METHOD FOR TREATING SARS-COV-2 INFECTIOUS DISEASE
20230406913 ยท 2023-12-21
Assignee
Inventors
Cpc classification
G01N2469/10
PHYSICS
International classification
Abstract
Disclosed are a composition for diagnosis of SARS-CoV-2 infectious disease, a diagnostic kit, a method for providing information for diagnosis, a method for screening a therapeutic agent, and a composition for prevention or treatment, wherein it is expected that the diagnosis or prognostic prediction of the severity of SARS-CoV-2 infectious disease can be attained and the control of expression and activity of TOX, which is a marker, can be advantageously used in the development of a therapeutic agent for SARS-CoV-2 infectious disease.
Claims
1. A method for treating SARS-CoV-2 infectious disease, the method comprising administering to a subject a composition comprising an agent for inhibiting expression or activity of thymocyte selection associated high mobility group box (TOX) protein or a polynucleotide encoding TOX protein.
2. The method of claim 1, wherein the agent for inhibiting expression or activity of TOX protein or a polynucleotide encoding TOX protein is an antibody or antigen-binding fragment thereof, a small-molecule compound, siRNA, shRNA, miRNA, or a combination thereof.
3. A method for treating a severe SARS-CoA-2 patient, the method comprising: measuring the protein level or mRNA expression level of thymocyte selection associated high mobility group box (TOX) in a biological sample isolated from a SARS-CoV-2 infected patient; comparing the measured protein level or mRNA expression level of TOX with that of a normal control group; and administering a composition for treatment of SARS-CoV-2 infection to a SARS-CoV-2 infected patient, from which a biological sample with a higher protein level or mRNA expression level of TOX than the normal control group is isolated.
4. The method of claim 3, wherein the biological sample is at least one selected from the group consisting of blood, plasma, serum, saliva, nasal mucus, sputum, capsular fluid, amniotic fluid, ascites, cervical or vaginal discharge, urine, and cerebrospinal fluid.
5. The method of claim 3, wherein the composition for treatment of SARS-CoV-2 infection comprises remdesivir, regdanvimab, ritonavir-boosted nirmatrelvir, molnupiravir, dexamethasone, hydrocortisone, prednisolone, methylprednisolone, tocilizumab, baricitinib, convalescent plasma, casirivimab, imdevimab, sotrovimab, bamlanivimab, etesevimab, or a combination thereof.
6. The method of claim 3, wherein the composition for treatment of SARS-CoV-2 infection comprises an agent for inhibiting expression or activity of thymocyte selection associated high mobility group box (TOX) protein or a polynucleotide encoding TOX protein.
7. The method of claim 6, wherein the agent for inhibiting expression or activity of TOX protein or a polynucleotide encoding TOX protein is an antibody, a small-molecule compound, siRNA, shRNA, or a combination thereof.
Description
BRIEF DESCRIPTION OF THE DRAWINGS
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DETAILED DESCRIPTION OF THE EXEMPLARY EMBODIMENTS
[0108] Hereinafter, the present disclosure will be described in more detail with reference to exemplary embodiments. These exemplary embodiments are provided only for the purpose of illustrating the present disclosure in more detail, and therefore, according to the purpose of the present disclosure, it would be apparent to a person skilled in the art that these examples are not construed to limit the scope of the present disclosure.
Preparative Example: Preparation of Plasma Samples
[0109] After patient information and blood samples were secured from SARS-CoV-2 (COVID-19) infected patients, the blood was analyzed. Specifically, 85 mild COVID-19 (Non-ICU) patients, 31 severe COVID-19 (ICU) patients, 31 COVID-19 (Discharged) patients discharged after hospitalization, and 20 normal controls were participated, and with respect to mild/severe COVID-19, severe patients were defined as patients who have received a respiratory intensive care in the intensive care unit (ICU) due to acute respiratory distress syndrome (ARDS) or sepsis, according to the ICU admission history. Plasma samples were prepared by centrifugation within 48 hours of whole blood collection at 2000 xg for 5 minutes.
[0110] The study protocol was approved by the clinical trial Institutional Review Board of Yeungnam University Hospital in Daegu (IRB Nos. 2018-05-022, 2020-03-057, and 2020-05-031-001), and informed consent was obtained from all the patients.
Example 1: Discovery of TOX as Biomarker for SARS-CoV-2 Infection
[0111] To determine the influence of SARS-CoV-2 infection on cellular and molecular changes, first, the blood of patients with various COVID-19 seventies (mild COVID-19 patients, severe COVID-19 patients, and COVID-19 patients discharged after hospitalization) was analyzed.
TABLE-US-00001 TABLE 1 Comparison of laboratory findings of patients with SARS-CoV-2 infection SARS-CoV-2 Non- SARS-CoV-2 Discharged P- Classification ICU (n = 85) ICU (n = 31) (n = 31) value White blood cell 6.1 3.2 8.9 3.3 5.6 1.1 0.001 count, 10.sup.9/L Neutrophil count, 10.sup.9/L 4.1 3.2 7.7 3.3 3.0 0.9 <0.001 Lymphocyte count, 10.sup.9/L 1.5 0.7 0.8 0.3 1.9 0.5 <0.001 Hemoglobin, g/dL 13.0 1.6 13.5 1.7 12.6 1.5 0.143 Platelets, 10.sup.9/L 245.0 107.9 186.7 64.9 258.6 75.0 0.069 Albumin, g/dL 3.9 0.5 3.0 0.3 4.0 0.4 <0.001 Alanine aminotransferase, 30.1 26.3 58.8 93.6 38.8 26.1 0.033 IU/L Aspartate aminotransferase, 37.5 26.6 100.3 97.0 28.4 9.3 <0.001 IU Total bilirubin, mg/dL 0.8 0.4 1.1 0.6 1.4 4.3 0.398 Blood urea nitrogen, mg/dL 14.6 9.0 20.0 11.4 10.8 3.1 0.001 Creatinine, mg/dL 0.8 0.5 1.0 0.3 0.7 0.2 0.107 Creatinine phosphokinase, 100.7 159.1 131.9 122.0 71.1 68.3 0.332 IU/L Lactate dehydrogenase, 555.5 184.0 1272.6 542.1 380.4 131.8 <0.001 IU/L C-reactive protein, mg/dL 4.2 6.7 17.7 9.5 0.4 1.1 <0.001 Data are presented as mean standard error of the mean. (one-way ANOVA).
[0112] As above, it was discovered during the finding of biomarkers for SARS-CoV-2 infection that the transcription factor TOX was present in the serum of COVID-19 patients, and additional experiments were conducted on TOX as follows.
Example 2: Levels of TOX in SARS-CoV-2 Infected Patients
ELISA
[0113] Recombinant TOX protein (Abcam, ab160644) was diluted to 1 g/100 L, coated on Nunc-Immuno MicroWell 96-well plates, and incubated overnight at 4 C. Prior to use, the plates were washed 3 times with PBST and blocked with 3% BSA in PBS at 37 C. for 30 minutes. Primary antibody (TOX antibody, Cell signaling Technology, #99036) (1:2000 dilution) and SARS-CoV-2 infected patient plasma sample (20 g) were pre-incubated at 37 C. for 1 hour, and then the cultured sample was transferred to a peptide-coated plate and incubated at 37 C. for 1 hour. The plates were washed 5 times with PBST, incubated with secondary antibody (Cell signaling Technology, #7074) (1:5000 dilution) at 37 C. for 30 minutes, and then washed 5 times with PBST. The washed plates were treated with a 100 L/well TMB ELISA substrate for 10 minutes at 37 C. and then a 100 L/well stop solution was added. The detection was performed at 450 nm with an ELISA plate reader (Tecan, Austria).
PBMC Separation
[0114] Peripheral blood mononuclear cells (PBMCs) were separated by a Percoll density gradient (pH 8.5-9.5, Sigma-Aldrich, UK) with reference to Blood. 2014 Jan. 9; 123(2): 239-48. The separated PBMCs were suspended in RPMI 1640 medium (Sigma-Aldrich). A purity of 95% and a cell viability of 97% were confirmed by trypan blue staining.
Real-Time PCR
[0115] Herein, 1 g of total RNA was reverse transcribed into random hexamers by using expand reverse transcriptase (Roche) to generate cDNA in PBMCs. Real-time PCR was performed using the LightCycler FastStart DNA Master SYBR Green I by Roche Diagnostics GmbH, according to the manufacturer's protocol: 95 C. for 10 min (initial denaturation), 95 C. for 10 min (denaturation), 60 C. for 5 min (annealing), and 72 C. for 15 min (elongation), 45 cycles. The mRNA expression levels were determined according to the gene-specific standard curve.
Time-Course Analysis
[0116] While the separated PBMCs were incubated for 24 hours, the level of TOX secretion was analyzed by TOX ELISA, and the cell viability was analyzed by WST-1 reagent.
Computed Tomography
[0117] Lung tissues of SARS-CoV-2 patients were examined using computed tomography (CT) imaging.
[0118] As a result, as shown in
[0119] Furthermore, as shown in
[0120] Last, as shown in
[0121] These results indicate a correlation between the TOX protein level and the severity of COVID-19 disease.
Example 3: Effect of TOX on Inflammation of SARS-CoV-2 Infected Patients
[0122] PBMCs isolated from the patients with SARS-CoV-2 infectious disease in Example 2 were cultured with recombinant TOX protein at 37 C. for 24 hours. The supernatant was used for cytokine ELISA analysis, and the lysate was used for NF-B activity assay. All experiments were independently performed at least three times.
Cytokine ELISA
[0123] The cytokine concentrations in the plasma were quantified using commercially available ELISA kits according to the manufacturers instructions, and detected at 450 nm with an ELISA plate reader (Tecan). The kits used were as follows: Human IL-1 Quantikine ELISA kit (DLB50, R&D Systems, USA), Human IL-4 Quantikine ELISA kit (D4050, R&D Systems), Human IL-6 Quantikine ELISA kit (D6050, R&D Systems), Human IL-10 Quantikine ELISA Kit (D1000B, R&D Systems), Human IFN- Quantikine ELISA Kit (DIF50, R&D Systems), and Human TNF- Quantikine ELISA kit (DTAOOD, R&D Systems).
NF-B Activity Kit
[0124] NF-B activity was analyzed using an ELISA-based NF-B family transcription factor assay kit (43296; Active Motif, USA). Specifically, a nuclear extract (2 g) was incubated in 96-well plates, on which NF-B consensus oligonucleotides were immobilized, at 37 C. for 1 hour. The captured complex was incubated with NF-B primary antibody at 37 C. for 1 hour and then incubated with HRP-conjugated secondary antibody at 37 C. for 1 hour. The antibodies were detected by determining the optical density (OD) at 450 nm through a Tecan Spark microplate reader.
[0125] As a result, as shown in
[0126] These results indicate that TOX protein had a great effect on the reduction of inflammation reaction caused by SARS-CoV-2 infection.
Example 4: in Vivo TOX Effect
[0127] TOX antibody (ab237009) was administered to mice with abdominal sepsis induced by cecal ligation and puncture (CLP), and the survival rate of the mice was checked.
[0128] Specifically, C57BL/6 male mice (6-7-weeks-old, weighing 18-20 g) were purchased from Orient Bio and used after an acclimatization period of 12 days. Five mice per cage were raised with a 12:12 hour light/dark cycle in an environment of a temperature of 20-25 C. and a humidity of 40-45%. The mice were given a normal rodent diet, and water was freely supplied (ad libitum). Any animal was treated according to the Guide for Care and Use of Laboratory Animals published by KRIBB No. 1305021.
[0129] The CLP-induced sepsis mouse model was prepared with reference to Nat Protoc 4, 31-36 (2009). A2 cm midline incision was made to expose the cecum and adjoining small intestine. The cecum was then ligated tightly using 5.0 mm of a 3.0-silk suture from the cecal tip, punctured with a 22-gauge needle, and then gently squeezed to extrude feces from the perforation site. The cecum was then returned to the peritoneal cavity, and the laparotomy site was sutured using 4.0-silk. For sham operations, the cecum of the mice was surgically exposed, but not ligated or punctured, and then returned to the abdominal cavity.
[0130] The TOX antibody was administered two times (100 ng/ml or 200 ng/ml) 12 and 36 hours after CLP, and then the survival rate was checked every six hours. The blood was collected at 72 hours to investigate organ damage biomarkers in the blood. In addition, H&E staining was performed to investigate the lung tissue damage.
[0131] As a result, as shown in
[0132] These results indicate that TOX antibody had a great effect in the treatment of sepsis caused by SARS-CoV-2 infection.