DETECTION PROCEDURE OF A REPLICATING CORONAVIRUS

Abstract

The invention relates to an in vitro method for determining whether a sample contains a replicating coronavirus, said method comprising detecting a coronavirus Papain-like protease (PLpro) activity.

Description

Title of the invention: METHOD FOR DETECTING A REPLICATING CORONAVIRUS Field of invention

[0001] The invention relates to a rapid test for detecting the presence of a coronavirus in a sample. State of the art

[0002] The international community is currently facing a pandemic of acute respiratory syndrome due to the coronavirus designated SARS-CoV-2. The RT-PCR diagnostic test using nasopharyngeal swabs is the most widely used test for detecting the presence of this virus at the individual or population level. However, due to its technical characteristics (detection of the presence of RNA), this test cannot be used to detect in real time a person whether symptomatic or not, but contagious. Other individual tests exist, based on immunochromatography techniques or gene amplification techniques, but they do not provide an answer regarding the immediate contagiousness of the person.

[0003] Risk perception and risky behaviors are becoming important variables in predicting the spread of the pandemic. Providing affordable, reliable, and easy-to-use tools remains a challenge to assess actual risk at the individual level.

[0004] Systematic, high-performance rapid testing is essential to reduce the medical and economic impact of Covid-19. Summary of the invention

[0005] In this context, the inventors now propose a rapid and reliable test to detect the contagious risk.

[0006] More generally, the invention relates to an in vitro method for determining whether a sample contains a replicating coronavirus, in particular a SARS-Cov-2, said method comprising the detection of a Papain-like protease (PLpro) activity of coronavirus.

[0007] For this, the method preferably comprises contacting the sample with at least one substrate peptide of the PLpro protease, preferably detectably labeled. Preferably the activity of PLpro is revealed by a fluorescence test.

[0008] Another preferred subject of the invention is an in vitro method for determining whether a human subject is infected with a coronavirus, in particular a SARS-Cov-2, and is contagious, said method comprising contacting a biological sample of said subject, such as a saliva sample, with at least one PLpro protease substrate peptide, preferably detectably labeled with a fluorophore, said peptide comprising or consisting of the sequence XiLX2GG, where Xi and X2 are any amino acids, the same or different, the peptide preferably consisting of the sequence RLRGG.

[0009] The test allows the presence of PLpro to be revealed in less than 30 minutes. Figures

[0010] [Fig-1] is a graph showing the fluorescence assay of PLpro activity on the Z-RLRGG-AMC substrate, compared with PLpro activity on the ISG15-AMC substrate over a period of 60 minutes. Concentrations of the z-RLRGG-AMC peptide were increased by respecting an enzyme / substrate ratio of 1 / 200.

[0011] [Fig.2] is a graph that reports the measurement of the fluorescence emitted by the labeled substrate peptide in the presence of human saliva (3 samples) and PLPro and Triton X100, for a period of 30 minutes. Each saliva was tested in triplicate and the means and standard deviations are represented. Detailed description of the invention

[0012] The objective of the test is to detect the presence of a replicating coronavirus in saliva samples or any other sample, biological or non-biological.

[0013] By "replicative" or "replicating" 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 coronavirus, making it possible to reveal its replication activity, preferably by means of fluorescent labeling, in a liquid medium or not. It can be a self-test, that is to say a test that can be carried out without the help of a health professional.

[0014] Sample

[0015] The sample may be a biological or non-biological sample, for example a sample obtained from an inert support.

[0016] In a preferred embodiment, the biological sample may be a sample obtained from a human subject, adult or child, or from a non-human animal. The subject to be tested is preferably suspected or likely to be infected with a coronavirus. It may be a symptomatic subject (for example, presenting a fever and / or cough), or asymptomatic.

[0017] Preferred examples of a biological sample include saliva or a nasopharyngeal swab. Most advantageously, the biological sample is a saliva sample. For example, a saliva sample may be obtained by sputum (Sapkota et al., 2020) or using a sponge-type device or pipette aspiration or similar device. Any other biological fluid sample (such as blood or plasma) or tissue biopsy can also be tested.

[0018] Alternatively, the sample is an environmental sample, wastewater sample, or a food sample.

[0019] In another embodiment, the method makes it possible to verify whether an object or material is contaminated by a replicating coronavirus, and likely to represent an infectious risk. In a particular example, 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. For example, the object may be a hospital bed or a medical device having been used by a patient infected with a coronavirus.

[0020] 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 from the sample. A lysis buffer containing Triton X100 may for example be used.

[0021] Coronavirus

[0022] The test can detect the presence of any coronavirus, including SARS-CoV, MERS-CoV, or SARS-CoV-2. Preferably the coronavirus is SARS-CoV-2.

[0023] Furthermore, the method of the invention makes it possible to detect any variant of SARS-CoV-2. For example, the following SARS-CoV-2 viruses can be detected:

[0024] - Lineage B or Wuhan

[0025] -Lineage B. 1

[0026] - Lineage B.1.1.7 or British variant SARS-CoV-2 VOC 202012 / 01

[0027] - Lineage B. 1.351 or South African variant VOC-202012 / 02

[0028] - Lineage B. 1.1.207 or Nigerian variant

[0029] - Danish variant cluster 5 (AFVLspike)

[0030] - Lineage B.1.1.248 or Brazilian variant VOC-202101 / 02

[0031] - British B.1.525 lineage or Nigerian variant VUI-202102 / 03 (PHE)

[0032] - Lineage B. 1.526 or New York variant

[0033] - Lines B. 1.427 or B. 1.429 or Californian variant CAL.20C

[0034] - Variant 20A / 484Q (B. 1.617) or Indian variant

[0035] Preferably, the coronavirus is a SARS-CoV-2, and the sample is a saliva sample.

[0036] Papdin-like protease (PLpro) and substrate peptide

[0037] PLPro is a protease located in the non-structural protein 3 (NS3) of the coronavirus polyprotein (Osipiuk et al, 2021).

[0038] In vitro characterization of the enzymatic activities of PLpro revealed that this The latter can recognize and hydrolyze cellular ubiquitin-associated proteins (Ub) and the ubiquitin-like protein (UBL) ISG15 (interferon-induced gene 15), both carrying the recognition motif LXGG (SEQ ID NO: 1) at their C-terminus (Lindner et al., 2007; Lindner et al, 2005).

[0039] The method according to the invention preferably comprises bringing the sample into contact with at least one peptide substrate of the PLpro protease, preferably detectably labeled.

[0040] The substrate peptide may in particular comprise or consist of the sequence XiLX2 GG (SEQ ID NO: 10), where Xi and X2 are any amino acids, identical or different.

[0041] Preferably, X2 is a basic amino acid, preferably an arginine (R).

[0042] In a preferred embodiment, the substrate peptide consists of the sequence RLRGG (SEQ ID NO:2).

[0043] The method of the invention thus advantageously uses a PLPro substrate peptide, which comprises or consists of the sequence RLRGG. Preferably, it can be fused at the C-terminus to a GGG tripeptide, which is particularly useful in the case of colorimetric revelation.

[0044] Other substrate peptides comprise, or consist of, the sequence LXGG, X representing any amino acid, preferably a basic amino acid, such as arginine, lysine or histidine; or asparagine.

[0045] The substrate peptides may for example comprise the following motif, or consist of the following sequences:

[0046] RLRGG (SEQ ID NO:2) or RLRGG-GGG (SEQ ID NO:3)

[0047] ELNGG (SEQ ID NO: 4) or ELNGG-GGG (SEQ ID NO: 5)

[0048] RLKGG (SEQ ID NO: 6), or RLKGG-GGG (SEQ ID NO: 7)

[0049] SLKGG (SEQ ID NO: 8) or SLKGG-GGG (SEQ ID NO: 9)

[0050] Other substrate peptides useful in the invention are described in Rut et al, 2020.

[0051] In a preferred embodiment, the substrate peptide is used at a concentration ranging from 0.1 pM to 1 mM, preferably 0.1 to 500 pM, preferably 1 to 100 pM, more preferably about 1 to 50 pM.

[0052] Advantageously, the substrate peptide can be lyophilized, and suspended by adding the sample in the lysis buffer. In a preferred embodiment, the sample is a saliva sample which provides the liquid allowing the lyophilized substrate peptide to be released.

[0053] Revelation of enzymatic activity

[0054] The enzymatic activity of PLPro in the sample can be detected by any means known to those skilled in the art, in a liquid medium or not.

[0055] In a preferred embodiment, the test uses a PLpro substrate peptide which is detectably marked.

[0056] For this, the peptide is preferably itself carrying a detectable marker.

[0057] The term "detectable label" as used herein refers to a molecule or compound or group of molecules or group of compounds used to measure the enzymatic activity of PLpro. In some cases, the detectable label may be detected directly. In other cases, the detectable label may be part of a binding pair, which may then be subsequently detected. Signals from the detectable label may be detected by a variety of means and will depend on the nature of the detectable label. Detectable labels may include, but are not limited to, fluorescent moieties, colored substances (i.e., colorimetric moieties), or isotopes.Examples of means for detecting a detectable marker include, but are not limited to, spectroscopic, photochemical, biochemical, immunochemical, electromagnetic, radiochemical, or chemical means, such as fluorescence, chemifluorescence, or chemiluminescence, or any other suitable means.

[0058] In a preferred embodiment, the enzymatic activity is revealed by a fluorescence test. In this embodiment, the substrate peptide advantageously carries a fluorophore, preferably at its C-terminal end.

[0059] Advantageously, the substrate peptide carries a fluorophore at one end, and a protective group at the other.

[0060] Any fluorophore can be used, preferably a small organic fluorophore (less than 1kDa).

[0061] Preferably, a coumarin derivative, such as aminomethylcoumarin (AMC) or methylumbelliferone (MEU), may be used as the fluorophore. For example, peptide derivatives of 7-amino-4-methylcoumarin are fluorogenic labels that exhibit fluorescence emission at about 420-480 nm upon release of the fluorophore. Any fluorophore that allows detection in wavelengths ranging from 350 to 500 nm, preferably 420-480 nm, is also contemplated.

[0062] By way of non-limiting examples, derivatives of pyrene or naphthalene can also be used as fluorophores.

[0063] Preferably, the method thus comprises bringing the sample into contact with at least one substrate peptide of the PLpro protease carrying a fluorophore, the fluorophore being an aminomethyl coumarin (AMC) group, preferably carried at the C-terminal end of the substrate peptide.

[0064] In a particularly preferred embodiment, the substrate peptide is Z-RLRGG-(AMC), where (AMC) is aminomethyl coumarin, and Z is a carboxybenzyl protecting group.

[0065] A colorimetric test is also envisaged. By "colorimetry" is meant a method of chemical analysis having as its object the determination of the concentration of an element, by measuring the intensity of the coloration that it confers on the sample put into solution, or, if it is colorless or slightly colored, by that which it produces in the presence of a specific reagent giving a colored compound.

[0066] In this embodiment, the substrate peptide advantageously carries a colorimetric marker, preferably at its C-terminal end.

[0067] Among the colorimetric markers, we can cite the p-nitroanilide groups which release yellow nitroaniline with a maximum absorbance at 380 nm. 4-Aminoantipyrine is also a useful chromogen.

[0068] Tests using chromogenic substrates may include, for example, enzymatic tests, for example based on peroxidase, which rely on the ability to detect the disappearance of substrate or the formation of product. In another example, 2,2-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (or ABTS), a substrate for peroxidases, may be used. Other examples that may be mentioned are the chromogenic markers o-phenylenediamine, pyrogallol, or 3,3'-diaminobenzidine.

[0069] Kit or device

[0070] The invention also provides a kit useful for carrying out the test described above.

[0071] Such a kit may comprise

[0072] optionally a container intended to accommodate the sample to be tested

[0073] a container, which may be the container intended to accommodate the sample to be tested, comprising a lysis buffer, including for example at least one detergent or a mixture of detergents, intended to release the proteins from the sample;

[0074] and a container comprising a detectably labeled PLpro substrate peptide.

[0075] In another embodiment, all of the reagents may be contained in a single compartment, preferably in lyophilized form.

[0076] Preferably, when the sample to be tested is saliva, it is the addition of saliva which resuspends all of the reagents and initiates the reaction.

[0077] The examples and figures illustrate the invention without limiting its scope. Examples

[0078] Example 1: Detection of the enzymatic activity of PLpro by fluorescence measurement (substrate: ISG15-AMC)

[0079] 1.1. Fluorescence measurement

[0080] 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).

[0081] More precisely, the substrate protein used is FISG15, a protein of approximately 17 kDa for which the PLpro enzyme has a high affinity.

[0082] In order to verify that the PLpro enzyme is capable of cleaving the ISG15-AMC substrate, a fluorescence monitoring test over time was carried out and demonstrates the activity of the PLpro enzyme.

[0083] The fluorescence curve obtained also made it possible to detect significant activity in a time approximately equal to 11-12 minutes.

[0084] 1.2. Influence of dilution buffer and saliva on the activity of the PLpro enzyme

[0085] The impact of the dilution buffer on the enzyme activity was assessed using monitoring of the fluorescence emitted over time. Three combinations of buffers were tested

[0086] (1) HEPES

[0087] (2) HEPES + NaCl

[0088] (3) HEPES + NaCl + Tris(2 carboxyethyl)phosphine (TCEP).

[0089] The fluorescence monitoring curves showed an increase in the emitted fluorescence, with no significant difference between the dilution buffers tested. It can therefore be concluded that the dilution buffer has no impact on the activity of the PLpro enzyme.

[0090] The inventors then verified that saliva was not a factor that could influence the activity of PLpro.

[0091] Fluorescence, related to enzyme activity, was measured in the presence of saliva or HEPES buffer. Each solution tested contained the ISG15-AMC substrate concentrated at 1pM and the PLpro enzyme at 5 nM. The control did not contain any enzyme.

[0092] The curves obtained showed that the enzymatic activity of PLpro is maintained in the presence of human saliva.

[0093] Example 2: Detection of the enzymatic activity of PLpro by fluorescence measurement (substrate: Z-RLRGG-AMC)

[0094] 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.

[0095] [Fig.l] shows a very increased fluorescence for the same substrate / enzyme ratio.

[0096] [Fig.2] shows the very good results obtained with the saliva of two donors, in the presence of external PLpro, the response being significantly much higher than the controls, from 15 minutes of reaction. References

[0097] - Lindner, HA, Fotouhi-Ardakani, N., Lytvyn, V., Lachance, P., Sulea, T., Ménard, R., 2005. The papain-like protease from the severe acute respiratory syndrome co-ronavirus is a deubiquitinating enzyme. J. Virol. 79, 15199-15208.

[0098] - Lindner, HA, Lytvyn, V., Qi, H., Lachance, P., Ziomek, E., Ménard, R., 2007. Se- lectivity in ISG15 and ubiquitin recognition by the SARS coronavirus papain-like protease. Arch. Biochem. Biophys. 466, 8-14.

[0099] - Osipiuk, J., Azizi, SA., Dvorkin, S. et al. 2021.Structure of papain-like protease from SARS-CoV-2 and its complexes with non-covalent inhibitors. Nat Commun 12, 743

[0100] Ruth , W. , Lv , Z. , Zmudzinski , M. , Patchett , S. , Nayak , D. , Snipas , SJ , El Oualid , F. , . [ PMC free article ] [ PubMed ] Huang, TT, Bekes, M., Drag, M., Olsen, SK, bioRxiv.

[0101] - Sapkota, D., Spland, TM, Galtung, HK, Sand, LP, Giannecchini, S., To, KKW, Mendes-Correa, MC, Giglio, D., Hasséus, B., Braz-Silva, PH, J. Clin. Pathol.

Claims

Claims

1. An in vitro method for determining whether a sample contains a replicating coronavirus, said method comprising detecting coronavirus Papain-like protease (PLpro) activity by contacting the sample with at least one PLpro protease substrate peptide, wherein said substrate peptide consists of the sequence RLRGG and carries a fluorophore, the detection of PLpro activity being demonstrated by fluorescence measurement.

2. The method of claim 1, said substrate peptide carrying a fluorophore at one end, and a protecting group at the other.

3. Method according to one of claims 1 or 2, said fluorophore being an aminomethyl coumarin (AMC) group, preferably carried at the C-terminal end of the substrate peptide.

4. The method of claim 3, wherein the substrate peptide is Z-RLRGG-(AMC), where (AMC) is aminomethyl coumarin, and Z is a carboxybenzyl group.

5. Method according to one of claims 1 to 4, in which the coronavirus is a SARS-CoV-2.

6. A method according to one of claims 1 to 5, wherein the sample is a saliva sample.

7. An in vitro method for determining whether a human subject is infected with a coronavirus and is contagious, said method comprising detecting a coronavirus Papain-like protease (PLpro) activity by contacting a biological sample of said subject with at least one PLpro protease substrate peptide, wherein said substrate peptide consists of the sequence RLRGG and carries a fluorophore, the detection of the PLpro activity being demonstrated by fluorescence measurement.

8. The method of claim 7, wherein the sample is a saliva sample.

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