Application of CXCL11 as antiviral drug

Abstract

The invention discloses an application of CXCL11 as an antiviral drug, relates to the field of biological medicines, and in particular relates to a protein of an antiviral drug, and the protein as the antiviral drug can inhibit infection and replication of RSV (Respiratory Syndrome Virus). The invention also provides a technical enlightenment for taking a related host protein in a virus infection pathway as an antiviral drug research target, and an application related indication of the CXCL11 protein analogue to clinical symptoms and / or clinical indications caused by severe respiratory syncytial virus infection. The invention further provides an administration route of the CXCL11 serving as an antiviral drug, a dosage range of drug intervention and the like, and finally, the feasibility of the CXCL11 serving as the antiviral drug is further verified through drug intervention application after drug challenge in CXCL11 <- / -> mice.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and in particular to a protein of an antiviral drug. The protein can be used as an antiviral drug to inhibit the infection and replication of RSV. Background Art

[0002] CXCL11 (chemokine ligand 11), also known as interferon-induced T cell α chemokine (I-TAC) or interferon-γ-induced protein 9 (IP-9), is a protein belonging to the ELR-negative CXC chemokine family. It is expressed by a variety of immune cells under interferon (IFN) induction stimulation, where ELR-negative means that the three amino acid residues of glutamic acid, leucine and arginine are negative. CXCL11 can regulate the migration, differentiation and proliferation of immune cells by binding to receptors on cells such as CXCR3, CCR3 and CCR5. The naming of CXC and CC in these chemokines or receptors is based on the sequence structure of cysteine ​​residues in chemokine molecules. Therefore, the interaction between IFN-γ and CXCL11 plays an important immune function in the human body, especially in the chemotaxis and maturation of Th1 in Th (T-helper cells) and the regulation of the balance between Th1 / Th2. Respiratory syncytial virus (RSV) is the most common viral pathogen of acute lower respiratory tract infections (ALRTIs) in children worldwide and the leading cause of hospitalization for viral respiratory tract infections in infants and young children, especially for premature infants, children with congenital heart disease or immunodeficiency. Data show that the incidence of ALRTIs caused by RSV infection is about 3.1%, accounting for 18.7% of ALRTIs in children, and RSV is the most important pathogenic factor for hospitalized ALRTIs in Chinese children from 2014 to 2021 (Clinical characteristics and molecular epidemiology of humanmetapneumovirus in children with acute lower respiratory tract infections in China, 2017 to 2019: A multicentre prospective observational study, Zhao H et al., VirolSin, Vol. 37, No. 6, published on August 25, 2022). In recent years, considerable progress has been made in the development of RSV vaccines and preventive monoclonal antibodies. However, there is currently no highly effective and specific antiviral drug for the treatment of RSV infection. Therefore, the clinical treatment of RSV infection, especially severe RSV infection, mainly relies on supportive treatment.Studies have found that CXCL11 is significantly elevated in children with RSV infection and is associated with the severity of RSV disease. In addition, during RSV infection, the balance between Th1 / Th2 cells has a crucial impact on the severity and prognosis of the disease.

[0003] Antiviral drugs mostly target viral proteins. Due to the high mutation rate of the viral genome, the emergence of drug-resistant strains has become the biggest challenge facing existing antiviral drugs. In contrast, the mutation rate of human genes is low. Selecting host proteins related to viral adsorption into cells, nucleic acid replication, protein synthesis, and viral particle release and diffusion as potential drug targets provides a new idea for the development of antiviral drugs and is more conducive to avoiding the emergence of drug-resistant strains. Based on the background knowledge of CXCL11 and RSV infection, it can be inferred that moderate CXCL11 expression can regulate immune responses, help clear RSV, and reduce RSV damage to host cells. However, no research has yet clarified how to use CXCL11 as an antiviral drug specifically against respiratory syncytial virus infection, especially in children. Summary of the invention

[0004] The purpose of the present invention is to provide an application of CXCL11 as an antiviral drug, in particular to provide a host protein related to a viral infection pathway, which has an antiviral effect and can be used as an antiviral drug to inhibit RSV infection and replication.

[0005] To achieve the above object, the present invention provides the following technical solutions: In a first aspect, the present invention provides an antiviral drug protein for inhibiting respiratory syncytial virus infection, wherein the protein is a CXCL11 protein of the CXC chemokine family or an analog of the CXCL11 protein.

[0006] According to a preferred embodiment of the present invention, the protein of the antiviral drug has a sequence shown in any one of SEQ ID No. 1 and / or SEQ ID No. 2.

[0007] According to a preferred embodiment of the present invention, the protein of the antiviral drug is natural CXCL11 protein or recombinant CXCL11 protein, and further comprises a pharmaceutically acceptable stabilizer, carrier or additive.

[0008] According to a preferred embodiment of the present invention, the antiviral drug protein is configured as a dry powder inhaler, spray, aerosol or nasal drops for nasal administration.

[0009] According to a preferred embodiment of the present invention, the protein of the antiviral drug is used to suppress diseases and / or symptoms caused by severe respiratory syncytial virus infection in children.

[0010] In a second aspect, the present invention provides a use of an antiviral protein. The above-mentioned antiviral drug protein, as an antiviral drug, has corresponding indications including acute upper respiratory tract infection, acute lower respiratory tract infection, bronchiolitis, pneumonia, and tracheobronchitis caused by respiratory syncytial virus infection; or as an antiviral drug, has corresponding functional indications for clinical symptoms including nasal congestion, runny nose, cough, asthma, sneezing, rales, fever, loss of appetite, cyanosis, shortness of breath, difficulty breathing, and suffocation caused by respiratory syncytial virus infection.

[0011] According to a preferred embodiment of the present invention, the use of the antiviral protein is to inhibit respiratory syncytial virus infection caused by any one or more of the following clinical indications: poor general condition, accompanied by impaired consciousness, accompanied by hypoxemia, accompanied by hyperthermia or persistent hyperthermia for more than 5 days, signs of dehydration or anorexia, abnormalities in chest X-ray or chest CT, lung necrosis, lung abscess, and extrapulmonary complications.

[0012] In a third aspect, the present invention provides an antiviral drug, including the protein of the above-mentioned antiviral drug.

[0013] In a fourth aspect, the present invention provides a method for preparing an antiviral drug, which is prepared by directly extracting the protein of the antiviral drug from a living organism and adding pharmaceutically acceptable auxiliary ingredients; or obtaining the antiviral drug through cell culture, fermentation, and genetic engineering.

[0014] In a fifth aspect, the present invention provides an antiviral drug protein, the use of the antiviral protein, an antiviral drug, a method for preparing an antiviral drug, and its use in the preparation of drugs for inhibiting respiratory syncytial virus infection.

[0015] Beneficial effects of the present invention: The present invention develops a technical inspiration of using a host protein related to a viral infection pathway as a potential drug target, and on this basis provides a protein of an antiviral drug, which belongs to the chemokine family and is a CXCL11 protein or an analog of the CXCL11 protein. The RSV titer, RSV structural protein, RSV nucleic acid, cytokine or pathological section analysis of CXCL11-KO A549 cells and WT A549 cells, CXCL11- / - mice and WT mice after RSV infection respectively confirm that the deletion of the CXCL11 gene promotes RSV infection and replication and the inflammatory response induced by the infection, and the intervention of CXCL11 protein can effectively inhibit the weight loss of experimental animals, RSV replication in lung tissue, inflammatory infection in lung tissue, etc. Specifically, the CXCL11 protein is administered by intranasal inoculation at a dose of 0.05-0.3 mg rCXCL11 / kg body weight, which can significantly inhibit RSV infection and replication, and effectively resist RSV damage to lung tissue. The specific experiment also verifies that the CXCL11 protein exhibits multiple beneficial effects as an effective antiviral drug component.

[0016] Since CXCL11 protein belongs to the CXC chemokine family, it may also have the potential to enhance immune response. Therefore, these effects of CXCL11 protein are not only helpful in treating infected viruses, but may also be used to prevent viral infections other than RSV. These potential functions need to be further studied, as well as the mechanism of action of CXCL11 protein as an antiviral drug, the optimization of drug dosage, and the improvement of safety and effectiveness, so as to provide more scientific basis and effective means for the development of drugs for viral infections using CXCL11 protein. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 Figure 1 shows the experimental results of cell CXCL11 gene deficiency and RSV infection and replication differences, focusing on the comparison of the detection results of CXCL11-KO A549 cells and WT A549 cells infected with RSV A2 strain 24 hours and 48 hours later, respectively. A shows the qRT-PCR detection results of the relative expression of RSV N gene mRNA in the two cells; B shows the TCID of RSV titer 50 Detection results; C shows the Western blot test results of whether there is a specific binding reaction between the two cells and the corresponding antibodies of RSV N protein, RSV F protein and GAPDH. CXCL11-KO A549 cells are equivalent to CXCL11 "-" in the figure, and WTA549 cells (equivalent to CXCL11 "+" in the figure); the horizontal axis unit is hpi; Figure 1 The vertical axis in center A is the relative expression of RSV NmRNA; Figure 1The vertical axis of B is RSV titer, the unit is Log 10 TCID 50 / mL; Figure 1 The vertical axes in center C are the antibodies corresponding to RSV N protein, RSV F protein, and GAPDH added in Western blot experiments; Figure 2 This is the experimental result diagram of the difference between mouse CXCL11 deficiency and RSV infection, focusing on the comparison of CXCL11 - / - The test results of WT mice and WT mice within 7 days after being infected with RSV A2 strain, respectively. A shows a scatter plot of the daily change trend of the weight of mice within 7 days after the two types of mice were infected with RSV; B shows a bar graph of the RSV titer test results of the lung tissues of the two types of mice on the 4th and 7th days after RSV infection; C shows a photo of the pathological section analysis of the lung tissues of the two types of mice on the 4th and 7th days after RSV infection, with a scale of 500 µm; D and E show the volcano plots of the fold increase and P value of the cytokine test results of the lung tissues of the two types of mice on the 4th and 7th days after RSV infection; in D and E, the red dots indicate the expression of CXCL11 in the lung tissues of the two types of mice. - / - The fold increase of cytokine detection results in lung tissues of mice and WT mice was significantly increased. The blue dots indicate the expression of CXCL11 - / - The fold increase of cytokine detection results in lung tissues of mice and WT mice was significantly lower. The black dots indicate the expression of CXCL11 - / - There was no significant difference in the fold value changes of cytokine detection results in lung tissues of mice and WT mice; Figure 3 The present invention is used as an antiviral drug in CXCL11 - / - The intervention effect diagram of mice treated with nasal drops focuses on the difference in the direct intervention effect between the drug intervention group and the non-intervention control group; A is a line graph of the daily change trend of the mouse weight in the drug intervention group and the non-intervention control group within 4 days after RSV infection; B is a line graph of CXCL11 - / - A bar graph showing the RSV titer test results in lung tissue of mice on day 4 after RSV infection; C is CXCL11 - / - Photograph of pathological section analysis of lung tissue of mice on day 4 after RSV infection. Scale bar: 500 µm. DETAILED DESCRIPTION

[0018] Exemplary embodiments of the present invention are described in detail below in conjunction with the accompanying drawings. Unless otherwise specified, the same or similar terms represent the same concept, such as RSV=respiratory syncytial virus, wild-type (Wild-type, not genetically modified)=WT, N gene / protein=nucleocapsid gene / protein, F protein=fusion protein, hpi=hours postinfection, MOI=multiplicity of infection, antibody=Ab, etc.

[0019] In the detailed description below, for ease of explanation, many specific details are set forth to provide a comprehensive understanding of the embodiments of the present disclosure. In the detailed description below, for ease of explanation, many specific details are set forth to provide a comprehensive understanding of the embodiments of the present disclosure. The following examples are intended only to illustrate the present invention and should not be construed as limiting the scope of the present invention.

[0020] The experimental methods used in the following examples are conventional methods unless otherwise specified, and the materials and reagents used are all commercially available unless otherwise specified. - / -mice) were purchased from Beijing Weishanglide Biotechnology Co., Ltd.; original A549 cells without genetic modification or mutation (referred to as WT A549 cells), HEp-2 cells, and RSV A2 strain virus were all purchased from ATCC; A549 cells with CXCL11 gene knocked out (KO) (referred to as CXCL11-KO A549 cells) were purchased from Beijing Anbiqi Biotechnology Co., Ltd.; RNA extraction kit (full name: RNeasy Mini Kit, catalog number: 74104) was purchased from Qiagen; ultra-micro-spectrophotometer (NanoDrop series, NanoDrop Life Spectrophotometer), 7500 real-time fluorescence PCR instrument (full name: Applied Biosystems 7500 Real-Time PCR System), BCA protein detection kit (full name: Pierce™ BCA protein detection kit, catalog number 23225) were all purchased from Thermo Fisher Scientific; mouse 13 inflammatory cytokine joint detection V-type test plate (full name: LEGENDplexTM Mouse Anti-Virus Response Panel (13-plex) with V-Bottom Plate, Catalog No.: 740741) was purchased from BioLegend; the first-strand cDNA synthesis RT-PCR kit (full name: SuperScript™ III First-Strand Synthesis System, Catalog No.: 18080051) was purchased from Invitrogen; the TB Green fluorescent quantitative PCR kit (full name: TB Green® Premix Ex Taq™ kit, catalog number: RR430A) was purchased from Takara; immunostaining blocking solution (full name: QuickBlock™ immunostaining blocking solution, catalog number: P0260), anti-fluorescence quenching reagent (catalog number: P0131), protease inhibitor (catalog number P1010), phosphatase inhibitor (catalog number P1087), 6×SDS loading buffer (catalog number P0289), goat anti-rabbit IgG antibody (catalog number: A0208), goat anti-mouse IgG antibody (catalog number: A0216) were all purchased from Beyotime; anti-RSV primary antibody (full name: anti-respiratory syncytial virus primary antibody, catalog number: ab43812), mouse anti-RSV N protein antibody (N is nucleocapsid protein, catalog number: ab94806), mouse anti-RSV F protein antibody (F is a fusion protein, catalog number: ab43812) was purchased from Abcam; fluorescent secondary antibody (catalog number: 4408S) and rabbit anti-GAPDH protein antibody (catalog number: 5174S) were purchased from CST;ECL chemiluminescent solution (Cat. No.: D8220) was purchased from Solebao; chemiluminescent imager (model: MiniChemi610) was purchased from Beijing Saizhi Venture Technology Co., Ltd.; rCXCL11 is recombinant CXCL11 protein (Cat. No. HY-P700169AF) was purchased from MCE. However, it is obvious that one or more embodiments can be implemented without these specific details. If the specific conditions are not specified in the embodiments, they are carried out according to conventional conditions or conditions recommended by the manufacturer. ;

[0021] The molecular biology experimental methods not specifically described in the following embodiments are all carried out with reference to the specific methods listed in the book "Molecular Cloning Experiment Guide" (3rd edition) by J. Sambrook, or in accordance with the instructions for reagents and products. The present invention seeks to protect the CXCL11 protein as an antiviral drug, and its use or application in antiviral aspects, and does not include the process of blocking, alleviating or eliminating the cause or lesion in order to restore or gain health or reduce pain in living human or animal bodies; the purpose of studying the CXCL11 protein is to develop an antiviral drug, not for the purpose of treatment; the use or application of the CXCL11 protein in antiviral aspects is specifically reflected in the process of preparing and producing antiviral drugs, such as the [Functions and Indications] that need to be clearly recorded in the drug instructions. This method does not include a specific treatment process, so it does not have a therapeutic nature in essence. Therefore, the present invention does not belong to the diagnosis and treatment of diseases, and meets the basic requirements of the Patent Law for patent protection objects. The specific embodiments of the present invention are described in detail below, and the specific contents are as follows: Example 1, RSV infection cell test 1. Preparation of RSV suspension Since HEp-2 cells are the preferred permissive cells for RSV, HEp-2 cells were selected for RSV virus amplification. 5The cells were inoculated into a cell culture flask at a density of 10 cells / mL, and complete medium (10% FBS 1% PS low-glucose DMEM medium) was added, and the cells were placed in a 5% CO2, 37°C cell culture incubator for 16 hours; the complete medium was replaced with a virus amplification medium (2% FBS 1% PS Opti-MEM medium), and RSV A2 virus liquid was added at a ratio of MOI = 0.1 to infect HEp-2 cells, and the cells were continued to be placed in a 5% CO2, 37°C cell culture incubator for culturing. The pathological changes were observed daily until about 80% of the cells were cytopathic (about 72-96hpi), the cell culture flask was quickly placed at -80°C for freezing, and then quickly placed in a 37°C water bath for a round of freezing and thawing to fully release the virus, and the virus liquid after freezing and thawing was collected and centrifuged at 4°C, 2000 rpm for 10 minutes. The supernatant after centrifugation was aliquoted as RSV A2 virus suspension, which was quickly frozen in liquid nitrogen and stored in a -80°C refrigerator for use.

[0022] 2. RSV infection of cells Since A549 cells originate from the lower respiratory tract and are human alveolar basal epithelial cells of lung cancer, they contain highly unsaturated fatty acid lecithin, have a squamous morphology, and retain biological characteristics similar to the lower respiratory tract, A549 cells were selected as a cell model to simulate the infection and replication of RSV infected lower respiratory tract cells. WT A549 cells and CXCL11-KO A549 cells were inoculated in 12-well plates, added with complete medium (10% FBS 1% PSDMEM / F12 medium), and placed in a 5% CO2, 37°C cell culture incubator for 16 hours; the complete medium was replaced with virus maintenance medium (2% FBS 1% PS DMEM / F12 medium), and the RSV A2 strain virus suspension prepared above was added at a ratio of MOI=0.5 to infect the two cells respectively, and continued to be placed in a 5% CO2, 37°C cell culture incubator for culture. After culturing for 24 hours and 48 hours, the virus infection fluids were obtained. Total RNA and protein in the virus infection fluids corresponding to the two types of cells were extracted for detection, or the virus infection fluids were directly used to detect the virus titer.

[0023] 3. RSV RNA detection Extraction of total RNA from cells: Collect virus infection fluids of the above two types of cells at corresponding times (24h, 48h) after RSV infection, and use RNA extraction kit to extract total RNA according to the product instructions. Subsequently, use ultra-micro spectrophotometer to measure and calculate the concentration and purity of the extracted total RNA by A260 / A280 ratio. Dispense the extracted total RNA into RNase-free centrifuge tubes and immediately store at -80°C for use.

[0024] qRT-PCR: including reverse transcription PCR (RT-PCR) and fluorescent real-time quantitative PCR (Quantitative Real-Time PCR, qPCR). Use the first-strand cDNA synthesis RT-PCR kit according to the product instructions to complete RT-PCR to obtain cDNA; use the TB Green fluorescent quantitative PCR kit according to the product instructions, and use a 7500 real-time fluorescent PCR instrument to complete qPCR. According to the conservative sequence of the RSV N gene in GenBank, the primers RSV NF and RSV NR of the specific N gene were designed using Primer premier 5 primer design software, and the sequences shown in SEQ ID No.3-4 were verified and obtained through experiments. In order to improve the accuracy of analyzing the relative expression level of RNA of the N gene, GAPDH was used as an internal reference gene for standardization. The primers GAPDH-F and GAPDH-R of GAPDH are shown in SEQ ID No.5-6. The present invention compares the knockout of human and mouse CXCL11 protein sequences and the rCXCL11 intervention experiment to verify the necessity of the CXCL11 sequence for achieving antiviral function. The CXCL11 sequence and the above primer sequences are summarized in Table 1.

[0025] Table 1 Sequence Listing

[0026] qRT-PCR result analysis: During the entire amplification process, fluorescence signal acquisition, data storage and subsequent analysis are all automatically completed by the 7500 real-time fluorescence PCR instrument and its supporting software. The standardized test results are analyzed by 2 -△△CT The relative quantitative method was used to calculate and draw a bar graph of the relative expression of RSV N gene mRNA, such as Figure 1 As shown in A, the figure can intuitively reflect that whether it is 24 hours or 48 hours after infection, the expression of RSV N gene mRNA in CXCL11-KO A549 cells and WT A549 cells shows extremely significant differences (24 hours: "**" is equivalent to p<0.01, the difference is more significant; 48 hours: "***" is equivalent to p<0.001, the difference is very significant), and in CXCL11-KO A549 cells, the expression level of RSV N gene mRNA is significantly higher than that in WT A549 cells.

[0027] 4. RSV protein detection Protein detection mainly includes cell protein extraction and Western blot experiments. First, you need to pre-prepare protein lysis buffer. Prepare it according to the ratio of RIPA lysis buffer: protease inhibitor: phenylmethylsulfonyl fluoride (PMSF): phosphatase inhibitor = 100:1:1:2.

[0028] Extraction of cell protein: The virus infection solution of the above two types of cells at the corresponding time (24h, 48h) after RSV infection was washed twice with PBS to remove the culture medium and impurities. After centrifugation, an appropriate amount of the above-prepared protein lysis solution was added to the cell pellet. The usage amount was about 1×10 6 Add 100ul protein lysis buffer to each cell, vortex to mix the cell pellet and protein lysis buffer thoroughly, then place on ice for lysis for 30 min. During this period, vortex once every 10 min to make the lysis more complete. The lysis suspension was centrifuged at 12000 rpm and 4°C for 15 min, and the supernatant after centrifugation was collected. A small amount of supernatant was retained and the protein concentration of the supernatant was determined using the BCA protein detection kit according to the product instructions; 1 / 5 volume of 6×SDS loading buffer was added to the remaining supernatant, vortexed to mix evenly, and then placed in a 101°C constant temperature metal bath for 7-8 min to denature the protein. After protein denaturation, the cell protein suspension prepared from the two cells can be directly used as a sample for protein electrophoresis or stored at -20°C for later use.

[0029] Western blot experiment: Take the cell protein suspension prepared from the above two kinds of cells, balance the sample according to the protein concentration, and separate them by 10% polyacrylamide gel electrophoresis (SDS-PAGE). The electrophoresis condition is 90V constant voltage electrophoresis for about 80 minutes, until the target protein and the corresponding band of the marker can be clearly distinguished. After the electrophoresis, the constant voltage transfer is performed, and the condition is 90V transfer for 70 minutes. The protein is transferred to the nitrocellulose membrane, and then the membrane is blocked in 5% skim milk for 2 hours to reduce nonspecific binding. Incubate overnight at 4°C with 1:2000 dilution of mouse anti-RSV N protein antibody, 1:2000 dilution of mouse anti-RSV F protein antibody and 1:1000 dilution of rabbit anti-GAPDH protein antibody. After washing the membrane with PBST (PBS plus Tween-20), the secondary antibody is added with 1:3000 dilution of goat anti-rabbit IgG antibody and goat anti-mouse IgG antibody respectively and incubated at room temperature for 1 hour. Wash the membrane again with PBST. After adding highly sensitive ECL chemiluminescent solution, the image was collected using a chemiluminescent imager. The results are as follows: Figure 1 As shown in C, it is shown that CXCL11 deficiency can significantly promote the expression of RSV structural proteins (RSV N and RSV F proteins).

[0030] 5. RSV titer determination Sample pretreatment includes 4 steps: ① Freeze-thaw: Take out the 12-well plates with the corresponding time (24h, 48h) after the above RSV virus infects the two types of cells from the incubator, quickly place them at -80℃ for freezing, then take them out and place them in a 37℃ water bath for rapid thawing to complete a round of freeze-thaw, aiming to completely lyse the cells and fully release the virus particles; ② Transfer: Use a pipette to carefully transfer the cell lysate in the 12-well plate to a sterile Ep tube to avoid bubbles and contamination; ③ Centrifugation: Centrifuge the Ep tube at 2000 rpm at 4℃ for 10 minutes to remove cell debris and other impurities to obtain a relatively pure virus supernatant; ④ Collection: Use a pipette to carefully transfer the supernatant from the Ep tube to avoid aspirating the sediment at the bottom. The collected supernatant is the sample to be tested for titer detection.

[0031] Virus titer determination: TCID 50 Assay method, the cells are HEp-2 cells that are preferentially permissive to RSV. ① Prepare cells: Prepare one day in advance, evenly inoculate HEp-2 cells in a 96-well cell culture plate, place in a 5% CO2, 37°C cell culture incubator and culture until the cell growth density reaches 80-90% for virus infection. ② Virus dilution: The sample to be tested is the above-mentioned virus infection fluid, and the sample to be tested is diluted 10 times in a gradient using pure Opti-MEM medium, with a total of 8 dilution gradients, and the concentrations after dilution are recorded as 10 -1 ~10 -8; ③ Virus infection inoculation: transfer the above virus dilutions to 96-well plates respectively, inoculate 8 replicate wells at each dilution at 100 μL / well, and the negative control well is pure Opti-MEM medium at 100 μL / well; place the 96-well cell culture plate in a 5% CO2, 37°C cell culture incubator for culture; discard the virus solution after 2 hours of virus adsorption, wash 1-2 times with PBS to remove unadsorbed virus particles, replace the culture medium with virus maintenance medium (2% FBS 1% PS low-glucose DMEM culture medium), and continue to culture in a 5% CO2, 37°C cell culture incubator; ④ Fixation and staining: On the 7th day after infection, discard the culture supernatant, wash 1-2 times with PBS, discard PBS, add 4% paraformaldehyde to fix the cells at room temperature for 15 min; discard 4% paraformaldehyde, wash 1-2 times with PBS again, discard PBS, add immunostaining blocking solution and incubate at room temperature for 20 min to block nonspecific antigen sites; ⑤ Antibody incubation and fluorescence observation: discard the immunostaining blocking solution, add anti-RSV primary antibody, incubate overnight at 4°C to allow the primary antibody to fully bind to the RSV antigen; the next day, discard the primary antibody, wash with PBS 1-2 times and then discard the PBS, add fluorescent secondary antibody, incubate at room temperature in the dark for 2 hours to allow the fluorescent secondary antibody to fully bind to the primary antibody; discard the supernatant to remove unbound fluorescent secondary antibody, wash with PBS again 1-2 times and then discard the PBS; add anti-fluorescence quenching reagent and incubate at room temperature in the dark for 5 min to protect the fluorescent signal and reduce quenching; observe the fluorescence of each well under a fluorescence microscope, and record the number of positive fluorescence wells at each dilution; ⑥ Calculate the virus titer: by TCID 50 The virus titer was calculated by the formula lgTCID 50 =[(percentage of lesions above 50% - 50%) / (percentage of lesions above 50% - percentage of lesions below 50%)] × dilution factor + logarithm of dilution of lesions above 50%. RSV titer results are as follows Figure 1 As shown in B, it can be seen that the RSV virus titer in CXCL11-KO A549 cells is significantly higher than that in WT A549 cells.

[0032] The results of the related tests on A549 cells infected with RSV were summarized as follows: Figure 1 As shown in the figure, it can be seen that A549 cells in which the CXCL11 gene is knocked out can promote RSV infection and replication after being infected with RSV, especially in CXCL11-KO A549 cells, the mRNA expression level of RSV N gene is significantly increased, specifically: at 24 hours, "**" is equivalent to a p value <0.01, indicating that the RSV titer difference between the two cell types is more significant; at 48 hours, "*" is equivalent to a p value <0.05, indicating that there is still a significant difference in RSV titer between the two cell types.

[0033] Example 2: RSV infection mouse experiment 1. RSV infection in mice The experimental animals in this paper are mainly BALB / c mice, and the focus is on comparing WT mice and CXCL11 - / - The status changes of mice after being infected with RSV A2 strain included body weight, RSV titer in lung tissue, pathological sections of lung tissue, and cytokine detection in lung tissue.

[0034] 6-8 week old SPF grade WT mice and CXCL11 - / - The mice were transferred to an ABSL-2 biosafety laboratory environment for breeding, with 4-6 BALB / c mice per cage, ensuring adequate food and water supply, and were kept in this experimental environment for at least 3 days to fully adapt to the new environment. - / - The mice were randomly divided into two groups: a control group (9 mice in each group) and an infection group (18 mice in each group). All experimental mice were given an anesthetic (Avertin) at a concentration of 2.5% by intraperitoneal injection. The injection volume was calculated based on the weight of the mice and was 0.14 mL / 10 g. After the mice entered a deep anesthesia state, they were inoculated. The RSV A2 virus suspension prepared above was diluted with PBS to a virus concentration of 1×10 6 TCID 50 / mL was used as the inoculant for the infection group, with a nasal inoculation dose of 50 μL / mouse. The control group used PBS as the inoculant, with a nasal inoculation dose of 50 μL / mouse. The inoculated mice continued to be raised in an ABSL-2 environment, and the status of the BALB / c mice was monitored daily.

[0035] The weight change curve of mice in 7 consecutive days after infection is as follows Figure 2 As shown in A, in the infection group, "Wild-type" represents the WT mouse infection group, and "CXCL11 - / - ” indicates CXCL11 - / - Mouse infection group, visible: CXCL11 - / - The weight loss of mice after infection was significantly higher than that of WT mice, and CXCL11 was expressed on day 7 after infection. - / -The weight of the mice did not return to the pre-infection standard. Relatively speaking, although the weight of WT mice showed a downward trend after infection, it quickly recovered, especially on the 6th and 7th days after infection. As can be seen in the figure, "***" is equivalent to a p value <0.001, indicating that the difference in weight between the two types of mice is very significant. In addition, starting from the 6th day after infection, the weight of WT mice was significantly higher than the pre-infection standard. It can be seen that since the CXCL11 gene of WT mice was not knocked out, the extremely significant difference in weight is unlikely to be caused by random factors. This provides strong evidence that WT mice have the ability to resist the effects of RSV on mouse growth because they retain the CXCL11 gene.

[0036] Collect samples and pre-treatment of lung tissue groups: On the 4th and 7th days after infection, the two groups of mice were eye-removed and bled, and then the mice were killed by cervical dislocation. The mouse lung tissues were quickly collected and divided into 3 parts for immediate treatment as follows: the first part of the lung tissue was placed in a sterile grinding tube, and 500 μL of Opti-MEM medium pre-cooled at 4°C was added to completely cover the lung tissue blocks; then 2 sterile grinding beads were added, and the grinding tube was placed in a grinder, and the grinder parameters were set to 60 Hz. Grinding was performed twice, each time for 60 seconds, with an interval of 15 seconds between the two grindings; after the grinding was completed, the grinding tube was removed from the grinder and immediately centrifuged at 3000 rpm at 4°C for 10 minutes; the supernatant was carefully collected and spun using 0.22 The supernatant after filtration is the lung tissue homogenate, which can be used for RSV titer detection; the second part of the lung tissue is immediately fixed in a 4% paraformaldehyde solution to prepare a lung tissue fixed sample for subsequent pathological section analysis; the third part of the lung tissue is also placed in a sterile grinding tube, and the above-prepared protein lysate is added. The lung tissue lysate is prepared by grinding, centrifuging and filtering in a similar preparation method to the above-mentioned lung tissue homogenate, and is stored at -80°C for cytokine detection.

[0037] 2. RSV titer detection in lung tissue RSV titer detection: refer to the TCID for RSV titer determination above 50 The lung tissue homogenate prepared above was used as the test sample, and the concentrations after dilution were recorded as 10 -0.5 ~10 -2 , the results are as follows Figure 2 As shown in B, in the infection group, "Wild-type" is still used to represent the WT mouse infection group, and "CXCL11 - / - ” indicates CXCL11 - / -In the mouse infection group, it can be seen that: from the perspective of infection time, the RSV titer in the lung tissue of all BALB / c mice on the 7th day after infection seemed to be slightly higher than that on the 4th day after infection, but both WT mice and CXCL11 - / - There was no significant difference in the increase of RSV titer in lung tissue of mice after infection ("ns"=p>0.05 in the figure, indicating that the difference was not significant); at the same time point after infection, WT mice and CXCL11 - / - The changes in RSV titers in mouse lung tissues showed that CXCL11 was expressed on days 4 or 7 after infection. - / - The RSV titers of the WT mice and CXCL11 mice were significantly higher than those of the WT mice, and the RSV titers between the two groups showed significant differences. - / - The RSV titers of the two groups of mice were significantly different (p<0.05). - / - The difference in RSV titers between the two groups of mice was very significant (p<0.001), suggesting that the difference in RSV titers between the two types of mice on the fourth day after infection was extremely unlikely to be caused by random factors, once again providing strong evidence that WT mice have the ability to resist RSV replication in lung tissue because they retain the CXCL11 gene.

[0038] 3. Pathological section analysis of lung tissue Histopathological examination: The fixed lung tissue samples obtained by the above pretreatment were taken and prepared into lung tissue sections by paraffin embedding. The sections were stained with hematoxylin-eosin (H&E) to prepare pathological sections that can show the pathological changes of mouse lung tissue. The pathological changes of lung tissue were photographed and analyzed under a microscope. The pathological section analysis photos are as follows: Figure 2 As shown in C, in the infection group, "Wild-type" is still used to represent the WT mouse infection group, and "CXCL11 - / - ” indicates CXCL11 - / - Mouse infection group, it can be seen that compared with the WT mouse infection group, CXCL11 - / - The lung tissue of the mouse infection group showed more severe pathological changes such as alveolar interstitial thickening and subbronchial inflammatory cell infiltration.

[0039] 4. Cytokine detection in lung tissue Cytokine detection: The lung tissue lysate obtained by the above pretreatment was taken, and the protein concentration of the lung tissue lysate was first quantitatively detected by the BCA (Bicinchoninic Acid) method, and then detected by the flow cytometry microsphere method and the mouse 13 cytokine joint detection V-type test panel. The 13 cytokines tested included IFN-γ (interferon-γ), KC (keratinocyte-derived chemokine), TNF-α (tumor necrosis factor-α), MCP-1 (monocyte chemoattractant protein-1), p70 (may refer to a specific protein or cytokine, but specific confirmation is required in this context), RANTES (regulated expression and secretion of activated normal T cells), IL-1β (interleukin-1β), IP-10 (interferon-induced protein-10), GM-CSF (granulocyte-macrophage colony-stimulating factor), IL-10 (interleukin-10), IFN-β (interferon-β), IFN-α (interferon-α) and IL-6 (interleukin-6). The volcano diagram was drawn based on the quantitative detection results of the 13 cytokines combined test at different times (day 4 and day 7 after infection). Figure 2 Medium D and Figure 2 As shown in Figure E, in the volcano plot of cytokine analysis of lung tissues on days 4 and 7 after infection, the horizontal axis represents the expression of CXCL11 in the infected group. - / - The multiple value (based on Log2) of the quantitative results of a certain cytokine in the mouse infection group and the WT mouse infection group. The vertical axis represents the P value, which is used to measure the statistical significance between the detection values ​​of the 13 cytokines. Figure 2 Medium D and Figure 2 Comparison in E shows that: First, except for the TNF-α multiple increase on the 7th day after infection, the other quantitative detection values ​​of the 13 cytokines showed a significant increase in expression level (that is, the Log2 multiple increase was positive), especially IFN-γ among the cytokines, which showed an extremely significant and rapid increase in expression level on the 4th day after infection. The multiple increase based on Log2 was close to 8, which shows that the expression of IFN-γ increased by 2. 8 The P value was less than 0.0001, which further proved that the rapid increase in IFN-γ expression was statistically significant and could more truly reflect the role of cytokine IFN-γ in CXCL11. - / -There is a very significant difference in the rapid increase between the mouse infection group and the WT mouse infection group. However, on the 7th day after infection, the difference in the significance of most cytokines showed a downward trend, and it is inferred that the 4th day after infection may be the peak period of the expression of inflammatory factors in the mouse lungs. It can also be known that WT mice can resist a certain degree of RSV infection because the CXCL11 gene has not been knocked out, and the expression level of inflammatory factors is relatively low, making it easier to avoid the occurrence of severe symptoms after RSV infection, which provides positive technical inspiration for our search for antiviral drugs.

[0040] The results of the related tests on RSV infected BALB / c mice showed that CXCL11 - / - Compared with the WT mouse infection group, there were significant differences in the degree of weight loss, lung virus titer, severity of lung pathology, and expression of inflammatory cytokines in the mouse infection group.

[0041] Example 3: Effect of rCXCL11 as an antiviral drug Pre-quarantine qualified 6-8 week old SPF grade CXCL11 - / - The mice were transferred to an ABSL-2 biosafety laboratory environment for breeding, with 4-6 mice per cage, and adequate food and water supply. They were kept in this experimental environment for at least 3 days to fully adapt to the new environment. - / - The mice were randomly divided into two groups: a drug intervention group and a non-intervention control group without antiviral drug effective components, with 9 mice in each group. All experimental mice were given 2.5% anesthetic (Avertin) by intraperitoneal injection. The injection volume was calculated according to the weight of the mice, which was 0.14 mL / 10 g. After the mice entered a deep anesthesia state, they were challenged with the virus: the RSV A2 virus suspension prepared above was diluted with PBS to a virus concentration of 1×10 6 TCID 50 / mL was used as the challenge infection dose of the infection group to simulate the lower respiratory tract infection route. The challenge infection dose of 50 μL / mouse was given to the drug intervention group and the non-intervention control group by nasal drops; after the challenge infection, the intervention agent was inoculated three times at the same time as the challenge (0h), 1 day after the challenge (1d), and 2 days after the challenge (2d). The inoculation dose of the intervention agent was 20 μL / mouse each time. After each challenge and intervention treatment, the mice continued to be raised in an ABSL-2 environment, and the status of the BALB / c mice was monitored daily.

[0042] Inoculation method and intervention agent: In the early stage, BALB / c mice were used for preliminary screening of inoculation methods, including intraperitoneal injection, oral administration with drinking water, eye drops and nasal instillation (referred to as nasal drops). Comparison showed that nasal drops were relatively convenient, the dosage was accurate, and the difference between the control group and the control group after drug intervention was obvious. Therefore, nasal drops were preferred for the evaluation of specific intervention effects. Both the drug intervention group and the non-intervention control group were inoculated with intervention agents after RSV challenge. The non-intervention control group (RSV+PBS) used PBS as the intervention agent, and the drug intervention group (RSV+rCXCL11) used PBS buffer to dilute the recombinant CXCL11 protein (rCXCL11) to prepare the initial product of the antiviral drug as the intervention agent. The active ingredient of the antiviral drug was rCXCL11. The effective intervention dose obtained by the BALB / c mouse experimental screening was 0.05-0.3 mg rCXCL11 / kg body weight, preferably 0.1 mg rCXCL11 / kg body weight.

[0043] The weight change curve of mice in 4 consecutive days after virus infection is as follows Figure 3 As shown in A, it can be seen that the weight loss of mice in the drug intervention group was significantly lower than that of the non-intervention control group, especially on the 3rd and 4th days after the infection with the virus after three interventions, the weight loss of mice in the drug intervention group was significantly slowed down, and the difference between the drug intervention group and the non-intervention control group was more significant ("**" is equivalent to p<0.01). It can be seen that rCXCL11, as the effective component of the antiviral drug, can resist the weight loss caused by viral infection (especially RSV infection) to a certain extent after intervention treatment of experimental animals. Since the change in the weight of experimental animals is one of the important indicators for evaluating the efficacy of drugs, the fact that rCXCL11 can slow down the weight loss trend of mice as the effective component of the antiviral drug preliminarily indicates that rCXCL11 as the effective component of the antiviral drug may have a certain degree of inhibitory effect on viral replication and reducing the damage of the virus to animal cells, and may also prevent the further replication and spread of the virus, thereby reducing the degree of harm of the virus to the body's inflammatory response, and can be preliminarily determined as a candidate drug component with development potential.

[0044] On the 4th day after the challenge infection, the method of collecting samples and grouping lung tissues for pretreatment was referred to Example 2. The mouse lung tissues were collected and divided into 2 parts for immediate processing, and prepared into lung tissue homogenates for RSV titer detection and lung tissue fixed samples for pathological section analysis, respectively. The lung tissue fixed samples were further sliced ​​and stained to prepare lung tissue pathological sections. The RSV titer detection results corresponding to the drug intervention group and the non-intervention control group were as follows: Figure 3 As shown in B, the pathological section analysis photos are as follows Figure 3 As shown in C.

[0045] according to Figure 3The RSV titer test results of lung tissue shown in B show that compared with the non-intervention control group, the RSV titer of the lung tissue of mice in the drug intervention group was significantly decreased ("*" is equivalent to p<0.05, significant difference). It can be seen that the intervention agent prepared with rCXCL11 as the effective component of the antiviral drug can reduce the RSV titer of lung tissue, that is, it can effectively reduce the number of RSV in the lung tissue and inhibit the replication and spread of RSV in the lung tissue, which directly indicates that the effective ingredient rCXCL11 has potential antiviral activity, especially antiviral activity against RSV infection. It can be seen that the preparation of the effective ingredient rCXCL11 into an antiviral candidate drug provides strong data support for the subsequent antiviral drugs to enter clinical trials. Preliminary experiments showed that the highest intervention dose of rCXCL11 in BALB / c mice was 0.5 mg rCXCL11 / kg body weight, and no death of experimental animals occurred during the 30-day observation period when WT mice (6 mice) were given nasal drops of this intervention dose alone. It can be inferred that when the intervention dose is less than 0.5 mg rCXCL11 / kg body weight, it is at least within the safe dose range of rCXCL11 medication intervention. However, the maximum safe dose and minimum protective dose still need to be verified by more comprehensive animal experiments.

[0046] according to Figure 3 The pathological sections of lung tissues shown in C show that the pathological sections of lung tissues of mice in the non-intervention control group, which were not treated with rCXCL11 as an effective component of antiviral drugs, showed severe lung tissue infection, such as lung inflammatory cell infiltration, alveolar interstitial thickening, and subbronchial inflammatory infiltration, indicating that the mice in the non-intervention control group had significant lung inflammation and infection, and may be accompanied by abnormal lung structure and function; Relatively speaking, the pathological sections of lung tissues of mice in the drug intervention group after three interventions with rCXCL11 as an effective component of antiviral drugs were relatively normal, indicating that rCXCL11, as an effective component, can have a significant positive effect on resisting virus replication and inhibiting virus transmission against RSV, thereby indirectly avoiding or alleviating lung inflammation and damage caused by RSV infection. On the other hand, the relatively normal pathological sections in the drug intervention group further proved that using rCXCL11 as an effective component of antiviral drugs is safe, that is, it does not cause obvious damage or side effects to lung tissue. Since rCXCL11 can more effectively resist the lung inflammation caused by RSV infection, it can be speculated that rCXCL11 may also have potential immunomodulatory effects, which will require further in-depth research on the mechanism of action of rCXCL11.

[0047] In summary, by preparing rCXCL11 as an effective component of antiviral drugs into a drug intervention agent as a potential drug ingredient, the experimental data of comparing the weight loss, lung virus titer, and lung pathology severity between the drug intervention group and the non-intervention control group showed that natural CXCL11 protein or recombinant CXCL11 protein can be used as an effective component of antiviral drugs and applied to the preparation of drugs related to inhibiting respiratory syncytial virus infection. In addition, the CXCL11 gene of host cells can also be used as a modification target to promote their autonomous secretion of CXCL11 protein to resist RSV infection or other respiratory-related virus infection.

[0048] The specific embodiments of the present invention have been described in detail so that those skilled in the art will be easily understood. However, according to all the descriptions disclosed, different modifications or replacements can be made to those details, and these changes are within the scope of protection of the present invention. The full scope of the present invention is given by the attached claims and any equivalents thereof.

Claims

1. An antiviral drug protein for inhibiting respiratory syncytial virus infection, characterized in that: It is a CXCL11 protein of the CXC chemokine family or an analog of the CXCL11 protein.

2. The antiviral drug protein according to claim 1, characterized in that The protein has a sequence shown in any one of SEQ ID No.1 and / or SEQ ID No.

2.

3. The antiviral drug protein according to claim 1, characterized in that The protein is a natural CXCL11 protein or a recombinant CXCL11 protein, and further comprises a pharmaceutically acceptable stabilizer, carrier or additive.

4. The antiviral drug protein according to claim 1, characterized in that A dry powder inhaler, spray, aerosol or nasal drops formulated for nasal administration.

5. The antiviral drug protein according to claim 1, characterized in that For use in pediatric populations to suppress illness and / or symptoms caused by severe respiratory syncytial virus infection.

6. Use of an antiviral protein, characterized in that: The protein of the antiviral drug described in any one of claims 1 to 5, as an antiviral drug, has corresponding indications including acute upper respiratory tract infection, acute lower respiratory tract infection, bronchiolitis, pneumonia, and tracheobronchitis caused by respiratory syncytial virus infection; or as the clinical symptoms corresponding to the functions of the antiviral drug, it includes nasal congestion, runny nose, cough, asthma, sneezing, rales, fever, loss of appetite, cyanosis, shortness of breath, difficulty breathing, and suffocation caused by respiratory syncytial virus infection.

7. The use of the antiviral protein according to claim 6, characterized in that: It is used to inhibit respiratory syncytial virus infection caused by any one or more of the following clinical indications: poor general condition, accompanied by impaired consciousness, accompanied by hypoxemia, accompanied by very high fever or persistent high fever for more than 5 days, signs of dehydration or refusal to eat, abnormal chest X-ray or chest CT, lung necrosis, lung abscess, and extrapulmonary complications.

8. An antiviral drug, characterized in that: A protein comprising the antiviral drug according to any one of claims 1 to 5.

9. A method for preparing an antiviral drug, characterized in that: The antiviral drug of claim 1 is prepared by directly extracting the protein of the antiviral drug from an organism and adding pharmaceutically acceptable auxiliary ingredients; or the antiviral drug of claim 8 is obtained by cell culture, fermentation, and genetic engineering.

10. The antiviral protein of any one of claims 1 to 5, the use of the antiviral protein of any one of claims 6 to 7, the antiviral drug of claim 8, and the preparation method of the antiviral protein of claim 9 in the preparation of drugs for inhibiting respiratory syncytial virus infection.

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