CN116510020A - Use of substances which reduce the CCR2 content or activity for the treatment or prevention of fever with thrombocytopenia syndrome - Google Patents

Abstract

The invention discloses the use of a substance that reduces CCR2 content or activity for the treatment or prevention of fever with thrombocytopenia syndrome. The invention discovers that CCR2 is a target spot for preventing and treating the infection of the fever with thrombocytopenia syndrome virus, reveals that the CCR2 plays an important role in the infection and proliferation process of the fever with thrombocytopenia syndrome virus, can be used as the target spot for inhibiting the infection of the fever with thrombocytopenia syndrome virus, and has good research prospect when the CCR2 inhibitor is used as a candidate medicament for preventing and treating the fever with thrombocytopenia syndrome virus. The invention provides a theoretical basis for using CCR2 as a target point of fever with thrombocytopenia syndrome treatment, and has great application value for the treatment of fever with thrombocytopenia syndrome virus infection and the research and development of medicines for treating fever with thrombocytopenia syndrome virus infection.

Description

Use of substances which reduce the CCR2 content or activity for the treatment or prevention of fever with thrombocytopenia syndrome

Technical Field

The present invention relates to the biomedical field, and to the use of a substance that reduces CCR2 content or activity for the treatment or prevention of fever with thrombocytopenia syndrome.

Background

Fever with thrombocytopenia syndrome (Severe fever with thrombocytopenia syndrome, SFTS) is a new onset of toxic hemorrhagic fever with a mortality rate varying from 12% to 50%. The causative agent of this disease is Dabiebanda virus (Dabiebandavirus), also known as SFTSV (SFTS virus), belonging to the genus Bandavirus (Phenuiviridae) of the family of the Cortinariaceae. The major transmission vehicle for fever with thrombocytopenia syndrome is ticks, and there are many cases where people pass through intimate contact with patient secretions. Due to the high mortality rate, complex pathogenic mechanisms and widely distributed transmission media, the disease has attracted high attention from the international society, and SFTS is one of ten infectious diseases that is urgently studied by the world health organization. At present, clinical treatment schemes for SFTS patients mainly adopt symptomatic support treatment, and no specific antiviral treatment medicines or protective vaccines are available in batches. Therefore, the method can clearly define the target spot related to the fever with thrombocytopenia syndrome virus infection, is helpful for the research and the application of antiviral therapeutic drugs, and has great significance for preventing the outbreak of public health events.

C-C chemokine receptor 2 (C-C motif chemokine receptor, CCR 2) belongs to the G-protein coupled receptor superfamily members and is a receptor for monocyte chemotactic proteins 1-4 (MCP 1-4), MCP 1-4 being a chemoinducer of the pro-inflammatory response. CCR2 is the earliest chemocatalytic receptor for inflammatory monocytes and is expressed in T cells, dendritic cells and epithelial cells, and CCR2 mediates inflammatory cell migration and activation by engagement with ligands.

Disclosure of Invention

The invention aims to solve the technical problems of inhibiting infection or proliferation of fever with thrombocytopenia syndrome virus and treating or preventing fever with thrombocytopenia syndrome.

In order to solve the technical problems, the invention firstly provides the application of a substance for reducing the content or activity of CCR2 (Gene ID:729230, update date 2023-03-29) in preparing a product for treating or assisting in treating or preventing or assisting in preventing fever with thrombocytopenia syndrome.

In such applications, the CCR2 content or activity reducing substance may be a protein, polypeptide or small molecule compound that inhibits CCR2 protein synthesis or promotes CCR2 protein degradation or inhibits CCR2 protein function.

In particular, the CCR2 content or activity reducing substance may be a CCR2 antibody or a CCR2 inhibitor.

In one embodiment of the invention, the CCR2 antibody is anti-human CCR2 antibody (BioLegend, cat.357202).

The CCR2 inhibitor may be any substance that can bind to CCR2 protein and inhibit CCR2 protein activity, and as long as it inhibits CCR2 protein activity, it is within the scope of the present invention to include CCR2 antagonists, CCR2 inhibitors.

In the above application, the CCR2 inhibitor may be RS102895Hydrochloride (CCR 2 antagolist RS102895 Hydrochloride) or CCR2 antagonist 1 (CCR 2 antagolist 1).

The invention also provides application of a substance for reducing the expression quantity of the CCR2 Gene (Gene ID:729230, update date 2023-03-29) or a substance for knocking out the CCR2 Gene in preparing a product for treating or assisting in treating or preventing or assisting in preventing fever with thrombocytopenia syndrome.

In the above application, the substance for reducing the expression level of CCR2 gene may be an siRNA specifically recognizing CCR2 gene;

the CCR2 gene knockout substance may be a substance that knocks out CCR2 gene using a CRISPR-Cas9 system.

In one embodiment of the invention, the knockout of the CCR2 gene achieves that a partial sequence (WT sequence, SEQ ID No. 13) thereof is edited as M1 sequence (SEQ ID No. 14) and/or M2 sequence (SEQ ID No. 15).

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

In the above application, the siRNA may be an siRNA formed by SEQ ID No.1 and its reverse complement in the sequence table, or an siRNA formed by SEQ ID No.2 and its reverse complement.

The application of the substance for reducing the CCR2 content or activity in preparing a product for inhibiting or assisting in inhibiting fever with thrombocytopenia syndrome virus infection or proliferation also belongs to the protection scope of the invention.

The application of the substance for reducing the expression quantity of the CCR2 gene in preparing a product for inhibiting or assisting in inhibiting fever with thrombocytopenia syndrome virus infection or proliferation also belongs to the protection scope of the invention.

The application of the CCR2 gene knockout substance in preparing a product for inhibiting or assisting in inhibiting fever with thrombocytopenia syndrome virus infection or proliferation also belongs to the protection scope of the invention.

The application of a substance for treating or preventing the fever with thrombocytopenia syndrome or a substance for inhibiting the fever with thrombocytopenia syndrome virus infection or proliferation taking CCR2 as a target in preparing a product for treating or preventing the fever with thrombocytopenia syndrome or a product for inhibiting the fever with thrombocytopenia syndrome virus infection or proliferation also belongs to the protection scope of the invention.

The invention also provides a product, the active ingredient of the product is the substance for reducing the CCR2 content or activity, or the substance for reducing the expression level of the CCR2 gene or the substance for knocking out the CCR2 gene;

the function of the product is either b 1) or b 2) as follows:

b1 Treating or preventing fever with thrombocytopenia syndrome;

b2 Inhibiting infection or proliferation of febrile with thrombocytopenia syndrome virus.

Wherein the infection may be an infection of an animal cell, tissue or individual with a febrile with thrombocytopenia syndrome virus.

In the present invention, the product may be a drug or vaccine.

The invention discovers that CCR2 is a target spot which can be used for preventing and treating fever with thrombocytopenia syndrome virus infection. Firstly, the expression of the fever with thrombocytopenia syndrome virus gene can be obviously inhibited by knocking down the endogenous CCR2 expression of THP-1 cells through siRNA; the gene expression and replication of the fever with thrombocytopenia syndrome virus in the CCR2 knockout cell line are significantly inhibited; inhibitors and antibodies to CCR2 can inhibit infection of host cells by febrile thrombocytopenia syndrome virus; CCR2 knockout can obviously reduce the fatality rate of animals infected by the febrile thrombocytopenia syndrome virus, and CCR2 inhibitor can obviously improve the survival rate of animals infected by the febrile thrombocytopenia syndrome virus. The invention reveals that CCR2 plays an important role in the process of infection and proliferation of the fever with thrombocytopenia syndrome virus, can be used as a target point for inhibiting the infection of the fever with thrombocytopenia syndrome virus, and has good research prospect when the CCR2 inhibitor is used as a candidate drug for preventing and treating the fever with thrombocytopenia syndrome virus. The invention provides a theoretical basis for using CCR2 as a target point of fever with thrombocytopenia syndrome treatment, and has great application value for the treatment of fever with thrombocytopenia syndrome virus infection and the research and development of medicines for treating fever with thrombocytopenia syndrome virus infection.

The following detailed description of the invention is provided in connection with the accompanying drawings that are presented to illustrate the invention and not to limit the scope thereof. The examples provided below are intended as guidelines for further modifications by one of ordinary skill in the art and are not to be construed as limiting the invention in any way.

Drawings

FIG. 1 shows the inhibitory effect of knockdown of the CCR2 expression level of THP-1 cell line on infection and proliferation of febrile concomitant thrombocytopenia syndrome virus. A is qPCR for detecting virus RNA; b is western blotting to detect the expression level of CCR2 and NP. NC, CCR2 KD1, CCR2 KD2, ATF6 KD represent transfected siNC, CCR2-siRNA-1, CCR2-siRNA-2, siATF6, respectively.

FIG. 2 shows the effect of qPCR detection of viral RNA on inhibition of infection and proliferation of different strain strains of the febrile thrombocytopenia syndrome virus by CCR2-KO cell line.

FIG. 3 is the inhibitory effect of bone marrow derived macrophage BMDM on infection and proliferation of febrile concomitant thrombocytopenia syndrome virus in CCR2 knockout mice. A is the detection of CCR2 gene expression; b is viral load detection virus proliferation; CCR (CCR) ^-/- Represents CCR2Knockout mice.

FIG. 4 shows the inhibitory effect of CCR2 inhibitors on infection and proliferation of febrile concomitant thrombocytopenia syndrome virus. A is the detection result of RS102895hydrochloride treatment; b is the detection result of CCR2 antagonist 1 treatment; c is qPCR detection of RNA of RS102895 hydrochloride-treated virus; d is qPCR to detect CCR2 antagonist 1 treated viral RNA; e is a western blot of RS102895hydrochloride and CCR2 antagonist 1-treated virus in THP-1 cells; f is a Western blot of RS102895hydrochloride and CCR2 antagonist 1 treated virus in huh-7 cells. C. In D, vehicle shows the concentration of the inhibitor at 0, lanes from left to right on E and F show the results of the concentrations of RS102895hydrochloride at 0, 0.20, 0.39, 0.78, 1.56, 3.12, 6.25, 12.50, 25.00. Mu.M, respectively, and lanes from left to right on the lower graph show the results of the concentrations of CCR2 antagonist 1 at 0, 3.125, 6.25, 12.5, 25, 50, 100, 200, 400. Mu.M, respectively.

FIG. 5 shows the inhibitory effect of CCR2 antibodies on infection and proliferation of febrile concomitant thrombocytopenia syndrome virus. The viral load was detected by qPCR with probes, the antibody concentrations on the abscissa, with each concentration representing isotype control IgG treatment on the left and CCR2 antibody treatment on the right.

Figure 6 is that CCR2 knockout significantly reduced the mortality of mice from febrile concomitant thrombocytopenia syndrome virus infection. A is the weight change of mice after inoculation with the SFTSV virus, and the weight before inoculation is recorded as 100%; b is the change in survival rate.

Fig. 7 shows that treatment with CCR2 inhibitor RS102895 with febrile concomitant thrombocytopenia syndrome virus infection significantly improved survival in mice. A is a serum viral load detection result, V represents Vehicle, and RS represents RS102895; b is survival rate change.

Detailed Description

The experimental methods in the following examples, unless otherwise specified, are conventional methods, and are carried out according to techniques or conditions described in the literature in the field or according to the product specifications. Materials, reagents, instruments and the like used in the examples described below are commercially available unless otherwise specified. The quantitative tests in the following examples were each set up at least three replicates.

SEM was used for data quantification experimental standard deviations in the following examples, with significant differences between the data using t-test.

SFTSV (fever with thrombocytopenia syndrome virus, severe fever with thrombocytopenia syndrome virus) strain HBMC16 (Zhang, Y., shen, S., shi, J.et al.isolation, challenge, and phylogenic analysis of three new severe fever with thrombocytopenia syndrome bunyavirus strains derived from Hubei Province, china.Virol.sin.32,89-96 (2017) in the examples described below.

https:// doi.org/10.1007/s 12250-017-3953-3) which are available to the public from the applicant in accordance with national biosafety regulations, are used only in repeated experiments in connection with the invention and are not used for other purposes.

SFTSV strains HNXY2017-50, HNXY2017-66, WCH (Li H, zhang LK, li SF, 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/s 41422-019-0214-z) in the examples described below, which are available to the public from the applicant in accordance with the relevant regulations of national biosafety, are used only for the relevant experiments of the repeated invention, and are not used for other purposes.

CCR2 knockout mice: jackson laboratories, strain B6.129S4-Ccr2 ^tm1Ifc/J 。

The mononuclear cell line THP-1 of human peripheral blood, the human hepatoma cell line huh-7 and the African green monkey kidney cells Vero in the following examples are all products of American type culture Collection (American Type Culture Collection, ATCC).

Example 1 influence of changes in CCR2 expression levels on infection and proliferation of febrile concomitant thrombocytopenia syndrome virus.

The effect of CCR2 in host cells on infection with thrombocytopenia syndrome virus was studied by knocking down the expression level of CCR2 Gene (Gene ID:729230, update date 2023-03-29) in human peripheral blood mononuclear cell line THP-1 by CCR2 siRNA.

The method comprises the following steps: THP-1 cells, plated in 24 well plates, each1. Mu.L of siRNA (siRNA 1 or siRNA 2) and 1. Mu.L of LRNAiMAX (Invitrogen, cat.13778150) were added to the wells, 48. Mu.L of Opti-MEM (Gibco, cat.2085556), equal amounts of siNC were added to the negative control group, equal amounts of siATF6 were added to the positive control group, no siRNA was used as a blank (vehicle), the mixture was shaken and mixed, and the mixture was placed in an incubator at 37℃for culturing for 48 to 72 hours, the medium used was RPMI1640 medium (Gibco, cat.C11875500 BT), and the cell density was 10 ⁵ The siRNA concentration was 20. Mu.M per well. After the transfection, the cells were infected with SFTSV virus HBMC16, the SFTSV addition amount was MOI=5, and after incubation at 37℃for 1 hour, the medium was replaced with RPMI1640 medium containing 2% fetal bovine serum (Gibco, cat.10099-141C), and the cells were cultured in a 37℃incubator for 24 hours. The effect of viral replication was detected by real-time fluorescent quantitative PCR (dye-process qPCR) and western blot (western blot).

Wherein, the siRNA sequence is as follows:

CCR2-siRNA-1：5’-GGCTGTATCACATCGGTTATT-3’(SEQ ID No.1)；

CCR2-siRNA-2：5’-GAGGAUGGAAUAAUUUCCATT-3’(SEQ ID No.2)；

siNC：5’-UUCUCCGAACGUGUCACGUTT-3’(SEQ ID No.3)；

siATF6：5’-GCAGCAACCAAUUAUCAGUUU-3’(SEQ ID No.4)。

the primers used for the dye-process qPCR are as follows:

SFTSV-Forward：5’-CTCACTCATGCCCTCAACGA-3’(SEQ ID No.5)；

SFTSV-Reverse：5’-GATGAACTCACCAGCCCTGC-3’(SEQ ID No.6)；

GAPDH-Forward:5’-GAAGGTGAAGGTCGGAGTC-3’(SEQ ID No.7)；

GAPDH-Reverse:5’-GAAGATGGTGATGGGATTTC-3’(SEQ ID No.8)。

effect of real-time fluorescent quantitative PCR (dye-process qPCR) to detect intracellular SFTSV replication: extracting cell nucleic acid by using a cell total RNA extraction kit (Tiangen, cat.DP430-H), wherein the specific steps are carried out according to the specification; qPCR detection was performed using a real-time fluorescent quantitative PCR (qPCR) kit (TaKaRa, cat.066A), specific procedures were performed with reference to the kit instructions, and primers were as described above (SEQ ID Nos. 5-8).

Antibodies used for western blotting were as follows: CCR2 antibody (Gibco Invitrogen, cat.711255), NP rabbit serum polyclonal antibody (NP protein as antigen (either directly synthesized or obtained by cellular expression), immunized rabbit), murine anti-GAPDH monoclonal antibody (priley, cat.c1312-100).

NP protein: MSEWSRIAVEFGEQQLNLTELEDFARELAYEGLDPALIIKKLKETGGDDWVKDTKFIIVFALTRGNKIVKASGKMSNSGSKRLMALQEKYGLVERAETRLSITPVRVAQSLPTWTCAAAAALKEYLPVGPAVMNLKVENYPPEMMCMAFGSLIPTAGVSEATTKTLMEAYSLWQDAFTKTINVKMRGASKTEVYNSFRDPLHAAVNSVFFPNDVRVKWLKAKGILGPDGVPSRAAEVAAAAYRNL (SEQ ID No. 19).

As shown in FIG. 1, compared with the cells transfected with siNC and siATF6, the expression level of CCR2 is obviously reduced after CCR2-siRNA-1 and CCR2-siRNA-2 are transfected; compared with the cells transfected with siNC, the infection amount of SFTSV viruses and the expression of virus genes are obviously reduced after the transfection of CCR2-siRNA-1, CCR2-siRNA-2 and siATF6. The result shows that knocking down the endogenous CCR2 expression of THP-1 cells by siRNA can obviously inhibit the expression of SFTSV genes (namely NP proteins) and inhibit the proliferation of viruses.

Example 2 construction of CCR2 knockout cell lines by lentiviral transfection infection and proliferation assays of different strains of SFTSV in CCR2 knockout cell lines.

1. Constructing a recombinant plasmid: based on the CCR2 gene sequence, the sgRNA target sequence was designed as follows:

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

control-sgRNA target sequence: 5'-GTATTACTGATATTGGTGGG-3' (SEQ ID No. 10).

Synthesizing a target sequence, and inserting the synthesized target sequence into a vector after cutting a lentiCRISPR v.2 (Addgene, cat.52961) plasmid by using BsmBI enzyme to respectively obtain a recombinant plasmid lentiCRISPR-CCR2 for transcribing and targeting the CCR2 gene sgRNA and a control plasmid lentiCRISPR-CK.

2. Lentiviral vector construction: HEK-293T cells were plated in 10cm cell culture dishes at 37℃with 5% CO ₂ The density reached around 90% on overnight incubation until transfection the next day. Lipofectamine 2000 (Thermo Fisher, cat. 11668019) was usedThe DNA mixture was prepared from Opti-MEM (Gibco, cat.2085556), lentiCRISPR-CCR2 (or control plasmid lentiCRISPR-CK) and pMD2.G (Addgene Cat.12259), gently mixed, and left at room temperature for 20 minutes to form the DNA mixture. Then add it into a cell culture dish, mix gently, 5% CO at 37deg.C ₂ Culturing overnight. After 12-15 h, the culture medium in the dish was changed, and then 10mL of fresh serum-containing DMEM medium (Gibco, cat.11965092) was added. And (5) after 48-72 hours of transfection, collecting and filtering the supernatant containing the virus to obtain the lentivirus. Lentiviruses from lentiCRISPR-CCR2 were designated CCR2-sgRNA lentiviruses and lentiviruses from lentiCRISPR-CK were designated control lentiviruses.

3. Lentiviruses infect THP-1 cells and screen stably transformed cells: THP-1 cells were transfected with CCR2-sgRNA lentivirus or control lentivirus, and then cultured in RPMI1640 medium containing 4mg/ml puromycin for 7 days, and the monoclonal was selected for expansion culture. After extracting genomic DNA from the cells in the expanded culture, PCR amplification is performed by using a pre-designed sequencing primer, and the amplified product is sent to a sequencing company for sequencing, so as to obtain the THP-1 cells (marked as CCR2-KO cell line) with the CCR2 knockdown.

Sequencing primer: CCR2-detect-F:5'-GAGCGGTGAAGAAGTCACCA-3' (SEQ ID No. 11);

CCR2-detect-R：5’-CAGAAGCAAACACAGCCACC-3’(SEQ ID No.12)。

in the resulting cell line, part of the sequence in the CCR2 gene sequence (WT sequence, SEQ ID No. 13) was edited into the M1 sequence (SEQ ID No. 14) and the M2 sequence (SEQ ID No. 15), the resulting cell line was heterozygous.

WT sequence:

CTGGTCGTCCTCATCTTAATAAACTGCAAAAAGCTGAAGTGCTTGACTGACATTTACCTGCTCAACCTGGCCATCTCTG

ATCTGCTTTTTCTTATTACTCTCCCATTGTGGGCTCACTCTGCTGCAAATGAGTGGGTCTTTGGGAATGCAATGTG(SEQ ID No.13)；

m1 sequence: CTGGTCATCCTCATCTTTGGTCTTTGGGAATGCTGGTCTTTGGGAATGCAATGTG (SEQ ID No. 14);

m2 sequence: CTGGTCGTCCTCATCTTAATAAACTGCAAAAAGCTGAAGTGCTTGACTGACATTTACCTGCTCAACCTGGCCATCTCTG ATCTGCTTTTTCTTATTACTCTCCCATTGGGGCTCACTCTGCTGCAAATGAGTGGGTCTTTGGGAATGCAATGTG (SEQ ID No. 15).

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

The CCR2-KO cell lines were infected with four strains of SFTSV, HBMC16, HNXY2017-50, HNXY2017-66 and WCH, respectively, with a moi=0.1, a cell density of 2×10 ⁵ The culture medium used is RPMI1640 culture medium, after cells and viruses are incubated for 1h at 37 ℃, the culture medium is replaced by RPMI1640 culture medium containing 2% fetal bovine serum, and the culture is continued for 24 hours in a 37 ℃ incubator. Gene expression of SFTSV virus in cells was detected by real-time fluorescent quantitative PCR (dye-process qPCR) using THP-1 cells (WT) in which CCR2 gene was not knocked out as a control.

The method for detecting gene expression of intracellular SFTSV virus by real-time fluorescence quantitative PCR (dye method qPCR) is the same as in example 1.

The results are shown in FIG. 2, in which both the infection and proliferation process of SFTSV 4 strains in the CCR2 KO cell line were significantly inhibited compared to WT cells.

Example 3 detection of SFTSV infection and proliferation by mouse bone marrow derived macrophages BMDM of CCR2 KO.

1. Primary isolation culture of mouse bone marrow derived macrophages

Taking 6-week-old C57BL/6J mice (wild type mice, WT) and CCR2 knockout mice (CCR 2) ^-/- ) After euthanasia, the femur and tibia of the mice were removed, DMEM was aspirated with a 1mL syringe to flush out the cells in the bone marrow, filtered through a 40. Mu.M cell screen, and DMEM medium containing 10% FBS,1% diabody (Gibco, cat.15140-122) and 50ng/mL M-CSF (PeproTech, cat.315-02-10) was added at 37℃with 5% CO ₂ Culturing for 7 days to obtain the primary Bone Marrow Derived Macrophage (BMDM) with the purity of 90 percent. CCR2 expression of BMDM derived from CCR2 knockout mice was examined by flow cytometry (flow Antibody FITC anti-mouse F4/80Antibody,Biolegend,Cat.123107,PE anti-mouse CD192 (CCR 2) anti-body, biolegend, cat.150610), and as a result, as shown in FIG. 3A, CCR2 expression level of knockout mice was reduced as compared with WT mice.

2. Detection of SFTSV infection and proliferation by BMDM

The BMDM obtained by isolation culture was plated and infected with SFTSV strain HBMC16, the virus addition amount was MOI=0.1, and the cell density was 1.5X10 ⁵ The culture medium used is DMEM culture medium, after cells and viruses are incubated at 37 ℃ for 1h, the DMEM culture medium with 2% fetal bovine serum is replaced, and the cells are placed in a 37 ℃ incubator for culturing for 24h, and cell supernatants are collected. The gene expression of SFTSV virus in the cell supernatant was detected by real-time fluorescent quantitative PCR (probe method qPCR).

The primers used for the probe method qPCR are as follows:

SFTSV-S-Forward：5’-AGCCTAATTGGATATGTCAAATTGC-3’(SEQ ID No.16)；

SFTSV-S-Reverse：5’-CGGGTGAAGTGGCTGAAGG-3’(SEQ ID No.17)；

and (3) probe: 5'-6-FAM-AGCAGCAGCAGCAACCTCAGCAGC-BHQ1-3' (SEQ ID No. 18).

Detection of viral genes in cell supernatants: extracting nucleic acid from cell supernatant by using a viral RNA extraction kit (Tiangen, cat.DP315-R), wherein the specific steps are carried out according to the specification; qPCR was performed using the probe method qPCR kit (TaKaRa, cat.064A), specific steps were performed with reference to the kit instructions, and primers used were as described above (SEQ ID Nos. 16 to 18).

The results are shown in figure 3B, with significant inhibition of SFTSV infection and proliferation in BMDM of CCR2 knockout mice compared to WT mice.

Example 4 inhibition of infection and proliferation of SFTSV by CCR2 inhibitors and antibodies was tested.

1. CCR2 inhibitor treatment

In huh-7 cells, cytotoxicity was tested against two inhibitors of CCR2, RS102895Hydrochloride (CCR 2 antagonst RS102895 Hydrochloride) (MedChemExpress, cat.No.:HY-18611; CAS No.: 1173022-16-6) and CCR2 antagonist 1 (CCR 2 antagonst 1) (MedChemExpress, cat.No.:HY-112792; CAS No.:168 MedChemExpress, cat.No. -96-4). The method comprises the following specific steps:

huh-7 cells at 1X 10 ⁴ Inoculating the cells/wells into 96-well cell culture plate, placing into 37deg.C, 5% CO ₂ Adhering the culture boxes overnight, respectively1.56. Mu.M, 3.125. Mu.M, 6.25. Mu.M, 12.5. Mu.M, 25. Mu.M, 50. Mu.M, 100. Mu.M, 200. Mu.M, 400. Mu.M RS102895hydrochloride or CCR2 antagonist 1 were treated with 3 duplicate wells per group, and the control group was added with the same amount of Dimethyl Methylenewind (DMSO). After 24h of drug treatment, the OD was detected by staining with MTT kit (Solebao, cat. CA 1210) _450nm Cell viability was analyzed and tested for CC for RS102895hydrochloride as shown in figure 4, A, B ₅₀ CC for CCR2 antagonist 1 at 101.7. Mu.M ₅₀ 4150 μm. In subsequent experiments, both RS102895hydrochloride and CCR2 antagonist 1 were used at concentrations within safe and nontoxic ranges. IC for further inhibiting SFTSV against RS102895hydrochloride and CCR2 antagonist 1 ₅₀ The test was performed, and the results are shown in FIG. 4, A, B, IC with RS102895hydrochloride ₅₀ IC for CCR2 antagonist 1 at 3.061. Mu.M ₅₀ 37.76. Mu.M.

The mononuclear cell line THP-1 of human peripheral blood or the human liver cancer cell line huh-7 are plated, the culture medium used for THP-1 is RPMI1640 culture medium (Gibco, cat. C11875500 BT), the culture medium used for huh-7 is DMEM culture medium (Gibco, cat. 11965092), two inhibitors RS102895hydrochloride aiming at CCR2 and CCR2 antagonist 1 are added to treat THP-1 and huh-7 cells; the concentration of RS102895hydrochloride is set to 0, 0.20, 0.39, 0.78, 1.56, 3.12, 6.25, 12.50, 25.00. Mu.M, the solvent is RPMI1640 medium or DMEM medium containing 2% fetal bovine serum, and the cell density is 1×10 ⁵ A/hole; the concentration of CCR2 antagonist 1 was set to 0, 3.125, 6.25, 12.5, 25, 50, 100, 200, 400. Mu.M, the solvent was RPMI1640 medium or DMEM medium containing 2% fetal bovine serum, and the cell density was 1X 10 ⁵ A/hole; after addition of the inhibitor, incubation was carried out for 1h at 37℃and then infection with SFTSV strain HBMC16, SFTSV was added in an amount of MOI=0.1, after incubation for 1h at 37℃the DMEM medium was replaced with 2% FBS, and after further culture for 24h cells were harvested, the effect of inhibiting SFTSV replication was examined by using real-time fluorescent quantitative PCR (dye qPCR) and Western blotting of RS102895hydrochloride and CCR2 antagonist 1.

Effect of real-time fluorescent quantitative PCR (qPCR) to detect intracellular SFTSV replication: extracting cell nucleic acid by using a cell total RNA extraction kit (Tiangen, cat.DP430-H), wherein the specific steps are carried out according to the specification; qPCR was performed using a real-time fluorescent quantitative PCR (qPCR) kit (TaKaRa, cat.066A), specific procedures were performed with reference to the kit instructions, the dye-process primers used in example 1 (SEQ ID Nos. 5-8), and the Western blotting method was as described in example 1.

As shown in fig. 4C-F, the gene expression level of intracellular SFTSV was significantly reduced after treatment with two inhibitors of CCR2, RS102895hydrochloride and CCR2 antagonist 1, both inhibitors of CCR2 had a significant inhibitory effect on SFTSV replication, and this inhibitory effect was in a dose-dependent trend.

2. CCR2 antibody treatment

Mononuclear cell line THP-1 of human peripheral blood or human hepatoma cell line huh-7 were plated, RPMI1640 medium (Gibco, cat. C11875500 BT), DMEM medium (Gibco, cat. 11965092) was used as the medium for huh-7, anti-human CCR2 anti-ibody (BioLegend, cat. 357202) or isotype control IgG isotype control (BioLegend, cat. 400201) was added to the medium for CCR2, the antibody concentration was set to 0.05, 0.5, 5 μg/ml, and the cell density was 1×10 ⁵ A/hole; the antibody was added and incubated at 37℃for 1 hour, then the cells were infected with SFTSV strain HBMC16 in an amount of MOI=1 or 5, and the cell supernatants were harvested 24 hours after the virus infection to detect the viral load of SFTSV.

Detection of viral load: extracting nucleic acid from cell supernatant by using a viral RNA extraction kit (Tiangen, cat.DP315-R), wherein the specific steps are carried out according to the specification; qPCR was performed using the probe method qPCR kit (TaKaRa, cat.064A), and specific steps were performed with reference to the kit instructions, using the primer probes as in example 3 (SEQ ID Nos. 16 to 18).

As shown in fig. 5, the supernatant viral load was significantly reduced after CCR2 antibody treatment, indicating that antibodies against CCR2 also have significant inhibitory effect on SFTSV infection and proliferation, further indicating that CCR2 is an important target for SFTSV infection and proliferation.

Example 5 CCR2 knockout mouse model SFTSV infection was tested.

The method comprises the following steps: female mice (CCR 2) with CCR2 knockdown at 5 weeks of age raised in SPF environment ^-/- ) 11 and wild C57BL/6J small11 mice (WT) were intraperitoneally injected with type I interferon blocking antibody (IFNAR 1) (BioXCell, cat.BE0241), 1.7 mg/mouse, pretreated for 24 hours, and then intraperitoneally inoculated with 100. Mu.L/mouse SFTSV strain HBMC16 virus (2X 10) ⁴ FFU/mL), weight of mice was weighed daily and the survival status of mice was observed, and if the weight of mice was reduced by 25%, the mice were considered dead.

The results are shown in fig. 6, where mice lost weight after the first day of SFTSV infection, but gradually lost weight after day 6 of infection, where CCR2 knockout mice lost weight faster and survived significantly more than WT mice, indicating that CCR2 gene knockout significantly improved survival of SFTSV infected mice.

Example 6 detection of therapeutic effect of CCR2 inhibitors on SFTSV infected mice.

5 weeks old wild type C57BL/6J mice bred in SPF environment were used, and 5 control groups were set up, 13 SFTSV+Vehicle groups, and 13 SFTSV+RS102895 groups. Groups of mice were intraperitoneally injected with type I interferon blocking antibody (IFNAR 1), 1.7 mg/mouse, pre-treated for 24h; then, mice in the SFTSV+Vehicle group and the SFTSV+RS102895 group were inoculated intraperitoneally with 100. Mu.L/mouse (2X 10) of the SFTSV strain HBMC16 virus ⁴ FFU/mL), sftsv+rs102895 group mice were gavaged once daily with 15 mg/kg/day gavage RS102895hydrochloride (MedChemExpress) (dissolved in 20% sbe- β -CD), sftsv+vehicle group mice were gavaged with equal volumes of 20% sbe- β -CD per day; the control group was not subjected to any treatment. Mice were weighed daily and observed for survival, and 50 μl of tail vein blood was collected for serum viral load detection on days 3 and 5 of SFTSV virus infection, and the mice were considered dead if their body weight was reduced by 25%.

Serum viral load detection: the detection method was the same as in example 3.

As a result, as shown in fig. 7, the serum viral load was significantly reduced on days 3 and 5 of viral infection in the sftsv+rs102895 group (treatment group) mice, and the survival rate of the treatment group was significantly improved, compared to the solvent group (sftsv+vehicle).

The present invention is described in detail above. It will be apparent to those skilled in the art that the present invention can be practiced in a wide range of equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the invention and without undue experimentation. While the invention has been described with respect to specific embodiments, it will be appreciated that the invention may be further modified. In general, this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains.

Claims (10)

1. Use of a substance that reduces CCR2 content or activity for the preparation of a product for the treatment or prevention of fever with thrombocytopenia syndrome.

2. The use according to claim 1, characterized in that: the material for reducing the CCR2 content or activity is protein, polypeptide or small molecule compound for inhibiting the synthesis of CCR2 protein or promoting the degradation of CCR2 protein or inhibiting the function of CCR2 protein.

3. Use according to claim 1 or 2, characterized in that: the CCR2 content or activity reducing substance is a CCR2 antibody or a CCR2 inhibitor.

4. Use of a substance that reduces the amount of CCR2 gene expression or a substance that knocks out CCR2 gene for the preparation of a product for the treatment or prevention of fever with thrombocytopenia syndrome.

5. The use according to claim 4, characterized in that: the substance for reducing the expression quantity of the CCR2 gene is siRNA for specifically recognizing the CCR2 gene;

the material for knocking out the CCR2 gene is a material for knocking out the CCR2 gene by using a CRISPR-Cas9 system.

6. The use according to claim 5, characterized in that: the siRNA is formed by SEQ ID No.1 and a reverse complementary sequence thereof in a sequence table, or formed by SEQ ID No.2 and a reverse complementary sequence thereof.

7. Use of a CCR2 content or activity reducing substance according to any of claims 1-3 for the preparation of a product for inhibiting fever with thrombocytopenia syndrome virus infection or proliferation.

8. Use of the CCR2 gene expression level reducing agent according to any one of claims 4 to 6 or the CCR2 gene knockout agent according to claim 4 or 5 for the preparation of a product for inhibiting fever with thrombocytopenia syndrome virus infection or proliferation.

9. Use of a substance for treating or preventing fever with thrombocytopenia syndrome or a substance for inhibiting fever with thrombocytopenia syndrome virus infection or proliferation with CCR2 as a target for the preparation of a product for treating or preventing fever with thrombocytopenia syndrome or a product for inhibiting fever with thrombocytopenia syndrome virus infection or proliferation.

10. A product comprising as an active ingredient the CCR 2-reducing agent according to any one of claims 1 to 3, or the CCR2 gene expression level reducing agent according to any one of claims 4 to 6, or the CCR2 gene knockout agent according to claim 4 or 5;

the function of the product is either b 1) or b 2) as follows:

b1 Treating or preventing fever with thrombocytopenia syndrome;

b2 Inhibiting infection or proliferation of febrile with thrombocytopenia syndrome virus.