Application of substances that reduce CCR2 levels or activity in the treatment or prevention of fever with thrombocytopenia syndrome

Abstract

This invention discloses the application of substances that reduce CCR2 content or activity in the treatment or prevention of fever with thrombocytopenia syndrome (FPS). This invention discovers that CCR2 is a target for the prevention and treatment of FPS virus infection, revealing that CCR2 plays an important role in the infection and proliferation of FPS virus and can serve as a target for inhibiting FPS virus infection. Utilizing CCR2 inhibitors as candidate drugs for the prevention and treatment of FPS virus shows great research potential. This invention provides a theoretical basis for using CCR2 as a target for the treatment of FPS and has significant application value for the treatment of FPS virus infection and the development of drugs for treating FPS virus infection.

Description

Technical Field

[0001] This invention relates to the biomedical field and the use of substances that reduce CCR2 content or activity in the treatment or prevention of fever with thrombocytopenia syndrome. Background Technology

[0002] Severe fever with thrombocytopenia syndrome (SFTS) is a newly emerging viral hemorrhagic fever with a mortality rate ranging from 12% to 50%. The causative agent is Dabiebandavirus (SFTSV), belonging to the genus Bandavirus in the family Phenuiviridae. Ticks are the primary vector for transmission of SFTS, although there have been multiple reports of human-to-human transmission through close contact with the secretions of infected individuals. Currently, clinical treatment for SFTS primarily focuses on symptomatic and supportive care; there are no approved specific antiviral drugs or protective vaccines. Therefore, identifying the targets associated with viral infection in SFTS will contribute to the development and application of antiviral drugs and is of great significance for preventing the outbreak of public health emergencies.

[0003] CC motif chemokine receptor 2 (CCR2) belongs to the G protein-coupled receptor superfamily and is the receptor for monocyte chemoattractant proteins 1–4 (MCP1–4), which are chemical inducers of pro-inflammatory responses. CCR2 is the earliest chemocatalytic receptor on inflammatory monocytes and is expressed on T cells, dendritic cells, and epithelial cells. Through ligand binding, CCR2 mediates the migration and activation of inflammatory cells. Summary of the Invention

[0004] The technical problem to be solved by this invention is how to inhibit viral infection or proliferation of fever with thrombocytopenia syndrome, and how to treat or prevent fever with thrombocytopenia syndrome.

[0005] To address the aforementioned technical problems, this invention first provides the application of substances that reduce the content or activity of CCR2 (Gene ID:729230, updated 2023-03-29) in the preparation of products for the treatment or adjuvant treatment or prevention or adjuvant prevention of fever with thrombocytopenia syndrome.

[0006] In the above applications, the substance that reduces CCR2 content or activity can be a protein, polypeptide, or small molecule compound that inhibits CCR2 protein synthesis, promotes CCR2 protein degradation, or inhibits CCR2 protein function.

[0007] Specifically, the substance that reduces CCR2 content or activity may be a CCR2 antibody or a CCR2 inhibitor.

[0008] In one embodiment of the present invention, the CCR2 antibody is an anti-human CCR2 antibody (BioLegend, Cat. 357202).

[0009] The CCR2 inhibitor can be any substance that can bind to the CCR2 protein and inhibit its activity. Any substance that can inhibit the activity of the CCR2 protein is within the scope of this invention, including CCR2 antagonists and CCR2 inhibitors.

[0010] In the above applications, the CCR2 inhibitor may be RS102895 hydrochloride or CCR2 antagonist 1.

[0011] The present invention also provides the use of substances that reduce the expression level of the CCR2 gene (Gene ID: 729230, updated on 2023-03-29) or substances that knock out the CCR2 gene in the preparation of products for the treatment or adjuvant treatment or prevention or adjuvant prevention of fever with thrombocytopenia syndrome.

[0012] In the above applications, the substance that reduces the expression level of the CCR2 gene can be siRNA that specifically recognizes the CCR2 gene;

[0013] The substance that knocks out the CCR2 gene can be a substance that knocks out the CCR2 gene using the CRISPR-Cas9 system.

[0014] In one embodiment of the present invention, knocking out the CCR2 gene results in a portion of its sequence (WT sequence, SEQ ID No. 13) being edited into an M1 sequence (SEQ ID No. 14) and / or an M2 sequence (SEQ ID No. 15).

[0015] In one embodiment of the present invention, the sgRNA target of the CCR2 gene is the DNA fragment shown in SEQ ID No. 9, which is knocked out using the CRISPR-Cas9 system.

[0016] In the above applications, the siRNA may be the siRNA formed by SEQ ID No. 1 and its reverse complementary sequence in the sequence listing, or the siRNA formed by SEQ ID No. 2 and its reverse complementary sequence.

[0017] The application of substances that reduce CCR2 content or activity in the preparation of products that inhibit or assist in the inhibition of viral infection or proliferation of fever with thrombocytopenia syndrome is also within the scope of protection of this invention.

[0018] The application of the substance that reduces CCR2 gene expression in the preparation of products that inhibit or assist in the inhibition of viral infection or proliferation of fever with thrombocytopenia syndrome is also within the scope of protection of this invention.

[0019] The application of the substance that knocks out the CCR2 gene in the preparation of products that inhibit or assist in the inhibition of viral infection or proliferation of fever with thrombocytopenia syndrome is also within the scope of protection of this invention.

[0020] The use of substances targeting CCR2 for the treatment or prevention of fever with thrombocytopenia syndrome, or substances that inhibit viral infection or proliferation of fever with thrombocytopenia syndrome, in the preparation of products for the treatment or prevention of fever with thrombocytopenia syndrome, or in the preparation of products that inhibit viral infection or proliferation of fever with thrombocytopenia syndrome, is also within the scope of protection of this invention.

[0021] The present invention also provides a product, wherein the active ingredient of the product is the substance that reduces CCR2 content or activity, or the substance that reduces CCR2 gene expression, or the substance that knocks out the CCR2 gene;

[0022] The product has the following functions: b1) or b2):

[0023] b1) Treatment or prevention of fever with thrombocytopenia syndrome;

[0024] b2) Inhibit viral infection or proliferation in fever with thrombocytopenia syndrome.

[0025] The infection may be an infection of animal cells, tissues, or individuals by the fever with thrombocytopenia syndrome virus.

[0026] In this invention, the product may be a drug or a vaccine.

[0027] This invention discovers that CCR2 is a potential target for the prevention and treatment of fever with thrombocytopenia syndrome (FPS) virus infection. Firstly, knocking down endogenous CCR2 expression in THP-1 cells via siRNA significantly inhibits FPS virus gene expression; both gene expression and replication of FPS virus are significantly suppressed in CCR2 knockout cell lines; CCR2 inhibitors and antibodies can inhibit FPS virus infection of host cells; CCR2 knockout significantly reduces the mortality rate of FPS virus-infected animals, while CCR2 inhibitors significantly improve the survival rate of FPS virus-infected animals. This invention reveals that CCR2 plays a crucial role in the infection and proliferation of FPS virus and can serve as a target for inhibiting FPS virus infection. Utilizing CCR2 inhibitors as candidate drugs for the prevention and treatment of FPS virus shows great promise for research. This invention provides a theoretical basis for using CCR2 as a target for the treatment of fever with thrombocytopenia syndrome, and has significant application value for the treatment of viral infections with fever with thrombocytopenia syndrome and the development of drugs for treating viral infections with fever with thrombocytopenia syndrome.

[0028] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way. Attached Figure Description

[0029] Figure 1 To investigate the inhibitory effect of knocking down CCR2 expression in the THP-1 cell line on viral infection and proliferation of fever with thrombocytopenia syndrome (FPS). A shows qPCR detection of viral RNA; B shows Western blotting detection of CCR2 and NP expression levels. NC, CCR2 KD1, CCR2 KD2, and ATF6 KD represent transfections with siNC, CCR2-siRNA-1, CCR2-siRNA-2, and siATF6, respectively.

[0030] Figure 2 The inhibitory effect of CCR2-KO cell line on infection and proliferation of different strains of fever with thrombocytopenia syndrome virus was detected by qPCR using viral RNA.

[0031] Figure 3 The inhibitory effect of bone marrow-derived macrophages (BMDM) from CCR2 knockout mice on viral infection and proliferation in fever with thrombocytopenia syndrome (FTS). A shows the detection of CCR2 gene expression; B shows the detection of viral load and viral proliferation; CCR... - / -This indicates a CCR2 knockout mouse.

[0032] Figure 4 The inhibitory effects of CCR2 inhibitors on the infection and proliferation of virus in patients with fever and thrombocytopenia syndrome (FTS) are shown in the following figures: A: Detection results of RS102895 hydrochloride treatment; B: Detection results of CCR2 antagonist 1 treatment; C: RNA detection of RS102895 hydrochloride-treated virus by qPCR; D: RNA detection of CCR2 antagonist 1-treated virus by qPCR; E: Western blotting of proteins from viruses treated with RS102895 hydrochloride and CCR2 antagonist 1 in THP-1 cells; F: Western blotting of proteins from viruses treated with RS102895 hydrochloride and CCR2 antagonist 1 in huh-7 cells. In C and D, "Vehicle" indicates that the inhibitor concentration is 0. In E and F, the lanes in the upper figure, from left to right, represent the results of RS102895 hydrochloride concentrations of 0, 0.20, 0.39, 0.78, 1.56, 3.12, 6.25, 12.50, and 25.00 μM, respectively. In the lower figure, the lanes, from left to right, represent the results of CCR2 antagonist 1 concentrations of 0, 3.125, 6.25, 12.5, 25, 50, 100, 200, and 400 μM, respectively.

[0033] Figure 5 This study demonstrates the inhibitory effect of CCR2 antibody on viral infection and proliferation of fever with thrombocytopenia syndrome virus (FPS). Viral load was detected by probe-based qPCR, with the x-axis representing antibody concentration. For each concentration, the left side represents isotype control IgG treatment, and the right side represents CCR2 antibody treatment.

[0034] Figure 6 CCR2 knockout significantly reduced mortality in mice infected with SFTSV virus. A represents the change in body weight of mice after inoculation with SFTSV virus, with the body weight before inoculation recorded as 100%; B represents the change in survival rate.

[0035] Figure 7 The CCR2 inhibitor RS102895 significantly improved the survival rate of mice infected with fever with thrombocytopenia syndrome. A represents the serum viral load test results, V represents Vehicle, and RS represents RS102895; B represents the change in survival rate. Detailed Implementation

[0036] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials, reagents, instruments, etc., used in the following examples are all commercially available. All quantitative experiments in the following examples were performed in at least three replicates.

[0037] In the following examples, the standard deviation of the quantitative experiments was measured using SEM, and the t-test was used to determine the significance of differences between data.

[0038] The SFTSV (severe fever with thrombocytopenia syndrome virus) strain HBMC16 in the following examples is described in Zhang, Y., Shen, S., Shi, J. et al. Isolation, characterization, and phylogenic analysis of three new severe fever with thrombocytopenia syndrome bunyavirus strains derived from Hubei Province, China. Virol. Sin. 32, 89–96 (2017).

[0039] (https: / / doi.org / 10.1007 / s12250-017-3953-3), the public may obtain the biological material from the applicant in accordance with the relevant national biosafety regulations. The biological material is only for repeating the relevant experiments of this invention and may not be used for other purposes.

[0040] The SFTSV strains HNXY2017-50, HNXY2017-66, and WCH (Li H, Zhang LK, LiSF, et al. Calcium channel blockers reduce severe fever with thrombocytopenia syndrome virus (SFTSV) related fatality. Cell Res. 2019; 29(9):739-753. doi:10.1038 / s41422-019-0214-z) in the following examples can be obtained from the applicant by the public in accordance with the relevant national biosafety regulations. These biological materials are only for repeating the relevant experiments of this invention and cannot be used for other purposes.

[0041] CCR2 knockout mice: Jackson laboratory, strain B6.129S4-Ccr2 tm1Ifc / J .

[0042] The human peripheral blood mononuclear cell line THP-1, human liver cancer cell line huh-7, and African green monkey kidney cells Vero in the following examples were all products of the American Type Culture Collection (ATCC).

[0043] Example 1: Effect of altered CCR2 expression on viral infection and proliferation of fever with thrombocytopenia syndrome.

[0044] The expression level of the CCR2 gene (Gene ID: 729230, updated 2023-03-29) in the human peripheral blood mononuclear cell line THP-1 was knocked down by CCR2 siRNA to investigate the effect of intracellular CCR2 on viral infection with fever with thrombocytopenia syndrome.

[0045] Methods: THP-1 cells were seeded in 24-well plates. Each well contained 1 μL of siRNA (siRNA1 or siRNA2), 1 μL of RNAiMAX (Invitrogen, Cat. 13778150), and 48 μL of Opti-MEM (Gibco, Cat. 2085556). For the negative control group, an equal amount of siNC was added to each well. For the positive control group, an equal amount of siATF6 was added to each well. A vehicle without siRNA was used as a blank control. After thorough mixing, the cells were incubated at 37°C for 48–72 h in RPMI 1640 medium (Gibco, Cat. C11875500BT) at a cell density of 102. 5 Cells were transfected with siRNA at a concentration of 20 μM per well. After transfection, cells were infected with SFTSV virus HBMC16 at an MOI of 5. After incubation at 37°C for 1 h, the medium was replaced with RPMI 1640 medium containing 2% fetal bovine serum (Gibco, Cat. 10099-141C), and the cells were incubated at 37°C for 24 h. The viral replication efficiency was detected by real-time quantitative PCR (dye-based qPCR) and Western blot.

[0046] The siRNA sequence is as follows:

[0047] CCR2-siRNA-1: 5'-GGCTGTATCACATCGGTTATT-3' (SEQ ID No. 1);

[0048] CCR2-siRNA-2: 5'-GAGGAUGGAAUAAUUUCCATT-3' (SEQ ID No. 2);

[0049] siNC: 5'-UUCUCCGAACGUGUCACGUTT-3' (SEQ ID No. 3);

[0050] siATF6: 5'-GCAGCAACCAAUUCAGUUU-3' (SEQ ID No. 4).

[0051] The primers used for dye-based qPCR are as follows:

[0052] SFTSV-Forward: 5'-CTCACTCATGCCCTCAACGA-3' (SEQ ID No. 5);

[0053] SFTSV-Reverse: 5'-GATGAACTCACCAGCCCTGC-3' (SEQ ID No. 6);

[0054] GAPDH-Forward:5'-GAAGGTGAAGGTCGGAGTC-3' (SEQ ID No. 7);

[0055] GAPDH-Reverse: 5'-GAAGATGGTGATGGGATTTC-3' (SEQ ID No. 8).

[0056] The effect of real-time quantitative PCR (dye-based qPCR) on the detection of intracellular SFTSV replication: Cellular nucleic acid extraction was performed using a total RNA extraction kit (Tiangen, Cat.DP430-H), with specific steps following the manufacturer's instructions; qPCR detection was performed using a real-time quantitative PCR (qPCR) kit (TaKaRa, Cat.066A), with specific steps following the manufacturer's instructions, and the primers used were as described above (SEQ ID No. 5-8).

[0057] The antibodies used for Western blotting of proteins are as follows: CCR2 antibody (Gibco Invitrogen, Cat. 711255), NP rabbit serum polyclonal antibody (obtained by immunizing rabbits with NP protein as antigen (which can be directly synthesized or obtained through cell expression), and mouse anti-GAPDH monoclonal antibody (Prilele, Cat. C1312-100).

[0058] NP protein: MSEWSRIAVEFGEQQLNLTELEDFARELAYEGLDPALIIKKLKETGGDDWVKDTKFIIVFALTRGNKIVKASGKMSNSGSKRLMALQEKYGLVERAETRLSITPVRVAQSLPTWTCAAAAAL KEYLPVGPAVMNLKVENYPPEMMCMAFGSLIPTAGVSEATTKTLMEAYSLWQDAFTKTINVKMRGASKTEVYNSFRDPLHAAVNSVFFPNDVRVKWLKAKGILGPDGVPSRAAEVAAAAYRNL(SEQ ID No.19).

[0059] The results are as follows Figure 1 As shown, compared with cells transfected with siNC and siATF6, the expression level of CCR2 was significantly decreased after transfection with CCR2-siRNA-1 and CCR2-siRNA-2. Compared with cells transfected with siNC, the infectivity of SFTSV virus and the expression of viral genes were significantly decreased after transfection with CCR2-siRNA-1, CCR2-siRNA-2, and siATF6. These results indicate that knocking down endogenous CCR2 expression in THP-1 cells using siRNA significantly inhibits the expression of the SFTSV gene (i.e., NP protein) and suppresses viral proliferation.

[0060] Example 2: Construction of CCR2 knockout cell lines by lentiviral transfection and detection of infection and proliferation of different SFTSV strains in CCR2 knockout cell lines.

[0061] 1. Recombinant plasmid construction: Based on the CCR2 gene sequence, an sgRNA target sequence was designed, the specific sequence of which is as follows:

[0062] CCR2-sgRNA target sequence: 5'-GCAGCAGAGTGAGCCCACAA-3' (SEQ ID No. 9);

[0063] Control sgRNA target sequence: 5'-GTATTACTGATATTGGTGGG-3' (SEQ ID No. 10).

[0064] The target sequence was synthesized, and after digesting the lentiCRISPR v.2 (Addgene, Cat.52961) plasmid with BsmBI, the synthesized target sequence was inserted into the vector to obtain the recombinant plasmid lentiCRISPR-CCR2, which transcribes the CCR2 gene sgRNA, and the control plasmid lentiCRISPR-CK, respectively.

[0065] 2. Lentiviral vector construction: HEK-293T cells were plated in 10cm cell culture dishes and incubated overnight at 37°C with 5% CO2 until the cell density reached approximately 90% at transfection the next day. A DNA mixture was prepared using lipofectamine 2000 (Thermo Fisher, Cat. 11668019), Opti-MEM (Gibco, Cat. 2085556), lentiCRISPR-CCR2 (or the control plasmid lentiCRISPR-CK), and pMD2.G (Addgene Cat. 12259). The mixture was gently mixed and incubated at room temperature for 20 minutes to allow the DNA mixture to form. This mixture was then added to cell culture dishes, gently mixed, and incubated overnight at 37°C with 5% CO2. After 12–15 hours, the culture medium in the dishes was replaced, and 10 mL of fresh serum-containing DMEM medium (Gibco, Cat. 11965092) was added. 48–72 hours after transfection, the supernatant containing the virus was collected, filtered, and the lentivirus was obtained. The lentivirus obtained from lentiCRISPR-CCR2 is designated as CCR2-sgRNA lentivirus, and the lentivirus obtained from lentiCRISPR-CK is designated as control lentivirus.

[0066] 3. Lentiviral infection of THP-1 cells and selection of stable transfected cells: THP-1 cells were transfected with CCR2-sgRNA lentivirus or control lentivirus, and then cultured in RPMI 1640 medium containing 4 mg / ml puromycin for 7 days. Single clones were selected for expansion culture. Genomic DNA was extracted from the expanded cultured cells, and PCR amplification was performed using pre-designed sequencing primers. The amplification products were sent to a sequencing company for sequencing to obtain CCR2 knockout THP-1 cells (denoted as CCR2-KO cell line).

[0067] Sequencing primers: CCR2-detect-F: 5'-GAGCGGTGAAGAAGTCACCA-3' (SEQ ID No. 11);

[0068] CCR2-detect-R: 5'-CAGAAGCAAACACAGCCACC-3' (SEQ ID No. 12).

[0069] In the resulting cell lines, a portion of the CCR2 gene sequence (WT sequence, SEQ ID No. 13) was edited into M1 sequence (SEQ ID No. 14) and M2 sequence (SEQ ID No. 15), and the resulting cell lines were heterozygous.

[0070] WT sequence:

[0071] CTGGTCGTCCTCATCTTAATAAACTGCAAAAAGCTGAAGTGCTTGACTGACATTTACCTGCTCAACCTGGCCATCTCTG

[0072] ATCTGCTTTTTCTTATTACTCTCCCATTGTGGGCTCACTCTGCTGCAAATGAGTGGGTCTTTGGGAATGCAATGTG (SEQ ID No. 13);

[0073] M1 sequence: CTGGTCATCCTCATCTTTGGTCTTTGGGAATGCTGGTCTTTGGGAATGCAATGTG (SEQ ID No. 14);

[0074] M2 sequence: CTGGTCGTCCTCATCTTAATAAACTGCAAAAAGCTGAAGTGCTTGACTGACATTTACCTGCTCAACCTGGCCATCTCTG ATCTGCTTTTTCTTATTACTCTCCCATTGGGGCTCACTCTGCTGCAAATGAGTGGGTCTTTGGGAATGCAATGTG (SEQ ID No. 15).

[0075] 4. Infection and proliferation detection of different SFTSV lines in CCR2 knockout cell lines

[0076] CCR2-KO cell lines were infected with four different SFTSV strains: HBMC16, HNXY2017-50, HNXY2017-66, and WCH. The virus loading was at an MOI of 0.1, and the cell density was 2 × 10⁻⁶. 5 Cells were incubated per well with RPMI 1640 medium. After incubation at 37°C for 1 hour, the medium was replaced with RPMI 1640 medium containing 2% fetal bovine serum, and the cells were incubated at 37°C for another 24 hours. Real-time quantitative PCR (dye-based qPCR) was used to detect SFTSV virus gene expression in the cells, with THP-1 cells (WT) without CCR2 gene knockout serving as a control.

[0077] The method for detecting intracellular SFTSV virus gene expression by real-time quantitative PCR (dye-based qPCR) is the same as in Example 1.

[0078] The results are as follows Figure 2 As shown, compared with WT cells, the infection and proliferation of all four SFTSV strains in the CCR2 KO cell line were significantly inhibited.

[0079] Example 3: Detection of SFTSV infection and proliferation by BMDM macrophages derived from the bone marrow of CCR2 KO mice.

[0080] I. Primary isolation and culture of mouse bone marrow-derived macrophages

[0081] Six-week-old C57BL / 6J mice (wild-type, WT) and CCR2 knockout mice (CCR2 knockout mice) were used. - / - After euthanasia, femurs and tibias of mice were harvested. Cells were flushed from the bone marrow using 1 mL of DMEM via a syringe, filtered through a 40 μM cell sieve, and cultured in DMEM medium containing 10% FBS, 1% penicillin antibody (Gibco, Cat. 15140-122), and 50 ng / mL M-CSF (PeproTech, Cat. 315-02-10). The cells were cultured at 37°C with 5% CO2 for 7 days to obtain 90% pure primary bone marrow-derived macrophages (BMDM). Flow cytometry was used to detect CCR2 expression in BMDM derived from CCR2 knockout mice (flow cytometry antibodies: FITC anti-mouse F4 / 80 Antibody, Biolegend, Cat. 123107; PE anti-mouse CD192 (CCR2) Antibody, Biolegend, Cat. 150610). The results are as follows: Figure 3 As shown in Figure A, the expression level of CCR2 was reduced in knockout mice compared to WT mice.

[0082] II. Detection of SFTSV infection and proliferation by BMDM

[0083] The BMDM cells obtained from the isolation culture were plated and infected with the SFTSV strain HBMC16 at an MOI of 0.1 and a cell density of 1.5 × 10⁻⁶ cells / year. 5 Cells were incubated per well with DMEM medium. After incubating the cells and virus at 37°C for 1 hour, the medium was replaced with DMEM medium containing 2% fetal bovine serum, and the cells were incubated at 37°C for 24 hours. The cell supernatant was then collected. The gene expression of SFTSV virus in the cell supernatant was detected using real-time quantitative PCR (qPCR).

[0084] The primers used for probe-based qPCR are as follows:

[0085] SFTSV-S-Forward: 5'-AGCCTAATTGGATATGTCAAATTGC-3' (SEQ ID No. 16);

[0086] SFTSV-S-Reverse: 5'-CGGGTGAAGTGGCTGAAGG-3' (SEQ ID No. 17);

[0087] Probe: 5'-6-FAM-AGCAGCAGCAGCAACCTCAGCAGC-BHQ1-3' (SEQ ID No. 18).

[0088] Detection of viral genes in cell supernatant: Nucleic acid was extracted from cell supernatant using a viral RNA extraction kit (Tiangen, Cat.DP315-R), following the instructions in the manufacturer's manual. qPCR detection was performed using a probe-based qPCR kit (TaKaRa, Cat.064A), following the instructions in the manufacturer's manual. The primers used were as described above (SEQ ID No.16-18).

[0089] The results are as follows Figure 3 As shown in Figure B, compared with WT mice, the infection and proliferation of SFTSV in BMDM of CCR2 knockout mice were significantly inhibited.

[0090] Example 4: Detection of the inhibitory effects of CCR2 inhibitors and antibodies on SFTSV infection and proliferation.

[0091] I. CCR2 Inhibitor Treatment

[0092] In huh-7 cells, the cytotoxicity of two CCR2 inhibitors, RS102895 hydrochloride (MedChemExpress, Cat. No.: HY-18611; CAS No.: 1173022-16-6) and CCR2 antagonist 1 (MedChemExpress, Cat. No.: HY-112792; CAS No.: 1683534-96-4), was assessed. The specific steps are as follows:

[0093] Huh-7 cells were administered at a dose of 1×10⁻⁶. 4 Cells were seeded per well in 96-well cell culture plates and incubated overnight at 37°C with 5% CO2. Cells adhered to the plates and were treated with 1.56 μM, 3.125 μM, 6.25 μM, 12.5 μM, 25 μM, 50 μM, 100 μM, 200 μM, and 400 μM RS102895 hydrochloride or CCR2 antagonist 1, respectively, with three replicates per group. The control group received the same amount of dimethyl sulfoxide (DMSO). After 24 h of treatment, OD was measured using an MTT assay kit (Solepro, Cat. CA1210). 450nmAnalyze cell viability; tests show that... Figure 4 As shown in Figures A and B, the CC of RS102895 hydrochloride... 50 The CCR2 antagonist 1 has a concentration of 101.7 μM. 50 The concentration was 4150 μM. In subsequent experiments, the concentrations of RS102895 hydrochloride and CCR2 antagonist 1 were both within the safe and non-toxic range. Further studies investigated the IC50 of RS102895 hydrochloride and CCR2 antagonist 1 in inhibiting SFTSV. 50 The test was conducted, and the results were as follows: Figure 4 As shown in Figures A and B, the IC50 of RS102895 hydrochloride is... 50 The IC50 of CCR2 antagonist 1 was 3.061 μM. 50 The value is 37.76 μM.

[0094] Human peripheral blood mononuclear cell line THP-1 or human hepatocellular carcinoma cell line huh-7 were plated. THP-1 cells were cultured in RPMI 1640 medium (Gibco, Cat. C11875500BT), and huh-7 cells were cultured in DMEM medium (Gibco, Cat. 11965092). Both THP-1 and huh-7 cells were treated with two CCR2 inhibitors: RS102895 hydrochloride and CCR2 antagonist 1. The concentrations of RS102895 hydrochloride were set at 0, 0.20, 0.39, 0.78, 1.56, 3.12, 6.25, 12.50, and 25.00 μM. The solvent used was RPMI 1640 medium or DMEM medium containing 2% fetal bovine serum. The cell density was 1 × 10⁻⁶ cells / year. 5 Cells / well; the concentration of CCR2 antagonist 1 was set at 0, 3.125, 6.25, 12.5, 25, 50, 100, 200, and 400 μM, and the solvent used was RPMI 1640 medium or DMEM medium containing 2% fetal bovine serum, with a cell density of 1 × 10⁻⁶ cells / well. 5 Cells were incubated at 37°C for 1 hour after the inhibitor was added, and then infected with SFTSV strain HBMC16 at an MOI of 0.1. After incubation at 37°C for 1 hour, the medium was replaced with DMEM medium containing 2% FBS and cultured for another 24 hours. Cells were then harvested and the effects of RS102895 hydrochloride and CCR2 antagonist 1 on inhibiting SFTSV replication were detected by real-time quantitative PCR (dye-based qPCR) and Western blotting.

[0095] The effect of real-time quantitative PCR (qPCR) on intracellular SFTSV replication: Cellular nucleic acid extraction was performed using a total RNA extraction kit (Tiangen, Cat.DP430-H), with specific steps following the manufacturer's instructions; qPCR detection was performed using a real-time quantitative PCR (qPCR) kit (TaKaRa, Cat.066A), with specific steps following the manufacturer's instructions. The dye primers used are described in Example 1 (SEQ ID No. 5-8), and the Western blotting method was the same as described in Example 1.

[0096] The results are as follows Figure 4 As shown in C-F, treatment with two CCR2 inhibitors, RS102895 hydrochloride and CCR2 antagonist 1, significantly reduced the gene expression of SFTSV in cells. Both CCR2 inhibitors significantly inhibited SFTSV replication, and this inhibitory effect showed a dose-dependent trend.

[0097] II. CCR2 antibody treatment

[0098] Human peripheral blood mononuclear cell line THP-1 or human hepatocellular carcinoma cell line huh-7 were plated. THP-1 was cultured in RPMI 1640 medium (Gibco, Cat. C11875500BT), and huh-7 was cultured in DMEM medium (Gibco, Cat. 11965092). Anti-human CCR2 antibody (BioLegend, Cat. 357202) or isotype control IgG (BioLegend, Cat. 400201) was added to the plates at concentrations of 0.05, 0.5, and 5 μg / ml, with a cell density of 1 × 10⁻⁶ cells / ml. 5 Cells / well; after adding antibody, incubate at 37°C for 1 hour, then infect with SFTSV strain HBMC16. The amount of SFTSV added is MOI = 1 or 5. After 24 hours of virus infection, collect cell supernatant to detect the viral load of SFTSV.

[0099] Viral load detection: Nucleic acid was extracted from cell supernatant using a viral RNA extraction kit (Tiangen, Cat.DP315-R), following the instructions in the manufacturer's manual. qPCR detection was performed using a probe-based qPCR kit (TaKaRa, Cat.064A), following the instructions in the manufacturer's manual. The primers and probes used were the same as in Example 3 (SEQ ID No.16-18).

[0100] The results are as follows Figure 5As shown, the viral load in the supernatant was significantly reduced after treatment with CCR2 antibody, indicating that the antibody against CCR2 also has a significant inhibitory effect on SFTSV infection and proliferation, further demonstrating that CCR2 is an important target for SFTSV infection and proliferation.

[0101] Example 5: Detection of SFTSV infection in CCR2 knockout mouse model.

[0102] Methods: Five-week-old female CCR2 knockout mice (CCR2 knockout mice) housed in an SPF environment were used. - / - Eleven wild-type C57BL / 6J mice (WT) and one hundred and eleven wild-type C57BL / 6J mice were pretreated with type I interferon blocking antibody (IFNAR1) (BioXCell, Cat.BE0241) at 1.7 mg / mouse via intraperitoneal injection for 24 h, followed by intraperitoneal inoculation with 100 μL / mouse of SFTSV strain HBMC16 virus (2 × 10⁻⁶). 4 The mice were weighed daily and their survival status was observed. If a mouse's weight decreased by 25%, it was considered dead.

[0103] The results are as follows Figure 6 As shown, the body weight of mice decreased on the first day after SFTSV infection, but gradually recovered after the sixth day of infection. The CCR2 knockout mice recovered their body weight faster and had a significantly higher survival rate than WT mice, indicating that CCR2 gene knockout can significantly improve the survival rate of SFTSV-infected mice.

[0104] Example 6: Detection of the therapeutic effect of CCR2 inhibitor on SFTSV-infected mice.

[0105] Five-week-old wild-type C57BL / 6J mice housed in an SPF environment were divided into three groups: a control group (n=5), an SFTSV+Vehicle group (n=13), and an SFTSV+RS102895 group (n=13). Each group of mice was pretreated 24 h by intraperitoneal injection of type I interferon blocking antibody (IFNAR1), 1.7 mg / mouse. Then, mice in the SFTSV+Vehicle and SFTSV+RS102895 groups were intraperitoneally inoculated with 100 μL / mouse of the SFTSV strain HBMC16 virus (2 × 10⁻⁶ mcg). 4Mice in the SFTSV+RS102895 group were administered RS102895 hydrochloride (MedChemExpress) (dissolved in 20% SBE-β-CD) by gavage at 15 mg / kg / day once daily. Mice in the SFTSV+Vehicle group were administered an equal volume of 20% SBE-β-CD by gavage daily. The control group received no treatment. Mice were weighed daily and their survival status was observed. 50 μL of tail vein blood was collected on days 3 and 5 of SFTSV infection for serum viral load testing. Mice were considered dead if their body weight decreased by 25%.

[0106] Serum viral load detection: The detection method is the same as in Example 3.

[0107] The results are as follows Figure 7 As shown, compared with the solvent group (SFTSV+Vehicle), the serum viral load of mice in the SFTSV+RS102895 group (treatment group) was significantly reduced on days 3 and 5 of viral infection, and the survival rate of the treatment group was significantly improved.

[0108] The present invention has been described in detail above. Those skilled in the art will recognize that the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. While specific embodiments have been provided, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein.

Claims

1. The use of substances that reduce CCR2 content or activity in the preparation of products for the treatment or prevention of fever with thrombocytopenia syndrome; the products are pharmaceuticals; The substance that reduces CCR2 content or activity is a CCR2 antibody or a CCR2 inhibitor; The CCR2 inhibitor is RS102895 hydrochloride or CCR2 antagonist 1, wherein the CAS No. of CCR2 antagonist 1 is 1683534-96-4.

2. The use of substances that reduce CCR2 gene expression or knock out the CCR2 gene in the preparation of products for the treatment or prevention of fever with thrombocytopenia syndrome; the products are drugs; The substance that reduces the expression level of the CCR2 gene is a siRNA that specifically recognizes the CCR2 gene. The siRNA is the siRNA formed by SEQ ID No. 1 and its reverse complementary sequence in the sequence listing, or the siRNA formed by SEQ ID No. 2 and its reverse complementary sequence. The substance used to knock out the CCR2 gene is a substance used to knock out the CCR2 gene using the CRISPR-Cas9 system. The sgRNA target of the CRISPR-Cas9 system for knocking out the CCR2 gene is the DNA fragment shown in SEQ ID No.

9.

3. The use of the substance that reduces CCR2 content or activity as described in claim 1 in the preparation of a product for inhibiting viral infection or proliferation of fever with thrombocytopenia syndrome; the product is a drug.

4. The use of the substance that reduces CCR2 gene expression or knocks out the CCR2 gene as described in claim 2 in the preparation of a product that inhibits viral infection or proliferation of fever with thrombocytopenia syndrome; the product is a drug.

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