Marker for novel coronavirus specific antibodies or combinations comprising the same

Abstract

The present application relates to a biomarker (i.e. glycosylated proline) or a combination comprising the same for identifying the level of SARS-CoV-2 antibodies, or use of a reagent for determining the level of glycosylated proline or a combination comprising glycosylated proline in a biological sample in the manufacture of a kit. The present application also relates to a pharmaceutical composition and methods of use thereof. By detecting the level of the biomarker, the level of SARS-CoV-2 antibodies can be assessed / identified. By reducing the concentration of glycosylated proline (e.g. using an inhibitor of glycosylated proline, e.g. a DPP4 inhibitor), the level of neutralizing antibodies can be increased and the rate of decline of neutralizing antibodies can be slowed, and the time for which neutralizing antibodies persist can be extended.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biotechnology, in particular to a biomarker for identifying the level of SARS-CoV-2 antibody, and use of a reagent for determining the level of glycylproline or a combination comprising glycylproline in a biological sample in the preparation of a kit. The present application also relates to a pharmaceutical composition and its pharmaceutical use. BACKGROUND

[0002] The novel coronavirus (SARS-CoV-2) belongs to the coronavirus, which is a large virus family. Currently known, coronavirus can cause cold and more serious diseases such as Middle East respiratory syndrome (MERS) and severe acute respiratory syndrome (SARS).

[0003] After recovery from novel coronavirus infection, the body can produce corresponding antibodies in a certain short period of time, which can resist the reinfection of novel coronavirus. However, with the passage of time, the titer of antibodies will gradually decrease after maintaining for a period of time, so as to disappear. Thus, patients with novel coronavirus reinfection and asymptomatic infected persons can appear clinically. Vaccination of new crown vaccine can help the human body produce neutralizing antibodies against viruses, but with the passage of time, the amount of neutralizing antibodies in the human body will slowly decrease. Therefore, after a certain period of time, booster vaccination is needed to achieve the effect of strengthening immunity.

[0004] For the above two groups of people (novel coronavirus pneumonia recovery subjects or subjects after vaccination of new crown vaccine), the titer of novel coronavirus specific antibody is an important indicator for measuring the immunity of the subjects in the body. How to conveniently and quickly evaluate the level of new crown specific antibody through biomarkers is an important scientific problem and a technical problem that needs to be solved urgently. SUMMARY

[0005] In order to solve the above problems, the applicant of the present application finds and verifies the biomarker (i.e., glycylproline) and the combination comprising the same (i.e., glycylproline, butyric acid, acylcarnitine (C18:1), acylcarnitine (C18:0) and lysine) which can be used to evaluate the level of new crown specific antibody. Further, in the body of the subject, by reducing the concentration of glycylproline (for example, using an inhibitor of glycylproline, for example, DPP4 inhibitor), the level of neutralizing antibody is improved, the speed of decline of neutralizing antibody is slowed down, and the time of retention of neutralizing antibody is prolonged. Thus, the present application is completed.

[0006] Accordingly, in a first aspect, the present application provides a kit for identifying or distinguishing the level of SARS-CoV-2 antibodies in a subject, the kit comprising reagents for determining the level of glycylproline or a combination comprising glycylproline in a biological sample.

[0007] In certain embodiments, the combination comprising glycylproline further comprises one or more biomarkers selected from the group consisting of butyric acid, acylcarnitine (C18:1), acylcarnitine (C18:0) and lysine.

[0008] In certain embodiments, the combination comprising glycylproline is glycylproline, and 1, 2, 2 or 4 biomarkers selected from the group consisting of butyric acid, acylcarnitine (C18:1), acylcarnitine (C18:0) and lysine.

[0009] In certain embodiments, the combination comprising glycylproline is glycylproline, butyric acid, acylcarnitine (C18:1), acylcarnitine (C18:0) and lysine.

[0010] In certain embodiments, the kit comprises a first reagent or combination of reagents for determining the level of glycylproline in the subject.

[0011] In certain embodiments, the kit further comprises a second reagent or combination of reagents for determining the level of butyric acid in the subject, a third reagent or combination of reagents for determining the level of acylcarnitine (C18:1) in the subject, a fourth reagent or combination of reagents for determining the level of acylcarnitine (C18:0) in the subject; and a fifth reagent or combination of reagents for determining the level of lysine in the subject.

[0012] In certain embodiments, the CAS number of the glycylproline is: 704-15-4. In certain embodiments, the EINECS number of the glycylproline is: 211-880-2.

[0013] In certain embodiments, the CAS number of the lysine is: 56-87-1. In certain embodiments, the EINECS number of the lysine is: 200-294-2.

[0014] In certain embodiments, the CAS number of the butyric acid is: 107-92-6. In certain embodiments, the EINECS number of the butyric acid is: 203-532-3.

[0015] In certain embodiments, the CAS number of the acylcarnitine (C18:0) is: 25597-09-5.

[0016] In certain embodiments, the reagent (e.g., first, second, third, fourth, fifth, or combination of reagents) determines the level of glycylproline or combination comprising glycylproline in the biological sample by liquid chromatography tandem mass spectrometry (LC-MS / MS), assay of chromatography and / or mass spectrometry, fluorescence assay, electrophoresis, immunoaffinity, hybridization, immunochemistry, ultraviolet spectroscopy (UV), fluorescence analysis, radiochemical analysis, near infrared spectroscopy (near IR), nuclear magnetic resonance spectroscopy (NMR), light scattering analysis (LS), and turbidimetry.

[0017] In certain embodiments, the reagent determines the level of glycylproline or combination comprising glycylproline in the biological sample by liquid chromatography tandem mass spectrometry.

[0018] In certain embodiments, the kit further comprises reagents and / or consumables for liquid chromatography tandem mass spectrometry, or any combination thereof.

[0019] In certain embodiments, the reagents and / or consumables for liquid chromatography tandem mass spectrometry are selected from the group consisting of a chromatography column, acetonitrile, ammonium acetate, ammonium formate, formic acid, glycylproline standard, isopropanol, methanol, or any combination thereof.

[0020] In a second aspect, the present application provides use of a reagent for determining the level of glycylproline or combination comprising glycylproline in a biological sample in the manufacture of a kit for identifying or differentiating the level of SARS-CoV-2 antibodies in a subject.

[0021] In certain embodiments, the combination comprising glycylproline further comprises one or more biomarkers selected from the group consisting of butyric acid, acylcarnitine (C18:1), acylcarnitine (C18:0), and lysine.

[0022] In certain embodiments, the combination comprising glycylproline is glycylproline, and 1, 2, 2, or 4 biomarkers selected from the group consisting of butyric acid, acylcarnitine (C18:1), acylcarnitine (C18:0), and lysine.

[0023] In certain embodiments, the combination comprising glycylproline is glycylproline, butyric acid, acylcarnitine (C18:1), acylcarnitine (C18:0), and lysine.

[0024] In certain embodiments, the kit comprises a first reagent or combination of reagents for determining the level of glycylproline in a subject.

[0025] In certain embodiments, the kit further comprises a second reagent or combination of reagents for determining the butyric acid level of the subject, a third reagent or combination of reagents for determining the acylcarnitine (C18:1) level of the subject, a fourth reagent or combination of reagents for determining the acylcarnitine (C18:0) level of the subject; and a fifth reagent or combination of reagents for determining the lysine level of the subject.

[0026] In certain embodiments, the glycylproline has a CAS number of: 704-15-4. In certain embodiments, the glycylproline has an EINECS number of: 211-880-2.

[0027] In certain embodiments, the lysine has a CAS number of: 56-87-1. In certain embodiments, the lysine has an EINECS number of: 200-294-2.

[0028] In certain embodiments, the butyric acid has a CAS number of: 107-92-6. In certain embodiments, the butyric acid has an EINECS number of: 203-532-3.

[0029] In certain embodiments, the acylcarnitine (C18:0) has a CAS number of: 25597-09-5.

[0030] In certain embodiments, the reagent (e.g., first, second, third, fourth, fifth, or combination of reagents) determines the level of glycylproline or combination comprising glycylproline in the biological sample by liquid chromatography tandem mass spectrometry (LC-MS / MS), assay of chromatography and / or mass spectrometry, fluorescence assay, electrophoresis, immunoaffinity, hybridization, immunochemistry, ultraviolet spectroscopy (UV), fluorescence analysis, radiochemical analysis, near infrared spectroscopy (near IR), nuclear magnetic resonance spectroscopy (NMR), light scattering analysis (LS), and turbidimetry.

[0031] In certain embodiments, the reagent determines the level of glycylproline or combination comprising glycylproline in the biological sample by liquid chromatography tandem mass spectrometry.

[0032] In certain embodiments, the kit further comprises reagents and / or consumables for liquid chromatography tandem mass spectrometry, or any combination thereof.

[0033] In certain embodiments, the reagents and / or consumables for liquid chromatography tandem mass spectrometry are selected from the group consisting of a chromatography column, acetonitrile, ammonium acetate, ammonium formate, formic acid, glycylproline standard, isopropanol, methanol, or any combination thereof.

[0034] In certain embodiments, the kit is used to distinguish or identify: (1) a subject who has not been infected with SARS-CoV-2, (2) a subject who has been infected with SARS-CoV-2 and carries antibodies, and (3) a subject who has been infected with SARS-CoV-2 and antibodies have waned; or,

[0035] the kit is used to distinguish or identify: (1) a subject who has not been vaccinated with a SARS-CoV-2 vaccine, (2) a subject who has been vaccinated with a SARS-CoV-2 vaccine and carries antibodies, and (3) a subject who has been vaccinated with a SARS-CoV-2 vaccine and antibodies have waned.

[0036] In certain embodiments, the biological sample is selected from the group consisting of whole blood (e.g., peripheral blood), plasma, serum, or any combination thereof.

[0037] In certain embodiments, the subject is a mammal (e.g., a mouse, a human).

[0038] In a third aspect, the present application provides a pharmaceutical composition comprising an inhibitor of glycylproline and a vaccine of SARS-CoV-2.

[0039] In certain embodiments, the inhibitor of glycylproline is a DPP4 inhibitor.

[0040] In certain embodiments, the DPP4 inhibitor is selected from the group consisting of sitagliptin, vildagliptin, saxagliptin, alogliptin, linagliptin, gemigliptin, teneligliptin, or any combination thereof.

[0041] In certain embodiments, the vaccine of SARS-CoV-2 is selected from the group consisting of a nucleic acid vaccine (e.g., a DNA vaccine, an mRNA vaccine), a protein vaccine, a pseudovirus vaccine, a viral vaccine (e.g., an inactivated viral vaccine), or any combination thereof.

[0042] In certain embodiments, the vaccine of SARS-CoV-2 is prepared from an inactivated or reduced-activity strain of SARS-CoV-2.

[0043] The sequence of the SARS-CoV-2 strain can be obtained from the China National Center for Bioinformation (CNCB), National Genomics Science Data Center (NGDC), or the NCBI nucleic acid database GenBank. In certain embodiments, the full genome sequence of the SARS-CoV-2 strain is numbered as MT019529, MT019530, MT019531, MT019532, or MT019533 in NCBI.

[0044] In this context, since glycyl-proline can be produced by dipeptidyl peptidase (DPP4), the concentration of glycyl-proline can be reduced by using a DPP4 inhibitor. Since proline peptidase can degrade glycyl-proline, the degradation of glycyl-proline can be prevented by using a proline peptidase inhibitor N-carbobenzyloxy-L-proline, thereby increasing the concentration of glycyl-proline in the blood.

[0045] Since DPP4 inhibitors are often used as clinical anti-diabetes type 2 drugs, they can be directly used in human populations to change the concentration of glycyl-proline, thereby regulating the level of coronavirus-specific neutralizing antibodies, after safety evaluation.

[0046] In certain embodiments, the use of a pharmaceutical composition as described hereinbefore in the manufacture of a medicament for producing or increasing the level of antibodies to SARS-CoV-2 in a subject; or, for slowing down the decline or extinction of the level of antibodies to SARS-CoV-2 in a subject.

[0047] In certain embodiments, the subject is a mammal (e.g., a mouse, a human).

[0048] In certain embodiments, the subject is infected with SARS-CoV-2, or has been vaccinated with SARS-CoV-2. In certain embodiments, the subject has diabetes type II.

[0049] In certain embodiments, the pharmaceutical composition is administered to the subject by administering the inhibitor of glycyl-proline to the subject before, after or simultaneously with administering a SARS-CoV-2 vaccine to the subject.

[0050] In certain embodiments, the pharmaceutical composition is administered to the subject by administering the inhibitor of glycyl-proline to the subject after administering a SARS-CoV-2 vaccine to the subject.

[0051] In a fourth aspect, the present application provides a method for distinguishing or identifying a subject who has not been infected with SARS-CoV-2, a subject who has been infected with SARS-CoV-2 and carries antibodies, and a subject who has been infected with SARS-CoV-2 and the antibodies have subsided, comprising:

[0052] (1) obtaining a biological sample comprising a biomarker or a combination thereof from a subject;

[0053] (2) determining the level of the biomarker or the combination thereof in the biological sample; and

[0054] (3) differentiating or identifying a subject who has never been infected with SARS-CoV-2, a subject who has been infected with SARS-CoV-2 and carries antibodies, and a subject who has been infected with SARS-CoV-2 and antibodies wane, based on the level of the biomarker or the combination thereof;

[0055] wherein the biomarker is glycylproline.

[0056] In certain embodiments, the combination of biomarkers is glycylproline, butyric acid, acylcarnitine (C18:1), acylcarnitine (C18:0), and lysine.

[0057] In certain embodiments, the biological sample is selected from the group consisting of whole blood (e.g., peripheral blood), plasma, serum, or any combination thereof.

[0058] In certain embodiments, the subject is a mammal (e.g., mouse, human).

[0059] In a fifth aspect, the present application provides a method of differentiating or identifying a subject who has never been vaccinated with SARS-CoV-2 vaccine, a subject who has been vaccinated with SARS-CoV-2 vaccine and carries antibodies, and a subject who has been vaccinated with SARS-CoV-2 vaccine and antibodies wane, comprising:

[0060] (1) obtaining a biological sample comprising a biomarker or a combination thereof from a subject;

[0061] (2) determining the level of the biomarker or the combination thereof in the biological sample; and

[0062] (3) differentiating or identifying a subject who has never been infected with SARS-CoV-2, a subject who has been infected with SARS-CoV-2 and carries antibodies, and a subject who has been infected with SARS-CoV-2 and antibodies wane, based on the level of the biomarker or the combination thereof;

[0063] wherein the biomarker is glycylproline.

[0064] In certain embodiments, the combination of biomarkers is glycylproline, butyric acid, acylcarnitine (C18:1), acylcarnitine (C18:0), and lysine.

[0065] In certain embodiments, the biological sample is selected from the group consisting of whole blood (e.g., peripheral blood), plasma, serum, or any combination thereof.

[0066] In certain embodiments, the subject is a mammal (e.g., a mouse, a human).

[0067] In certain embodiments, in step (3), the subject who has not been infected with SARS-CoV-2, the subject who has been infected with SARS-CoV-2 and carries antibodies, and the subject who has been infected with SARS-CoV-2 and the antibodies have waned, or the subject who has not been vaccinated with SARS-CoV-2 vaccine, the subject who has been vaccinated with SARS-CoV-2 vaccine and carries antibodies, and the subject who has been vaccinated with SARS-CoV-2 vaccine and the antibodies have waned, are distinguished or identified by comparing the level of the biomarker or the combination thereof with a reference value.

[0068] In certain embodiments, the reference value is the level or range of the biomarker or the combination thereof in a biological sample obtained from a normal population. In certain embodiments, the reference value is the level or range of the biomarker or the combination thereof in a biological sample obtained from a subject who has been infected with SARS-CoV-2 and carries antibodies. In certain embodiments, the reference value is the level or range of the biomarker or the combination thereof in a biological sample obtained from a subject who has been infected with SARS-CoV-2 and the antibodies have waned.

[0069] In certain embodiments, in step (3), the level of the biomarker or the combination thereof is subjected to ridge regression using elastic net regression to obtain a prediction model; then, the prediction model is used to distinguish or identify the subject who has not been infected with SARS-CoV-2, the subject who has been infected with SARS-CoV-2 and carries antibodies, and the subject who has been infected with SARS-CoV-2 and the antibodies have waned, or the subject who has not been vaccinated with SARS-CoV-2 vaccine, the subject who has been vaccinated with SARS-CoV-2 vaccine and carries antibodies, and the subject who has been vaccinated with SARS-CoV-2 vaccine and the antibodies have waned.

[0070] Definitions of terms

[0071] In the present application, unless otherwise specified, the scientific and technical terms used herein have the meanings commonly understood by a person of ordinary skill in the art. Also, the molecular genetic, nucleic acid chemistry, chemical, molecular biology, biochemistry, cell culture, microbiology, cell biology, genomics, and recombinant DNA, etc. procedures used herein are in accordance with conventional methods widely used in the corresponding field. At the same time, in order to better understand the present application, the definitions and explanations of related terms are provided as follows.

[0072] As used herein, the term "biomarker" refers to a biochemical indicator that can mark changes or possible changes in the function of systems, organs, tissues, cells, and subcellular structures, with very wide use. Biomarkers can be used for disease diagnosis, disease staging, or to evaluate the safety and effectiveness of new drugs or new therapies in target populations.

[0073] As used herein, "severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)", also known as "novel coronavirus", belongs to its beta coronavirus genus, and is a single-stranded positive-sense RNA virus containing envelope. The genomic sequence of SARS-CoV-2 is known to those skilled in the art, which can be found in, for example, GenBank: MN908947. SARS-CoV-2 contains at least three membrane proteins, including surface spike protein (S), integral membrane protein (M) and membrane protein (E). The receptor of SARS-CoV-2 is the same as that of SARS-CoV, which is the angiotensin-converting enzyme 2 (ACE2) on the host cell specifically bound by the receptor binding domain (RBD) on the S protein, which plays a crucial role in the process of viral infection of cells.

[0074] As used herein, the terms "novel coronavirus" and "SARS-CoV-2" have the same meaning and can be used interchangeably.

[0075] As used herein, the term "novel coronavirus pneumonia" refers to pneumonia caused by infection with the novel coronavirus, and both have the same meaning and can be used interchangeably.

[0076] As used herein, the term "metabolomics" refers to a technique for detecting metabolites with a molecular weight of less than 1000 daltons using chromatography-mass spectrometry technology.

[0077] As used herein, the term "subject" includes, but is not limited to, various animals, particularly mammals, such as humans, mice.

[0078] As used herein, the term "glycylproline", English name "glycylproline", abbreviated as "Gly-pro", its molecular formula is C7H 12N2O3. In certain embodiments, the glycylproline has a CAS number of: 704-15-4. In certain embodiments, the glycylproline has an EINECS number of: 211-880-2. In certain embodiments, glycylproline can be produced by dipeptidyl peptidase (DPP4) catalysis.

[0079] As used herein, the term "butyric acid", has a molecular formula of C4H8O2. In certain embodiments, butyric acid has a CAS number of 107-92-6. In certain embodiments, butyric acid has an EINECS number of: 203-532-3.

[0080] As used herein, the term "acylcarnitines", is a kind of amino acid-like substance, the main role of acylcarnitines is the transport and mitochondrial oxidation of fatty acids. In certain embodiments, the C18 in acylcarnitines (C18:1) and acylcarnitines (C18:0) refers to the number of carbon chains contained, and 1 or 0 refers to the number of carbon-carbon double bonds (C=C) contained.

[0081] As used herein, the term "acylcarnitines (C18:0)" refers to acylcarnitines containing 18 carbon fatty acid residues and 0 carbon-carbon double bonds (C=C), and the English abbreviation is CAR(C18:0). In certain embodiments, the acylcarnitines (C18:0) has a CAS number of: 25597-09-5.

[0082] As used herein, the term "acylcarnitines (C18:1)" refers to acylcarnitines containing 18 carbon fatty acid residues and 1 carbon-carbon double bond (C=C), and the English abbreviation is CAR(C18:1). There is one carbon-carbon double bond (C=C) in acylcarnitines (C18:1), and due to the diversity of the position of the carbon-carbon double bond (C=C), acylcarnitines (C18:1) exist as isomers. In certain embodiments, acylcarnitines (C18:1) refers to acylcarnitines with one carbon-carbon double bond (C=C).

[0083] As used herein, the term "lysine", English name "Lysine (Lys)", has a molecular formula of H2N(CH2)4CH(NH2)COOH. In certain embodiments, the lysine has a CAS number of 56-87-1. In certain embodiments, the lysine has an EINECS number of: 200-294-2.

[0084] As used herein, the term "Receiver Operating Characteristic Curve" or "ROC curve" is a plot of the true positive rate (sensitivity) versus the false positive rate (1 - specificity) that illustrates the trade-off between sensitivity and specificity for a binary classifier system. As used herein, the term "Receiver Operating Characteristic Curve" or "ROC curve" is a plot of the true positive rate (sensitivity) versus the false positive rate (1 - specificity) that illustrates the trade-off between sensitivity and specificity for a binary classifier system. In some embodiments, the diagnostic accuracy of a ROC curve is represented by the area under the curve. The area under the curve is referred to as "Area Under Curve" or "AUC". Generally, the AUC is between 0 and 1. If the sensitivity of a diagnostic test is 1, and the false positive rate is 0.

[0085] As used herein, the term "DPP4 inhibitor" refers to a class of drugs that treat type 2 diabetes. DPP4 inhibitors (DPP4 inhibitors) have similar inhibition rates of DPP4 at their respective therapeutic doses. They all have high oral bioavailability and high safety, and can be directly administered to subjects.

[0086] Advantages of the invention

[0087] The present application provides a biomarker (i.e., glycylproline) capable of evaluating / identifying the level of SARS-CoV-2 antibody and a combination comprising the same (i.e., the combination of glycylproline, butyric acid, acylcarnitine (C18:1), acylcarnitine (C18:0), lysine), and the level of the biomarker or the combination comprising the same can be detected to evaluate / identify the level of SARS-CoV-2 antibody.

[0088] Further, by detecting the level of the biomarker or the combination comprising the same, it is possible to identify / distinguish between (1) a subject who has never been infected with SARS-CoV-2, (2) a subject who has been infected with SARS-CoV-2 and carries antibodies, and (3) a subject who has been infected with SARS-CoV-2 and whose antibodies have subsided; or (1) a subject who has never been vaccinated with SARS-CoV-2, (2) a subject who has been vaccinated with SARS-CoV-2 and carries antibodies, and (3) a subject who has been vaccinated with SARS-CoV-2 and whose antibodies have subsided.

[0089] In addition, by reducing the concentration of glycylproline in the subject (e.g., using an inhibitor of glycylproline, such as a DPP4 inhibitor), it is possible to increase the level of neutralizing antibodies, slow down the rate of decline of neutralizing antibodies, and prolong the time of retention of neutralizing antibodies.

[0090] Embodiments of the present application will be described in detail below with reference to the accompanying drawings and examples, but those skilled in the art will understand that the following drawings and examples are only used to illustrate the present application, and are not a limitation on the scope of the present application. According to the following detailed description of the drawings and preferred embodiments, various objects and advantages of the present application will become apparent to those skilled in the art. BRIEF DESCRIPTION OF DRAWINGS

[0091] Figure 1 ROC curve; wherein, Figure 1 A shows the ROC curve of five markers (glycyl-proline, butyric acid, acylcarnitine (C18:1), acylcarnitine (C18:0), lysine) for distinguishing between COVID-19 recovered and antibody carriers (CA group), COVID-19 recovered but antibody subsided (CO), Figure 1 B shows the ROC curve of the combination of the five markers.

[0092] Figure 2 shows the screening and verification results of the biomarkers; wherein, Figure 2 A shows the concentration of glycyl-proline in the plasma of healthy volunteers (H group), COVID-19 recovered and antibody carriers (CA group), and COVID-19 recovered but antibody subsided (CO); Figure 2 B shows the grouping of healthy mice after COVID-19 vaccination using glycyl-proline (Gly-pro), the inhibitor of proline peptide enzyme N-benzyloxy carbonyl-L-proline (Cbz-pro), glycyl-proline combined with DPP4 inhibitor sitagliptin (Gly-pro & sitagliptin) intervention, and no intervention (RBD control group); Figure 2 C shows the experimental results of the above four groups (RBD, Gly-pro, Cbz-pro and Gly-pro & sitagliptin) one week after vaccination and drug intervention; Figure 2 D shows the experimental results of the above four groups (RBD, Gly-pro, Cbz-pro and Gly-pro & sitagliptin) two weeks after vaccination and drug intervention; Figure 2 E shows the experimental results of the above four groups (RBD, Gly-pro, Cbz-pro and Gly-pro & sitagliptin) three weeks after vaccination and drug intervention; Figure 2 F shows the experimental results of the above four groups (RBD, Gly-pro, Cbz-pro and Gly-pro & sitagliptin) four weeks after vaccination and drug intervention.

[0093] Figure 3This study demonstrated, through further sorting of immune cell subsets in animal experiments, that glycylproline can modulate antibody-mediated immune responses. Germinal center B cells (GC B cells, Figure 3 A), Follicular helper T cells (Tfh, Figure 3 B) and plasma cells Figure 3 The levels of C) are regulated by glycylproline. Detailed Implementation

[0094] The invention will now be described with reference to the following embodiments, which are intended to illustrate the invention (and not limit it).

[0095] Unless otherwise specified, the experiments and methods described in the embodiments are performed in accordance with conventional methods well known in the art and described in various references. For example, conventional techniques such as immunology, biochemistry, chemistry, molecular biology, microbiology, cell biology, genomics, and recombinant DNA used in this invention can be found in Sambrook, Fritsch, and Maniatis, *Molecular Cloning: A Laboratory Manual*, 2nd edition (1989); *Current Protocols in Molecular Biology* (edited by F.M. Ausubel et al., 1987); the *Methods in Enzymology* series (academic publishing company): *PCR2: A PRACTICAL APPROACH* (edited by M.J. MacPherson, BD. Hames, and GR. Taylor, 1995); and *Animal Cell Culture*. CELLCULTURE (edited by R.R. Freshney (1987)).

[0096] Furthermore, unless specific conditions are specified in the examples, conventional conditions or conditions recommended by the manufacturer should be followed. Reagents or instruments whose manufacturers are not specified are all commercially available conventional products. Those skilled in the art will understand that the examples are described by way of illustration and are not intended to limit the scope of protection claimed by the invention. All disclosures and other references mentioned herein are incorporated herein by reference in their entirety.

[0097] Example 1. Acquisition of metabolomic samples

[0098] 1.1 Blood collection

[0099] Three groups of plasma samples were collected from healthy volunteers, COVID-19 recovered subjects with antibodies, and COVID-19 recovered subjects without antibodies. The COVID-19 recovered subjects with antibodies were collected from the plasma samples of COVID-19 recovered subjects who had symptoms for 40-70 days and were discharged. Among them, the COVID-19 recovered subjects without antibodies were all negative for COVID-19 specific IgG serum detection. The COVID-19 specific IgG detection method is as follows: 100 μL of diluted plasma is obtained by diluting the derivative reagent with plasma (1:100 to 1:800), and the diluted plasma is added to the pre-cooled plate and incubated at 37°C for 1 hour. After washing, 100 μL of horseradish peroxidase (HRP) combined with RBD protein specific to new coronavirus is added to each well, and incubation is continued at 37°C for 30 minutes. After washing, the optical density (OD) value is detected at a wavelength of 450 nm. Excess plasma samples are stored in a -80°C refrigerator.

[0100] In this study, there were 17 COVID-19 recovered subjects with antibodies, 30 COVID-19 recovered subjects without antibodies, and 35 healthy volunteers. The COVID-19 recovered subjects had no symptoms of diabetes and had a relatively consistent hospitalization time. All blood samples were collected after overnight fasting, collected with EDTA anticoagulant tubes, and stored at low temperature after collection for detection.

[0101] 1.2 Inactivation of metabolomics samples

[0102] Since the samples we used were collected from COVID-19 recovered subjects, there was no need to inactivate the virus again in the P3 laboratory. Only experimental operations were required in the P2 laboratory.

[0103] Example 2. Plasma metabolomic experiment

[0104] (1) Plasma sample extraction: The standard mixed solution was prepared into eight different concentrations, quality control samples (mixed with all samples) and blank solvent, and actual samples, each sample was taken 20 μL and placed in a 96-well plate. 120 μL of standard mixed solution was added to each well, the microplate was covered with aluminum foil, and the rotation speed was 650 rpm at 10°C for 20 minutes. After centrifugation at 4000g for 20 minutes, 30 μL of supernatant was taken to a new 96-well plate. 3-nitrophenylhydrazine derivative reagent was added at 30°C for 60 min at a rotation speed of 1200 rpm. After derivatization, 330 μL of pre-cooled 50% methanol aqueous solution was added to each well, shaken at 1200 rpm at 10°C for 5 min, and centrifuged at 4°C for 30 min at 4000g. Finally, the supernatant was transferred to a new 96-well plate and detected as soon as possible.

[0105] (2) Chromatography, mass spectrometry collection conditions:

[0106] Chromatography was performed using ultra-high performance liquid chromatography (Shimpack UFLC SHIMADZU CBM30A). The main liquid chromatography conditions included: pre-column: Waters ACQUITY UPLC BEH C18 1.7 μm, 2.1 mm*5 mm; column: Waters ACQUITY UPLC BEH C18 1.7 μm, 2.1 mm*100 mm. Mobile phase: A phase was ultrapure water (0.1% formic acid was added); B phase was acetonitrile / isopropanol (70:30, v / v). Elution gradient: 0 to 1.0 min, 5% B; 1 to 11.0 min, 5% to 78% B; 11.0 to 13.5 min, 78% to 95% B; 13.5 to 14.0 min, 95% to 100% B; 14.0 to 16.0 min, 100% B; 16.0 to 16.1 min, 100% to 5% B; 16.1 to 18.0 min, 5% B. Flow rate: 0.4 mL / min, column temperature 40 °C, injection volume 5 μL.

[0107] Mass spectrometry was performed using triple quadrupole mass spectrometry (AB SCIEX QTRAP 6500+). The main mass spectrometry conditions included: electrospray ion source and desolvation temperature were 150 °C and 550 °C, respectively. Mass spectrometry voltage: 5500 V (+), -4500 V (-). Ion source gas I: 55 psi, gas II: 60 psi, curtain gas: 25 psi. Collision-induced ionization parameters: high. In the triple quadrupole, each ion pair was scanned and detected according to the optimized declustering voltage and collision energy.

[0108] (3) Quality control (QC) settings:

[0109] 10 μL of each sample was taken and mixed to prepare a quality control sample. When detecting the samples, a quality control sample was injected after every ten samples according to the sample order.

[0110] Example 3. Processing of data

[0111] Mass spectrometry data processing was performed using software Analyst 1.6.3. The repeatability of metabolite extraction and detection was confirmed by total ion chromatogram (TIC) and MRM peak. According to the retention time (RT) and mass-to-charge ratio (m / z), the metabolites were qualitatively and quantitatively analyzed in the metabolite library derived from 3-nitrophenylhydrazine derivatization.

[0112] The metabolite chromatographic peak area is exported by data processing software, and the positive and negative ions in the metabolite are removed by the software package. The metabolites with a coefficient of variation value greater than 0.5 are removed by calculating the coefficient of variation value in the quality control sample. If the metabolite can be detected in both cation and anion ionization modes, the mode with smaller metabolite coefficient of variation value is taken.

[0113] Metabolites or lipids detected in a single group in the mass spectrum are deleted if the number is less than 80%, and the missing values are filled using the missing value filling function in the R package MetaboAnalyst, using the median.

[0114] Example 4. Main data calculation methods and analysis methods

[0115] The analysis statistics of the data are mainly performed by Microsoft office excel and Graphpad prism software. For the differential analysis of metabolites, T test and difference fold (FC, Fold change) are used for judgment. Statistical significance analysis uses one-tailed T test or fisher exact test, and p<0.05 is considered statistically significant. Other calculation methods adopted include Z-score, etc. Pathway analysis of metabolomics and lipidomics uses R package MetaboAnalyst.

[0116] Through the above analysis, the levels of the following 5 biomarkers showed significant differences in the three groups of people (healthy volunteers, COVID-19 recovered subjects with antibodies, and COVID-19 recovered subjects with antibody resolution). Among them,

[0117] The concentration of glycylproline in the plasma of healthy volunteers (H) is 0.185±0.138 micromole; the concentration of glycylproline in the plasma of COVID-19 recovered subjects with antibodies (CA) is 0.593±0.451 micromole; the concentration of glycylproline in the plasma of COVID-19 recovered subjects with antibody resolution (CO) ranges from 0.825±0.302 micromole.

[0118] The concentration of butyric acid in the plasma of healthy volunteers (H) is 0.034±0.021 micromole; the concentration of butyric acid in the plasma of COVID-19 recovered subjects with antibodies (CA) is 0.022±0.021 micromole; the concentration of butyric acid in the plasma of COVID-19 recovered subjects with antibody resolution (CO) ranges from 0.040±0.016 micromole.

[0119] The concentration of acylcarnitine (C18:1) in the plasma of healthy volunteers (H) was 0.63 ± 0.24 μmol; the concentration of acylcarnitine (C18:1) in the plasma of recovered COVID-19 patients carrying antibodies (CA) was 0.46 ± 0.19 μmol; and the concentration of acylcarnitine (C18:1) in the plasma of recovered COVID-19 patients with antibody deterioration (CO) ranged from 0.28 ± 0.16 μmol.

[0120] The concentration of acylcarnitine (C18:0) in the plasma of healthy volunteers (H) was 0.62 ± 0.24 μmol; the concentration of acylcarnitine (C18:0) in the plasma of recovered COVID-19 patients carrying antibodies (CA) was 0.48 ± 0.21 μmol; and the concentration of acylcarnitine (C18:0) in the plasma of recovered COVID-19 patients with antibody deterioration (CO) ranged from 0.28 ± 0.15 μmol.

[0121] The concentration of lysine in the plasma of healthy volunteers (H) was 243.72 ± 51.05 μmol; the concentration of lysine in the plasma of recovered COVID-19 patients carrying antibodies (CA) was 201.16 ± 33.12 μmol; and the concentration of lysine in the plasma of recovered COVID-19 patients with antibody deterioration (CO) ranged from 244.18 ± 54.52 μmol.

[0122] Furthermore, in order to analyze the diagnostic efficacy of the above five biomarkers in differentiating the three population groups, we conducted ROC analysis (data processing and analysis methods are as described above).

[0123] Specific results are as follows Figure 1 As shown, where, Figure 1 A represents the ROC curves of five biomarkers (glycylproline, butyric acid, acylcarnitine (C18:1), acylcarnitine (C18:0), and lysine) used to distinguish between COVID-19 recovered patients with antibodies (CA group) and COVID-19 recovered patients with antibody decline (CO group). Figure 1 B is the ROC curve of the combination of five markers.

[0124] The results showed that the area under the ROC curve (AUC) of each of the five biomarkers individually was high (AUC above 0.75), far exceeding that of the random effect (AUC of 0.5). The area under the ROC curve (AUC) of the combination of the five biomarkers was particularly outstanding, with an AUC of 0.94.

[0125] These results indicate that the five biomarkers—glycylproline, butyric acid, acylcarnitine (C18:1), acylcarnitine (C18:0), and lysine—can generate high-performance predictive models, whether used individually or in combination, and can be used to identify three population groups. The combined effect of the five biomarkers is particularly outstanding.

[0126] Example 5. Method of animal experiment for COVID-19 vaccination

[0127] Among the above five markers, the difference of Gly-pro between CA and CO is more significant, so Gly-pro is selected for further experimental analysis. The level of Gly-pro is shown in FIG. 1A. Figure 2 As shown in FIG. 1A, the experimental results show that the concentration of Gly-pro in the plasma of healthy volunteers (H), COVID-19 recovered subjects carrying antibodies (CA) and COVID-19 recovered subjects but with antibody subsidence (CO) has a significant difference, and the difference is stable, proving that Gly-pro can be used to indicate COVID-19 subjects and can be used as a biomarker to distinguish the above three groups of people.

[0128] 1. Neutralizing antibody level

[0129] To confirm whether Gly-pro can regulate the level of COVID-19 specific neutralizing antibodies, we plan to adjust the level of COVID-19 neutralizing antibodies by interfering with Gly-pro. Since Gly-pro can be produced by DPP4 catalysis, the level of Gly-pro can be reduced by using sitagliptin (DPP4 inhibitor). Since Gly-pro can be degraded by proline peptidase, the level of Gly-pro can be increased by using N-carbobenzyloxy-L-proline (proline peptidase inhibitor).

[0130] Therefore, we use SARS-CoV-2 RBD protein fragment 50 μg to intraperitoneally inject 48 6-week-old BALB / c female mice. The mice are randomly divided into four groups (12 mice in each group), and the immunization experiment mode diagram of the four groups of mice inoculated with SARS-CoV-2 RBD is shown in FIG. 2A. Figure 1 As shown in FIG. 2A: one group is injected with Gly-pro (3.7 μmol / kg / day) every day (corresponding to the Gly-pro group of FIG. 2B), one group is injected with Gly-pro (3.7 μmol / kg / day) every day and orally administered with sitagliptin (10 mg / kg / day) (corresponding to the Gly-pro & sitagliption group of FIG. 2B), one group is intraperitoneally injected with proline peptidase inhibitor N-carbobenzyloxy-L-proline (60 mg / kg / day) (corresponding to the Cbz-pro group of FIG. 2B), and the other group is intraperitoneally injected with PBS as a blank control (corresponding to the RBD group of FIG. 2B). The body weight of all mice is measured every week, and the mice are bled one week, two weeks, three weeks and four weeks after immunization, and the OD value is detected at a wavelength of 450 nm to detect the level of neutralizing antibodies. Figure 1 Figure 2 Figure 2 Figure 2

[0131] 2. Immune response level ​​​​

[0132] The present example further illustrates that gly-pro can regulate antibody immune response by sorting immune cell subsets (germinal center B cells (GC B cells), follicular helper T cells (Tfh), and plasma cells) in animal experiments. It is noted that germinal center B cells take one to several weeks to mature and more efficiently present antigens to follicular helper T cells, and antibody-producing plasma cells are also differentiated from germinal center B cells.

[0133] Therefore, we used SARS-CoV-2 RBD protein fragments 50 μg single dose intraperitoneal injection of 48 6-week-old BALB / c female mice, and after two weeks of mouse immunization, germinal center B cells, follicular helper T cells and plasma cells were sorted and detected from lymph and spleen. Germinal center B cells and follicular helper T cells were detected in lymph, and plasma cells were detected in spleen. Corresponding markers were selected as the basis for cell sorting in flow sorting, and about 10,000 cells were sorted for quantitative analysis in each sample. CD45+ B220+ CD95+ GL-7+ corresponds to germinal center B cells, CD45+ CD4+ CD185+ PD-1+ corresponds to follicular helper T cells, and B220+ CD27+ CD138+ corresponds to plasma cells. The data show the mean and sampling error, and each group has 5 mice. *, p < 0.05; **, p < 0.01; ****, p < 0.0001.

[0134] The results are shown in Figure 3 As shown, injection of gly-pro (Gly-pro group) or N-carbobenzyloxy-L-proline (Cbz-pro group) can significantly down-regulate the level of immune cell subsets, but the combined administration of gly-pro and sitagliptin (Gly-pro & S group) can reverse the negative effect of gly-pro.

[0135] The above experimental results suggest that the neutralizing antibody level of mammals vaccinated with the new crown vaccine is indeed regulated by gly-pro, and the level of gly-pro in subjects with different antibody levels is different, suggesting that gly-pro can be used to identify or distinguish subjects with different antibody levels vaccinated with the new crown vaccine. Considering that the clinical use of DPP4 inhibitors can down-regulate the level of gly-pro, it is speculated that taking DPP4 inhibitors can help maintain or increase the neutralizing antibody level of patients recovered from COVID-19 or vaccinated with the new crown vaccine.

[0136] Although the specific embodiments of the present application have been described in detail, those skilled in the art will understand that various modifications and changes can be made to the details based on all the teachings disclosed herein, and these changes are within the scope of protection of the present application. The entire scope of the present application is given by the appended claims and any equivalents thereof.

Claims

1. Use of reagents for determining the level of glycylproline or combinations containing glycylproline in biological samples in the preparation of a kit for identifying or differentiating the level of SARS-CoV-2 antibodies in a subject.

2. The use according to claim 1, wherein the combination containing glycylproline further comprises one or more biomarkers selected from the following: butyric acid, acylcarnitine C18:1, acylcarnitine C18:0 and lysine.

3. The use according to claim 1, wherein the combination containing glycylproline is glycylproline, butyric acid, acylcarnitine C18:1, acylcarnitine C18:0 and lysine.

4. The use according to claim 1, wherein the kit comprises a first reagent or combination of reagents for determining glycylproline levels in a subject.

5. The use according to claim 1, wherein the kit further comprises a second reagent or combination of reagents for determining the subject's butyrate level, a third reagent or combination of reagents for determining the subject's acylcarnitine C18:1 level, a fourth reagent or combination of reagents for determining the subject's acylcarnitine C18:0 level, and a fifth reagent or combination of reagents for determining the subject's lysine level.

6. The use as described in claim 1, wherein, The reagent is used to determine the level of glycylproline or combinations containing glycylproline in the biological sample by the following methods: chromatographic and / or mass spectrometry, electrophoresis, immunoaffinity, hybridization, immunochemistry, ultraviolet spectroscopy (UV), fluorescence analysis, radiochemical analysis, near-infrared spectroscopy (near IR), nuclear magnetic resonance spectroscopy (NMR), light scattering analysis (LS), and turbidimetry.

7. The use according to claim 1, wherein the reagent is used to determine the level of glycylproline or a combination containing glycylproline in the biological sample by liquid chromatography-tandem mass spectrometry.

8. The use as described in claim 1, wherein the kit further comprises reagents and / or consumables for liquid chromatography-tandem mass spectrometry, reagents and / or consumables for mass spectrometry, or any combination thereof.

9. The use according to claim 8, wherein the reagents and / or consumables for liquid chromatography-tandem mass spectrometry are selected from chromatographic columns, acetonitrile, ammonium acetate, ammonium formate, formic acid, glycylproline standards, isopropanol, methanol, or any combination thereof.

10. The use according to any one of claims 1-9, wherein, The kit is used to distinguish or identify: (1) subjects who have not been infected with SARS-CoV-2, (2) subjects who have been infected with SARS-CoV-2 and carry antibodies, and (3) subjects who have been infected with SARS-CoV-2 and whose antibodies have regressed; or, The kit is used to distinguish or identify: (1) subjects who have not been vaccinated against SARS-CoV-2, (2) subjects who have been vaccinated against SARS-CoV-2 and carry antibodies, and (3) subjects who have been vaccinated against SARS-CoV-2 and whose antibodies have subsided.

11. The use of claim 1, wherein, The biological sample is selected from whole blood, plasma, serum, or any combination thereof.

12. The use of claim 1, wherein the subject is a mammal.

13. The use of claim 1, wherein the subject is a mouse or a human.

14. Use of the pharmaceutical composition in the preparation of a medicament for generating or increasing the level of SARS-CoV-2 antibodies in a subject; or for slowing the decline or regression of SARS-CoV-2 antibody levels in a subject; wherein, The pharmaceutical composition contains an inhibitor of glycylproline and a vaccine against SARS-CoV-2.

15. The use as described in claim 14, wherein, The subjects were humans.

16. The use as described in claim 14, wherein the subject has been infected with SARS-CoV-2 or has been vaccinated against SARS-CoV-2.

17. The use according to claim 16, wherein the subject suffers from type 2 diabetes.

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