Biomarker IFIT3 for detecting severe coronavirus pneumonia and application thereof

Abstract

The invention discloses an interferon-induced protein (IFIT3) with a tetrapeptide repetitive sequence 3 for detecting an early circulating plasma biomarker of coronavirus severe pneumonia and application of the interferon-induced protein, and belongs to the technical field of medicine and pharmacology. The biomarker comprises IFIT3 (interferon isothiocyanate 3); the invention also discloses application of the biomarker in preparation of a diagnostic kit or a diagnostic reagent for distinguishing a new crown severe pneumonia patient from a normal person. The biomarker is screened out through a high-throughput sequencing expression profile analysis technology of leukocytes of critical disease COVID19 patients, has higher sensitivity and specificity as a molecular marker, proves that IFIT3 possibly participates in the attack of the new crown severe pneumonia, can be used as an early diagnostic biomarker and a new treatment target of the new crown severe pneumonia, and has a good application prospect. The method has great clinical value and practical significance for clinical early diagnosis of the coronavirus infection state, evaluation of related treatment targets and improvement of the life quality of patients.

Description

Technical Field

[0001] The present invention relates to the field of medical technology, and in particular to a biomarker for detecting severe COVID-19 and severe pneumonia, namely, an interferon-induced protein having a tetrapeptide repeat sequence 3, namely IFIT3, and its application. Background Art

[0002] Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which has affected millions of people worldwide, belongs to the Coronaviridae family, which includes severe acute respiratory syndrome coronavirus (SARS-CoV-1), Middle East respiratory syndrome coronavirus (MERS-CoV), and four seasonal coronaviruses that cause mild infections.

[0003] The clinical spectrum of severe acute respiratory syndrome coronavirus 2-associated pneumonia ranges from asymptomatic and mild to moderate, severe, and critical illness. The most common symptoms are fever, cough, headache, fatigue, dyspnea, loss of smell, and loss of taste. Approximately one-third of infected individuals are asymptomatic. Among symptomatic patients, 81% have mild to moderate symptoms, 14% develop severe symptoms (dyspnea, hypoxia, lung involvement on imaging); and 5% develop respiratory failure, ARDS, shock, or multiorgan failure. Worldwide, the case fatality rate for hospitalized patients ranges from 1-10% to 17%, with differences related to the variable size of the denominator, the number of individuals studied, demographics, the genetic background of the person, the functioning of the healthcare system, and viral mutations. Undetected cases during the first wave led to a significant underestimation of asymptomatic infections.

[0004] A common feature of critical illness is immune dysregulation and cytokine disturbances, with a sudden increase in proinflammatory cytokines and other inflammatory markers. This hyperinflammatory syndrome can lead to coagulopathy, oxidative stress, organ damage, and death, consistent with the fact that severe COVID-19 pneumonia is primarily a vascular disease rather than a purely respiratory illness. Multisystem inflammatory syndrome (MIS-C) rarely develops in children. Myocardial infarction (MIS-A) can also occur in adults.

[0005] Risk factors include older age, male sex, and comorbidities, including chronic lung disease, cardiovascular disease and hypertension, diabetes, obesity, and cancer, but also a person's genetic background. In addition, host genetic factors are considered risk factors for coronavirus infection or the course of severe COVID-19 pneumonia.

[0006] Many genes highlighted in genetic studies of severe COVID-19 are associated with key pathophysiological processes, including viral entry into cells, immune responses, and inflammatory responses. Notably, association studies for severe COVID-19 have been conducted primarily in Caucasian populations, whereas previous SARS association studies were primarily conducted in East Asian populations. Africans, South Asians, and South Americans remain underrepresented in genetic studies of severe COVID-19.

[0007] Based on COVID-19-related risk factors, retrospective predictions of future events have limited accuracy. There is an urgent need for simpler, more reliable diagnostic markers to confirm the likelihood of severe COVID-19 pneumonia. Therefore, seeking biomarkers with high sensitivity and specificity, ease of use, safety, and cost-effectiveness is of great practical significance for disease prevention and diagnosis. Summary of the Invention

[0008] The purpose of the present invention is to provide a biomarker IFIT3 for detecting severe COVID-19 and its application to solve the problems existing in the above-mentioned prior art. The biomarker is screened out by high-throughput sequencing expression spectrum analysis technology of leukocytes of critically ill COVID19 patients. It has good sensitivity and specificity and can be used as an early diagnostic biomarker and new therapeutic target for severe COVID-19, providing a new direction for the early clinical diagnosis of severe COVID-19, related therapeutic targets and long-term prognosis evaluation.

[0009] To achieve the above object, the present invention provides the following solutions:

[0010] A biomarker for detecting severe pneumonia caused by a coronavirus, characterized by comprising an interferon-induced protein having a tetrapeptide repeat sequence 3, namely IFIT3, wherein the early stage is within 7 days after the onset of symptoms, and the expression level of IFIT3 is detected by peripheral blood plasma samples, and its area under the ROC curve is ≥0.95.

[0011] Use of a reagent for detecting interferon-induced protein IFIT3 in the blood in developing a diagnostic tool for distinguishing patients with severe pneumonia caused by coronavirus from healthy people who are asymptomatic and have no inflammation, wherein the tool has a sensitivity of not less than 90% and a specificity of not less than 95%.

[0012] Optionally, the distinguishing method includes the following steps:

[0013] (1) Peripheral blood plasma samples were collected from the subjects to be tested, and peripheral plasma samples from healthy individuals without infection symptoms were used as controls. The mean IFIT3 content of the control group was X±Y (SD, standard deviation);

[0014] (2) using immunological detection methods to detect the content of IFIT3 in the plasma samples of the test subjects;

[0015] (3) If the IFIT3 content of the tested subject is 2 times the standard deviation higher than the mean of the control group, the patient is judged to have severe pneumonia caused by a coronavirus.

[0016] Optional immunological detection methods include ELISA or chemiluminescence.

[0017] A diagnostic kit for distinguishing patients with severe pneumonia caused by a coronavirus from healthy individuals, comprising:

[0018] (a) Monoclonal antibodies that specifically bind to IFIT3;

[0019] (b) detection buffer;

[0020] (c) a specification sheet, comprising validation data for the IFIT3 threshold (X+2Y) and the area under the ROC curve ≥ 0.95 as defined in claim 3. The present invention discloses the following technical effects:

[0021] This invention, by screening plasma biomarkers for early diagnosis of severe COVID-19 pneumonia, has great clinical value and practical significance for the early diagnosis of severe COVID-19 pneumonia, the evaluation of relevant therapeutic targets, and the improvement of quality of life. The screened IFIT3 was screened out through high-throughput sequencing expression profiling technology of white blood cells in critically ill human COVID-19 patients. As a molecular marker, it is more sensitive and specific, proving that IFIT3 may be involved in the pathogenesis of coronary heart disease and can serve as an early diagnostic biomarker and new therapeutic target for novel coronavirus infection. This provides a new direction for the early clinical diagnosis and long-term prognosis assessment of severe COVID-19 pneumonia. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0023] Figure 1 This is a volcano plot showing the difference between the transcriptome sequencing results of peripheral blood leukocytes from seven critically ill patients with coronavirus infection and seven normal controls. Upregulated and downregulated proteins were identified using the criteria of LogFC ≥ 1 & LogFC ≤ -1, P < 0.05. Red represents upregulated proteins, and blue represents downregulated proteins.

[0024] Figure 2 The box plot shows the expression of IFIT3 gene in 7 critically ill patients with coronavirus infection and 7 healthy controls (HC). In the coronavirus infection group, the expression of IFIT3 gene was significantly higher than that in the HC group (P value < 0.05), and the difference between the two was statistically significant.

[0025] Figure 3The ROC curve was obtained, and the area under the curve was 0.98. The results showed that IFIT3 as a biomarker could well distinguish 7 patients with coronary heart disease from 7 normal people (P<0.05). DETAILED DESCRIPTION

[0026] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0027] It should be understood that the terms described herein are intended only to describe particular embodiments and are not intended to limit the present invention. In addition, for numerical ranges herein, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. The intermediate value within any stated value or stated range, and each smaller range between any other stated value or intermediate value within the stated range, is also encompassed within the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded within the scope.

[0028] Unless otherwise indicated, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein may also be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of any conflict with any incorporated document, the contents of this specification shall prevail.

[0029] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments described herein without departing from the scope or spirit of the invention. Other embodiments will be apparent to those skilled in the art from the description of the invention. The description and examples are intended to be exemplary only.

[0030] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0031] Terminology Notes:

[0032] interferon-induced protein with tetratricopeptide repeat 3, IFIT3;

[0033] The incubation period of coronavirus is short, and its occurrence and development is a relatively rapid process. The whole process from health to development of severe new coronary pneumonia is often more inflammatory. At present, there is no rapid diagnostic molecular marker that can be used to assist in its differential diagnosis and risk assessment. In order to shorten the time from the onset of symptoms to receiving diagnosis and treatment, it is very necessary to analyze and measure the relevant factors in the pathogenesis of severe new coronary pneumonia, and to find reliable serum markers. To this end, the present invention discloses a biomarker for detecting severe new coronary pneumonia, and the biomarker is IFIT3. The biomarker is obtained by screening the difference in the transcriptome profiles of peripheral blood leukocytes of critically ill coronavirus-infected patients and normal healthy people. It can be used to distinguish patients with severe new coronary pneumonia from normal people, and can be used as an early diagnostic biomarker and a new therapeutic target for severe new coronary pneumonia. The purpose of the present invention is to provide a biomarker for detecting severe new coronary pneumonia with relatively high specificity and sensitivity and its application to solve the problems existing in the above-mentioned prior art. The present invention studies as follows:

[0034] Experimental Example 1: Transcriptomic Analysis of Differentially Expressed Genes in Patients with Severe COVID-19 and Healthy People

[0035] Using the GEO public database, we downloaded the GSE154998 gene expression dataset (containing transcriptome profiles of peripheral blood leukocytes from 7 critically ill coronavirus patients and 7 healthy controls). We used high-throughput sequencing technology to analyze differentially expressed genes between patients with severe COVID-19 and healthy controls:

[0036] 1. Genetic Difference Analysis

[0037] The data were analyzed using the R language Limma package. The differentially expressed genes were screened using the conditions |LogFC| > 1 and P < 0.05. A total of 195 differentially expressed genes were obtained (147 up-regulated genes and 48 down-regulated genes).

[0038] 2. PPI interaction and key gene screening:

[0039] The STRING network was used to construct a PPI functional network for differentially expressed genes. Cytoscape software was then used to screen key core genes within the PPI interaction network, ultimately identifying 10 HUB genes. Through literature review and analysis of relevant data, IFIT3 was selected as the top 1 core gene for further study.

[0040] 3. Verification of IFIT3 Expression

[0041] Gene expression of the IFIT3 gene was verified using gene expression matrix data. The results showed that there was a significant difference in IFIT3 expression between the case group and the control group (P < 0.05).

[0042] 4. Data Analysis

[0043] For transcriptomics data, R software (version number: versio4.3.3) was used to visualize the expression of IFIT3.

[0044] 2. Results Analysis

[0045] like Figure 1 As shown in the figure, this figure shows the differential analysis results of the data set GSE154998. The conditions |LogFC|>1, P<0.05 were used to screen differential genes, and a total of 195 differential genes (147 up-regulated genes and 48 down-regulated genes) were obtained.

[0046] like Figure 2 As shown in the figure, the expression level of IFIT3 as a biomarker in the normal control group and the severe COVID-19 pneumonia group. The results showed that there was a significant difference in IFIT3 between healthy people and patients with coronary heart disease (P < 0.001).

[0047] like Figure 3 As shown in the figure, this is the ROC curve of APCS, and the area under the curve is 0.98. The results show that IFIT3 as a biomarker can well distinguish 7 patients with new coronary pneumonia and 7 normal people (P<0.01).

[0048] Table 2 ROC analysis of IFIT3 for screening patients with COVID-19

[0049]

[0050] The embodiments described above are merely descriptions of preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Without departing from the spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by persons skilled in the art should fall within the scope of protection defined by the claims of the present invention.

Claims

1. An early biomarker for detecting severe pneumonia caused by a coronavirus, characterized in that: The invention comprises an interferon-induced protein having a tetrapeptide repeat sequence 3, namely IFIT3, wherein the early stage is within 7 days after the onset of symptoms, and the expression level of IFIT3 is detected by peripheral blood plasma samples, and the area under the ROC curve thereof is ≥0.

95.

2. Use of a reagent for detecting interferon-induced protein IFIT3 in blood in the development of a diagnostic tool, characterized in that: The diagnostic tool is used to distinguish patients with severe pneumonia caused by coronavirus from healthy people who are asymptomatic and have no inflammation, wherein the sensitivity of the tool is not less than 90% and the specificity is not less than 95%.

3. The use according to claim 2, characterized in that The distinguishing method comprises the following steps: (1) Peripheral blood plasma samples were collected from the subjects to be tested, and peripheral plasma samples from healthy individuals without infection symptoms were used as controls. The mean IFIT3 content of the control group was X±Y (SD, standard deviation); (2) using immunological detection methods to detect the content of IFIT3 in the plasma samples of the test subjects; (3) If the IFIT3 content of the tested subject is 2 times the standard deviation higher than the mean of the control group, the patient is judged to have severe pneumonia caused by a coronavirus.

4. The use according to claim 3, characterized in that The immunological detection method includes ELISA or chemiluminescence.

5. A diagnostic kit for distinguishing patients with severe pneumonia caused by a coronavirus from healthy individuals, characterized in that: include: (a) Monoclonal antibodies that specifically bind to IFIT3; (b) detection buffer; (c) a description comprising validation data for the IFIT3 threshold (X+2Y) and the area under the ROC curve ≥ 0.95 as defined in claim 3.

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