Classifications

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  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
  • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
  • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
  • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
  • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
  • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
  • C12Q1/70—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving virus or bacteriophage
  • C12Q1/701—Specific hybridization probes
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
  • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
  • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
  • C12Q1/6844—Nucleic acid amplification reactions
  • C12Q1/6851—Quantitative amplification
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61B—DIAGNOSIS; SURGERY; IDENTIFICATION
  • A61B10/00—Instruments for taking body samples for diagnostic purposes; Other methods or instruments for diagnosis, e.g. for vaccination diagnosis, sex determination or ovulation-period determination; Throat striking implements
  • A61B10/0045—Devices for taking samples of body liquids
  • A61B10/0051—Devices for taking samples of body liquids for taking saliva or sputum samples
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
  • C12Q2600/00—Oligonucleotides characterized by their use
  • C12Q2600/158—Expression markers

Definitions

• the present invention relates to the technical field of diagnostic methods and kits.
• the subject of the invention is methods and kits making it possible to determine whether or not a subject is suffering from an infection by a replicative respiratory virus.
• a PCR test only detects fragments of the DNA or RNA genome of the virus, and therefore the presence of a virus which is not active, that is to say which has not the ability to replicate in the subject's organism.
• the presence of symptoms in particular that can be described as mild (or weak), which could be associated with many infections, viral or bacterial, is also not a sign of the presence of a replicative respiratory virus.
• symptoms in particular that can be described as mild (or weak), which could be associated with many infections, viral or bacterial, is also not a sign of the presence of a replicative respiratory virus.
• it is sometimes difficult to associate symptoms such as fever, suspecting an infection, with the presence of a directly responsible replicating respiratory virus.
• Application WO 2019/236768 also proposes a method for detecting a respiratory infection in a subject, based on the level of expression of one or more genes of the subject (host gene).
• a very long list of host genes is given, among which certain ISGs appear, including IFIT1, IFIT2, IFIT3 and RSAD2, without these being presented as particularly advantageous to select.
• IFIT1, IFIT2, IFIT3 and RSAD2 include these being presented as particularly advantageous to select.
• the fact of knowing whether or not the subject is infected by a replicative respiratory virus is in no way studied in this patent application.
• the present invention relates to a method for determining in vitro or ex vivo the presence in a subject of an infection by a replicative respiratory virus, comprising a step i) of determination, in a test sample from of the mouth or nose of said subject, of the level of transcripts of at least one marker gene selected from the genes stimulated by interferons, known as ISG (hereinafter called ISG marker gene, or more simply marker gene or ISG, for the sake of simplicity).
• ISG marker gene hereinafter called ISG marker gene, or more simply marker gene or ISG, for the sake of simplicity.
• the level of transcripts of one or more marker genes chosen from: IFI27, IFI44L, IFIT1, RSAD2, ISG15, and SIGLEC1 is determined.
• the level of transcripts of all these marker genes can be determined.
• the level of transcripts of one or more marker genes chosen from: IFI27, IFI44L, IFIT1 and RSAD2 is determined.
• the level of transcripts of all the marker genes IFI27, IFI44L, IFIT1 and RSAD2 is determined.
• the level of transcripts can be determined for each marker gene independently, or a simultaneous determination of the different transcripts of all the marker genes used, the level of which is globally determined.
• data in particular a score as used in the examples
• the test sample is an oropharyngeal or nasopharyngeal sample or a saliva sample.
• step i) consists of determining the mRNA level of the said marker gene(s).
• the methods according to the invention can be implemented on a sample from a subject who exhibits symptoms of an infection, or on a sample from a subject who is asymptomatic.
• the method according to the invention is particularly advantageous, in the case of asymptomatic subjects or of subjects presenting symptoms which can be qualified as benign, such as fatigue, body aches, muscle pain, fever, respiratory symptoms, including cough without pneumonitis, headache, sore throat, malaise, nausea, vomiting, diarrhea, anosmia, or ageusia.
• the test sample can be obtained or taken at any time.
• the test sample may be obtained or collected before or after the onset of symptoms.
• the test sample can come from any type of subject, from a subject who has or has not been diagnosed with a respiratory virus, from a subject who is awaiting a result of detection or diagnosis of a respiratory virus, in particular by detection of DNA or RNA of said virus...
• the methods according to the invention can be carried out on a sample from a subject who does not have neutralizing autoantibodies directed against the interferon a and/or neutralizing autoantibodies directed against interferon w.
• the method according to the invention may comprise the following steps: i) determining the level of transcripts of at least one marker gene chosen from among the ISGs, for said test sample of the subject, ii) comparing the level of transcripts of said at least obtained marker gene, at a reference level.
• the methods according to the invention comprise a step iii) which is to conclude on the presence or not of an infection by a replicative respiratory virus in said subject. Such a conclusion is made using at least one threshold value.
• threshold value is meant a predetermined value which is relevant for draw a conclusion as to the presence or absence of an infection by a replicative respiratory virus in a subject.
• a comparison is carried out between a threshold value and the level of transcripts of at least one ISG marker gene determined for the test sample of said subject, or with data (in particular a score) obtained from the level of transcripts of at least an ISG marker gene determined for the test sample of said subject.
• Said data (or score) can, in particular, be obtained from the levels of transcripts of several ISG marker genes determined for the test sample of said subject.
• the level of transcripts of at least one ISG marker gene determined for the test sample of said subject or the data or the score obtained from the level of transcripts of at least one gene ISG marker determined for the test sample of said subject, is different, in particular greater than the threshold value, it is concluded that there is an infection by a replicative respiratory virus in said subject.
• step iii) it can be concluded that there is or is not an infection by a replicative respiratory virus in said subject, from the result of step ii), that is to say the comparison between the level of transcripts of said at least one marker gene obtained, and the reference level of said marker gene which then plays the role of threshold value.
• the level of transcripts of a single ISG marker gene is determined.
• the transcript level of several ISG marker genes is determined globally and this global level is compared to a global reference level.
• the reference level used as a threshold value, with which the level of transcripts of a marker gene is compared corresponds to the level of transcripts of said marker gene, in a reference subject, and in particular in a subject not infected with a replicative respiratory virus, or in a population of such subjects.
• Subjects not infected with a replicative respiratory virus include subjects who test positive for the presence of a respiratory virus, but for whom the virus is not replicative, and subjects not infected with a respiratory virus ( negative to a test for the presence of a virus respiratory).
• the reference level corresponds to the level of transcripts of said marker gene, in a subject showing no infection by a replicative respiratory virus, whether or not he is positive for a test for detecting a respiratory virus, and who, preferably does not possess neutralizing autoantibodies directed against interferon a and/or neutralizing autoantibodies directed against interferon w, or else the reference level corresponds to the level of transcripts of said marker gene in a population of such subjects showing no infection by a replicative respiratory virus, whether or not they are positive for a detection test for a respiratory virus, and who preferably do not possess neutralizing autoantibodies directed against interferon a and/or neutralizing autoantibodies directed against interferon w.
• the level of transcripts of a single marker gene chosen from among the ISGs can be determined in step i) and it is concluded that the presence of an infection by a virus replicative respiratory in said subject in the event of a difference with the reference level of said marker gene.
• the invention relates to a method for determining in vitro or ex vivo the presence in a subject of an infection by a replicative respiratory virus comprising the following steps: i) determining the level of transcripts of a single selected marker gene among the ISGs, in a test sample from the mouth or nose of said subject, ii) comparing the level of transcripts of said marker gene obtained, with a reference level, used as a threshold value, said reference level being the level of transcripts of said marker gene, in a reference subject, and in particular in a subject not infected with a replicative respiratory virus, who preferably does not possess neutralizing autoantibodies directed against interferon a and/or d neutralizing autoantibodies directed against interferon w, or in a population of such subjects, and iii) concluding the presence of an infection by a replicative respiratory virus in said subject in the event of a difference with said reference level of said marker gene.
• said difference corresponds to a level of transcripts of said marker gene for the test sample which is higher than that of the reference level.
• said marker gene selected from among the ISGs is preferably NFIT1 or NFI44L.
• step i) the transcript levels of several marker genes selected from among the ISGs can be determined in step i), for the test sample, and
• the level of transcripts of each marker gene determined for the test sample can be compared with the reference level of the said corresponding marker gene.
• the invention relates to a method for determining in vitro or ex vivo the presence in a subject of an infection by a replicative respiratory virus comprising the following steps: i) determining the levels of transcripts of several marker genes selected from among the ISGs for the test sample, in a test sample originating from the mouth or nose of said subject, ii) comparing the level of transcripts of each marker gene determined in the test sample with a reference level corresponding to said marker gene , used as a threshold value, for each marker gene considered, said reference level being the level of transcripts of said marker gene, in a reference subject, and in particular in a subject not infected with a replicative respiratory virus, which, preferably does not have neutralizing autoantibodies directed against interferon a and/or neutralizing autoantibodies directed against interferon w, or in a population of te ls subjects, and iii) concluding the presence of an infection by a replicative respiratory virus in said subject, in the event of a difference with the
• the level of transcripts of one or more marker genes chosen from: IFI27, IFI44L, IFIT1, RSAD2, ISG15 , and SIGLEC1 is determined.
• the level of transcripts of all these marker genes can be determined.
• the level of transcripts of one or more marker genes chosen from: IFI27, IFI44L, IFIT1 and RSAD2 is determined.
• the level of transcripts of all the marker genes IFI27, IFI44L, IFIT1 and RSAD2 is determined.
• step iii) it can be concluded that there is or is not an infection by a replicative respiratory virus in said subject, by implementing a intermediate step iibis) leading to data obtained from the level of transcripts of at least one marker gene selected from the ISGs, for the test sample.
• a comparison step ii) may be carried out during which the level of transcripts of each ISG marker gene is compared with a reference level of said marker gene, this comparison taking in particular the form of calculating a ratio, in particular the ratio between said level of transcripts of each marker gene on a reference level of said marker gene.
• An intermediate step iii) can then be carried out: this step can consist of calculating a score representative of the results of the comparisons carried out for each marker gene.
• a score is, for example, the median of all the ratios obtained (in particular, level of transcripts of each ISG marker gene for the test sample/reference level corresponding to said marker gene).
• the conclusion can then be made by comparing this score with a threshold value, in particular of 1.5, to conclude that there is or is not the presence of an infection by a replicative respiratory virus in the subject of interest. If the score obtained for the test sample is higher than this threshold value, in particular by 1.5, it will be concluded that there is presence of an infection by a replicative respiratory virus.
• the level of transcripts of one or more marker genes chosen from: IFI27, IFI44L, IFIT1, RSAD2, ISG15, and SIGLEC1 is determined.
• the level of transcripts of all these marker genes can be determined.
• the level of transcripts of one or more marker genes chosen from: IFI27, IFI44L, IFIT1 and RSAD2 is determined.
• the level of transcripts of all the marker genes IFI27, IFI44L, IFIT1 and RSAD2 is determined.
• a method for determining in vitro or ex vivo the presence in a subject of an infection by a respiratory virus replicative comprising the following steps: i) determining the levels of transcripts of several marker genes selected from among the ISGs, in a test sample from the mouth or nose of said subject, ii) comparing the level of transcripts of each marker gene determined in the test sample with a reference level corresponding to said marker gene, with calculation of the ratio between said level of transcripts on said reference level of said marker gene, iibis) calculating the median of all the ratios obtained in step ii), and iii ) conclude on the presence of an infection by a replicative respiratory virus in said subject, when said median calculated in step iibis) is greater than a threshold value, which is preferably equal at 1.5.
• the value of 1.5 can thus be considered as the threshold value used to issue the conclusion that there is or is not the presence of an infection by a replicative respiratory virus.
• the reference level used for the calculation of the ratio is the level of transcripts of said marker gene, in a subject not infected with a respiratory virus, or in a population of such subjects, said marker gene.
• a subject not infected with a respiratory virus is, in particular, a subject negative for a test for detecting the presence of a respiratory virus.
• the reference level used for calculating the ratio is the level of transcripts of said marker gene, in a subject not infected with a respiratory virus, not possessing neutralizing autoantibodies directed against interferon a and/ or neutralizing autoantibodies directed against interferon w or in a population of such subjects.
• the methods according to the invention can also comprise a step carried out before or concomitantly with step i), during which the test sample is subjected to a diagnostic test, for detection of DNA or RNA, in particular by PCR or RT-PCR, of a respiratory virus, and in particular of a SARS-CoV-2 virus.
• a diagnostic test for detection of DNA or RNA, in particular by PCR or RT-PCR, of a respiratory virus, and in particular of a SARS-CoV-2 virus.
• said diagnostic test is carried out prior to step i) and gives a positive result.
• the test sample is subjected to a diagnostic test, by detection of DNA or RNA, in particular by PCR or RT-PCR, of a respiratory virus chosen from the SARS-CoV-2 virus and its variants and the influenza viruses, in particular influenza A, B and C, said diagnostic test having given a positive result.
• a diagnostic test can also be implemented, prior to or concomitantly with step i), and the positive or negative result can be obtained before or after the determination of the presence or absence in said subject, of infection with a replicative respiratory virus.
• the determination of the level of transcripts of the marker gene(s) is carried out, in particular at the mRNA level.
• the determination of the level of transcripts is, in particular, carried out by hybridization, amplification or sequencing, and in particular by RT-qPCR.
• the level of transcripts of the marker gene(s) obtained can be a normalized value with respect to the level of transcripts of one or more housekeeping genes, in particular chosen from DECR1, HPRT1 and PPIB.
• the present invention also relates to a kit for determining in vitro or ex vivo the presence in a subject of an infection by a replicative respiratory virus, comprising:
• At least one threshold value stored on a medium readable by a computer and/or used in the form of a code executable by a computer configured to compare the level of transcripts of said marker gene, determined using the determination means or data obtained from said level of marker gene transcripts to said threshold value.
• the kit will generally include at least one means of determining the level of transcripts for each of them.
• the kit can comprise a threshold value and/or a reference level for each of them, stored(s) on a medium readable by a computer and/or used(s) in the form of code executable by a computer.
• the kit can also comprise an executable code making it possible to generate a global reference datum, from the comparison of each marker gene with its reference datum and allowing a comparison of this global datum with a threshold value.
• the comparison of the level of each marker gene to its reference level can take the form of calculating a ratio, as previously explained.
• said threshold value corresponds to the level of transcripts of said marker gene in a subject not exhibiting any infection by a replicative respiratory virus and who, preferably, does not possess neutralizing autoantibodies directed against interferon a and/or does not possess neutralizing autoantibodies directed against interferon w, or in a population of subjects exhibiting no infection by a replicative respiratory virus and who, preferably, do not possess neutralizing autoantibodies directed against interferon a and/or do not have neutralizing autoantibodies directed against interferon w.
• such a kit according to the invention further comprises at least one reference level for said marker gene, stored on a computer-readable medium and/or used in the form of a code executable by a computer configured to compare the level of transcripts of the marker gene determined by means of determining said reference level, with said reference level which is, preferably, the level of transcripts of said marker gene, in an uninfected subject by a respiratory virus, or in a population of such subjects.
• at least one reference level for said marker gene stored on a computer-readable medium and/or used in the form of a code executable by a computer configured to compare the level of transcripts of the marker gene determined by means of determining said reference level, with said reference level which is, preferably, the level of transcripts of said marker gene, in an uninfected subject by a respiratory virus, or in a population of such subjects.
• kits according to the invention further comprise at least one means for detecting a respiratory virus, and in particular at least one means for detecting a SARS-CoV-2 virus and/ or at least one means of detecting the influenza virus (influenza A, B or C).
• the present invention also relates to a kit for determining in vitro or ex vivo the presence in a subject of an infection by a replicative respiratory virus, comprising:
• At least one means for detecting a respiratory virus and in particular at least one means for detecting a SARS-CoV-2 virus.
• such a kit further comprises at least one threshold value stored on a computer-readable medium and/or used in the form of code executable by a computer configured to compare the level of transcripts said marker gene determined by the determining means, or data obtained from said transcript level of the marker gene, at said threshold value.
• said threshold value corresponds to the level of transcripts of said marker gene in a subject not exhibiting any infection by a replicative respiratory virus and who, preferably, does not possess neutralizing autoantibodies directed against interferon has and/or does not possess self- neutralizing antibodies directed against interferon w, or in a population of subjects showing no infection by a replicative respiratory virus and who, preferably, do not possess neutralizing autoantibodies directed against interferon a and/or do not possess no neutralizing autoantibodies directed against interferon w.
• such a kit according to the invention further comprises at least one reference level for said marker gene, stored on a computer-readable medium and/or used in the form of a code executable by a computer configured to compare the level of transcripts of the marker gene determined by means of determining said reference level, with said reference level which is, preferably, the level of transcripts of said marker gene, in a subject not infected with a respiratory virus, or in a population of such subjects.
• the detection kits according to the invention may also comprise a negative control sample, such as a sample making it possible to ensure the absence of contamination, and/or a positive control sample which corresponds to the level of transcripts of said marker gene at a concentration representative of the level of transcripts of a subject having an infection by a replicative respiratory virus or in a population of subjects having an infection by a replicating respiratory virus.
• a negative control sample such as a sample making it possible to ensure the absence of contamination
• a positive control sample which corresponds to the level of transcripts of said marker gene at a concentration representative of the level of transcripts of a subject having an infection by a replicative respiratory virus or in a population of subjects having an infection by a replicating respiratory virus.
• FIG. 3 Figure 3 shows the kinetic measurements of ISG NP (gray square) and blood (black square) scores after diagnosis of COVID-19.
• n 75 longitudinal samples from 23 patients.
• the Loess fit curves represent local polynomial regressions as determined by the Loess method.
• the 95% Cl is indicated (grey area).
• the black circles represent negative viral culture samples and the open circles the positive viral culture sample, the triangles correspond to positive samples in cell culture without cytopathic effect.
• Figure 5 is an ROC (Receiver Operating Curve) curve distinguishing the results of positive and negative SARS-CoV-2 viral cultures. Area under the curve and Youden's index are shown and represent the ability of the NP ISG type l/lll score to separate positive and negative virus cultures.
• ROC Receiveiver Operating Curve
• FIG. 6 represents the type I/III nasal ISG score measured after serial dilutions at 1/10 (10° to 10 -2 ) of NP samples (solid line, FilmArray®) as well as the detection of SARS-CoV-2 (Ct detection, dashed line, SARS-CoV-2 R-gene® kit).
• the lower gray zone indicates the threshold value of the nasal ISG type l/lll score known as normal (1.99) and the upper gray zone marks the value of 6.75 of the nasal ISG type l/lll score associated to a viral culture of a replicative virus.
• FIG. 7 compares the normalized nasal viral load and the NP ISG type l/lll score in patients with severe COVID-19, without anti-IFN autoantibodies (nAb) (open circles) or with autoantibodies (nAb). - neutralizing antibodies directed against IFN-a (black triangles).
• the gray area represents the viral load obtained for 90% of mild COVID-19 patients with a score nasal ISG type I/III > 6.75 (separation value associated with a viral culture of a replicative virus).
• FIG. 8 is an ROC curve discriminating between the positive and negative result of the viral PCR for the IFN type l/III score.
• the area under the curve, Youden's index and cut-off point are shown and represent the ability of the nasal IFN type I/III score to discriminate PCR positive from negative samples.
• FIG. 10 highlights graphs representing (A) IFI27 mRNA expression level versus viral PCR, (B) IFI44L mRNA expression level versus viral PCR, ( C) RSAD2 mRNA expression level as a function of viral PCR and (D) IFIT1 mRNA expression level as a function of viral PCR.
• the level of expression of the mRNAs is expressed in log2 of the ratio between the OR of the ISG and the median OR of this same ISG of a population of VS.
• Figure 12 is an ROC curve discriminating between the positive and negative result of the viral culture of the influenza virus for the nasal IFN l/III score. The area under the curve, the Youden index and the cutoff point are shown and represent the ability of nasal IFN l/III score and viral load to differentiate between positive and negative cultures.
• the term “subject” designates a mammal and, preferably, a human being.
• Said subject, who is a human being can be a patient who has come into contact with a health professional, such as a doctor (for example, a general practitioner) or a medical structure or a health establishment (for example, a hospital or a clinic, and more particularly an emergency department, an intensive care unit, an intensive care unit or a continuing care unit, or a medical structure for the elderly, of the EHPAD type).
• a health professional such as a doctor (for example, a general practitioner) or a medical structure or a health establishment (for example, a hospital or a clinic, and more particularly an emergency department, an intensive care unit, an intensive care unit or a continuing care unit, or a medical structure for the elderly, of the EHPAD type).
• sample from a subject's mouth or nose it can be a sample taken from the subject's nose or mouth or an exhaust air sample. by the subject.
• the sample may be a nasal sample or a saliva sample, but is preferably an oropharyngeal or nasopharyngeal sample.
• Oropharyngeal or nasopharyngeal samples are obtained in particular by taking a sample using a swab, respectively at the back of the mouth or nose of a subject.
• replicative virus is meant a virus which is capable of multiplying in said subject.
• a subject infected with a replicative virus is a transmission vector for such a virus, to another subject.
• the concept of replicative virus excludes virus fragments, which have lost their ability to reproduce. Such virus fragments will give a positive result, during a test for detecting the DNA or RNA of the virus, but do not have the capacity to replicate in the subject, who therefore cannot be a transmission vector of the corresponding virus.
• the risk of transmission of the virus is high, which is not the case in the absence of the possibility of replication.
• the capacity of a virus to multiply (replicate) and therefore its replicative character can be defined by the fact that the latter multiplies (increase in viral load), which can result in the observation of a cytopathic effect when it is cultured, or a part of the test sample taken which is likely to contain such a replicative virus is cultured, on epithelial cells, such as Vero cells.
• Vero cells that are particularly suitable for testing the replication capacity of a virus are in particular those deposited under the ATCC number CCL-81®, or else the Vero E6 cells deposited under the ATCC number CRL-1586®.
• a cytopathic effect can, in particular, be materialized by the presence of lysis plaques, which is characteristic of the presence of replicative viral particles.
• a subject infected with a replicative virus is a transmission vector for such a virus to another subject
• respiratory virus means a virus which infects the respiratory tract and/or the lungs. Such viruses are typically found in samples taken from the nose, throat and/or mouth of a subject, in particular in nasal or nasopharyngeal samples (which require a sample taken deeper in the nose), oral samples -pharyngeal (which requires a swab at the back of the throat) or saliva.
• respiratory viruses include seasonal coronaviruses, the SARS-CoV-2 virus, whatever its variants, the influenza virus (influenza A, B and C), the respiratory syncytial virus (RSV ), rhinoviruses, metapneumoviruses, parainfluenza viruses and adenoviruses...
• the Brazilian variant P.1 is a descendant of the B.1.1.28 lineage (which circulated extensively in the state of Rio de Janeiro and probably emerged in Brazil in February 2020).
• 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, K417T mutations.
• the South African variant called 501Y.V2 (lineage B.1.351) also contains different mutations including three, K417N, E484K and N501Y, located in the RBD domain of the spike protein, the receptor binding domain.
• the respiratory virus is chosen from the SARS-CoV-2 viruses, the influenza virus (influenza A, B and C), the respiratory syncytial virus (RSV), the rhinoviruses, the metapneumoviruses, parainfluenza viruses and adenoviruses, and preferably from among the SARS-CoV-2 viruses and its variants, and the influenza virus (influenza A, B and C).
• the respiratory virus is chosen from the SARS-CoV-2 virus and its variants.
• the respiratory virus is chosen from the influenza A, B and C viruses, preferably it is the influenza B virus.
• Interferons are among the first molecules produced in the body, after recognition of a pathogen by the receptors of innate immunity. Interferons are widely described in the literature. Reference may be made, for example, to Manry J. et al., 2012 and Hertzorg PJ et al., 2016. Human IFNs have been classified into three types, depending on the receptors they use, their homology and their chromosomal location . There are 17 type 1 IFNs: 13 subtypes of IFN-a, and IFN-b, e, k and w. The genes encoding these IFNs are located on chromosome 9 and bind to the same receptor composed of the IFNAR1 and IFNAR2 subunits.
• IFN-g the only type II IFN, is the product of a gene located on chromosome 12, and uses a receptor composed of IFN-yR1 and IFN-yR2 subunits.
• Type III IFNs have been described more recently and correspond to the cytokines: IL-28A, IL-28B and IL-29 (also called IFN-A2, IFN-A3 and IFN-A1).
• the genes encoding these IFNs are located on chromosome 19. Interferons regulate the expression of several hundred genes, the so-called interferon-stimulated genes (or ISGs).
• IFNs interferon receptors
• JFNARs interferon receptors
• JNK1 Janus kinase 1
• Tyk2 tyrosine kinase 2
• IRF9 interferon-stimulated response elements
• IFNARs interferon receptors
• JNK1 Janus kinase 1
• Tyk2 tyrosine kinase 2
• IRF9 interferon-stimulated response elements
• Secreted type III IFNs bind to another receptor, consisting of IFNLR and IL10RB, and yet activate the same ISGF-3, inducing widely overlapping ISGs (Lazear, H. M etal., 2019).
• IFN-I and III are involved as the first line of defense against infection, since they promote virus clearance, induce tissue repair, and stimulate the adaptive immune response.
• ISG genes By way of example of ISG genes, mention may be made of ADAR, BST2, CHUK, DDX58, EIF2AK2, FOS, GBP2, HLA-A, HLA-B, HLA-C, HLA-E, IFI35, IFI6, IFIH1 , IFIT2, IFIT3, IFITM1 , IFITM2, IFITM3, IFNA14/16, IFNA2, IFNA4/7/10/17/21 , IFNA5, IFNA6, IFNA8, IFNAR1 , IFNAR2, IFNB1 , IKBKB, IKBKE, IKBKG, IRF1, IRF3, IRF4 , IRF7, IRF9, JAK1 , JUN, MAP3K7, MAPK14, MAPK8, MAVS, MX1 ,
• the ISGs chosen from IFI27, IFI44L, IFIT1, RSAD2, ISG15 and SIGLEC1 are preferred in the context of the invention. Their sequences are given in the ENSEMBL and NCBI databases. These ISGs are, in particular, stimulated by type I and III interferons.
• IFI27, IFI44L, RSAD2 and IFIT 1 which are four ISGs regulated through ISRE elements dependent on IFN-stimulated gene factor 3 (ISGF3), are even more preferred within the scope of the invention.
• the level of transcripts of one or more marker genes of the ISG type chosen from: IFI27, IFI44L, IFIT 1, RSAD2, ISG15, and SIGLEC1 (Picard C, etal., 2018).
• neutralizing antibody an antibody which neutralizes the effect of the target against which it is directed.
• auto-antibodies antibodies generated by the subject himself directed against self-proteins. Such autoantibodies can be generated in the absence of infection.
• symptom means any type of symptom, for example symptoms which may be qualified as mild, such as fatigue, body aches, fever, respiratory symptoms, such as cough without pneumopathy, headache, anosmia or ageusia, or even severe symptoms, which require hospitalization in intensive care or respiratory assistance.
• SARS-CoV-2 a classification of SARS-CoV-2 infections (for people who test positive for SARS-CoV-2, by PCR test or antigen test), asymptomatic to critical was established based on symptoms (Trouillet-Aimpuls, S. etal., 2020 and https://www.covid19treatmentguidelines.nih.gov/overview/clinical-spectrum/):
• the methods according to the invention can be implemented on samples from subjects having an infection with SARS-CoV-2, which is asymptomatic, presymptomatic, benign or moderate.
• a “comparison” or a verification of a “difference” between two values or levels can be carried out by any known technique, and in particular by any automated technique, carried out by a computer or assisted by a computer. Comparing or verifying a “difference” may involve calculating a ratio or a difference.
• the issue of a conclusion as to the presence or not of an infection by a replicative respiratory virus in the subject from which the test sample originates, can also be carried out by any automated, computer-implemented or computer-assisted technique.
• the “median” of a set of values is the value located in the middle of the lowest value and the highest value of the set of values.
• test for detecting a respiratory virus diagnostic test for a respiratory virus or test for detecting the presence of an infection by a respiratory virus, is meant any test known to those skilled in the art making it possible to bring such a conclusion, in particular tests for detecting the DNA or RNA of said respiratory virus, in particular PCR tests or even antigenic tests.
• the invention proposes to use the level of transcripts of at least one marker gene selected from the genes stimulated by interferons, called ISG, determined in a test sample from the mouth or nose of a subject, to determine whether or not said subject has a replicative respiratory virus infection.
• the invention proposes methods for determining in vitro or ex vivo the presence in a subject of an infection by a replicative respiratory virus, comprising a step i) of determination, in a test sample originating from the mouth or nose of said subject, the level of transcripts of at least one marker gene selected from the genes stimulated by interferons, called ISG.
• the level of transcripts of at least one marker gene selected from among the genes stimulated by interferons, called ISGs is used to determine whether or not said subject is suffering from an infection by a replicative respiratory virus. .
• a conclusion as to the possible presence of an infection by a replicative respiratory virus in said subject is delivered by taking into account the level of transcripts of a gene ISG marker, or even several levels of transcripts of different ISG marker genes.
• the conclusion can be made on this sole basis (i.e. from the level of transcripts of a single ISG marker gene or from several levels of transcripts of different ISG marker genes) or by taking into account other genes than ISGs, although this is not preferred within the scope of the invention.
• the determination of the level of transcripts is done by quantitative detection of one or more transcript(s) of the gene, and in particular of the messenger RNA (mRNA) type, in the sample of interest ( whether it is the test sample or a reference sample).
• the determination of the level of transcripts of an ISG therefore involves a quantitative measurement representative of the quantity of transcripts of said ISG, in particular the quantity of mRNA of said ISG, in the test sample.
• transcript means the RNAs, and in particular the messenger RNAs (mRNAs), resulting from the transcription of the gene. More precisely, the transcripts are the RNAs produced by the transcription of a gene followed by the post-transcriptional modifications of the pre-RNA forms.
• the measurement of the level of transcripts of the same gene can comprise the level of identical or different transcripts.
• the determination of the level of transcripts relates to the determination of the level of mRNA of said gene.
• the detection can be carried out by a direct method, by any method known to those skilled in the art making it possible to determine the presence of said transcript in the 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.
• Numerous methods exist for the detection of nucleic acids see for example Kricka et al., 1999, and Relier GH et al., 1993).
• Gene expression can in particular be measured by Reverse Transcription-Polymerase Chain Reaction or RT-PCR, preferably by quantitative RT-PCR or RT-qPCR (for example using FilmArray® technology), by sequencing (including high-level sequencing throughput) or by hybridization techniques (for example with hybridization microchips or by techniques of the NanoString® nCounter® type).
• RT-PCR Reverse Transcription-Polymerase Chain Reaction
• sequencing including high-level sequencing throughput
• hybridization techniques for example with hybridization microchips or by techniques of the NanoString® nCounter® type.
• a technique without RNA or DNA extraction can be implemented, which is the case with the FilmArray® technique (Poritz et al. 2011).
• Determining the level of transcripts of a gene makes it possible to determine the quantity of one or more transcripts of said gene present in the sample of interest or to give a derived value therefrom which is representative of the quantity of transcripts present in the sample of interest.
• a derived value representative of the quantity can, for example, be the absolute concentration, calculated using a calibration curve obtained from successive dilutions of a solution of amplicons of known concentration. It can also correspond to the normalized and/or calibrated value of the quantity of transcript, such as the CNRQ (Calibrated Normalized Relative Quantity), (Hellemans et al., 2007), which integrates the values of a reference sample (or a calibrator) and one or more housekeeping genes (also called reference genes).
• housekeeping genes include DECR1, HPRT1, PPIB, RPLP0, PPIA, GLYR1, RANBP3, 18S, GAPDH, ACTB, ABCF1, ALAS1, GUSB, HPRT1, MRPS7, NMT1, NRDE2, OAZ1 genes. , PGK1, SDHA, STK1 11P and TBP.
• the level of transcripts of a selected marker gene determined for the subject's test sample is compared to a level reference for said marker gene, which in certain embodiments is also used as a threshold value to bring a conclusion as to the presence or not of an infection by a replicative respiratory virus, in the subject of interest.
• Such a comparison can be carried out in different ways, and this in a manner known to those skilled in the art.
• it is also possible, for the comparison to calculate a ratio between the level of transcripts of a selected marker gene and the reference level for said marker gene, in particular before carrying out a comparison with the threshold value used.
• the level of transcripts of the selected marker gene(s) is normalized with respect to the level of transcript one or more housekeeping genes (or reference genes), as known to those skilled in the art; more preferably using one or more of the following housekeeping genes: DECR1 (chromosomal location of the gene according to GRCh38/hg38: chr8:90,001,352-90,053,633), HPRT1 (chromosomal location of the gene according to GRCh38/hg38: chrX :134,452,842-134,520,513) and PPIB (chromosomal location of the gene according to GRCh38/hg38: chr15:64, 155,812-64, 163,205).
• housekeeping genes or reference genes
• the reference level used is also normalized beforehand, in the same way.
• the normalization whether for the reference level or for the level of transcripts of the test sample, is carried out before the comparison, in particular before the calculation of a ratio between the level of transcripts of the test sample and the level reference.
• this normalization may be taken into account for the choice of the threshold value.
• the method according to the invention includes the determination of the level of transcripts of the or housekeeping genes used for standardization.
• the level of transcripts of a single marker gene selected from the ISGs can be determined in step i) and it can be concluded that there is an infection by a replicative respiratory virus. in said subject, if the comparison of the level of transcripts of said ISG marker gene with a predetermined threshold value, shows that there is a difference with said given or predetermined threshold value corresponding to said marker gene. In particular, said difference corresponds to a level of transcripts for the test sample which is greater than that corresponding to the threshold value.
• said threshold value corresponds to a reference level of said marker gene which is the level of transcripts of said marker gene in a subject not showing any infection by a replicative respiratory virus and who, preferably, does not have any neutralizing auto-antibodies directed against interferon a and/or does not possess neutralizing auto-antibodies directed against interferon w, or in a population of subjects showing no infection by a replicative respiratory virus and who, preferably , do not possess neutralizing autoantibodies directed against interferon a and/or do not possess neutralizing autoantibodies directed against interferon w.
• said difference between the level of transcripts of the selected ISG marker gene determined in the test sample and the reference level of said ISG marker gene corresponds to the fact that the level of transcripts of said marker gene ISG determined in the test sample is increased, or even significantly increased, in particular by at least 10%, by at least 20%, by at least 30%, by at least 40%, by at least 50%, at least 60%, at least 70%, at least 80% or at least 90% compared to the reference level of said ISG.
• the increase considered relevant to bring a conclusion will be a function of the threshold value considered, and in particular of the reference level used as threshold value, and will be adapted accordingly by those skilled in the art.
• the threshold value corresponds to the reference level of said ISG marker gene which is the level of transcripts of said ISG marker gene in a subject not exhibiting any infection by a replicative respiratory virus and who, preferably, does not possess auto - neutralizing antibodies directed against interferon a and/or does not possess neutralizing autoantibodies directed against interferon w, or in a population of subjects not exhibiting no infection with a replicative respiratory virus and who preferably do not possess neutralizing autoantibodies directed against interferon a and/or do not possess neutralizing autoantibodies directed against interferon w, he may suffice that the level of transcripts of the selected ISG determined in the test sample is simply greater than the threshold value.
• a different reference level in particular the level in subjects not suffering from a respiratory infection or more generally healthy subjects (without infection of any kind, as are in particular blood donors )
• the difference with respect to the threshold value must be more significant, in particular according to one of the aforementioned % increases.
• An increase may be deemed significant, after applying a statistical method well known to those skilled in the art, such as the Student or Wilcoxon test. Such a method is notably used in the examples.
• said marker gene selected from among the ISGs can be any ISG gene given in the present description, but is preferably IFIT 1 or NFI44L.
• the comparison can be carried out by comparing the level of transcripts of each marker gene selected with a given or predetermined threshold value for each marker gene considered. It is also possible to make a more global comparison. In particular, it is possible to obtain the level of transcripts of several marker genes and to compare the level of overall transcripts of these marker genes with a single overall threshold value. It is also possible to carry out a comparison of the level of transcripts of each marker gene selected with a threshold value given for each marker gene.
• said threshold value corresponds to a reference level of said marker gene which is the level of transcripts of said marker gene in a subject not exhibiting any infection by a replicative respiratory virus and who, preferably, does not possess no neutralizing autoantibodies directed against interferon a and/or does not possess neutralizing autoantibodies directed against interferon w, or in a population of subjects having no infection with a replicative respiratory virus and who preferably do not possess neutralizing autoantibodies directed against interferon a and/or do not possess neutralizing autoantibodies directed against interferon w.
• step i) the transcript levels of several marker genes selected from among the ISGs can be determined in step i), for the test sample, and
• the level of transcripts of each marker gene determined in the test sample is compared with the threshold value of the said corresponding ISG marker gene.
• said difference corresponds to a level higher than that of the threshold value.
• the level of transcripts of said ISG marker gene determined in the the test sample is increased, or even increased significantly, in particular by at least 10%, by at least 20%, by at least 30%, by at least 40%, by at least 50%, at least 60%, at least 70%, at least 80% or at least 90% with respect to the threshold value considered for said ISG.
• the methods according to the invention may comprise the determination of the level of transcripts of 1 to 71 marker genes chosen from those given in the present description, and, preferably of the level of transcripts of 1 to 6, marker genes chosen by the ISGs, and in particular chosen from IFI27, IFI44L, IFIT 1, RSAD2, ISG15 and SIGLEC1.
• a comparison may be performed by calculating a ratio of the transcript level of said selected marker gene determined for the test sample to a corresponding baseline level for said marker gene. It is also possible that such a comparison is made by calculating the ratio of the reference level of said selected marker gene to the level of transcripts of said selected marker gene determined for the test sample.
• an overall ratio corresponding to the median of said ratios can be calculated and used to make a conclusion as to the presence or absence of an infection by a replicative respiratory virus in said subject.
• the median of all the ratios obtained is then calculated and it is concluded that the presence of infection by a replicative respiratory virus is present in said subject, when said median is greater than a threshold value, which may for example be equal to 1.5, preferably greater than 2, 3, 4, 5, or 6.
• a threshold value which may for example be equal to 1.5, preferably greater than 2, 3, 4, 5, or 6.
• the reference level of said marker gene for calculating the ratios may be the level of transcripts of said marker gene, in a subject not infected with a respiratory virus, or in a population of such subjects of said marker gene.
• a subject not infected with a respiratory virus is, in particular, a subject negative for a test to detect the presence of a respiratory virus.
• the reference level used for calculating the ratio is the level of transcripts of said marker gene, in a subject not infected with a respiratory virus, not possessing neutralizing autoantibodies directed against interferon a and/or neutralizing autoantibodies directed against interferon w or in a population of such subjects.
• the methods according to the invention may comprise the determination of the level of transcripts for 1 to 71, and preferably for 1 to 6, marker genes chosen by the ISGs, and in particular chosen ) among IFI27, IFI44L, IFIT1, RSAD2, ISG15 and SIGLEC1.
• the method according to the invention comprises: i) determining the levels of transcripts of several marker genes selected from IFI27, IFI44L, IFIT1, RSAD2, ISG15 and SIGLEC1, for the test sample, ii) for each marker gene for which the level of transcripts in the test sample is determined in step i), the ratio between said level of transcripts on the reference level of said marker gene, is calculated, iii) the median of all the ratios obtained is then calculated and it is concluded that there is an infection by a replicative respiratory virus in said subject, when said median is greater than the threshold value which may for example be equal to 1.5, preferably greater than 2 , 3, 4, 5, or 6.
• step i) it is possible to determine in step i) and to use in steps ii) and iii), the levels of transcripts of all the marker genes IFI27, IFI44L, IFIT1 and RSAD2 , or even of all the marker genes IFI27, IFI44L, IFIT1, RSAD2, ISG15 and SIGLEC1.
• the levels of transcripts of the IFI27, IFI44L, IFIT1 and RSAD2 genes are determined and used as marker genes. That is, the conclusion regarding the presence or absence in the subject of an infection with a replicative respiratory virus does not take into account the level of transcripts of another marker gene.
• the threshold value or the reference level are, in general, values determined upstream which are available during the implementation of the method according to the invention.
• the threshold value is a value which has previously been judged to be relevant for obtaining a conclusion as to the presence or absence of an infection by a replicative respiratory virus.
• the reference level corresponds to the level of transcripts for the marker gene considered, in a reference subject or a reference population.
• a reference sample is of the same type as the test sample, namely a sample from the mouth or nose of a subject and, preferably, an oropharyngeal or nasopharyngeal sample, or saliva.
• a reference subject can be a subject not infected with a replicative respiratory virus, that is to say, in particular a subject detected positive for the presence of a respiratory virus but for which the virus is not replicative. , or a subject not infected with a respiratory virus (in particular not positive for a test for detecting a respiratory virus); a subject not infected with a respiratory virus (in particular not positive for a test to detect a respiratory virus); a healthy subject, that is to say without infection of any kind, as blood donors are in particular...
• the reference sample (coming from a so-called reference subject) for determining a reference level used as a threshold value for the level of transcripts of a marker gene is, advantageously, a sample coming from a subject (called reference subject) not infected with a replicative respiratory virus, that is to say, in particular a subject detected positive for the presence of a respiratory virus but for which the virus is not replicative, or a subject not infected with a respiratory virus (in particular not positive for a test for detecting a respiratory virus), or a mixture of such samples.
• the reference sample (coming from a so-called reference subject) to determine the reference level used for calculating a ratio or a derived value or score as previously described is, advantageously, a sample from a subject (called reference subject) not infected with a respiratory virus (in particular not positive for a test for detecting a respiratory virus), or a mixture of such samples.
• the reference subjects from which the reference samples come do not possess neutralizing autoantibodies directed against interferon a and/or do not possess neutralizing autoantibodies directed against interferon w.
• Such verification can be done from a blood sample, in particular serum of the subject concerned, by any known antibody detection technique, in particular an ELISA technique.
• the reference sample can also be a sample from such a subject, treated ex vivo with an agent stimulating the immune system (such as LPS or lipopolysaccharide).
• the reference sample can also be a mixture of untreated sample(s) and samples treated ex vivo with an immune system stimulating agent.
• level of reference transcripts when the latter is the level of transcripts of a reference population, this may be the mean or the median of the various levels of transcripts measured for said population, i.e. i.e. the mean or median of the transcript levels measured in the reference samples.
• the method according to the invention may also comprise the detection of DNA or RNA, of one or more respiratory viruses, and in particular of SARS-CoV-2 or one of its variants (Mommert, M. et al., 2020).
• detection techniques are known to those skilled in the art. It is possible to use a non-quantitative, quantitative or semi-quantitative detection technique. In particular, the use of an amplification, hybridization or sequencing technique adapted to the DNA or RNA of the virus sought may be used.
• the invention also relates to a kit comprising means for determining the level of transcripts of one or more ISG marker genes such as defined in the context of the invention.
• Such means make it possible to quantitatively determine the level of transcripts of said selected gene in the test sample.
• the level of transcripts determined may not be equal to the quantity of transcripts present in the test sample, but may be a derived value, representative of the quantity of transcripts present in the test sample. Indeed, conventionally, the determination may include a step of amplification and/or normalization and/or calculation of a ratio...
• kit is meant a set of products and/or tools to be used together to obtain, in particular, the determination of the level of transcripts of one or more ISG marker genes as defined in the context of the invention.
• the necessary products or tools may or may not be grouped together in the same kit or device.
• kits according to the invention may comprise means of amplification and/or detection for one or more ISG marker genes, such as those mentioned above, in particular chosen from IFI27, IFI44L, IFIT1, RSAD2, ISG15, and SIGLEC1.
• the kits according to the invention may comprise amplification and/or detection means for each selected ISG marker gene, and in particular which are specific to said marker gene.
• the kits according to the invention may also comprise at least one means of determination, making it possible to determine the level of transcripts of a housekeeping gene.
• the kits according to the invention may comprise means of amplification and/or detection for one or more housekeeping genes (preferably selected from the list consisting of: DECR1, HPRT1 and PPIB).
• the kits according to the invention may comprise amplification and/or detection means for each housekeeping gene selected, and in particular which are specific to said housekeeping gene.
• the means for determining the level of transcripts comprise, in particular, one or more amplification product(s) or tool(s) and/or one or more detection product(s) or tool(s).
• the kit comprises one or more oligonucleotides, allowing the amplification and/or the detection of a transcript of said gene, in particular an amplification primer or pair of primers and/or at least one probe for detecting a transcript of said gene.
• primer or “amplification primer” is understood to mean an oligonucleotide or nucleotide fragment which may consist of 5 to 100 nucleotides, preferably of 15 to 30 nucleotides, and possessing a hybridization specificity with a nucleotide sequence target, under conditions determined for the initiation of an enzymatic polymerization, for example in an enzymatic amplification reaction of the target nucleotide sequence.
• primers consisting of two primers.
• probe or “hybridization probe” means an oligonucleotide or nucleotide fragment typically consisting of 5 to 100 nucleotides, preferably 15 to 90 nucleotides, even more preferably 15 to 35 nucleotides, possessing a hybridization specificity under determined conditions to form a hybridization complex with a target nucleotide sequence.
• the probe also includes a reporter (such as a fluorophore, an enzyme or any other detection system), which will allow the detection of the target nucleotide sequence.
• the target nucleotide sequence can 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.
• mRNA messenger RNA
• cDNA complementary DNA
• Primer and probe sequences suitable for determining the level of transcripts of each IFI27, IFI44L, IFIT1, RSAD2, ISG15 and SIGLEC1 gene are, in particular, described in M. Bergallo et al., 2020.
• hybridization is meant the process during which, under appropriate conditions, two oligonucleotides or 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 higher stringency.
• Stringency is defined in particular according to the base composition of a probe/target duplex, as well as by the degree of mismatch between two nucleic acids.
• the stringency can also be a function of the reaction parameters, such as the concentration and the type of ionic species present in the hybridization solution, the nature and the concentration of denaturing agents and/or the hybridization temperature.
• the stringency of the conditions under which a hybridization reaction must be carried out will mainly depend 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.
• 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.
• enzymatic amplification reaction is meant a process generating multiple copies of a target nucleotide fragment, by the action of at least one enzyme.
• amplification reactions are well known to those skilled in the art and the following techniques may be mentioned in particular: PCR
• RT-PCR reverse transcription
• mRNA RNA messenger
• cDNA complementary DNA
• kits according to the invention may comprise means for detecting a respiratory virus, preferably SARS-CoV-2.
• a respiratory virus preferably SARS-CoV-2.
• such means correspond to a product or tool for detecting and/or amplifying the DNA or RNA of said virus.
• kits according to the invention can also comprise positive control means, in particular a positive control sample, making it possible to qualify the quality of the extraction of the RNA, the quality of any amplification process and/or 'hybridization.
• kits according to the invention are characterized in that they comprise a set of amplification and/or detection means which allow the detection and /or the amplification of at most 100 genes, preferably at most 90, preferably at most 80, preferably at most 70, preferably at most 60, preferably at most 50, preferably at most 40, preferably at most 30, preferably at most 20, preferably at most 10 genes, in particular at most 6, at most 5, at most 4, at most 3, or at most 2 genes, in total.
• amplification and/or detection means which allow the detection and /or the amplification of at most 100 genes, preferably at most 90, preferably at most 80, preferably at most 70, preferably at most 60, preferably at most 50, preferably at most 40, preferably at most 30, preferably at most 20, preferably at most 10 genes, in particular at most 6, at most 5, at most 4, at most 3, or at most 2 genes, in total.
• biomarker genes that is to say of an objectively measurable biological characteristic which represents an indicator of normal or pathological biological processes or pharmacological response to a therapeutic intervention.
• the biomarker genes therefore include the marker genes selected from the ISGs and the housekeeping genes described in the context of the invention.
• the biomarker gene may, in particular, be detectable at the mRNA level.
• biomarker which may also be present in a kit according to the invention, mention may be made of an endogenous or loci biomarker (such as a gene or a HERV/Human Endogenous RetroVirus) which is found in the chromosomal material of an individual, or an exogenous biomarker (such as a virus).
• endogenous or loci biomarker such as a gene or a HERV/Human Endogenous RetroVirus
• kits according to the invention comprise, as means for amplifying and/or detecting genes, exclusively means for amplifying and/or detecting genes of biomarker(s) consisting exclusively in ways for amplifying and/or detecting one or more ISG marker genes, means for amplifying and/or detecting one or more housekeeping genes and, optionally, means for amplifying and/or detecting one or more respiratory viruses.
• kits can therefore comprise a solid support comprising one or more oligonucleotide(s) suitable for determining the level of transcripts of the or each IGS marker gene, or even one or more oligonucleotide(s) suitable for determining the level of transcripts of the gene or of each housekeeping gene selected, or even one or more oligonucleotide(s) suitable for the detection of a respiratory virus such as SARS-CoV-2, as previously described.
• solid supports are well known to those skilled in the art, and in particular described in applications WO2008/140568 and WO2017/093672 to which reference may be made for more details.
• kits are intended to be implemented in an automated manner as regards the comparison with a threshold value and/or reference value.
• the kits are intended to be implemented in an automated manner with respect to the comparison of the level(s) of transcript(s) of the selected ISG marker gene(s), and the value(s) threshold used, or if necessary by using a reference level to calculate a datum (or a score) from the level(s) of transcript(s) of the selected ISG marker(s) gene(s) and compare it with a threshold value.
• they comprise, in addition to one or more threshold values, at least one reference level for said (or each) selected ISG marker gene, which is either stored on a medium readable by a computer, or intended to be used in the form of a code executable by a computer, in particular configured to compare the level of transcripts of the ISG marker gene determined for the test sample, with said reference level.
• the invention also relates to the use of a kit as described in the context of the invention to determine the possible presence in a subject of an infection by a replicative respiratory virus.
• the use will aim to determine the presence or absence in a subject of a replicative respiratory virus, chosen from replicative seasonal coronaviruses, replicative SARS-CoV-2 viruses (regardless of the variant concerned), influenza viruses (influenza A, B and C), respiratory syncytial virus (RSV), rhinoviruses, metapneumoviruses, parainfluenza viruses and adenoviruses.
• a replicative respiratory virus chosen from replicative seasonal coronaviruses, replicative SARS-CoV-2 viruses (regardless of the variant concerned), influenza viruses (influenza A, B and C), respiratory syncytial virus (RSV), rhinoviruses, metapneumoviruses, parainfluenza viruses and adenoviruses.
• kits according to the invention are particularly suitable for determining the presence or absence in a subject of a replicative respiratory virus chosen from replicative SARS-CoV-2 viruses, whatever the variant concerned, and the influenza virus.
• the in vitro or ex vivo determination of the presence in a subject of an infection by a replicative respiratory virus is solely based on the determination, in a test sample from of the mouth or nose of said subject, of the level of transcripts of one or more marker gene(s) selected from among the genes stimulated by interferons, known as ISG.
• ISG the level of transcripts of one or more marker gene(s) selected from among the genes stimulated by interferons
• marker gene(s) selected from among the genes stimulated by interferons called ISGs, one or more other ( s) marker gene(s), for example, selected from type I interferon genes.
• additional marker gene(s) mention may be made of genes coding for the various proteins of type I, II and/or III interferons.
• the invention also relates to the methods for determining in vitro the presence in a subject of an infection by a replicative respiratory virus, as defined in the present description, which further comprise the treatment of a respiratory virus infection.
• the treatment is initiated as soon as when it is concluded that said subject has an infection with a replicative respiratory virus.
• the treatment can also be initiated, once it is concluded that the said subject has an infection with a replicative respiratory virus and that a respiratory virus present has been identified. In this case, the treatment will be directed against the identified respiratory virus. Even if there is no certainty that the respiratory virus whose presence has been identified and the respiratory virus present which is replicative are identical, the presumption is strong because the probability that the subject is infected with two different respiratory viruses is particularly reduced.
• the treatment can be initiated, as soon as the subject exhibits symptoms of a respiratory infection and it is concluded that the subject is present with an infection by a replicative respiratory virus.
• the treatment consists of the administration of a suitable antiviral drug.
• antiviral treatment mention may be made of Iopinavir®, Ritonavir®, recombinant interferons, in particular interferon beta, alpha and lambda.
• Iopinavir® Iopinavir®
• Ritonavir® recombinant interferons, in particular interferon beta, alpha and lambda.
• Many therapeutic treatments for COVID-19 are currently being tested (Canedo-Marroquin, G. etal., 2020).
• the invention also relates to methods for determining in vitro the presence in a subject of an infection by a replicative respiratory virus, as defined in the present description, which further comprise the establishment of measures of isolation, confinement and/or obligation to wear a mask for said subject.
• measures can be put in place, pending the result as to the determination of an infection by a replicative respiratory virus, and, possibly be lifted, in the event of the conclusion that there is no presence in said subject, infection with a replicative respiratory virus. It is also possible that such measures will be put in place, in the event of the conclusion that there is presence in the said subject, of an infection by a replicative respiratory virus.
• the invention also relates to a method for determining in vitro the presence in a subject of an infection by a replicative respiratory virus, as defined in the present description, which comprises, in addition to the steps described above, the setting in place or not, or the discontinuation of isolation, confinement and/or mask-wearing measures for said subject, depending on the conclusion made by said method, as to the presence or absence of an infection by a replicative respiratory virus, in said subject.
• the methods according to the invention may also include a subject management step, depending on the result given by the method.
• a subject identified as having a respiratory infection by a replicative virus, and in particular by SARS-coV-2, may be subject to a treatment protocol or to measures of isolation, confinement and/or the obligation to wear a mask.
• the present invention is illustrated in a non-limiting manner by the following examples.
• Table 1 summarizes the demographic data and the main clinical characteristics of the cohort of subjects who were the subject of the study: healthcare professionals (HCW) with mild symptoms of COVID-19 (COVID -19) and patients with critical COVID-19 admitted to an intensive care unit (ICU) (critical COVID-19). HCWs with mild COVID-19 were not hospitalized and were pneumonia-free.
• BMI body mass index
• IQR interquartile
• N/A not applicable
• a prospective longitudinal cohort study was performed at the University Hospital of Lyon, France (Hospices Civils de Lyon, HCL), encompassing HCWs with symptoms suggestive of SARS-CoV-2 infection (at least one of the following symptoms: fever, respiratory symptoms, headache, anosmia, ageusia - Trouillet-Aimpuls, S. etal. 2020a).
• the diagnosis of COVID-19 was confirmed in all patients by qRT-PCR (cobas® SARS-CoV-2 test, Roche Diagnostics, Basel, Switzerland).
• the nasopharyngeal (NP) swab used was either in Copan universal transport medium (UTM-RT®) or in a tube of Cobas® PCR medium.
• the FilmArray® prototype (in the form of a pocket) which was used makes it possible to determine the level of transcripts of four ISGs (protein 27 inducible by interferon alpha (IFI27), protein analogous to interferon-induced protein 44 (IFI44L), interferon-induced protein with tetratricopeptide repeats (IFIT1), and protein 2 containing radical S-adenosyl methionine domain (RSAD2)) and three housekeeping genes (hypoxanthine phosphoribosyl transferase 1 (HPRT1), peptidylpropyl isomerase B (PPIB), 2,4-dienoyl-CoA reductase 1 (DECR1)), for signal normalization.
• IFI27 protein 27 inducible by interferon alpha
• IFI44L protein analogous to interferon-induced protein 44
• IFIT1 interferon-induced protein with tetratricopeptide repeats
• RRSAD2 protein 2 containing radical S-aden
• NP sample or PAXgeneTM blood were tested with the prototype IFN (Tawfik, DM etal., 2020) according to the instructions of the maker. Briefly, the pouches were hydrated with the hydration solution provided with the kit. PAXgeneTM NP or blood samples were mixed with 800 ⁇ l of the sample buffer supplied with the kit and injected directly into the bag and passed through FilmArray® 2.0 and FilmArray® Torch instruments (BioFire Diagnostics, LLC., Known Lake City, UT). The results were delivered in less than an hour. Using a research version of the instrument, real-time quantification cycle (Cq) values and post-amplification melting curves were measured for each assay.
• Cq real-time quantification cycle
• the serum concentrations of IFN-a2 (fg/ml) from the plasma were determined by an ultrasensitive test (SIMOA®) using a commercial kit for the quantification of IFN-a2 (QuanterixTM, Lexington, MY).
• the assay was based on a three-step protocol using an HD-1 analyzer (Quanterix) and required a turnaround time of 3 hours from sampling to results.
• Interferon lambda was determined by an assay using a commercial kit for the quantification of IFN-1 (mesoscale discovery).
• the SARS-CoV-2 load was determined from an NP sample using the SARS-CoV-2 R-gene® kit (bioMérieux, Lyon, France). Briefly, nucleic acid extraction was performed from 0.2 ml of an NP sample on NUCLISENS® easyMAG® and by means of amplification using a Biorad CFX96 thermocycler. Viral load was quantified using four internally developed quantification controls (QS) targeting the SARS-CoV-2 N gene: QS1 to QS4 respectively at 2.5.10 6 ,
• NPs positive by RT-PCR were inoculated on confluent Vero cells (ATCC CCL-81®) with Eagle's minimal essential medium (EMEM) supplemented with 2% penicillin-streptomycin, 1% L-glutamine, and 2% inactivated fetal bovine serum.
• EMEM Eagle's minimal essential medium
• the plates were incubated at 33°C with 5% CO2 for 96 hours. Cytopathic effects (CPE) were monitored daily; samples were harvested when positive, while negative samples at 96 hours were subcultured onto new plates.
• RNA from supernatants was extracted by an automated MGISP-960 workstation using the MGI Easy magnetic bead viral DNA/RNA extraction kit (MGI Tech ⁇ , Marupe, Lithuania), and detection of SARS- CoV-2 was performed using the TaqPath(tm) COVID-19 CE-IVD RT-PCR kit on a QuantStudio(tm) 5 system (Applied Biosystems, Thermo Fisher Scientific, Waltham, USA).
• the presence of anti-SARS-CoV-2 antibodies was assessed using the Wantai Ab assay which detects total antibodies against the receptor binding domain (RBD) of protein S.
• the presence of anti-IFN- ⁇ antibodies was studied using a commercially available kit (Thermo-Fisher) (David, G et al., 2021).
• the neutralizing capacity of a patient's serum towards IFN- ⁇ was then evaluated as previously described (Bastard, P. et al., 2021).
• Nonparametric Wilcoxon-Mann-Whitney tests and Spearman correlation were performed for all comparisons, unless otherwise stated.
• the minimum p-value of the non-parametric significance test (Wilcoxon rank sum) was used applied iteratively to the ISG score, separated on standardized nasal viral load thresholds ranging from minimum (0.60) to maximum (9.00) detected values with a step of 0.01.
• the advantage of this technique is that it is semi-automated and also designed for clinical routine, giving a score derived from the quantification of four ISGs regulated through ISRE elements dependent on gene factor 3 stimulated by a IFN (ISGF3), namely IFI27, IFI44L, RSAD2 and IFIT1 (Fêtandez, N. et al., 2018).
• This score was analyzed during diagnosis in 23 FICWs infected with SARS-CoV-2, and compared to the same score obtained from blood samples and the serum level of IFN-a2 from the same patients.
• FIG. 7 demonstrates that there is a significant correlation between the score associated with the levels of transcripts of the various type I/III ISGs (called ISG score) obtained for the nano-pharyngeal samples and the viral load.
• ISG score the score associated with the levels of transcripts of the various type I/III ISGs
• the nasal ISG type l/l II score is lower in patients with neutralizing type I anti-IFN autoantibodies.
• nasal viral load the viral load in the nasopharyngeal samples
• ISG score the score ISG in nasopharyngeal specimens
• Anti-IFN-a and/or anti-IFN-w autoantibodies were detected in the serum of 6 of 19 severely affected patients and in none of the mildly symptomatic patients (Figure 7).
• patients with anti-IFN-1 autoantibodies had very low nasal ISG scores, regardless of viral load, except for one patient who had autoantibodies against IFN-a2 only, and for which it was assumed that IFN-w compensated for the blockade of IFN a2.
• NP samples 2017-2018
• HCL Livings Civils de Lyon
• NP swabs from healthy volunteers were recruited according to the same protocol as example 1.
• the RESPIFERON study was approved by the ethics committee for biomedical research and by the personal protection committee. CPC.
• Authorization to use the samples was granted on the basis of non-objection by the subjects directly concerned or their legal guardians, if applicable, after having informed them by mail. Anonymization of the subjects' personal data was carried out prior to their transfer to the laboratory.
• the viral load was quantified for each NP sample.
• the nucleic acids were extracted using the NucliSens® easyMAGTM automated extractor (bioMérieux, Marcy l'Etoile). The extraction was carried out from 200 pL of NP samples which were deposited in a lysis buffer. At the end incubation for 10 minutes at room temperature, 50 ⁇ l of magnetic silica were added before starting the extraction.
• RNA was reverse transcribed and amplified using the Influenza A/B R-gene® ready-to-use real-time RT-PCR kit.
• the reaction required 10 pL of eluate, 15 pL of amplification premix containing the reagents necessary for the PCR and 0.15 pL of reverse transcriptase (diluted in 10th).
• the amplification was launched on the Bio-Rad CFX96 following a specific protocol, namely 5 min at 50° C. for the reverse transcription followed by 15 min at 95° C. to activate the Taq polymerase, and finally 45 cycles which alternate: 10sec at 95°C for denaturation, 40sec at 60°C for hybridization, and 25sec at 72°C for elongation.
• the viral load was quantified using a range of r-gene standards (QS1 to QS4 containing respectively 10 5 , 10 4 , 10 3 and 10 2 copies/pL of plasmid).
• the CELL Control r-gene® kit was used firstly to check the presence of cells in the sample and quantify them by detecting the HPRT 1 housekeeping gene, and secondly to normalize the charge.
• This kit contains 2 quantification standards, QS1 and QS2 respectively at 10 4 and 10 3 copies/pL of DNA. Data were retrieved, processed and analyzed using CFX Maestro software.
• the viral load was normalized by dividing the number of copies of viral RNA/PCR by the number of cells/PCR and is expressed in Iog10 of the number of copies per 10 6 cells.
• the viral culture, the collection of the samples, the sampling of the culture supernatants and the extraction of the RNAs from said supernatants are as described in example 1.
• the detection of the influenza virus was carried out using the Influenza A/B R-gene® kit (bioMérieux, Marcy l'Etoile).
• Influenza virus infection is associated with increased nasal IFN response
• an IFN l/III score based on the expression of 4 ISGs in the nasal mucosa measured by RT-qPCR using the IFN FilmArray® BioFire® pocket prototype was calculated.
• This nasal IFN l/III score informative as to the local IFN response, turned out to be significantly higher (p ⁇ 0.0001) for the positive PCR samples, therefore from individuals infected with the virus of the Influenza, in comparison to PCR negative samples corresponding to uninfected individuals.
• ROC Receiveiver Operating Characteristic
• the level of expression of the ISG mRNAs, expressed in log2 of the QR ratios, was estimated for each NP sample from the results obtained with the IFN FilmArray® BioFire® pocket prototype as explained previously (see material and method).
• Nasal INF response reflects active influenza virus infection
• Figure 12 thus highlights the good performance of the IFN l/III score in predicting the replicative capacity of the GRB virus with an area under the curve of 0.89 with a cut-off point at 16.08, for a sensitivity of 0, 93 and a specificity of 0.86.
• this example confirms that the measurement of the level of transcripts of genes stimulated by interferons, known as ISG, made it possible to effectively and reliably detect the presence of an infection by a replicative respiratory virus, such as for example SARS-CoV-2 or the influenza virus, thus providing a new tool for the clinician to quickly identify patients at risk of viral transmission, and alternatively to avoid quarantine measures for patients who are not, or no longer, a possible source of contamination.
• a replicative respiratory virus such as for example SARS-CoV-2 or the influenza virus

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