Application of anti-inflammatory factor loaded recombinant attenuated coxsackie B3 virus in preparation of medicine for treating traumatic craniocerebral injury

Abstract

The invention belongs to the technical field of disease drugs, and particularly relates to application of a recombinant attenuated coxsackie B3 virus loaded with an anti-inflammatory factor in preparation of drugs for treating traumatic craniocerebral injury. The virus can regulate and control a peripheral immune phenotype capable of inhibiting inflammation, after severe craniocerebral injury of a mouse, a host immune system is remodeled through single intraperitoneal injection treatment, a treatment effect is achieved in improving an immune microenvironment in the acute stage of TBI, the neuroprotective effect is observed in the early stage and the late stage of TBI, and safety is achieved.

Description

Technical Field

[0001] The present invention belongs to the technical field of disease medicines, and specifically relates to the use of a recombinant attenuated Coxsackie B3 virus loaded with an anti-inflammatory factor in the preparation of a medicine for treating traumatic craniocerebral injury. Background Art

[0002] Central nervous system injury triggers a dynamic neuroinflammatory response, mediated by a neuroinflammatory cascade driven by activation of resident and infiltrating immune cells. Following primary injury, dysregulated inflammatory cascades are implicated in the maintenance of a proinflammatory microenvironment, leading to the development of secondary neurodegeneration and persistent neurological dysfunction. Due to the multifaceted nature of CNS injury, clinically effective therapies for conditions such as traumatic brain injury (TBI), spinal cord injury (SCI), and stroke have proven elusive to develop. Currently, there are no treatments that adequately address the chronic inflammatory component of secondary CNS injury. Existing studies have developed selective immunomodulatory approaches, such as by ameliorating proinflammatory M1 responses and promoting anti-inflammatory M2 active tissue remodeling, exerting neuroprotective effects, suggesting the feasibility of treating TBI through immune intervention.

[0003] The interaction between pathogen-associated immunogens and the host immune system can shape a new immune environment for the host. Different types of pathogens or the same pathogen with different variants have different immune system shaping abilities, which can have beneficial or harmful effects on the host; for example, HIV infection can cause severe damage to the host immune system, especially a reduction in CD4+T cells. This change in the immune environment makes the host more susceptible to other infections; different genotypes and mutations of hepatitis B virus can lead to different immune responses; some variants may lead to chronic infection, while others may be cleared by the host immune system; different subtypes of influenza virus can trigger different immune responses; for example, the immune responses caused by H1N1 and H3N2 subtype influenza viruses are different, which may be related to their antigenic variations.

[0004] Based on this characteristic of pathogens, existing studies use specific pathogens, such as attenuated strains and inactivated pathogens, to develop preventive or therapeutic vaccines against specific pathogens. In addition, the immune microenvironment shaped by pathogens may have specific therapeutic effects on some diseases that are not related to pathogens, such as oncolytic viruses that promote tumor clearance by regulating the tumor immune microenvironment. Adenoviruses can eliminate tumor cells by infecting tumor cells and triggering cell lysis, releasing tumor antigens, and activating the host's immune system. Adenoviruses (AAVs) can also be genetically engineered to express cytokines or other immunomodulatory molecules to enhance anti-tumor immune responses, and are widely used in gene therapy, especially the treatment of genetic diseases. These viruses and virus-related vectors can also have potential and applications in the treatment of chronic inflammatory diseases, autoimmune diseases, cardiovascular diseases and other diseases. Summary of the invention

[0005] The present invention finds that a recombinant attenuated Coxsackie B3 virus (rCVB3(mu)-TGFβ1) loaded with an anti-inflammatory factor (TGF-β1) can significantly treat traumatic brain injury with high safety.

[0006] In order to achieve the above object, the present invention can adopt the following technical solutions:

[0007] In one aspect, the present invention provides a recombinant attenuated Coxsackie B3 virus loaded with an anti-inflammatory factor, wherein the anti-inflammatory factor is TGF-β1.

[0008] Another aspect of the present invention provides a composition for treating traumatic brain injury, which comprises the recombinant attenuated Coxsackie B3 virus loaded with anti-inflammatory factors of the present invention.

[0009] Another aspect of the present invention provides a drug for treating traumatic brain injury, which comprises the recombinant attenuated Coxsackie B3 virus loaded with anti-inflammatory factors of the present invention or the composition of the present invention.

[0010] In another aspect, the present invention provides a use of CVB3 and / or the recombinant attenuated Coxsackie B3 virus loaded with anti-inflammatory factors of the present invention or the composition of the present invention in the preparation of a medicament for treating traumatic brain injury.

[0011] Preferably, the above application includes one or more combinations of the following applications:

[0012] (a) Use of the recombinant attenuated Coxsackie B3 virus loaded with anti-inflammatory factors or the composition of the present invention in the preparation of a drug for increasing the absorption rate of cerebral edema on the affected side;

[0013] (b) Use of the recombinant attenuated Coxsackie B3 virus loaded with anti-inflammatory factors or the composition of the present invention in the preparation of a drug for reducing apoptosis of affected-side brain tissue cells.

[0014] Preferably, the above application includes: application of CVB3 and / or the recombinant attenuated Coxsackie B3 virus loaded with anti-inflammatory factors in the present invention or the composition in the present invention in the preparation of a drug for improving nerve function.

[0015] Preferably, in the above application, the neural function includes memory ability and / or motor ability.

[0016] Preferably, in the above application, the dosage form of the drug includes injection, tablet, powder or patch.

[0017] The beneficial effects of the present invention include at least: the recombinant attenuated Coxsackie B3 virus loaded with anti-inflammatory factor (TGF-β1) provided by the present invention can regulate a peripheral immune phenotype that inhibits inflammation. After severe craniocerebral injury in mice, a single intraperitoneal injection treatment reshapes the host immune system and plays a therapeutic effect in improving the immune microenvironment in the acute phase of TBI. Neuroprotective effects are observed in both the early and late stages of TBI, and the recombinant attenuated Coxsackie B3 virus loaded with anti-inflammatory factor (TGF-β1) is more effective than the engineered attenuated Coxsackie B3 virus; in addition, the recombinant attenuated Coxsackie B3 virus loaded with anti-inflammatory factor (TGF-β1) has significant safety. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1A Schematic diagram for the construction of rCVB3(mu)-TGFβ1 vector;

[0019] Figure 1B This is the construction status of rCVB3(mu)-TGFβ1 vector;

[0020] Figure 1C The immunoblotting results of rCVB3(mu)-TGFβ1 protein are shown;

[0021] Figure 1D Immunofluorescence display of rCVB3(mu)-TGFβ1;

[0022] Figure 1E It is the plaque-tropic property of rCVB3(mu)-TGFβ1;

[0023] Figure 1F Schematic diagram of the safety evaluation experiment of rCVB3(mu)-TGFβ1 in normal mice;

[0024] Figure 1G Mice infected with rCVB3(mu)-TGFβ1 showed no pathological lesions;

[0025] Figure 2A The weight monitoring change curve of mice in each group (n=6 / group);

[0026] Figure 2B The representative results of H&E staining of heart and pancreatic tissue sections of mice in each group, some acinar cells in the red marked area are covered with fat;

[0027] Figure 2C Blood glucose level monitoring of mice in each group on the 3rd and 10th day (n=4 / group);

[0028] Figure 2D Physical signs of mice treated with high dose (2X105TCID50 / 100ul) of rCVB3(mu)-TGFβ1 for 280 days (n=1);

[0029] Figure 3A The images are representative results of the MRI examination of the mouse brain on the 1st and 7th days and the statistical analysis results of the edema absorption rate on the side of craniocerebral injury (n=6 / group);

[0030] Figure 3B This is the statistical analysis result of the edema absorption rate on the affected side of craniocerebral injury detected by MRI on the 1st and 7th day of each group of mice (n=6 / group);

[0031] Figure 3C Representative images of TUNEL staining of brain tissue sections of mice in each group;

[0032] Figure 3D Statistical analysis results of the percentage of TUNEL-positive cells (n=6);

[0033] Figure 4A Representative results of immunofluorescence labeling of M2 (IBA1+ARG1) microglia-specific proteins in mouse brain tissue sections;

[0034] Figure 4B Statistical analysis results of the percentage of M2-type microglia (n=3-5 / group);

[0035] Figure 5A The Morris water maze test results 50 days after TBI, representative path diagrams of mice in different groups in the spatial exploration test;

[0036] Figure 5B is the time spent in the target quadrant in the spatial exploration trial;

[0037] Figure 5C It is the number of times the target platform is passed in the space exploration test;

[0038] Figure 5D is the swimming speed between the groups. Statistical comparison was performed using one-way analysis of variance;

[0039] Figure 2C , Figure 3B , Figure 3D , Figure 4B , Figure 5B , Figure 5C and Figure 5D Data are presented as mean ± SEM. (*p<0.05, **p<0.01). DETAILED DESCRIPTION

[0040] The examples are provided to better illustrate the present invention, but the present invention is not limited to the examples. Therefore, those skilled in the art may make non-essential improvements and adjustments to the implementation scheme according to the above invention content, which still fall within the protection scope of the present invention.

[0041] The terms used herein are only used to describe specific embodiments and are not intended to limit the present disclosure. Unless the context has a significantly different meaning, expressions in the singular include expressions in the plural. As used herein, it should be understood that terms such as "include", "have", "include" are intended to indicate the presence of features, numbers, operations, components, parts, elements, materials or combinations. The terms of the present invention are disclosed in the specification, and are not intended to exclude the possibility that one or more other features, numbers, operations, components, parts, elements, materials or combinations thereof may exist or may be added. As used herein, " / " may be interpreted as "and" or "or", depending on the circumstances.

[0042] The embodiment of the present invention provides a recombinant attenuated Coxsackie B3 virus loaded with an anti-inflammatory factor, wherein the anti-inflammatory factor is TGF-β1, which may also be referred to as rCVB3(mu)-TGFβ1 hereinafter.

[0043] It should be noted that the recombinant attenuated Coxsackie B3 virus loaded with anti-inflammatory factors in the present invention may comprise the sequence shown in SEQ ID NO:1.

[0044] It should also be noted that in the treatment of tumors, a naturally occurring or genetically modified tumor lytic virus (OV) has been developed as an immunotherapy. Among them, the CVB3 virus has also been developed as a tumor fusion virus, and its damage-related molecular pattern can effectively induce the host immune regulatory response. In addition, CVB3 (mu) can also be used as a carrier for delivering immunomodulators, such as carrying cytokines, to obtain a cytokine-equipped CVB3, and further change the host immune microenvironment. The present invention clarifies the safety and immune system regulation properties of CVB3 (mu) and rCVB3 (mu)-TGFβ1 in a normal mouse animal model, wherein CVB3 (mu) upregulates TGF-β1, and rCVB3 (mu)-TGFβ1 can regulate the expression of CCR5 in mouse brain tissue. The vector does not significantly induce a pro-inflammatory response, but instead has a positive regulation on the regulation of anti-inflammatory effects. The present invention found that the CVB3 (mu) virus strain has pancreatic tissue targeting in the host, can be used as an effective immunogen to stimulate local immune responses in different tissues and reshape the host immune environment. The immune response induced by this attenuated strain seems to be non-IFN-γ enhanced. The immune regulation pattern of CVB3 (mu) in normal mice shows an anti-inflammatory feature, with high expression of TGF-β1.

[0045] It is also important to note that the neuroinflammatory response to acute central nervous system (CNS) injury is a multifactorial and dynamic process that includes both acute and chronic phases mediated by resident and infiltrating immune cells. Any effective treatment for conditions such as traumatic brain injury (TBI), spinal cord injury (SCI), and stroke must address this complex landscape. Single chemical immunomodulatory drugs often exhibit both neuroprotective and neurotoxic effects in the CNS injury microenvironment, explaining why several immunosuppressive therapeutic agents, including methylprednisolone, and erythropoietin, have failed to significantly improve outcomes in clinical trials of TBI and SCI. Inhibition of early inflammatory responses may even exacerbate injury, as demonstrated by increased edema in mice treated with methylprednisolone after SCI. Following CNS injury, the acute inflammatory process plays a crucial role in establishing a microenvironment that is favorable for neural repair and regeneration. Furthermore, acute immunosuppression fails to address the chronic inflammatory cascade that leads to the development of secondary neurodegeneration and long-term neurological dysfunction. Neurocentric treatments such as free radical scavengers and N-methyl-D-aspartate antagonists have also been studied in CNS injuries, but the results are poor, suggesting that simply trying to protect neurons from secondary brain injury may not be therapeutically feasible. Instead, a more promising approach may be to try to change secondary neuroinflammation and the damaged immune microenvironment so that it promotes neuroprotection and regeneration. Remodeling the host immune system is a complex task involving multiple methods and strategies, including the manipulation of immune cells, intervention of cytokines and chemokines, etc. The present invention uses a strong immunogen based on attenuated coxsackievirus to regulate the distribution and composition patterns of immune cells, cytokines and chemokines, and remodel the immune system environment that promotes neuroprotection and regeneration. In this study, the feasibility of treating severe TBI with a single intraperitoneal injection of CVB3 (mu) was demonstrated. By regulating the immune system, an anti-inflammatory immune microenvironment phenotype in the injured area was obtained, which reduced the progression of edema and played a neuroprotective role. On the 14th day of TBI, the proportion of M2 microglia around the site of craniocerebral injury increased. There were also significant changes in the activation and distribution of astrocytes. Cell apoptosis and autophagy around the injury site were reduced. On the 50th day, the cognitive ability of mice was improved, and the memory and motor ability of mice were improved. After observation until the 40th week, the mice still survived healthily, and it did not affect the normal life span of mice. Different time points of TBI represent different stages of inflammatory development. We focused on several different time points, including the acute, middle and late stages of TBI, so we involved the observation of acute and chronic inflammation. The results showed that a single dose of CVB3 (mu) seemed to have achieved good therapeutic effects at different stages. We did not observe chronic inflammation of TBI in the late stage of TBI.CVB3(mu)-based immunotherapy is different from current single chemical inflammation-suppressing drugs. It does not directly and nonspecifically inhibit inflammation, but attempts to change the microenvironment of secondary neuroinflammation and injury and promote neuroprotection and regeneration. Considering that the virus is cleared by the host immune system within a few days of administration, the long-term protective effect of CVB3(mu) may be a positive feedback caused by immune regulation in the early stage, which can regulate chronic immune re-reaction and thus bring about significant and lasting clinical recovery.

[0046] It should also be noted that TGF-β1, as an anti-inflammatory cytokine, has powerful immunomodulatory and neuroprotective effects. For example, regulatory TregT cells can promote neuroprotection and repair by releasing the anti-inflammatory cytokine TGF-β1. TGF-β1 cytokine has attracted much attention due to its powerful immunomodulatory ability. However, systemic administration can lead to severe toxic reactions, which limits their clinical application. In previous studies, it was found that the combined effect of CVB3(mu) and TGF-β1 exerted a neuroprotective effect in a severe TBI mouse model by intraperitoneal injection. A new targeted TGF-β signaling proposed by us can show better efficacy than CVB3(mu) for TBI. This is a different treatment method from intracranial pioneering administration (ICV) or AAV targeting neuron-specific cell TGF-β signaling. The present invention uses rCVB3 (mu) to load TGF-β1. In TBI mice, rCVB3 (mu)-TGFβ1 can play a protective role, and the effect shows better than CVB3 (mu) in the absorption rate of brain edema, apoptosis and autophagy of brain cells in the craniocerebral injury area, regulation of M2 microglia and astrocytes. In the present invention, two low-dose and high-dose viruses were used to treat severely injured TBI mice, both showing significant therapeutic effects, and positively correlated with the dose used. However, a unique fatty pancreas was observed in the pancreas of mice in the rCVB3 (mu)-TGFβ1 treatment group, although the pancreatic function of the mice was not impaired and no obvious adverse side effects were observed. No fatty pancreas was observed in normal mice with rCVB3 (mu)-TGFβ1, and the reason why rCVB3 (mu)-TGFβ1 caused fatty pancreas in TBI mice needs further study in the future. The experimental results in the present invention show that CVB3 (WT) and CVB3 (mu) viruses can reside in multiple tissues and organs of mice, but only CVB3 (WT) causes pathological damage to the pancreas and heart of mice, and CVB3 may target pancreas and heart infection. Our experimental data preliminarily show that CVB3 (mu) does not affect the physical signs and survival of mice in normal mice and TBI mice, and does not cause pathological and functional damage to the pancreas and heart of mice. In particular, when abnormal blood sugar levels are increased in TBI mice, CVB3 (mu) treatment maintains normal blood sugar levels in TBI mice (maintaining blood sugar levels may be the result of protection of damaged nerves).

[0047] An embodiment of the present invention provides a composition for treating traumatic brain injury, which comprises the recombinant attenuated Coxsackie B3 virus loaded with anti-inflammatory factors of the present invention.

[0048] It should be noted that the recombinant attenuated Coxsackie B3 virus loaded with anti-inflammatory factors in the present invention can be used in combination with other effective ingredients with the same efficacy to improve the therapeutic effect.

[0049] The embodiment of the present invention further provides a drug for treating traumatic brain injury, which comprises the recombinant attenuated Coxsackie B3 virus loaded with anti-inflammatory factors of the present invention or the composition of the present invention.

[0050] It should be noted that the recombinant attenuated Coxsackie B3 virus loaded with anti-inflammatory factors and the above-mentioned composition of the present invention can be added with excipients to prepare different medicines for treating traumatic brain injury, and the excipients are substances well known in the art.

[0051] The embodiment of the present invention also provides a use of the recombinant attenuated Coxsackie B3 virus loaded with anti-inflammatory factors or the composition of the present invention in the preparation of a drug for treating traumatic brain injury.

[0052] In some specific examples, the above application includes one or more combinations of the following applications:

[0053] (a) Use of the recombinant attenuated Coxsackie B3 virus loaded with anti-inflammatory factors or the composition of the present invention in the preparation of a drug for increasing the absorption rate of cerebral edema on the affected side;

[0054] (b) Use of the recombinant attenuated Coxsackie B3 virus loaded with anti-inflammatory factors or the composition of the present invention in the preparation of a drug for reducing apoptosis of affected-side brain tissue cells.

[0055] In some specific examples, the above applications include: use of the recombinant attenuated Coxsackie B3 virus loaded with anti-inflammatory factors in the present invention or the composition in the present invention in the preparation of a drug for improving neurological function.

[0056] In some specific examples, in the above applications, the neural function includes memory ability and / or motor ability.

[0057] In order to better understand the present invention, the content of the present invention is further explained below in conjunction with specific examples, but the content of the present invention is not limited to the following examples.

[0058] In the following examples, the rCVB3(mu)-TGFβ1 virus used in the experiment was obtained by reverse genetics using Vero cell rescue based on the re-prepared pCVB3(mu)-TGFβ1; the cell culture medium used in the cell transfection experiment was DMEM medium, the ligated plasmid vector transformation experiment was used for the engineered Escherichia coli TOP10, and the DH5α competent strain was purchased from Tiangen Biochemical Co., Ltd. (Beijing); the obtained strain was identified by plaque identification of the virus's plaque characteristics and virus titer, and was packaged and frozen in a -80°C refrigerator.

[0059] In the following examples, male Balb / c mice, 8–10 weeks old, were purchased from Beijing Weitong Lihua Biotechnology Co., Ltd. (Beijing, China). The experimental protocol of this study was ethically approved by the Animal Care and Utilization Committee of Shantou University Medical College (approval numbers: SUMC2021-133 and SUMC2022-413). The experiments were performed in accordance with the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. All mice were housed in a temperature-controlled (20 ± 2 °C), humidity-controlled (55 ± 5%), 12-h light / dark cycle enclosure with free access to food and water.

[0060] In the following example, the experimental model of traumatic brain injury was constructed as follows: 8-10 week old Balb / c male mice were used to construct a mouse animal model of traumatic brain injury (TBI), and mice were anesthetized with 1% sodium pentobarbital (35 mg / kg). A 4 mm diameter craniotomy was performed in the left parietal bone behind the coronal suture and next to the sagittal suture using a high-speed electric drill. An electric craniocerebral spinal cord injury impactor was used to cause a 2 mm deep cortical contusion on a 4 mm diameter cranial wound.

[0061] In the following example, the MRI experimental method is as follows: a 7.0T MRI scanner (Agilent Technologies Co. Ltd., CA,

[0062] MRI scans of mice were performed using a MRI scanner (Small Animal Instruments, Inc., USA). After anesthesia with isoflurane, the mice were fixed on the fixing system of the MRI scanner. MRI was performed on the 5th and 15th days after TBI. During MRI data acquisition, the body temperature and respiratory rate of the mice were observed with the help of an MRI-compatible monitoring device (Small Animal Instruments, Inc., USA). The T2-weighted (T2w) imaging parameters are as follows: TR = 3000ms, field of view = 35mm×35mm, TE = 34ms, slice thickness = 1mm, imaging matrix = 192×192, intersection gap = 0.05mm, flip angle = 90, average = 3. The experimental data were analyzed using MATLAB software.

[0063] In the following example, the Morris water maze method is as follows: The pool is divided into 4 quadrants, and the platform is placed in the center of one of the quadrants. The animal (rat or mouse) is placed in the water with its head facing the pool wall, and the placement position is randomly selected from one of the four starting positions of east, west, south, and north. Record the time (s) for the animal to find the underwater platform. In the first few training sessions, if this time exceeds 120s, guide the animal to the platform. Let the animal stay on the platform for 10s. Remove the animal and wipe it dry. If necessary, place the animal under a 150W incandescent lamp for 5min and return it to the cage. Each animal is trained 4 times a day, with an interval of 15-20min between two training sessions, and the training is continuous for 5 days. Exploration training, the day after the last acquisition training, the platform is removed and 120s of exploration training is started. The animal is placed in the water from the opposite side of the original platform quadrant. The time the animal spends in the target quadrant (the quadrant where the platform was originally placed) and the number of times it enters the quadrant are recorded as a test indicator of spatial memory. The day after the exploratory training, the 4-day counter-position training was started. The platform was placed in the opposite quadrant of the original platform, and the method was the same as the acquisition training. The training was performed 4 times a day. The time to find the platform, the swimming distance, and the swimming speed were recorded each time.

[0064] In the following example, the immunofluorescence method of paraffin tissue sections is as follows: mice are anesthetized with 1% sodium pentobarbital and perfused transcardially with saline and 4% paraformaldehyde (PFA) in 0.1 mol / L phosphate buffer. The brain is collected, fixed with paraformaldehyde, and cut into 5 μm slices. For subsequent immunofluorescence. The slices are dewaxed and hydrated. Antigen retrieval is performed by microwave retrieval (0.01 mol / lctrate buffer, 95 degrees for 30 minutes). The slices are cooled naturally to room temperature and washed three times with phosphate buffered saline (PBS). 0.1% TritonX-100 is punched at room temperature for 10 minutes. Wash and add tissue anti-fluorescence quencher A for 30 minutes. Block with 5% goat serum for 40 minutes, remove the serum and add primary antibodies. The primary antibodies used are ARG1 (Proteintech, 10016741, 1:200) and iba1 (abcam, AB283319, 1:200) at 4 degrees overnight. Incubate with secondary antibodies CY3 (beyotime, A0516, 1:500) and 488 (beyotime.A0428. 1:500) at room temperature for 3 hours. After rinsing with PBS, counterstain the cell nucleus with DAPI. Add anti-fluorescence quencher B for 5 minutes, and finally seal the slide with anti-fluorescence quencher.

[0065] In the following example, the H&E staining method is as follows: after the tissue sample is fixed, it is dehydrated, then embedded in paraffin and sliced; after slicing, it is first stained with hematoxylin dye to make the cell nucleus blue-purple, and then rinsed with tap water to differentiate the staining; then it is briefly differentiated with 1% hydrochloric acid alcohol, and then the acid reaction is fully washed away with tap water; next, it is stained with eosin dye to make the cytoplasm and extracellular matrix pink, and then it is dehydrated with 70%, 95% and 100% ethanol in turn, and finally it is transparentized with xylene and sealed with neutral gum. The prepared slices are observed under a microscope.

[0066] In the following example, the Western blot method is as follows: take out the treated tissue and add RIPA, homogenize and centrifuge to take the supernatant, add Loading buffer and PMSF after protein quantification, boil and denature, freeze in a -80 degree refrigerator for later use. In the formal experiment, configure the appropriate gel according to the size of the target protein, separate the concentrated gel at 80V, 20min, and then separate the separation gel at 110V, 60min. Transfer the membrane at 220MA, 60min, block with 5% skim milk powder, and add the corresponding primary antibody. Then add the secondary antibody and incubate at room temperature for 2 hours. Finally, use the ECL chemiluminescent gel imaging system to expose and visualize the detection.

[0067] In the following example, the method for identifying recombinant viruses by immunofluorescence experiment is as follows: Cell preparation: Vero cells were inoculated at an appropriate concentration on a single-well chamber slide and cultured overnight. The next day, the culture medium was discarded and 10 2 TCID50 / 100μl virus solution, incubate in an incubator for 1h, wash three times with 1X preheated PBS, add fresh culture medium and continue to culture for 48 hours until cytopathic effect is observed. Fixation: Use 4% paraformaldehyde to fix cells at room temperature for 5 minutes, wash cells three times with 37℃ preheated PBS after fixation, and observe whether the cell morphology remains intact under the microscope. Blocking: Add 1% BSA to block at room temperature for 1 hour. Primary antibody incubation: Dilute the primary antibody solution to an appropriate concentration, incubate the primary antibody at room temperature for 2 hours, and then wash three times with PBS washing solution after the end. Secondary antibody incubation: Add CY3-labeled anti-rabbit IgG fluorescent secondary antibody or 488-labeled anti-mouse IgG fluorescent secondary antibody diluted 1:200 and incubate at room temperature for 40 minutes, and wash three times with PBS after the incubation. During the incubation of secondary antibody, pay attention to avoid light to prevent fluorescence quenching. Labeling cell nuclei: Use Hoechst (1:1000) to label cell nuclei, incubate at room temperature for 10 minutes, and then wash three times with PBS. Sealing: Use the immunofluorescence antifade sealing solution produced by Biyuntian to seal the slides, and avoid bubbles during the sealing process. Photography: Take photos under a fluorescence microscope.

[0068] In the following example, the plaque analysis method of the recombinant virus is as follows: Take the recombinant virus rCVB3(mu)-TGFβ1 and make a 10-fold gradient dilution of the virus: take a dilution of 10 -3 , 10 -4 , 10 -5 , 10 -6 , 10 -7 , 10 -8 , respectively inoculated in 100% monolayer Vero cells; adsorbed in a 37°C, 5% CO2 incubator for 1 hour, washed 3 times with PBS; added 2 ml of 1X DMEM containing 0.9% low-melting point agarose to maintain the culture medium; added 4% paraformaldehyde diluted in PBS after 2.5 days, fixed at room temperature for 1 hour; removed the low-melting point agar with running water, added crystal violet staining solution, and fixed at room temperature for 30 minutes; washed away the residual stain with running water, and observed the morphology and diameter of the plaques.

[0069] In the following example, the method for detecting the expression of recombinant viral protein by immunoblotting is as follows: rCVB3(mu)-TGFβ1 recombinant virus is added at 10 2 TCID50 / 100ul was inoculated into a 6-well cell culture plate with 80% confluence of Vero cells. After adsorption in a 37°C, 5% CO2 incubator for 1h, the cells were washed 3 times with PBS, and 2ml of DMEM maintenance solution containing 10% FBS was added. The cells were cultured in a 37°C, 5% CO2 incubator until the cells were completely diseased. The supernatant of the culture medium was removed, RIPA was added and placed on ice to lyse the protein, and then 50μl of 2×SDS-PAGE protein loading buffer was added; the lysate was collected and placed in boiling water for 10min to fully denature; 20μl of the sample was loaded onto SDS-PAGE gel for electrophoresis to separate the protein. Transfer: immerse the PVDF membrane in methanol to activate it until it is transparent; immerse the filter paper in transfer buffer; remove the PVDF and filter paper, use a transfer instrument to transfer the membrane at a constant current of 220mA for 1h, and then place the membrane in a 10% skim milk powder shaker at room temperature for one hour to block; rinse with TBST 3 times, 5min each time, add 1:1000 diluted anti-flag mouse monoclonal antibody, and incubate at 4℃ overnight; rinse with TBST 3 times, 15min each time, add 1:1000 diluted HRP-labeled rabbit anti-mouse secondary antibody, incubate on a shaker at room temperature for 1h, then wash with TBST 3 times, 15min each time, and finally add exposure solution on the PVDF membrane for color development; take pictures with a universal chemiluminescence system.

[0070] In the following examples, the statistical analysis of experimental data was performed using GraphPadPrism8 software, and ANOVA analysis of variance and LSD multiple comparison analysis methods were used for data that conformed to normal distribution and heterogeneous variance. Brown-Forsythe and Weich analysis of variance tests were used for data that did not conform to normal distribution. Nonparametric Kruskal-Wallis rank analysis of variance and Dunn's test methods were used for data that did not conform to normal distribution. Experimental data are expressed as mean ± SEM. P < 0.05 was considered statistically significant.

[0071] 1. Construction and related tests of recombinant attenuated Coxsackie B3 virus loaded with anti-inflammatory factors (pCVB3(mu)-TGFβ1)

[0072] (I) Construction of pCVB3(mu)-TGFβ

[0073] (1) PCR to obtain TGF-β1 expression gene

[0074] The TGF-β1 monomer required for constructing the rCVB3(mu)-TGFβ1 recombinant virus is a protein of 12.5 kDa, and the expressed gene size is 336 bp. The experiment uses a high-fidelity DNA polymerase (for PCR segmented amplification; the PCR reaction system is shown in Table 1 below.

[0075] Table 1 PCR reaction system

[0076] 2X High PCR mix 25μl Forwardprimer 2μl Reverseprimer 2μl cDNA 2μl <![CDATA[ddH2O]]> 19μl Total 50μl

[0077] The PCR reaction parameters were set as follows: template pre-denaturation at 95°C for 3 min; cycle parameters: 95°C for 15 s, 58°C for 15 s, 72°C for 1 min, for a total of 35 cycles; and finally extension at 72°C for 5 min to complete the PCR reaction.

[0078] Agarose gel electrophoresis and gel recovery of amplified nucleic acid products were performed as follows: 1% agarose gel was prepared with 1X TAE electrophoresis buffer pre-added with SYBR safe nucleic acid dye, and then electrophoresed at 110V for 30min after sample loading. After the gel was completely melted, it was equilibrated to room temperature, 1 / 3 volume of isopropanol was added, and then added to the spin column, centrifuged at 12000rpm for 1min, the centrifuge was discarded, 500ul of membrane wash solution was added, and centrifuged at 12000rpm for 1min. The washing procedure was repeated once, the wash solution was discarded, centrifuged at 12000rpm for 2min, the liquid was shaken off, and it was placed at room temperature for 6min to allow the ethanol to evaporate completely, and then 30ul of TB buffer without RNA / DNase was added and eluted at 12000rpm for 2min.

[0079] (2) Enzyme digestion

[0080] The amplified DNA product and the pCV vector were digested with Cla I and Stu I endonucleases. The digestion system is shown in Table 2 below.

[0081] Table 2 Enzyme digestion system

[0082] Enzyme digestion of DNA substrate 1 μg Cla I 1μl Stu I 1μl 10X buffer 5μl <![CDATA[ddH2O]]> up to 50μl Total 50μl

[0083] The enzyme digestion system was placed in a water bath at 37°C overnight for full digestion. The digested product was again subjected to agarose gel electrophoresis, the nucleic acid in the gel was recovered to purify the digested product, and the concentration and purity of the final product were recorded by a microplate reader.

[0084] (3) Construction of full-length cDNA cloning plasmid

[0085] In the experiment, pCVB3 (mu) and the cDNA cloning vector of TGF-β1 were used to connect and fuse the protein gene sequence through the multiple cloning sites Stu I and Cla I. The ratio of the vector to the inserted gene fragment was 1:3. T4 ligase was used, and the reaction system was shown in Table 3 below.

[0086] Table 3 Reaction system for full-length cDNA cloning plasmid construction

[0087] pCV 50ng TGF-β1DNA 150ng T4 Ligase 0.5μl (one unit) 10X buffer 1μl <![CDATA[ddH2O]]> up to 10μl Total 10μl

[0088] Use E. coli TOP10 competent cells to transform the ligation product after overnight at 4°C: add 5 μl of the ligation product to 50 μl of TOP10 competent cells (competent cells are frozen and thawed on ice), incubate on ice for 30 min; heat shock in a 42°C water bath for 90 s, incubate on ice for 2 min; add 600 μl of LB medium without antibiotics, shake and culture at 37°C for 1 h; apply the bacterial solution on a plate containing AMP + The culture medium was placed on an LB agar plate containing antibiotics and inverted in a 37°C incubator for about 24 hours. After single colonies were formed, colonies were selected and positive clones were identified by colony PCR.

[0089] (4) Clone identification and plasmid extraction

[0090] Single clones were selected to form colonies, and the length of the inserted fragment in the vector was confirmed by colony PCR. The primers used in PCR were the vector upstream primer W4F and the inserted fragment TGF-β1 fragment downstream primer. The PCR product was identified by electrophoresis. The colonies with positive colony PCR were expanded and cultured in 5 ml Amp+LB liquid medium at 37°C overnight with shaking. The plasmid was extracted using the GIAGEN plasmid extraction kit.

[0091] (5) Double enzyme digestion and sequencing to identify plasmid

[0092] The plasmid was identified by restriction endonuclease digestion, and the identification system is shown in Table 4 below.

[0093] Table 4 Identification system

[0094] Plasmids 5μl(2ug) Cla I 1μl Stu I 1μl 10×NEB buffer 1μl Rehydrate to 10μl Total 10μl

[0095] The reaction was carried out in a water bath at 37°C for 2 h, and the enzyme-digested fragments were detected by 1% agarose gel electrophoresis. The plasmids with correct enzyme digestion results were sent to Guangzhou Aiji Biotechnology Co., Ltd. for sequencing, and the sequencing results were compared and analyzed.

[0096] (6) Transfection of Vero cells to rescue recombinant virus

[0097] The experiment used lipo2000 liposome transfection method to introduce pCVB3(mu)-TGFβ1 plasmid into Vero cells. The transfection steps are as follows: First, Vero cells were inoculated into 6-well cell culture plates and cultured to 90% density using DMEM complete medium containing 10% FBS fetal bovine serum. Take 10μl Lipofectamine2000 and 125μl Opti-MEM and mix them thoroughly, let them stand at room temperature for 5 minutes, and at the same time, mix 3μg plasmid with 125μl Opti-MEM; mix the two tubes of mixed solution thoroughly and let them stand at room temperature for 20 minutes; discard the cell culture medium and add 1.5ml Opti-MEM blood culture medium; add the plasmid and liposome mixture to the cells and place them in a 37℃, 5% CO2 incubator for culture. During this period, observe the cytopathic effect. When the cells show cytopathic effect, collect the cell suspension; centrifuge at 12000rpm 4℃ for 30 minutes, and collect the supernatant containing the virus.

[0098] (7) Recombinant virus amplification and propagation

[0099] The collected virus supernatant was amplified as follows: 10 μl of the supernatant of the primary virus solution was inoculated into a 25 cm plate cultured with Vero cells at 90% density. 2 The cells were placed in a cell culture flask and then adsorbed in a cell culture incubator at 37°C for 1 hour. After washing three times with preheated PBS, 5 ml of DMEM medium containing 2% FBS was added and cultured for 48 hours. During this period, the cytopathological effects were observed. When 90% of the cells were centrifuged at 12,000 rpm for 30 minutes at 4°C, the supernatant containing the virus was collected as the first-generation virus (P1) and frozen at -80°C. The TCID50 of the virus was determined according to the same method, that is, 10 viruses were added for each passage. 2 TCID50 / 100ul and frozen at -80°C. Virus samples were frozen in a -80°C freezer.

[0100] (8) Virus purification and concentration

[0101] Vero cell culture, inoculate Vero cells in 25T cell culture dishes to 100% coverage; inoculate rCVB3 (mu) -TGFβ1 in Vero cells until complete virus transformation, freeze in -80 ° C refrigerator, freeze and thaw three times repeatedly to completely release the virus in the cells, and collect about 15 ml of virus lysis supernatant in the same steps; collect the virus, transfer the virus solution to a centrifuge tube and centrifuge at 4 ° C at the highest speed for 30 minutes, collect the supernatant, and filter it once with a 0.45 μm filter to remove cell debris; ultrafiltration centrifugation to purify and concentrate the virus, add 15 ml of the virus supernatant to an ultrafiltration centrifuge tube (PALL life Sciences Macrosep Centrifugal Devices, 1000K, purple 6 / pkg OD990C36) in the sample collector (Sample Reservoir) The specific steps are as follows: 1) Insert the filter membrane paddle firmly into the bottom of the sample tube; 2) Connect the filtrate receiving tube to the bottom of the sample tube; 3) Use a pipette to draw 15 ml of the virus supernatant sample to the non-membrane side of the sample tube. Put the lid on the sample tube. Place the ultrafiltration device in a cradle rotor centrifuge; 4) Centrifuge at 3220g 4°C for 90 minutes to achieve the desired concentrate volume; 5) Remove the filtrate collection tube and discard the filtrate; 6) Concentrate the sample at least 10 times (for example, 15 ml is concentrated to 1.5 ml); 7) Exchange the buffer with saline and concentrate it 10 times again; 8) Repeat this procedure 3 to 5 times to remove 95 to 99% of the salt or buffer, and finally use TCID50 to determine the virus titer.

[0102] (9) TCID50 determination of virus titer

[0103] Cell preparation: Vero cells were inoculated in 96-well plates and cultured overnight. The virus was diluted 10 times in a row, for a total of 11 dilutions. The virus was inoculated in 96-well plate culture cells, with 8 replicates for each dilution. The cells inoculated with the virus were placed in a cell culture incubator for continued culture, and the cell lesions were observed and recorded under a microscope after 48 hours. TCID50 was calculated using the Reed-Muench method.

[0104] (II) rCVB3(mu)-TGFβ1 test

[0105] The schematic diagram of pCVB3(mu)-TGFβ1 construction is shown in Figure 1A As shown, pCVB3(mu)-TGFβ1 was constructed as Figure 1B The protein immunoblotting and immunofluorescence were shown in Figure 1C and 1D As shown, the plaque-loving properties are Figure 1E shown.

[0106] TBI mice were constructed using the above method, and pCVB3(mu)-TGFβ1 was administered to mice orally, with saline as the control, and the virus titer was 1X10 5 TCID50 / 100ul; on the 5th day after infection, no live virus was detected in the pancreas, heart, large intestine, small intestine, spleen, lung, kidney, muscle, serum and other tissues (Table 5), so it is believed that the strain will only exist in the mouse body for no more than 5 days.

[0107] Table 5 Detection of dynamic distribution of virus tissues after mice were infected with rCVB3(mu)-TGFβ1

[0108]

[0109] Note: “+” means live virus detected; “-” means no live virus detected

[0110] In addition, the schematic diagram of the safety evaluation experiment of rCVB3(mu)-TGFβ1 in normal mice is shown in Figure 1F As shown, the heart and pancreas tissue pathological sections of mice on day 14 were examined. The results of H&E tissue sections showed that rCVB3(mu)-TGFβ1 did not cause myocarditis, pancreatitis, and acinar atrophy ( Figure 1G )

[0111] 2. Experimental Design

[0112] TBI mice were constructed using the above method. 24 hours after TBI, mice were randomly assigned to the following 4 groups: sham + saline group; TBI + saline group; TBI + CVB3 (mu) group; TBI + rCVB3 (mu) -TGFβ1 group; each group had 6 mice; the virus titer was 1X10 5 TCID50 / 100ul; perform the following test.

[0113] 3. Recombinant Coxsackievirus CVB3(mu) and rCVB3(mu)-TGFβ1 have no toxic side effects and have safety characteristics in severe TBI mouse model

[0114] A controllable impactor was used to strike the right cerebral cortex tissue at a depth of 2 mm to establish a severe craniocerebral injury (TBI) model in mice. The weight change data of mice from 1 to 14 days after TBI were recorded. The results showed that the vital signs and condition of the mice were good. Compared with the sham operation control group (sham + saline group), the weight of the experimental group mice decreased after TBI, reaching the lowest point on the second day, and then began to recover. Among them, the weight gain rate of the TBI group and the treatment group was greater than that of the sham operation control group; after the 8th day, the weight gain of the mice in the TBI and rCVB3 (mu) -TGFβ1 administration groups began to be higher than that of the sham operation control group ( Figure 2A ).

[0115] In order to evaluate the safety of attenuated Coxsackievirus B3 and its vector in TBI mice, the heart and pancreatic tissue pathological sections of mice on the 15th day were detected. The results of H&E tissue sections showed that rCVB3(mu)-TGFβ1 did not cause cardiac pathological damage to normal and TBI mice, nor did it cause myocarditis, pancreatitis and acinar atrophy. Among them, a small amount of fatty pancreas was observed in the TBI mice in the rCVB3(mu)-TGFβ1 treatment group ( Figure 2B ).

[0116] In order to further clarify the effects of CVB3(mu) and rCVB3(mu)-TGFβ1 on the heart and pancreatic function of mice, the blood glucose level in the fasting serum of mice was detected (blood glucose was detected using the ARCHITECT c16000 clinical chemistry analyzer), as follows: The blood glucose levels of TBI mice were monitored on the 3rd and 10th days, respectively. The results showed that the blood glucose of TBI mice continued to rise after TBI, with a significant difference on the 10th day, while the blood glucose levels of mice in the CVB3(mu) and rCVB3(mu)-TGFβ1 treatment groups were stable and similar to those of mice in the sham operation control group. The results suggest that TBI mice have impaired blood glucose control ability due to craniocerebral injury, while mice in the CVB3(mu) and rCVB3(mu)-TGFβ1 treatment groups maintained normal blood glucose regulation function ( Figure 2C ).

[0117] In addition, the physical signs after injection of high-dose rCVB3(mu)-TGFβ1 were observed for a long time. At 280 days, the mice treated with high-dose rCVB3(mu)-TGFβ1 still maintained good health signs (see Figure 2D ).

[0118] The above results show that rCVB3(mu)-TGFβ1 has no obvious toxic side effects in normal and TBI mice, and does not affect the survival of mice, whether in the early stages of infection or over a longer period of time, showing safety characteristics.

[0119] IV. Experiment on reducing the volume of brain edema and apoptosis of neurons in the affected side of the brain by recombinant Coxsackievirus CVB3(mu) and rCVB3(mu)-TGFβ1 in a severe TBI mouse model

[0120] In order to clarify the therapeutic effect of rCVB3(mu)-TGFβ1 on severe TBI mice, the mouse brain was tested by MRI. The results showed that rCVB3(mu)-TGFβ1 could significantly promote the absorption rate of cerebral hematoma ( Figure 3A and Figure 3B ).

[0121] In addition, in order to clarify the neuroprotective effect of rCVB3(mu)-TGFβ1 on severe TBI mice, TUNEL staining was performed on the brain tissue sections of mice on the 5th day of administration. The results showed that the number of apoptotic cells around the brain injury area of ​​mice in the rCVB3(mu)-TGFβ1 treatment group was significantly reduced, which was significantly different from the sham operation control group ( Figure 3C-Figure 3D ).

[0122] The above results collectively indicate that rCVB3(mu)-TGFβ1 promotes the absorption rate of lateral cerebral edema and reduces the apoptosis of neuronal cells in the affected side of the brain in a severe TBI mouse model, and has a significant TBI therapeutic effect.

[0123] 5. Recombinant Coxsackievirus CVB3(mu) and rCVB3(mu)-TGFβ1 regulated the proportion of M2 microglia and astrocytes in a severe TBI mouse model

[0124] Microglia play an important role in the occurrence and development of TBI-induced neuroinflammation. The distribution characteristics of M1 and M2 microglia in the area surrounding traumatic brain injury in mice on the 14th day were detected. The results showed that there was no significant difference in the proportion of M1 microglia after TBI compared with the sham operation control group, while the proportion of M2 microglia in mice treated with CVB3(mu) and rCVB3(mu)-TGFβ1 increased significantly. Among them, the proportion of M2 microglia in mice treated with rCVB3(mu)-TGFβ1 was higher than that in mice treated with CVB3(mu), which was statistically significant, indicating that TGF-β1 plays an important role in inducing M2 microglia. Figure 4A and Figure 4B ).

[0125] VI. Recombinant Coxsackievirus CVB3 and rCVB3-TGFβ1 Improved Behavioral Cognition in a Severe TBI Mouse Model

[0126] On the 50th day, the behavioral changes of mice were observed, and the cognitive function was evaluated by water maze test. The results showed that compared with the sham group, TBI caused obvious neurological damage, which was manifested by a shorter stay time on the platform and almost inability to find the platform (P<0.01). Mice treated with low-dose and high-dose rCVB3(mu)-TGFβ1 maintained neurological function, and the time to find and stay on the platform was not significantly different from that of the sham group mice. The individual differences in the high-dose group were relatively large ( Figure 5B The number and speed of finding the platform were consistent with the trend of the time spent on the platform, and mice in the CVB3(mu)-TGFβ1-treated group achieved better performance ( Figure 5A , Figure 5B , Figure 5C and Figure 5D ). As expected, mice treated with low and high doses of rCVB3(mu)-TGFβ1 showed better behavioral performance in the MWM test after TBI than mice in the TBI group (P<0.05). These findings suggest that treatment with low and high doses of rCVB3(mu)-TGFβ1 mitigated neurological injury and improved neurological outcomes after TBI. The treatment groups achieved improvements in memory and motor abilities.

[0127] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solution of the present invention can be modified or replaced by equivalents without departing from the purpose and scope of the technical solution of the present invention, which should be covered by the scope of the claims of the present invention.

Claims

1. A recombinant attenuated Coxsackie B3 virus loaded with an anti-inflammatory factor, wherein the anti-inflammatory factor is TGF-β1.

2. A composition for treating traumatic brain injury, characterized in that: It comprises the recombinant attenuated Coxsackie B3 virus loaded with anti-inflammatory factors as described in claim 1.

3. A drug for treating traumatic brain injury, characterized in that: It comprises the recombinant attenuated Coxsackie B3 virus loaded with anti-inflammatory factors as described in claim 1 or the composition as described in claim 2.

4. Use of the recombinant attenuated Coxsackie B3 virus loaded with anti-inflammatory factors according to claim 1 or the composition according to claim 2 in the preparation of a medicament for treating traumatic brain injury.

5. The use according to claim 4, characterized in that: Applications include one or a combination of the following: (a) Use of the recombinant attenuated Coxsackie B3 virus loaded with anti-inflammatory factors according to claim 1 or the composition according to claim 2 in the preparation of a drug for increasing the absorption rate of cerebral edema on the affected side; (b) Use of the recombinant attenuated Coxsackie B3 virus loaded with anti-inflammatory factors as claimed in claim 1 or the composition as claimed in claim 2 in the preparation of a drug for reducing apoptosis of affected-side brain tissue cells.

6. The use according to claim 4, characterized in that: Applications include: application of the recombinant attenuated Coxsackie B3 virus loaded with anti-inflammatory factors as described in claim 1 or the composition as described in claim 2 in the preparation of a drug for improving nerve function.

7. The use according to claim 6, characterized in that: Neurological function includes memory and / or motor skills.

8. The use according to any one of claims 4 to 7, characterized in that The medicine comes in the form of an injection, tablet, powder, or patch.

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