, 395, 497, https: / / doi.Org / 10.1016 / S0140-6736(20)30183-5;

[0206] EZ Ong, et al.,”A Dynamic Immune Response Shapes COVID-19 Progression” Cell Host Microbe, 2020, 27 (6), 879, https: / / doi.Org / 10.1016 / j.chom.2020.03.021;

[0207] F. Venet et al., « Longitudinal assessment of IFN-I activity and immune profile in critically ill COVID-19 patients with acute respiratory distress syndrome”; Crit Care, 2021 , 25 (1 ), 140, https: / / doi.org / 10.1186 / s13054-021 -03558-w;

[0208] Hadjadj, J. et al. “Impaired type I interferon activity and inflammatory responses in severe COVID-19 patients”. Science 369, 718-724 (2020);

[0209] M. S. Abers, et al., “An immune-based biomarker signature is associated with mortality in COVID-19 patients” JCI Insight, 2021 , 6 (1 ):e144455, https: / / doi.Org / 10.1172 / jci. insight.144455;

[0210] Guardela et al., “50-gene risk profiles in peripheral blood predict COVID-19 outcomes: A retrospective, multicenter cohort study” EBioMedecine, 2021 , https: / / doi.Org / 10.1016 / j.ebiom.2021 .103439;

[0211] Y. Levy et al., “CD177, a specific marker of neutrophil activation, is associated with coronavirus disease 2019 severity and death” Iscience, 2021 , 24(7):102711 DOI: 10.1016 / j. isci.2021 .102711 ;

[0212]

[0169] G. Canedo-Marroquîn et al., “SARS-CoV-2: Immune Response Elicited by Infection and Development of Vaccines and Treatments” Front. Immunol, 2020, 11 :569760; DOI 10.3389 / fimmu.2020.569760;

[0213] Chawla, N. V et al.,. SMOTE: synthetic minority over-sampling technique. J. Artif. Int. Res. 16, 321-357 (2002);

[0214] Ozenne B. et al, “The precision-recall curve overcame the optimism of the receiver operating characteristic curve in rare diseases”. J Clin Epidemiol 68, 855- 859, doi: 10.1016 / j.jclinepi.2O15.02.010 (2015);

[0215] Greenwell, et al., “A Simple and Effective Model-Based Variable Importance Measure”. ArXiv abs / 1805.04755 (2018) ;

Claims

Claims

1. An in vitro or ex vivo method for determining the risk of death in a subject infected with a respiratory virus, said method comprising the following steps: a) measuring in a biological sample of said subject the level of expression of the ADGRE3 gene, b) comparing the level of expression of the ADGRE3 gene measured in step a) with a predetermined reference value.

2. Method according to claim 1, characterized in that the respiratory virus is SARS-CoV-2 or one of its variants.

3. Method according to claim 1 or 2, characterized in that it also comprises a step c) of conclusion as to the increased risk of death in said subject on the basis of the result of the comparison when an under-expression of said gene at the mRNA level is highlighted.

4. Method according to one of claims 1 to 3, characterized in that the reference value corresponds to the average of the level of expression of ADGRE3 obtained from biological samples from a population of subjects not showing any infection by a respiratory virus or to the average of the level of expression of ADGRE3 obtained from biological samples from a population of subjects infected by a respiratory virus and who are known to have survived after infection, in particular in the 28 days following admission to a healthcare establishment.

5. Method according to one of claims 1 to 4, characterized in that it further comprises a step of measuring in the biological sample of said subject the expression of at least one other additional gene chosen from C3AR1, CD177, OAS2, CIITA, IL-10, IL1 R2, CD74, TDRD9 and their combinations.

6. Method according to claim 5, characterized in that it further comprises a step of measuring in the biological sample of said subject the expression of at least one other additional gene chosen from C3AR1, CD177, OAS2, CIITA, IL-10, IL1 R2 and combinations thereof, and preferably among C3AR1, CIITA, CD177, IL-10 and combinations thereof.

7. Method according to one of claims 1 to 6, characterized in that the expression of the gene(s) is measured at the mRNA level.

8. Method according to one of claims 1 to 7, characterized in that the biological sample is a blood sample, preferably a whole blood sample.

9. Method according to one of claims 1 to 8, characterized in that the expression is measured by amplification, sequencing or hybridization.

10. Method according to one of claims 1 to 9, characterized in that the expression is measured by amplification via RT-PCR, preferably RT-qPCR.

11. Method according to one of claims 1 to 10, characterized in that the expression is normalized relative to the expression of one or more housekeeping genes.

12. Kit for the in vitro or ex vivo measurement of the expression of ADGRE3 in a biological sample comprising means for determining the level of expression of ADGRE3 in said sample, said means being chosen from amplification primers or probes.

13. Kit according to claim 12, characterized in that it comprises a positive control sample calibrated to contain the quantity of ADGRE3 which corresponds to the quantity or concentration representative of the level of expression measured at the mRNA level in a pool of samples from subjects not presenting an infection by a respiratory virus, and / or a negative control sample calibrated to contain the quantity of ADGRE3 which corresponds to the average quantity measured in a pool of samples from subjects who did not survive following an infection by a respiratory virus.

14. Kit according to claim 12 or 13, characterized in that it comprises means for determining the level of expression of at least one other additional gene selected from C3AR1, CD177, OAS2, CIITA, IL-10, IL1 R2, CD74, TDRD9 and combinations thereof, preferably selected from C3AR1, CD177, CIITA, I L-10, IL1 R2 and combinations thereof, and more preferably selected from C3AR1, CIITA, IL-10 and combinations thereof.

15. Use of the kit according to one of claims 12 to 14 for determining the risk of death, preferably the risk of death within 28 days after admission to a healthcare establishment, of a subject infected with a respiratory virus such as SARS-CoV-2 or one of its variants.

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