Classifications

• • 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
  • C12Q1/34—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase
  • C12Q1/37—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase involving peptidase or proteinase
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
  • G01N33/582—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with fluorescent label
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
  • G01N2333/005—Assays involving biological materials from specific organisms or of a specific nature from viruses
  • G01N2333/08—RNA viruses
  • G01N2333/165—Coronaviridae, e.g. avian infectious bronchitis virus

Definitions

• the invention relates to a rapid test for detecting the presence of a replicating virus in a sample.
• the international community is currently facing a pandemic of acute respiratory syndrome due to the coronavirus designated SARS-CoV-2.
• SARS-CoV-2 coronavirus
• the diagnostic test by RT-PCR from nasopharyngeal swabs has been the most widespread test to detect the presence of this virus at the individual or population level.
• this test cannot be used to detect a person in real time, whether they are symptomatic or not, but contagious.
• Other individual tests exist, based on immunochromatography techniques or on gene amplification techniques, but they do not provide an answer concerning the immediate contagiousness of the person.
• the inventors now propose a rapid and reliable test for detecting the contagious risk.
• the invention relates to an in vitro method for determining whether a sample contains a replicating virus, in particular a coronavirus, such as a SARS-Cov-2, said method comprising the detection of a Papain-like protease activity ( PLpro) of coronavirus.
• Replicating virus is any virus that expresses a PLpro.
• the method comprises bringing the sample into contact with at least one detectably labeled PLpro protease substrate peptide. More specifically, the activity of PLpro is revealed by a fluorescence test.
• the sample is a saliva sample, the presence of which has been shown to potentiate the fluorescence signal.
• the test allows the presence of PLpro to be revealed in less than 30 minutes, advantageously less than 15 minutes.
• Figure 1 is a graph which shows the fluorescence assay of PLpro activity on the Z-RLRGG-AMC substrate, in comparison with the activity of PLpro on the ISG15-AMC substrate for a period of 60 minutes.
• concentrations of the Z-RLRGG-AMC peptide were increased while respecting an enzyme/substrate ratio of 1/200.
• FIG. 2 is a graph which reports the measurement of the fluorescence emitted by the labeled substrate peptide in the presence of human saliva (3 samples) and of PLPro and of Triton X100, for a period of 30 minutes. Each saliva was tested in triplicate and the means and standard deviations are represented.
• Figure 3 is a graph that shows the potentiating effect of saliva on the observed fluorescence intensity.
• the graph follows the assay of the activity of PLpro at a concentration of 50 nM on the substrate peptide Z -RLRGG-AMC at a concentration of 25 mM. Controls do not include enzyme or substrates.
• Figure 4 is a graph showing the effect of 4 ions in the form of CaC, MgC, KCl and NaCl on the activity of PLpro against the substrate peptide Z-RLRGG-AMC.
• the activity of PLpro (10 nM) on the Z-RLRGG-AMC substrate (50 mM) is monitored over time.
• a sample without ion serves as a reference.
• Figure 5 is a graph showing the effect of Ca 2+ at different concentrations (from 0.2 to 20 mM).
• the activity of PLpro (5 nM) on the Z-RLRGG-AMC substrate (50 mM) is monitored over time.
• a sample without ion serves as a reference.
• the objective of the test is to detect the presence of a virus, in particular a replicative coronavirus, in saliva samples or any other sample, biological or non-biological.
• replicative or “in replication” it is meant that the virus produces virions, and multiplies.
• the test is based on the detection of the enzymatic activity of the papain-like protease (PLpro) of the virus, in particular a coronavirus, making it possible to reveal its replication activity, preferably by means of fluorescence labeling, in liquid medium or not.
• PLpro papain-like protease
• the test can be self-testing, i.e. a test that can be performed without the help of a healthcare professional.
• the sample can be a biological or non-biological sample, for example a sample obtained from an inert support.
• the biological sample may be a sample obtained from a human subject, adult or child, or from a non-human animal, especially a non-human mammal, preferably a domestic animal. It can be for example a pet such as a cat or a dog. It may still be a farm animal. A preferred example is a ruminant such as cattle. According to other aspects, the mammal can be chosen from sheep, goats, pigs, or equines.
• the subject to be tested is preferably suspected or likely to be infected with a coronavirus or another virus exhibiting PLpro activity. It can be a symptomatic subject (for example presenting a fever and/or cough, in humans), or asymptomatic.
• a biological sample examples include saliva or a nasopharyngeal swab.
• the biological sample is a saliva sample.
• a saliva sample can be obtained by sputum (Sapkota et al., 2020) or using a sponge-type device or pipette aspiration or similar device. You can also test any other sample of body fluid (such as blood or plasma) or tissue biopsy.
• the sample is an environmental, sewage or food sample.
• the method makes it possible to check whether an object or a material is contaminated by a virus, in particular a coronavirus, in replication, and likely to represent an infectious risk.
• the sample is obtained by taking a sample from an object, material, or machine, which has been or is likely to have been in contact with an infectious subject.
• the object may be a hospital bed or a medical device that has been used by a patient infected with coronavirus.
• the sample is preferably brought into contact with a lysis buffer, comprising for example at least one detergent or a mixture of detergents, intended to release the membrane proteins of the sample.
• a lysis buffer containing Triton X100 can for example be used.
• the lysis buffer can also contain a mucolytic agent, such as N-acetyl-cysteine (Nac) for example.
• the test can detect the presence of any virus with PLpro activity, i.e. any virus that expresses PLpro.
• the virus is a coronavirus, especially a SARS-CoV, MERS-CoV, or SARS-CoV-2.
• the coronavirus is SARS-CoV-2.
• the method of the invention makes it possible to detect any variant of SARS-CoV-2.
• SARS-CoV-2 viruses are detectable: - Lineage B or Wuhan
• the coronavirus is SARS-CoV-2
• the sample is a saliva sample.
• the coronavirus is an FCoV feline coronavirus.
• the virus to be detected is a picornavirus, preferably an aphthovirus (responsible for foot-and-mouth disease), more preferably a ruminant aphthovirus.
• PLPro is a protease located in nonstructural protein 3 (NS3) of the coronavirus polyprotein (Osipiuk et al, 2021).
• the method according to the invention preferably comprises bringing the sample into contact with at least one substrate peptide of the protease PLpro, preferably labeled in a detectable manner.
• the substrate peptide consists of the sequence RLRGG (SEQ ID NO:2).
• the substrate peptide is used at a concentration ranging from 0.1 mM to 1 mM, preferably 0.1 to 500 mM, preferably 1 to 100 mM, more preferably about 1 to 50 mM.
• the substrate peptide can be freeze-dried, and suspended by adding the sample to the lysis buffer.
• the sample is a saliva sample which provides the liquid to release the lyophilized substrate peptide.
• a cofactor of the enzymatic reaction between the papain-like protein (PLpro) and the substrate peptide consisting of the sequence RLRGG (SEQ ID NO: 2) is added.
• the substrate peptide in the presence of divalent cations, in particular Ca 2+ cations (provided for example in the CaC form).
• the salts are used at a concentration between 5 and 40 mM, preferably 20 mM.
• a concentration of 20 mM CaC offers a potentiating effect on the enzymatic reaction.
• the substrate peptide is placed in the presence of a reaction medium comprising a buffer, supplemented with a lysing agent such as Triton X100, Nac, and CaC, preferably before the addition of saliva.
• the enzymatic activity of PLPro in the sample can be detected by any means known to those skilled in the art, in liquid medium or not.
• the assay uses a PLpro substrate peptide that is detectably labeled.
• the peptide itself preferably carries a detectable label.
• detectable marker refers to a molecule or compound or group of molecules or group of compounds used to measure the enzymatic activity of PLpro.
• the detectable label can advantageously be detected directly.
• the enzymatic activity is revealed by a fluorescence test.
• the substrate peptide carries a fluorophore, preferably at its C-terminal end.
• the substrate peptide bears a fluorophore at one end, and a protective group at the other.
• Any fluorophore can be used, preferably a small organic fluorophore (less than 1 kDa).
• a coumarin derivative such as aminomethylcoumarin (AMC) or methylumbelliferone (MEU)
• AMC aminomethylcoumarin
• MEU methylumbelliferone
• the peptide derivatives of 7-amino-4-methylcoumarin are fluorogenic markers which exhibit fluorescence emission at approximately 420-480 nm upon release of the fluorophore. Any fluorophore allowing detection in wavelengths ranging from 350 to 500 nm, preferably 420 - 480 nm, is also envisaged.
• derivatives of pyrene or naphthalene can also be used as fluorophores.
• the method thus comprises bringing the sample into contact with at least one substrate peptide of the protease PLpro bearing a fluorophore, the fluorophore being an aminomethyl coumarin (AMC) group, preferably carried at the C end. -terminal of the substrate peptide.
• AMC aminomethyl coumarin
• the substrate peptide is Z-RLRGG-(AMC), where (AMC) is aminomethyl coumarin, and Z is a carboxybenzyl protecting group.
• the invention also provides a useful kit for carrying out the test described above.
• kit may optionally comprise a container intended to receive the sample to be tested a container, which may be the container intended to receive the sample to be tested, comprising a lysis buffer, including for example at least one detergent or a mixture of detergents, intended to release proteins from the sample; and a container comprising the PLpro substrate peptide, detectably labeled with a fluorophore.
• a container intended to receive the sample to be tested a container, which may be the container intended to receive the sample to be tested, comprising a lysis buffer, including for example at least one detergent or a mixture of detergents, intended to release proteins from the sample; and a container comprising the PLpro substrate peptide, detectably labeled with a fluorophore.
• all of the reagents may be contained in a single compartment, preferably in lyophilized form.
• the lysis buffer also comprising Nac and CaC.
• the sample to be tested is saliva
• it is the addition of the saliva which resuspends all of the reagents and initiates the reaction.
• the test can thus be carried out entirely in a single step after taking the sample.
• G saliva sample is deposited without delay in the container described above and brought into contact instantly with the reaction medium at room temperature indoors.
• the container is then agitated by inversion (typically 3 to 5 times) and the reaction proceeds without intervention for approximately 15 minutes.
• the container is brought into contact with the fluorescence detector which may or may not be of portable format, the reading wavelength of which is that specific to the fluorescence marker selected.
• the fluorescence reading is instantaneous and gives a positive or negative signal depending on the detection threshold identified by the substrate peptide carrying a fluorophore.
• Example 1 Detection of the enzymatic activity of PLpro by fluorescence measurement
• the enzymatic activity of PLpro was tested using a substrate protein of the enzyme, presenting at its C-terminal end a fluorophore, aminomethyl coumarin (AMC). More specifically, the substrate protein used is ISGI 5, a protein of approximately 17 kDa for which the PLpro enzyme has a high affinity.
• the fluorescence curve obtained also made it possible to detect a significant activity in a time approximately equal to 11-12 minutes.
• Example 1 The fluorescence measurement obtained in Example 1 with the ISG15-AMC substrate was compared with another substrate: Z-RLRGG-AMC which allows advantageous use at higher concentrations.
• Figure 1 shows a very increased fluorescence for the same substrate/enzyme ratio.
• Figure 2 shows the very good results obtained with the saliva of three donors, in the presence of external PLpro, the response being significantly detectable, from 10 minutes of reaction. Each test was repeated three times using 50 ⁇ L of saliva containing the PLP to be detected. The mean and standard deviations for these three donors are shown in Figure 2
• Example 3 Impact of saliva on the fluorescence signal (substrate: Z-RLRGG-AMC)
• Example 4 Assay of the enzymatic activity of PLpro by measuring fluorescence under the influence of different ions.
• a fluorescence measurement was performed on the Z-RLRGG-AMC substrate by testing the influence of different ions diluted in dilution buffer (HEPES).
• HEPES dilution buffer
• ions were thus tested, provided in the form of MgC, CaC, KCl and NaCl.
• the PLpro is at a concentration of 10 nM and the substrate Z-RLRGG-AMC at a concentration of 50 mM.
• Triton X100 [0.001%] was also added for virus lysis. Normalization of enzyme activity was performed relative to controls. Saliva was also retained when measuring PLpro activity.
• the Ca 2+ cation provides the best reaction potentiating effect after 15 minutes.
• the inventors also tested the influence of Ca 2+ cations on the enzymatic activity of PLpro at different concentrations of CaC (from 0.2 to 20 mM) (FIG. 5).
• the PLpro is at a concentration of 5 nM and the substrate Z-RLRGG-AMC at a concentration of 50 mM.
• Triton X100 [0.001%] was also added for virus lysis. Increasing the ion concentration increases the potentiating effect on the activity of PLpro.

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• 2021
  • 2021-06-04 FR FR2105925A patent/FR3123658A1/fr active Pending
• 2022
  • 2022-06-03 WO PCT/FR2022/051066 patent/WO2022254164A1/fr active Application Filing
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