Determining the risk of death of a subject infected with a respiratory virus by measuring the expression level of the cd74 gene

Abstract

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, for example SARS-CoV-2, comprising a measurement, in a biological sample of said subject, of the level of expression of the CD74 gene; the invention also relates to associated kits.

Description

Title of the invention: Determination of the risk of death of a subject infected by a respiratory virus by measuring the level of expression of the CD74 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 risk of death of a subject infected with a respiratory virus, in particular with a respiratory virus such as SARS-CoV-2 or one of its variants. PRIOR TECHNIQUE

[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] Despite this, in severe COVID-19 patients, the immune response can be defined by impaired inflammatory and immune responses with marked lymphopenia, elevated neutrophil and monocyte counts, decreased monocyte HLA-DR expression, moderate plasma cytokine storm, inadequate type I interferon signaling response, and downregulation of IFN-stimulated genes (ISG) (F. Venet et al., 2021).

[0006] Over the months, several research groups have tried to identify the risk factors characteristic of a severe course of COVID-19 disease in order to identify patients at high risk of developing said severe forms.

[0007] For example, in a study combining about fifty clinical characteristics and about two hundred high-level immunological characteristics dimensionality, three distinct immunotypes associated with disease severity have been described (Mathew et al., 2020).

[0008] In a large immune assessment study combining cellular data obtained by flow cytometry, soluble immune markers (multiplex cytokine analysis), RNA expression (Nanostring) and serology (ELISA), a central immune signature of peripheral blood in patients with COVID-19 was discovered, which could help identify immunopathology parameters, correlate with disease severity but also anticipate clinical progression (Laing et al., 2020).

[0009] More recently, it has also been established that the longitudinal trajectories of 11 circulating immune-based biomarkers could be associated with patient mortality when increased (10) or decreased (1), thus providing initial evidence that immune-based biomarkers could provide early warning of the outcome of COVID-19 patients (Abers et al., 2021). Gene expression profiles have also been described to predict the outcome of COVID-19 patients (Guardela B et al., 2021).

[0010] In this context, the biomarker CD177 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 (Lévy Y et al., 2021).

[0011] Nevertheless, and in view of the global prevalence, there is still a need to identify new biomarkers offering other alternatives to effectively predict the risk of mortality in subjects infected with respiratory viruses, particularly respiratory viruses responsible for COVID-19, in order to be able to adapt treatment, preferably early, through guided therapies and thus improve their chances of survival. SUMMARY OF THE INVENTION

[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, comprising a step of measuring, in a biological sample of said subject, the level of expression of the CD74 gene, and optionally the expression of at least one other additional gene chosen from TDRD9, IL1 R2 and / or CD177.

[0013] Advantageously, the method according to the invention may also further comprise the measurement of the expression of one or more additional genes involved in the host response such as those listed in Table 2.

[0014] The method according to the invention thus makes it possible in a completely advantageous manner to determine the risk of death of a subject infected by a respiratory virus, in particular a respiratory virus such as SARS-CoV-2 or one of its variants.

[0015] The conclusion of the risk of death is established in particular by comparing the level of expression of CD74 with that of a reference value which may correspond to the average of the level of expression of CD74 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 CD74 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 infection.

[0016] In particular, based on the result of comparing the level of CD74 expression with a reference value, a conclusion regarding the presence of an increased risk of death in the subject is made as soon as a decrease in expression is identified.

[0017] Advantageously, the method according to the invention comprises the determination of the expression of the CD74 gene and the additional IL1 R2 gene. Thus, the method according to the invention comprises the following steps of: measuring the level of expression of the CD74 gene and the additional IL1 R2 gene, comparing the level of expression of said genes for said biological sample or a value derived from this quantity, to a predetermined threshold value corresponding to the average of the level of expression of said genes 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 said genes obtained from biological samples from a population of subjects infected by a respiratory virus and known to have survived after infection, in particular within 28 days following infection, and concluding as to the risk of death, from the result of the comparison.

[0018] The process thus makes it possible to conclude that there is an increased risk of death for the subject when under-expression of CD74 and over-expression of IL1 R2 is demonstrated in the biological sample to be tested.

[0019] Advantageously, the method according to the invention comprises determining the expression of the CD74 gene and the additional genes IL1 R2 and CD177. Thus, the method according to the invention comprises the following steps: measuring the level of expression of the CD74 gene and the additional genes IL1 R2 and CD177. comparing the level of expression of said genes for said biological sample or a value derived from this quantity, to a predetermined threshold value corresponding to the average of the level of expression of said genes 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 said genes obtained from biological samples from a population of subjects infected by a respiratory virus and known to have survived after infection, in particular within 28 days following infection, and concluding as to the risk of death, from the result of the comparison.

[0020] The process thus makes it possible to conclude that there is an increased risk of death for the subject when under-expression of CD74 and over-expression of IL1 R2 and CD177 is demonstrated in the biological sample to be tested.

[0021] Another subject of the invention relates to a kit for the in vitro or ex vivo measurement of the expression of CD74 in a biological sample comprising means for determining the level of expression of CD74 in said sample. The kit may also comprise means for determining the level of expression of at least one other additional gene selected from TDRD9, IL1 R2 and CD177, and means for determining at least one gene involved in the host response such as those listed in Table 2.

[0022] Advantageously, the kit may in particular comprise a negative control sample calibrated to contain the quantity of CD74 which corresponds to the quantity or concentration representative of the level of expression measured in a pool of samples from subjects not presenting an infection by a respiratory virus, and / or a positive control sample calibrated to contain the quantity of CD74 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.

[0023] Another subject of the invention relates to the use of the kit for determining the risk of death, preferably the risk of death within 28 days post-infection, of a subject infected with a respiratory virus such as SARS-CoV-2 or one of its variants. DESCRIPTION OF FIGURES

[0024] Figure 1 is a plot of variable importance in different machine learning models used to predict 28-day mortality in the RICO subcohort. CD74, TDRD9, and ILR2 were identified in all 5 models, and CD74 performed well. CD177 was not captured by the Lasso model but was the fourth marker of interest identified by cumulative variable importance.

[0025] Figure 2 shows violin plots representing the expression of CD74 and additional genes associated with mortality identified by the machine learning models. P-values ​​were calculated using a Wilcoxon test. On the left are healthy volunteers, in the middle are patients alive at day 28 after admission, and on the right are those who died before day 28.

[0026] Figure 3 depicts the associations between CD74, CD177, TDRD9, and I L1 R2 mRNA levels measured at admission and the occurrence of death within 28 days of admission. Cumulative events unadjusted for death were obtained with Kaplan-Meier estimates. Patients were stratified into 2 groups based on the Youden threshold determined for each mRNA for the prediction of 28-day mortality in the total patient population at admission. The p-value of the Log-Rank test is shown. DETAILED DESCRIPTION OF THE INVENTION

[0027] Certain terms and expressions used in the context of the invention are detailed below.

[0028] 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, comprising a step of measuring, in a biological sample of said subject, the level of expression of the CD74 gene.

[0029] Surprisingly, it was found that measuring CD74 gene expression could determine the 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.

[0030] This is all the more surprising since in the same infected subject, the standard immunological parameters measured on admission to intensive care and used as references by practitioners, namely monocytic HLA-DR and interleukins Plasma IL-6 and IL-10, are not significantly associated with mortality at 28 days after admission. This demonstrates the value of measuring the level of expression of the CD74 gene and additional genes according to the invention.

[0031] The method which is the subject of the invention has the advantage of being able to easily assess the risk of death of a subject, for example a patient admitted to the intensive care unit or to the emergency room, by having a directly measurable biomarker and the measurement of which can be carried out directly in the healthcare establishment receiving them or in a nearby laboratory. In addition, the measurement of the CD74 biomarker, or of the other additional biomarkers of the invention, is entirely suitable for being carried out by automated analysis machines or by so-called rapid tests.

[0032] A biomarker (or "marker") is 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.

[0033] The CD74 gene is located on chromosome 5 and encodes the MHC II y chain, or fragment li (or ly), also known as CD 74 (Cluster of Differentiation 74). It is the invariant fragment li, a polypeptide that allows the formation, maturation, and transport of MHC II during its formation in antigen-presenting cells. More specifically, CD74 is heavily involved in antigen presentation and CD4+ T cell activation (Cresswell, 1994). It also serves as a cell surface receptor for the cytokine macrophage migration inhibitory factor (MIF) (Farr et al., 2020), which, when bound to the encoded protein, initiates survival pathways and cell proliferation.

[0034] The CD74 gene sequence is available from NCBI under reference NC_000005.10

[0035] 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.

[0036] Advantageously, the method according to the invention makes it possible to determine the risk of death in a subject within 28 days following the day on which the infection is confirmed, also called post-infection days, especially by a respiratory virus detection test.

[0037] 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.

[0038] 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 unit, in an intensive care unit (ICU) or in a continuing care unit, and in particular a patient in an ICU.

[0039] 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.

[0040] 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 serious 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 CD74.

[0041] 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.

[0042] 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 / ).

[0043] 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 contains twelve amino acid mutations in the spike protein, in particular the N501Y, E484K, K417T mutations. The South African variant (Beta variant) called 501Y.V2 and belonging to the B.1.351 lineage also contains different mutations, including three, K417N, E484K and N501Y, located in the RBD domain of the spike protein, the receptor binding domain.The Indian variant, dubbed the Delta variant, 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.

[0044] 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.

[0045] 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.

[0046] By "biological sample" we mean any sample from a subject, and which may be of different natures, such as blood or its derivatives, sputum, urine, stool, 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, placental tissue, gastrointestinal tract tissue, genital tract tissue, or central nervous system tissue.

[0047] 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 peripheral blood mononuclear cells (or PBMC, containing B lymphocytes, T lymphocytes, NK cells, dendritic cells and monocytes), B cell subpopulations, purified monocytes, or neutrophils.

[0048] 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.

[0049] 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.

[0050] 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.

[0051] 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 CD74 gene.

[0052] 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.

[0053] The term "transcript" refers to RNA, and in particular messenger RNA (mRNA), produced by gene transcription. More precisely, transcripts are the RNAs produced by the transcription of a gene followed by post-transcriptional modifications of the pre-RNA forms.

[0054] According to a preferred embodiment, the measurement of the expression level of CD74, and possibly that of one or more other additional genes as defined 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 CD74 gene therefore concerns the determination of the mRNA level of said gene.

[0055] 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, in particular 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).

[0056] 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 BiomarkTM 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.

[0057] According to a particular embodiment, the measurement of the expression of CD74 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.

[0058] According to a particular embodiment, the level of expression of CD74 is measured by quantitative RT-qPCR detection of CD74 mRNA transcripts.

[0059] Measuring the expression level allows to determine the quantity of one or more CD74 transcripts in the biological sample or also to give a derived value.

[0060] Thus, according to a particular embodiment, the level of expression of the CD74 gene is a value derived from the quantity of its transcripts. 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. It 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). As examples of genes of household, we can cite the genes DECR1, HPRT1, PPIB, RPLPO, PPIA, GLYR1, RANBP3, 18S, B2M, TBP, GAPDH and ACTB.

[0061] Generally speaking, in the method according to the invention, whatever its embodiments, the level of expression of CD74, 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.

[0062] According to a particular embodiment, the expression of CD74 can in fact be 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, 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 and ACTB, and more preferably, selected from DECR1, HPRT1 and PPIB.

[0063] 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 the 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.

[0064] In the case where the level of CD74 transcripts 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.

[0065] According to a particular embodiment, the level of expression of the CD74 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 transcripts, in particular mRNA, of said CD74 gene with a predetermined reference threshold value, shows that there is a difference with said reference threshold value corresponding to said CD74 gene. In particular, said difference corresponds to a level of transcripts for the biological sample to be tested which is lower than that corresponding to the threshold value.

[0066] Advantageously, according to a first variant, said threshold value corresponds to a reference expression level of said CD74 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 threshold value can also correspond to the average of the expression level of CD74 transcripts obtained from biological samples from a population of subjects not presenting any infection by a respiratory virus.

[0067] According to a second variant, the threshold value corresponds to a reference expression level of the CD74 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 28 days following infection. According to this variant, the threshold value may also correspond to the average expression level of CD74 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 28 days following infection.

[0068] Preferably, said difference between the level of transcripts of the CD74 gene determined in the test sample and the reference level of said gene (threshold 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 level, or reference threshold value, of said CD74 gene.

[0069] The reduction considered relevant to provide a conclusion will depend on the threshold value considered, and in particular on the reference level used as the reference threshold value, and may be adapted by a person skilled in the art. In particular, if the reference threshold value corresponds to the reference level of said CD74 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, or in a subject or population of subjects presenting an infection but having survived within 28 days following the infection, it may be sufficient for the level of CD74 transcripts determined in the test sample to be simply lower than the said reference threshold value.

[0070] 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 CD74 is highlighted in the biological sample to be tested.

[0071] By "underexpression" is meant a significant decrease in the level of expression compared to a reference threshold value. A person skilled in the art is able to determine the statistical test to be used to determine this reference threshold value with which the level of expression of CD74 must be compared. The exemplary embodiments present one of the possible methods.

[0072] According to this particular embodiment, the method can thus comprise the following steps: - determine the level of expression of the CD74 gene by measuring the quantity of at least one CD74 transcript in the biological sample of the subject infected with respiratory virus, - 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 on the presence of an increased risk of death, based on the result of the comparison.

[0073] According to this embodiment, the reference sample may be, for example, a sample from one subject or a mixture of samples from several subjects, said subjects being uninfected by a respiratory virus. In this case, the conclusion on the presence of an increased risk is made when the comparison of the quantity of transcripts with the reference value highlights a significant decrease.

[0074] The reference value may in particular correspond to an average value of the level of expression of CD74 measured from several samples, each from different subjects not infected by a respiratory virus.

[0075] 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 CD74.

[0076] 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.

[0077] Within the scope of the invention, the issuing of a conclusion as to the risk of death in the subject from whom the test sample is taken may also be carried out by any automated technique, carried out by a computer or assisted by a computer.

[0078] The method as described above, in all its embodiments, comprises, in addition to the step of measuring the expression of the CD74 gene, a step of measuring, in the biological sample of the subject, the expression of at least one, two, or three additional gene(s) selected from the following genes: TDRD9, IL1 R2 and CD177.

[0079] The reference values ​​or predetermined threshold value of the expression level of the additional genes can be determined in the same manner as the CD74 gene and as previously described.

[0080] In particular, for all embodiments of the present description, the reference value of the additional genes may advantageously correspond to the level of transcripts, preferably at the mRNA level, of said genes in a subject not presenting any infection by a respiratory virus or presenting an infection by a respiratory virus but having survived in the 28 days following the infection. The threshold value may also correspond to the average of the level of transcripts, preferably at the mRNA level, of said genes in a population of subjects not presenting any infection by a respiratory virus or presenting an infection by a respiratory virus but having survived in the 28 days following the infection.

[0081] The chromosomal locations of the additional genes are given in Table 1 below:

[0082] [Table 1]

[0083] According to a preferred variant of this embodiment, the method comprises a step of measuring, in the biological sample of the subject, the expression of the additional gene IL1 R2. According to this variant, it can be concluded that there is a risk of death, in particular an increased risk, in said subject when the comparison of the level of mRNA transcripts of the CD74 gene with a predetermined threshold value shows that there is a decrease in the level of expression or under-expression, and when the comparison of the level of mRNA transcripts of the additional gene IL1 R2 with a predetermined threshold value shows that there is an increase in the level of expression or over-expression.

[0084] According to another variant of this embodiment, the method comprises a step of measuring, in the biological sample of the subject, the expression of the additional gene TDRD9. According to this variant, it can be concluded that there is a risk of death, in particular an increased risk, in said subject when the comparison of the level of mRNA transcripts of the CD74 gene with a predetermined threshold value shows that there is a decrease in the level of expression or under-expression, and when the comparison of the level of mRNA transcripts of the additional gene TDRD9 with a predetermined threshold value shows that there is an increase in the level of expression or over-expression.

[0085] According to another variant of this embodiment, the method comprises a step of measuring, in the biological sample of the subject, the expression of the additional gene CD177. According to this variant, it can be concluded that there is a risk of death, in particular an increased risk, in said subject when the comparison of the level of mRNA transcripts of the CD74 gene with a predetermined threshold value shows that there is a decrease in the level of expression or under-expression, and when the comparison of the level of mRNA transcripts of the additional gene CD177 with a predetermined threshold value shows that there is an increase in the level of expression or over-expression.

[0086] According to a preferred variant of this embodiment, the method comprises a step of measuring, in the biological sample of the subject, the expression of the additional genes TDRD9 and IL1 R2. According to this variant, it can be concluded that there is a risk of death in said subject when the comparison of the level of mRNA transcripts of the CD74 gene with a predetermined threshold value shows that there is a decrease in the level of expression or under-expression, when the comparison of the level of mRNA transcripts of the additional gene TDRD9 with a predetermined threshold value shows that there is an increase in the level of expression or over-expression, and when the comparison of the level of mRNA transcripts of the additional gene IL1 R2 with a predetermined threshold value shows that there is an increase in the level of expression or over-expression.According to this preferred variant, increased expressions of TDRD9 and IL1 R2 and decreased expression of CD74 are significantly associated with the death of the subject within 28 days. following respiratory virus infection. Still according to this variant, the chances of survival of the subject are approximately 90% when the expressions of TDRD9 and IL1 R2 are reduced and the expression of CD74 is increased.

[0087] According to another variant of this embodiment, the method comprises a step of measuring, in the biological sample of the subject, the expression of the additional genes TDRD9 and CD177. According to this variant, it can be concluded that there is a risk of death, in particular an increased risk in said subject when the comparison of the level of mRNA transcripts of the CD74 gene with a predetermined threshold value shows that there is a decrease in the level of expression or under-expression, when the comparison of the level of mRNA transcripts of the additional gene TDRD9 with a predetermined threshold value shows that there is an increase in the level of expression or over-expression, and when the comparison of the level of mRNA transcripts of the additional gene CD177 with a predetermined threshold value shows that there is an increase in the level of expression or over-expression.

[0088] According to another variant of this embodiment, the method comprises a step of measuring, in the biological sample of the subject, the expression of the additional genes CD177 and IL1 R2. According to this variant, it can be concluded that there is a risk of death, in particular an increased risk, in said subject when the comparison of the level of mRNA transcripts of the CD74 gene with a predetermined threshold value shows that there is a decrease in the level of expression or under-expression, and when the comparison of the level of mRNA transcripts of the additional genes CD177 and IL1 R2 with predetermined threshold values ​​shows that there is an increase in the levels of expression or over-expression of said additional genes.

[0089] According to another variant of this embodiment, the method comprises a step of measuring, in the biological sample of the subject, the expression of the additional genes TDRD9, CD177 and IL1 R2. According to this variant, it can be concluded that there is an increased risk of death in said subject when the comparison of the level of mRNA transcripts of the CD74 gene with a predetermined threshold value shows that there is a decrease in the level of expression or under-expression, and when the comparison of the level of mRNA transcripts of the additional genes TDRD9, CD177 and IL1 R2 with the predetermined threshold values ​​shows that there is an increase in the level of expression or over-expression of said additional genes.

[0090] According to a particular embodiment, the method can thus comprise the following steps: - determine the level of expression of the CD74 and TDRD9 genes by measuring the quantity of at least one transcript of CD74 and TDRD9 in the subject's biological sample, - compare the quantity of said transcripts determined for said biological sample or a value derived from this quantity, to a predetermined reference value obtained from one or more reference samples, and - establish a conclusion regarding the risk of death, based on the result of the comparison.

[0091] According to this embodiment, the method makes it possible to conclude that there is a risk of death of the subject when an overexpression of TDRD9 and an underexpression of CD74 is demonstrated in the biological sample to be tested.

[0092] According to a preferred embodiment, the method can thus comprise the following steps: - determine the level of expression of the CD74 and IL1 R2 genes by measuring the quantity of at least one transcript of said genes in the subject's biological sample, - comparing the quantity of said transcripts determined for said biological sample, or a value derived from this quantity, to a reference value of said genes obtained from one or more reference samples, or to a predetermined threshold value, and - conclusion regarding the risk of death, based on the result of the comparison.

[0093] According to this embodiment, the method makes it possible to conclude that there is an increased risk of death of the subject when an under-expression of CD74 and an over-expression of IL1R2 is demonstrated in the biological sample to be tested.

[0094] According to another preferred embodiment, the method can thus comprise the following steps: - determine the level of expression of the CD74 gene and the additional genes IL1R2 and CD177, in particular by measuring the quantity of at least one transcript of said genes in the subject's biological sample, - comparing the quantity of said transcripts determined for said biological sample, or a value derived from this quantity, to a reference value for each of the genes from one or more reference samples, or to a predetermined threshold value, and - conclusion regarding the risk of death, based on the result of the comparison.

[0095] According to this embodiment, the method makes it possible to conclude that there is an increased risk of death of the subject when an under-expression of CD74 and an over-expression of IL1 R2 and CD177 is demonstrated in the biological sample to be tested.

[0096] According to another preferred embodiment, the method can thus comprise the following steps: - determine the level of expression of the CD74 gene and the additional genes IL1 R2, CD177 and TDRD9, in particular by measuring the quantity of at least one transcript of said genes in the subject's biological sample, - comparing the quantity of said transcripts determined for said biological sample, or a value derived from this quantity, to a reference value for each of the genes from one or more reference samples, or to a predetermined threshold value, and - conclusion regarding the risk of death, based on the result of the comparison.

[0097] According to this embodiment, the method makes it possible to conclude that there is an increased risk of death of the subject when an under-expression of CD74 and an over-expression of IL1 R2, CD177 and TDRD9 is demonstrated in the biological sample to be tested.

[0098] According to a particular 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 post-infection of the subject. Preferably, the risk of death corresponds to the risk of death of the subject within 7 days, 14 days, 21 days, 22 days, 23 days, 24 days, 25 days, 26 days, 27 days, or 28 days post-infection of the subject. More preferably, the risk of death corresponds to the risk of death within 28 days post-infection. The number of days post infection is defined as the number of days following the identification of the subject as being positive for a respiratory virus infection.

[0099] The method as described above, in all its embodiments, may also comprise, in addition to the step of measuring the expression of the gene(s) described above, a step of measuring in the biological sample of the subject the expression of one or more other additional genes, said genes being involved in the host response and chosen from the panel of genes presented in Table 2 below.

[0100] [Table 2]

[0101] Another subject of the invention relates to a kit for the in vitro or ex vivo measurement of the level of expression of the CD74 gene in a biological sample of a subject, said kit comprising at least one means for determining the level of expression of CD74 in said biological sample.

[0102] The means for determining the level of expression of the CD74 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.

[0103] 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.

[0104] The term "primer" or "amplification primer" means a nucleotide fragment which may consist of 5 to 100 nucleotides, preferably 15 to 30 nucleotides, and which has a specificity for hybridization 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.

[0105] A 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.

[0106] 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 DNA complementary DNA (cDNA) obtained by reverse transcription of said mRNA. When it is desired to target several different biomarkers (e.g. genes), several different probes are preferably used, each preferably having the capacity to hybridize specifically with a different biomarker.

[0107] By "hybridization" is meant the process during 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, that is to say the rigor of the operating conditions. The hybridization is all the more specific as it is carried out at a higher stringency. 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 may also be a function of 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 is to be carried out will depend primarily on the hybridization probes used. All of these data are well known, and appropriate conditions can be determined by those skilled in the art.

[0108] 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.

[0109] According to a particular embodiment, the kit further comprises at least one threshold value data stored on a computer-readable medium, for example a bar code, and / or used in the form of a code executable by a computer configured to compare the level of CD74 transcripts determined using the determination means, or data obtained from said level of CD74 gene transcripts, to said threshold value.

[0110] According to this embodiment, the threshold value may correspond to the transcript level, preferably the mRNA level, of CD74 in a subject having no infection by a respiratory virus or having an infection by a respiratory virus but having survived within 28 days of infection. The threshold value may also correspond to the average of the transcript level, preferably the mRNA level, of CD74 in a population of subjects with no respiratory virus infection or with respiratory virus infection but having survived within 28 days of infection.

[0111] According to another embodiment, the kit further comprises at least one reference level for said marker gene, 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 CD74 transcripts determined by the means of determining said reference level, with said reference level which is, preferably, the level of CD74 transcripts, in a subject not infected with a respiratory virus, or in a population of such subjects.

[0112] According to another embodiment, the kit further comprises a negative control sample which is a sample calibrated to contain the amount of CD74 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 positive control sample which is a sample calibrated to contain the amount of CD74 which corresponds to the average amount measured in a pool of samples from subjects known not to have survived, in particular within 28 days, following a respiratory virus infection. By "calibrated sample" is meant a sample prepared so as to comprise the amount or concentration of CD74 representative of a population of subjects with a respiratory virus infection or of subjects not with a respiratory virus infection.

[0113] According to another embodiment, the kit further comprises a negative control sample which is a sample calibrated to contain the amount of CD74 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 28 days following infection, and / or a positive control sample which is a sample calibrated to contain the amount of CD74 which corresponds to the average amount measured in a pool of samples from subjects known not to have survived, in particular within 28 days, following infection with a respiratory virus.

[0114] The kit, in all its embodiments, may also comprise at least one additional means for determining the level of expression of additional genes TDRD9, IL1 R2 and / or CD177, in said biological sample. According to this embodiment, the kit may comprise, in the same manner as defined above, a threshold value of the expression level of the additional genes and / or control samples (positive or negative) of said additional genes. The means for determining the expression level may be specific tools or reagents as defined above, for example primers or probes.

[0115] According to this embodiment, the kit may comprise, in the same manner as defined above, a threshold 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 above, for example primers or probes.

[0116] According to a particular embodiment, the kit, in all its embodiments, may also comprise, in addition to a means for determining the level of expression of CD74 and possibly additional genes TDRD9, IL1 R2 and / or CD177, at least one other additional means for determining the level of expression of one or more genes involved in the host response, from a biological sample of a subject. The genes involved in the host response are as defined in the panel of genes presented in Table 2.

[0117] The means for determining the level of expression may be specific tools or reagents as defined above, for example primers or probes.

[0118] Another object concerns the use of an in vitro or in vivo measurement kit as defined above to determine the risk of death in a subject infected with a respiratory virus.

[0119] According to a particular embodiment, the kit according to the invention, by the additional measurement of the level of expression of a panel of genes involved in the host response, can advantageously be used, in addition to determining the risk of death following infection by a respiratory virus, for determining the immune status of a subject in order to define in particular whether said subject is immunocompetent or immunocompromised.

[0120] 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.

[0121] 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.

[0122] Treatment may involve administering a suitable antiviral medication. Examples of antiviral treatments include Iopi navir®, 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-Marroquin, G. et al., 2020).

[0123] 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.

[0124] 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 target genes CD74, IL1 R2, and CD177 and optionally, also the expression of the additional target gene TDRD9, - measure the expression of said target genes.

[0125] The reagents specific for the expression products are selected from amplification primers, hybridization probes or antibodies, and are as defined previously.

[0126] 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 28 days.

[0127] 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 CD74 gene in said biological sample, - compare the level of CD74 gene expression with a reference value obtained from patients infected with a respiratory virus and who survived, in which, if the level of CD74 gene expression is lower than the reference value, it is determined that the patient has an increased risk of death.

[0128] According to a particular embodiment, the method also comprises measuring the expression level of one or more additional genes as defined previously 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 on the basis of the result of the comparison of the expression levels of all the genes measured and as defined previously.

[0129] Finally, another subject relates to a method comprising the quantitative measurement, in particular by RT-qPCR, of the mRNA of the CD74, IL1 R2 and CD177 genes in a biological blood sample from a subject infected with a respiratory virus such as SARS-CoV-2.

[0130] According to a particular embodiment, the method further comprises the quantitative measurement of the mRNA of the TDRD9 gene, in particular by RT-qPCR.

[0131] The present invention is illustrated in a non-limiting manner from the following examples. Examples Materials and Methods

[0132] Description of the two patient cohorts used

[0133] The RICO cohort is a prospective observational clinical study. From this cohort, a subcohort of 53 patients (hereinafter RICO subcohort) was recruited between March and May 2020 from three intensive care units of university hospitals (Hospices Civils de Lyon, Lyon, France). All patients had SARS-CoV-2 pulmonary infection confirmed by RT-PCR testing.

[0134] Briefly, the inclusion criteria were as follows: (1) male or female > 18 years, (2) hospitalization in intensive care units (ICU) for SARS-CoV-2 pneumonia, (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) collection within the first 24 hours after admission to ICU (D0) 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 subcohort, samples were collected on day 0 (within 48 hours of ICU admission), representing 50 patients. Patients had a mean age of 64 years [InterQuartile Range, 52-70] and a mixed distribution of men and women (80 / 20).

[0137] REALISM Cohort: A subset of 34 RNA samples from healthy volunteers was obtained retrospectively from the REALISM cohort, a single-center, longitudinal observational study. REALISM samples were collected between December 2015 and June 2018. This subcohort was used as a control dataset. Healthy volunteers were 63.5 years old [IQR, 52.0-70.0] and had a standard distribution of males and females (50 / 50).

[0138] Measurements of immunological markers

[0139] The following immunological markers are common to both subcohorts: T cell count, CD4+ T cell count, CD8+ T cell count, mHLA-DR (Ab / C), plasma IL-6, and plasma IL-10. Other immunological marker measurements were performed only in the RICO subcohort.

[0140] Plasma concentrations of IL-6, TNF-α, IFN-γ, and IL-10 were measured by Simpleplex® technology using the ELLA instrument according to the manufacturer's instructions. Plasma concentrations of IFNα2 were determined by singlemolecule array (SIMOA) on an HD-1 analyzer (Quanterix) using a commercial kit for quantification of IFN-α2. IFN content was obtained using nCounter® analysis technology (NanoString Technologies, Seattle) by calculating the median of the normalized count of the following 6 interferon-stimulated genes (ISGs): SIGLEC1, IFI27, IFI44L, IFIT1, ISG15, and RSAD2. Immunophenotyping of the T cell subpopulation was performed on a Beckman Coulter automated volumetric flow cytometer (Aquios CL). HLA-DR expression by monocytes was performed using antibodies from Beckman-Coulter and BD Biosciences (San Jose, USA).Monocyte HLA-DR expression was determined using the Anti-H LA-DR / Anti-Monocyte Quantibrite assay (BD Biosciences, San Jose, USA). The total number of antibodies bound per cell, expressed as Ab / C, was quantified using calibration with a standard curve determined with BD Quantibrite phycoerythrin (PE) beads (BD Biosciences).

[0141] Blood samples were collected in PAXgene® tubes (ref. 762165, PreAnalytiX GmbH Hombrechtikon Switzerland) at JO (48h post UCI admission).

[0142] Total RNA was extracted from PAXgene™ tubes using the Maxwell® 16 LEV simplyRNA Blood Kit (Promega) according to the manufacturer's guidelines. RNA quantity was determined using a NanoVue (Biochrom).

[0143] Then, 200 ng of total RNA was tested using a FilmArray® pouch optimized to detect a panel of genes involved in the host response, including CD74, by nested PCR. The pouches were analyzed on the FilmArray® Torch instrument (BioFire®, USA) which automatically performs the steps of nucleic acid extraction, reverse transcription and qPCR.

[0144] Normalized expression values ​​of the markers (relative to the reference genes DECR1, HPRT1 and PPIB) were calculated and used for the analyses.

[0145] Statistical analysis

[0146] Qualitative data were reported as numbers or concentrations and quantitative data were reported as median [IQR range]. Clinical or immunological characteristics of volunteers in the RICO subcohort and healthy volunteers (REALISM cohort) were compared using the nonparametric Mann-Whitney-Wilcoxon test for continuous variables or Fisher's exact test or chi-square test for categorical variables. The significance level was set at 5% for two-sided tests. Statistical analyses were performed using R software, version 4.0.4. Data were centered to perform unsupervised principal component analysis.

[0147] The correlation between gene expression markers and immunological parameters was assessed using Spearman's p-rank correlation scores. The entire gene panel of the FilmArray® pouch was used, without applying feature selection, to build five 28-day survival prediction models, namely: Elastic net, LASSO, Ridge regression, random forest (rf), and Partial Least Square Discriminant Analysis (PLS) using the CARET package (version 6.0-88). Hyperparameters were tuned and models were fitted using cross-validation (k-fold=3, number of replicates=10) in the RICO subcohort.

[0148] In summary, among the 5 machine learning algorithms evaluated, Elastic net (a=0.1, Δ=0.89898) was the best performer with an area under the ROC curve (AUC) of 0.675 [confidence interval], LASSO (a=1, Δ=0.112) ranked second with an AUC of 0.671, then PLS (ncomp=1) with an AUC of 0.668, Ridge regression model (a=0, Δ=1) was fourth with an AUC of 0.650 and finally, Random Forest (mtry=5) came last in terms of performance with an AUC of 0.63.

[0149] The biomarkers of interest were identified by the importance of the variables carried by each model in the occurrence of the 28-day mortality event. These biomarkers identified by the machine learning algorithms are the same as those that significantly result from the univariate analysis of association with mortality. at 28 days. Patients were stratified into 2 groups based on the Youden threshold, determined for each biomarker, for the prediction of 28-day mortality in the total patient population at admission and unadjusted cumulative events of death were obtained with Kaplan-Meier estimates.

[0150] Ethics

[0151] The REALISM study protocol was approved by the IRB (Southeast II Personal Protection Committee approval number 2015-42-2). This clinical study was also registered on clinicaltrials.gov (NCT02638779). Informed consent was obtained from each patient and / or from next of kin. Patients or legal representatives were informed of the study and their right to refuse participation.

[0152] 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). The committee waived the need for written informed consent because this was an observational study, presenting a low risk to patients, and no specific procedures, other than routine blood sampling, were required.

[0153] Both cohorts comply with the Declaration of Helsinki, the principles of good clinical practice and French law on the protection of personal data. Results

[0154] Clinical characteristics at admission to the intensive care unit

[0155] The characteristics of the RICO subcohort patients and recruited healthy volunteers are presented in Table 3 below. The patients were hospitalized in 3 hospitals in Lyon during the period that coincided with the first wave of COVID-19 cases in France (March - May 2020).

[0156] Briefly and as previously mentioned, 80% of patients were male. Patients were admitted to hospital with a median of 8 days after the presentation of the first symptoms [IQR, 5-10], with no significant difference between survivors and non-survivors at 28 days. 44% of patients had at least one comorbidity, with diabetes being the most common (13 / 22 patients). Patients had a median body mass index (BMI) (kg / m3) of 29.33 [IQR, 26.58-31.54], with no difference between survivors and non-survivors.

[0157] In terms of disease severity, patients had high PaCh / FiCh [median: 134; IQR: 90.5-191] and SAPS II score [median: 33.5; IQR: 24.5-43.25]. Among the 50 patients, 11 (22%) died by day 28.

[0158] Of note, non-survivors had a significantly higher SAPS II score [median: 39; IQR: 34-53.5] compared to survivors [median: 32; IQR: 22- 39.5] (p-value = 0.023).

[0159] Overall, survivors spent a median of 30 days [IQR, 13.25- 62.5] in hospital, including 10 days [IQR, 3-40] in intensive care. Half of this RICO sub-cohort developed secondary infections (52%), with no difference between survivors and non-survivors.

[0160] [Table 3] No- Patients Surviving at 28 days (n=50) Surviving at p (n=39) 28 days value (n=11) Demography Age 64 [52-70] 63 [51-69.5] 67 [60-78] 0.096 Gender, Male 40 (80%) 30 (76.92%) 10 (90.91%) 0.424 29.33 29.41 28.28 BMI [kg / m 31 0.292 [26.58-31.54] [26.86-32.2] [24.45-29.91] BMI > 30 kg / m 3 19 (38%) 16 (41.03%) 3 (27.27%) 0.498 Comorbidities None 37 (74%) 30 (76.92%) 7 (63.64%) Diabetes With 3 (6%) 1 (2.56%) 2 (18.18%) deterioration 0.197 sweet Without deterioration 10 (20%) 8 (20.51%) 2 (18.18%) organic >=1 22 (44%) 16 (41.03%) 6 (54.55%) Comorbidities 0.503 0 28 (56%) 23 (58.97%) 5 (45.45%) Charlson Score 0 [0-2] 0 [0-1] 1 [0-2] 0.243 Symptoms on admission Delay between the first 8 [5-10] 8 [5.5-10.5] 6 [4-7] 0.079 symptoms [days] Severity score SOFA Score 0.068 SAPS II Score 33.5 32 39 0.023 [24.5-43.25] [22-39.5] [34-53.5] PaCh / FICh ratio at 134 a 152.5 b 120 0.429 admission [90.5-191] [88.5-204.75] [101-135.5] Antiviral therapy Hydroxychloroquine 22 (62.86%) 17 (60.71%) 5 (71.43%) 0.689 Lopinavir / ritonavir 5 (14.29%) 5 (17.86%) 0 (0%) 0.559 Lopinavir / ritonavir + Interferon 5 (14.29%) 4 (14.29%) I (14.29%) 1 P Remdesivir 1 (2.86%) 1 (3.57%) 0 (0%) 1 Organ support Mechanical ventilation at 30 (60%) 20 (51.28%) 10 (90.91%) 0.033 admission Mechanical ventilation 19 (38%) 13 (33.33%) 6 (54.55%) 0.293 invasive Vasoactive drugs 19 (38%) 13 (33.33%) 6 (54.55%) 0.293 Substitution treatment 10 (20%) 7 (17.95%) 3 (27.27%) 0.671 renal Follow up Days in ICU 9 [4-29.75] 10 [3-40] 8 [6-19] 0.716 30 [13.25- Days in Hospital 21 [13-56] 15 [7.5-20] 0.007 62.5] 28-day mortality 11 (22%) 0 (0%) II (100%) <0.001 90-day mortality 14 (28.57%) 3 (7.89%) 11 (100%) <0.001 Infections acquired in ICU 26 (52%) 19 (48.72%) 7 (63.64%) 0.501

[0161] Medians and interquartile ranges [Q1-Q3] are shown for continuous variables, while numbers and percentages are shown for categorical variables. Patients in the COVID-19 study 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. Data were compared using the non-parametric Mann-Whitney test for continuous variables or the exact test Fisher's coefficients for categorical variables. ' Three patients did not present PaCh / FiCh, which resulted in 47 measurements (out of 50) in the column of all patients and 36 measurements (out of 39) in 28-day survivors.

[0162] Association between biomarker expression and clinical status of patients

[0163] The relationship between FilmArray® pocket transcriptomic markers and different clinical outcomes was analyzed.

[0164] First, the association with patient severity at admission, as reflected by clinical scores, was studied to confirm the potential of biomarkers. It was found that CD74 was negatively correlated with the SAPSII score (p = -0.30).

[0165] On the other hand, increased expression of TDRD9, CD177, and IL1 R2 was positively correlated with SAPSII score (TDRD9: p = 0.46, CD177: p = 0.5, and IL1 R2: P = 0.47).

[0166] Association between biomarker expression and 28-day patient mortality

[0167] Using 5 different machine learning approaches, 4 genes associated with the prediction of 28-day mortality were identified. These are CD74, TDRD9, IL1 R2 and CD177 (Figure 1).

[0168] These four markers are differentially expressed in univariate analysis between surviving and non-surviving patients (Table 4).

[0169] [Table 4] ORIQR [95% Cl] IQR p value 4.221 TDRD9 2.01 0.007 [1,591-13,621] 4.085 CD177 3.58 0.027 [1.315-16.548] 0.202 CD74 1.14 0.007 [0.054-0.569] 3.548 IL1 R2 2.28 0.010 [1.448-10.424] mHLA-DR 1.072 7335 0.868 [Ac / C] [0.435-2.402] CD3 T cells 0.26 362 0.050 [cells / pL] [0.054-0.816] Plasma IL-6 1.338 142 0.187 [pg / pL] [1.034-2.3] Plasma IL-10 1.116 16 0.221 [pg / pL] [0.945-1.407]

[0170] The association between 28-day survival status and transcriptomic markers or classical immune parameters was performed by implementing univariate logistic regression models and interquartile range (IQR) odds ratios (ORs) were extracted. To allow comparison between models, the calculated odds ratios (ORs) for each immune and transcriptomic parameter were standardized to an increment from the first to the third quartile. P values ​​< 0.05 are highlighted in bold.

[0171] Specifically, CD74 was significantly downregulated in non-surviving patients compared to surviving patients.

[0172] Regarding the expression level of additional genes, IL1 R2 and CD177 were significantly upregulated in non-surviving patients compared to surviving patients, while TDRD9 was significantly upregulated in non-surviving patients compared to survivors and to a greater extent compared to healthy volunteers.

[0173] On the other hand, the immunological parameters measured at admission (monocyte HLA-DR, plasma IL-6 and IL-10) were not significantly associated with the survival status of patients at 28 days, thus demonstrating the full predictive potential of CD74 and the additional markers according to the invention.

[0174] Based on the observation that age and invasive mechanical ventilation are associated with severity in patients with COVID-19 disease (cdc.gov / coronavirus / 2019-nCoV), these parameters were used as confounders in a multivariate analysis [3a]. We observed that the biomarker CD74 and the three additional biomarkers TDRD9, IL1 R2 and CD177 remained significantly associated with survival and presented an interquartile odds ratio (OR) of 5.35 for TDRD9, 0.22 for CD74, 3.26 for IL1 R2 and 5.22 for CD177 (results not shown).

[0175] Through Kaplan-Meier curves with the expression of each gene using the Youden threshold (predicting patient survival at 28 days following infection) to distinguish low / high expression, it was identified that TDRD9 expressions élevé , CD74 faible , CD177 élevé and IL1 R2 élevéwere significantly associated with death over a 28-day period, while the chances of survival were approximately 90% for patients with TDRD9 expressions faible , CD74 élevé , CD177 faible and IL1 R2 faible (Figure 3). These results thus demonstrate once again the predictive value of the biomarkers according to the invention for determining the risk of death of patients. infected with a respiratory virus of the SARS-Cov-2 type, in particular the risk of death at 28 days.

[0176] The results obtained above with the CD74 gene, and the additional genes were confirmed from the RICO cohort, more precisely in a second sub-cohort of 306 patients recruited between August 2020 and August 2021 in the three intensive care units of university hospitals (Hospices Civils de Lyon, Lyon, France). BIBLIOGRAPHICAL REFERENCES

[0177] 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;

[0178] E. Z. 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 ;

[0179] 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;

[0180] D. Mathew, et al., « Deep immune profiling of COVID-19 patients reveals distinct immunotypes with therapeutic implications” ; Science, 2020, 369 (6508), https: / / doi.Org / 10.1126 / science.abc8511 ;

[0181] A. G. Laing et al., “A dynamic COVID-19 immune signature includes associations with poor prognosis”, Nat Med, 2020, 26 (10), 1623, https: / / doi.org / 10.1038 / s41591-020- 1038-6.

[0182] 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;

[0183] 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;

[0184] 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 ;

[0185] G. Canedo-Marroquin 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.

Claims

CLAIMS 1. In vitro or ex vivo method for determining the risk of death in a subject infected with a respiratory virus, comprising a step of measuring, in a biological sample of said subject, the level of expression of the CD74 gene.

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 any one of claims 1 to 3, characterized in that it further comprises the following steps of: comparing the level of expression of the CD74 gene for said biological sample or a value derived from this quantity, to a predetermined threshold value, and establishing a conclusion as to the risk of death, from the result of the comparison 4. Method according to claim 3, characterized in that the reference threshold value corresponds to the average of the level of expression of the gene 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 said gene 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 infection.

5. Method according to any 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 IL1 R2, TDRD9 and / or CD177, preferably IL1 R2 and CD177.

6. Method according to claim 5, characterized in that it also comprises a step of measuring, in the biological sample of the subject, the expression of the two additional genes IL1 R2 and CD177, and the comparison of the expression levels with predetermined threshold values ​​for each of said additional genes.

7. Method according to claim 6, characterized in that it is concluded that there is an increased risk of death in said subject when comparing the level of expression of the gene CD74 with the predetermined threshold value shows that there is underexpression, and when comparing the expression level of the additional genes IL1R2 and CD177 with the predetermined threshold values, shows that there is overexpression.

8. Method according to any 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 any one of claims 1 to 8, characterized in that the level of expression is measured at the messenger RNA (mRNA) level.

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

11. Method according to claim 9 or 10, characterized in that the expression is normalized relative to the expression of one or more housekeeping genes.

12. Method according to one of claims 1 to 11, characterized in that it also comprises a step of measuring in the biological sample of the subject the expression of one or more other additional genes, said genes being involved in the host response and chosen from the panel of genes listed below: Kit for the in vitro or ex vivo measurement of the expression of CD74 in a biological sample comprising means for determining the level of expression of CD74 in said sample, said means being primers or probes, said kit being used for determining the risk of death of a subject infected by a respiratory virus, preferably SARS-CoV-2 or one of its variants. Kit for its use according to claim 13, characterized in that it comprises a negative control sample comprising the quantity of CD74 which corresponds to the quantity or the concentration representative of the level of expression measured in a pool of samples of subjects not presenting an infection by a respiratory virus, and / or a positive control sample comprising the quantity of CD74 which corresponds to the average quantity measured in a pool of samples of subjects who did not survive following an infection by a respiratory virus. Kit for its use according to claim 13 or 14, characterized in that it comprises means for determining the level of expression of at least one other additional gene selected from TDRD9, IL1 R2 and CD177.Kit for its use according to any one of claims 13 to 15, characterized in that it comprises means for determining the level of expression of one or more additional genes involved in the host response as defined in claim 10. Kit for its use according to any one of claims 13 to 16, characterized in that the risk of death corresponds to the risk of death within 28 days post-infection.

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