Application of a recombinant attenuated Coxsackie B3 virus loaded with anti-inflammatory factors in the preparation of drugs for treating traumatic brain injury.

Abstract

This invention belongs to the field of disease drug technology, specifically relating to the application of a recombinant attenuated Coxsackie B3 virus loaded with anti-inflammatory factors in the preparation of a drug for treating traumatic brain injury (TBI). This virus can regulate an anti-inflammatory peripheral immune phenotype. In mice with severe TBI, a single intraperitoneal injection remodeled the host immune system, demonstrating a therapeutic effect in improving the immune microenvironment during the acute phase of TBI. Neuroprotective effects were observed in both early and late TBI stages, and the virus exhibits good safety.

Description

Technical Field

[0001] This invention belongs to the field of disease drug technology, specifically relating to the application of a recombinant attenuated Coxsackie B3 virus loaded with anti-inflammatory factors in the preparation of a drug for treating traumatic brain injury. Background Technology

[0002] Central nervous system (CNS) injury triggers a dynamic neuroinflammatory response, mediated by a neuroinflammatory cascade activated by resident and infiltrating immune cells. Following primary injury, a poorly regulated inflammatory cascade is associated with maintaining a pro-inflammatory microenvironment, leading to secondary neurodegeneration and the development of 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 difficult to develop. Currently, there are no adequate treatments to address the chronic inflammatory component of secondary CNS injury. Existing research has developed selective immunomodulatory approaches, such as promoting anti-inflammatory M2-type tissue remodeling by improving pro-inflammatory M1 responses, thus exerting neuroprotective effects and suggesting the feasibility of immunomodulatory intervention for TBI.

[0003] The interaction between pathogen-associated immunogens and the host immune system can shape a new immune environment in the host. Different types of pathogens or the same pathogen with different variants have different abilities to shape the immune system, producing beneficial or harmful effects on the host; for example, HIV infection can lead to severe damage to the host's immune system, especially a reduction in CD4+ T cells. This altered 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's immune system; different subtypes of influenza virus can trigger different immune responses; for example, the immune responses induced by H1N1 and H3N2 subtypes of influenza virus differ, which may be related to their antigenic variations.

[0004] Based on these characteristics of pathogens, current research uses specific pathogens, such as attenuated strains and inactivated pathogens, to develop preventative or therapeutic vaccines against specific pathogens. Furthermore, the immune microenvironment shaped by pathogens may have specific therapeutic effects on some diseases unrelated to the pathogen itself; for example, oncolytic viruses promote tumor clearance by modulating the tumor immune microenvironment. Adenoviruses can clear tumor cells by infecting them and inducing 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, thereby enhancing anti-tumor immune responses, and are widely used in gene therapy, particularly for the treatment of hereditary diseases. These viruses and virus-related vectors also have potential applications in the treatment of chronic inflammatory diseases, autoimmune diseases, and cardiovascular diseases. Summary of the Invention

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

[0006] To achieve the above objectives, the present invention can adopt the following technical solutions:

[0007] 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, comprising a recombinant attenuated Coxsackie B3 virus loaded with an anti-inflammatory factor as described in the present invention.

[0009] Another aspect of the present invention provides a medicament for treating traumatic brain injury, comprising a recombinant attenuated Coxsackie B3 virus loaded with an anti-inflammatory factor as described in the present invention, or a composition as described in the present invention.

[0010] In another aspect, the present invention provides the 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 applications include one or more combinations of the following applications:

[0012] (a) The 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 medicament to improve the absorption rate of edema in the affected side of the brain.

[0013] (b) The 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 medicament that reduces apoptosis of cells in the affected brain tissue.

[0014] Preferably, the above applications include: the 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 improving neurological function.

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

[0016] Preferably, in the above applications, the dosage form of the drug includes injections, tablets, powders, or patches.

[0017] The beneficial effects of this invention include at least the following: the recombinant attenuated Coxsackie B3 virus loaded with the anti-inflammatory factor (TGF-β1) provided by this invention can regulate an anti-inflammatory peripheral immune phenotype. After severe traumatic brain injury in mice, a single intraperitoneal injection can remodel the host immune system and exert a therapeutic effect in improving the immune microenvironment in the acute phase of TBI. Neuroprotective effects were observed in both the early and late stages of TBI. Moreover, the recombinant attenuated Coxsackie B3 virus loaded with the anti-inflammatory factor (TGF-β1) is more effective than engineered attenuated Coxsackie B3 virus. In addition, the recombinant attenuated Coxsackie B3 virus loaded with the anti-inflammatory factor (TGF-β1) has significant safety. Attached Figure Description

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

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

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

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

[0022] Figure 1E It exhibits plaque-attracting characteristics of rCVB3(mu)-TGFβ1;

[0023] Figure 1F This is a schematic diagram of the safety assessment experiment of rCVB3(mu)-TGFβ1 in normal mice.

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

[0025] Figure 2A The curves showing the changes in body weight of mice in each group (n = 6 / group) are shown.

[0026] Figure 2B Representative H&E staining results of heart and pancreas tissue sections from each group of mice; some acinar cells in the red-marked area show fat coverage.

[0027] Figure 2C Blood glucose levels were monitored in each group of mice on day 3 and day 10 (n = 4 / group);

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

[0029] Figure 3A Representative images of the mouse brain obtained by MRI on day 1 and day 7, and statistical analysis results of the absorption rate of edema on the affected side of the brain after traumatic brain injury (n=6 / group);

[0030] Figure 3B The statistical analysis results of the absorption rate of edema on the affected side of the brain injury in mice in each group were obtained by MRI on day 1 and day 7 (n=6 / group);

[0031] Figure 3C Representative images of TUNEL-stained brain tissue sections from each group of mice;

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

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

[0034] Figure 4B Statistical analysis results of the proportion of microglia in the M2 subtype (n = 3-5 animals / group);

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

[0036] Figure 5B The time spent in the target quadrant during a space exploration experiment;

[0037] Figure 5C The number of times the target platform is passed during a space exploration experiment;

[0038] Figure 5D The swimming speeds between groups were compared. One-way ANOVA was used for statistical comparisons.

[0039] Figure 2C , Figure 3B , Figure 3D , Figure 4B , Figure 5B , Figure 5C and Figure 5D In this study, data are expressed as mean ± SEM. (*p<0.05, **p<0.01). Detailed Implementation

[0040] The embodiments described are provided to better illustrate the present invention, but are not intended to limit the scope of the invention to the embodiments described. Therefore, non-essential improvements and adjustments made to the embodiments by those skilled in the art based on the above description are still within the scope of protection of the present invention.

[0041] The terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit this disclosure. Singular expressions include plural expressions unless they have a distinct meaning in the context. As used herein, it should be understood that terms such as “comprising,” “having,” “including,” are intended to indicate the presence of features, numbers, operations, components, parts, elements, materials, or combinations thereof. The terminology of the invention is disclosed in the specification and is not intended to exclude the possibility that one or more other features, numbers, operations, components, parts, elements, materials, or combinations thereof may be present or added. As used herein, “ / ” may be interpreted as “and” or “or,” depending on the context.

[0042] This invention provides a recombinant attenuated Coxsackie B3 virus loaded with an anti-inflammatory factor, TGF-β1, which may also be referred to as rCVB3(mu)-TGFβ1.

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

[0044] It should also be noted that in tumor treatment, naturally occurring or genetically modified tumor-lysing viruses (OVs) have been developed as an immunotherapy. CVB3 virus has also been developed as an oncolytic virus, and its damage-associated molecular pattern can effectively induce host immune regulatory responses. In addition, CVB3(mu) can also be used as a vector to deliver immunomodulators, such as carrying cytokines to obtain cytokine-equipped CVB3, further altering the host immune microenvironment. This invention has clarified the safety and immune system modulatory properties of CVB3(mu) and rCVB3(mu)-TGFβ1 in a normal mouse model. CVB3(mu) upregulates TGF-β1, and rCVB3(mu)-TGFβ1 can regulate CCR5 expression in mouse brain tissue. This vector does not significantly induce pro-inflammatory responses; instead, it positively regulates anti-inflammatory effects. This invention discovers that the CVB3(mu) virus strain has pancreatic tissue targeting in the host and can serve 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 appears to be non-IFN-γ enhanced. The immune regulation pattern of CVB3(mu) in normal mice shows an anti-inflammatory characteristic with high expression of TGF-β1.

[0045] It is also important to note that the neuroinflammatory response is a multifactorial and dynamic process of acute central nervous system (CNS) injury, encompassing 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 situation. Single chemoimmunomodulatory agents often exhibit both neuroprotective and neurotoxic effects in the CNS injury microenvironment, explaining why several immunosuppressive therapies, including methylprednisolone and erythropoietin, have failed to significantly improve outcomes in clinical trials for TBI and SCI. Suppressing the early inflammatory response may even exacerbate the injury, as evidenced 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 conducive to neurorepair and regeneration. Furthermore, acute immunosuppression fails to resolve the chronic inflammatory cascade that leads to secondary neurodegeneration and long-term neurological dysfunction. Neurocentric therapies such as free radical scavengers and N-methyl-D-aspartate antagonists have been investigated in CNS injury, but with limited success, suggesting that simply attempting to protect neurons from secondary brain injury may not be therapeutically feasible. Instead, a more promising approach may be to attempt to alter secondary neuroinflammation and the damaged immune microenvironment to promote neuroprotection and regeneration. Remodeling the host immune system is a complex task involving multiple methods and strategies, including manipulation of immune cells, intervention with cytokines and chemokines. This invention uses a potent immunogen based on attenuated Coxsackievirus to modulate the distribution and composition patterns of immune cells, cytokines, and chemokines, remodeling the immune system environment to promote neuroprotection and regeneration. In this study, the feasibility of treating severe TBI with a single intraperitoneal injection of CVB3(mu) was demonstrated. By modulating the immune system, an anti-inflammatory immune microenvironment phenotype was achieved in the injured area, reducing the progression of edema and exerting a neuroprotective effect. On day 14 of TBI, the proportion of M2 microglia around the site of brain injury was increased. Significant changes were also observed in the activation and distribution of astrocytes. Apoptosis and autophagy were reduced around the injury site. Cognitive abilities in mice were improved by day 50, with improvements in memory and motor skills. Mice remained healthy and alive until week 40, and their lifespan was not affected. Different time points in TBI represent different stages of inflammatory development. Several different time points were studied, including the acute, intermediate, and late phases of TBI, thus involving the observation of acute and chronic inflammation. The results showed that a single dose of CVB3 (mu) appeared to achieve good efficacy at different stages. We did not observe chronic inflammation in the late stage of TBI.Unlike current single chemical anti-inflammatory drugs, CVB3(mu)-based immunotherapy does not directly and nonspecifically suppress inflammation. Instead, it attempts to alter the microenvironment of secondary neuroinflammation and damage, 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 due to positive feedback caused by early immune regulation, which can modulate 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, possesses potent immunomodulatory and neuroprotective effects. For example, regulatory TregT cells can promote neuroprotection and repair by releasing the anti-inflammatory cytokine TGF-β1. TGF-β1 cytokines have attracted considerable attention due to their potent immunomodulatory capabilities. However, systemic administration can lead to severe toxic reactions, limiting their clinical application. In previous studies, the combined effect of CVB3(mu) and TGF-β1, administered via intraperitoneal injection, was found to exert neuroprotective effects in a mouse model of severe TBI. Our proposed novel approach targeting TGF-β signaling demonstrates superior efficacy against TBI compared to CVB3(mu). This is a therapeutic approach distinct from pioneering intracranial administration (ICV) or AAV targeting neuron-specific cellular TGF-β signaling. This invention utilizes rCVB3(mu) loaded with TGF-β1. In TBI mice, rCVB3(mu)-TGFβ1 exerts a protective effect, showing better efficacy than CVB3(mu) in regulating cerebral edema absorption, apoptosis and autophagy in brain cells in the traumatic brain injury area, and the regulation of M2 microglia and astrocytes. Both low-dose and high-dose viral therapies were used to treat severely injured TBI mice, both showing significant therapeutic effects positively correlated with the dosage. However, a unique fatty pancreas was observed in the pancreas of mice treated with rCVB3(mu)-TGFβ1, despite no impairment of pancreatic function and no significant adverse side effects. Fatty pancreas was not observed in normal mice with rCVB3(mu)-TGFβ1. The reason for the fatty pancreas observed in TBI mice with rCVB3(mu)-TGFβ1 requires further investigation. The experimental results of this 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, suggesting that CVB3 may target pancreatic and cardiac infection. Our preliminary experimental data show that CVB3(mu) does not affect the physical signs or survival of normal mice or TBI mice, and does not cause pathological or functional damage to the pancreas and heart. In particular, in TBI mice, where abnormally high blood glucose levels were observed, CVB3(mu) treatment maintained normal blood glucose levels (this maintenance of blood glucose may be a result of the protection of damaged nerves).

[0047] This invention provides a composition for treating traumatic brain injury, comprising a recombinant attenuated Coxsackie B3 virus loaded with an anti-inflammatory factor as described in this invention.

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

[0049] The present invention also provides a medicament for treating traumatic brain injury, comprising a recombinant attenuated Coxsackie B3 virus loaded with anti-inflammatory factors or a composition thereof.

[0050] It should be noted that the recombinant attenuated Coxsackie B3 virus loaded with anti-inflammatory factors and the above-mentioned composition in this invention can be prepared into different drugs for the treatment of traumatic brain injury by adding excipients. The excipients are substances known in the art.

[0051] The present invention also provides the 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 medicament for treating traumatic brain injury.

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

[0053] (a) The 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 medicament to improve the absorption rate of edema in the affected side of the brain.

[0054] (b) The 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 medicament that reduces apoptosis of cells in the affected brain tissue.

[0055] In some specific examples, the above applications include: the use of recombinant attenuated Coxsackie B3 virus loaded with anti-inflammatory factors in this invention or the composition in this invention in the preparation of medicaments that improve neurological function.

[0056] In some specific examples, the neural functions described above include memory and / or motor abilities.

[0057] To better understand the present invention, specific examples are provided below to further illustrate the content of the present invention, but the content of the present invention is not limited to the examples below.

[0058] In the following examples, the rCVB3(mu)-TGFβ1 virus used in the experiment was obtained by reverse genetics using Vero cells based on the reconstituted pCVB3(mu)-TGFβ1; the cell culture medium used in the cell transfection experiment was DMEM medium; the plasmid vector transformation experiment was performed on engineered E. coli TOP10; the DH5α competent strain was purchased from Tiangen Biotech Co., Ltd. (Beijing); the obtained strain was identified by plaque detection to determine the plaque characteristics and viral titer, and then aliquoted and frozen at -80℃.

[0059] In the following examples, male Balb / c mice, aged 8-10 weeks, were purchased from Vital River Biotechnology Co., Ltd. (Beijing, China). The experimental protocol of this study was ethically approved by the Animal Protection and Utilization Committee of Shantou University Medical College (Approval Nos.: SUMC2021-133 and SUMC2022-413). The experiments were conducted in accordance with the National Institutes of Health's "Guidelines for the Care and Use of Laboratory Animals." All mice were housed in temperature-controlled (20±2℃), humidity-controlled (55±5%), and light / dark cycles of 12 hours, with free access to food and water.

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

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

[0062] MRI scans were performed on mice in the USA. Mice were anesthetized with isoflurane and then fixed to the MRI scanner's fixation system. MRI scans were performed on days 5 and 15 after TBI. During MRI data acquisition, body temperature and respiratory rate were monitored using an MRI-compatible monitoring device (Small Animal Instruments, Inc., USA). T2-weighted (T2w) imaging parameters were as follows: TR = 3000 ms, field of view = 35 mm × 35 mm, TE = 34 ms, slice thickness = 1 mm, imaging matrix = 192 × 192, intersection gap = 0.05 mm, flip angle = 90°, mean value = 3. Experimental data were analyzed using MATLAB software.

[0063] In the following example, the Morris water maze method is implemented as follows: Divide the pool into four quadrants, with the platform placed in the center of one quadrant. Place the animal (rats or mice) headfirst into the water, randomly selecting one of four starting positions: east, west, south, or north. Record the time (in seconds) it takes for the animal to find the underwater platform. In the first few training sessions, if this time exceeds 120 seconds, guide the animal to the platform. Let the animal stay on the platform for 10 seconds. Remove the animal and dry it. If necessary, place the animal under a 150W incandescent lamp for 5 minutes and return it to its cage. Train each animal four times a day, with 15-20 minutes between training sessions, for 5 consecutive days. Exploration training: The day after the final acquisition training session, remove the platform and begin 120-second exploration training. Place the animal into the water from the opposite side of the original platform quadrant. Record the time the animal spends in the target quadrant (the quadrant where the platform was originally placed) and the number of times it enters that quadrant, using this as an indicator of spatial memory. The day after the exploration training ended, four days of alignment training began. The platform was placed in the quadrant opposite to the original platform's location, using the same method as the acquisition training. Training was conducted four times daily. The time to find the platform, swimming distance, and swimming speed were recorded each time.

[0064] In the following example, the immunofluorescence method for paraffin tissue sections was as follows: Mice were anesthetized with 1% sodium pentobarbital and perfused with the heart using physiological saline and 0.1 mol / L phosphate-buffered saline (PBS) containing 4% paraformaldehyde (PFA). The brain was collected, fixed with PFA, and cut into 5 μm sections for subsequent immunofluorescence. The sections were dewaxed and hydrated. Antigen retrieval was performed using microwave retrieval (0.01 mol / L trate buffer, 95°C for 30 min). The sections were allowed to cool naturally to room temperature and washed three times with PBS. They were then perforated with 0.1% Triton X-100 at room temperature for 10 min. After washing, tissue antifluorescence quencher A was added for 30 min. The sections were blocked with 5% goat serum for 40 min, the serum was removed, and primary antibodies were added. Primary antibodies used included ARG1 (Proteintech, 10016741, 1:200) and IBA1 (Abcam, AB283319, 1:200), and incubated overnight at 4°C. Incubate with secondary antibody CY3 (beyotime, A0516, 1:500) and 488 (beyotime, A0428, 1:500) at room temperature for 3 hours. After rinsing with PBS, counterstain the cell nuclei with DAPI. Add anti-fluorescence quencher B for 5 minutes, and finally mount with anti-fluorescence quencher.

[0065] In the following example, the H&E staining method is as follows: After fixing the tissue sample, it is dehydrated, then embedded in paraffin and sectioned; after sectioning, it is first stained with hematoxylin dye to make the cell nuclei blue-purple, and then rinsed with tap water to differentiate and stain; then it is briefly differentiated with 1% hydrochloric acid alcohol, and then thoroughly washed with tap water to remove the acidic reaction; next, it is stained with eosin dye to make the cytoplasm and extracellular matrix pink, then dehydrated with 70%, 95% and 100% ethanol in sequence, finally cleared with xylene, and mounted with neutral resin. The prepared sections are observed under a microscope.

[0066] In the following example, the Western blot method is as follows: Processed tissue is removed, added to RIPA, homogenized, centrifuged, and the supernatant is collected. After protein quantification, loading buffer and PMSF are added, followed by boiling for denaturation and freezing at -80°C for later use. For the actual experiment, a suitable gel is prepared according to the size of the target protein. The stacking gel is separated at 80V for 20 min, and the separating gel is separated at 110V for 60 min. Transfer is performed at 220 mA for 60 min, blocked with 5% skim milk powder, and the corresponding primary antibody is added. Secondary antibody is then added and incubated at room temperature for 2 hours. Finally, the membrane is exposed using an ECL chemiluminescent gel imaging system for visualization and detection.

[0067] In the following example, the method for identifying recombinant viruses using immunofluorescence assay is as follows: Cell preparation: Vero cells are seeded at an appropriate concentration on single-well glass slides and cultured overnight. The next day, the culture medium is discarded, and 10... 2 Incubate 50 / 100 μl of TCID50 virus solution in an incubator for 1 hour, wash three times with pre-warmed 1X PBS, add fresh culture medium, and continue culturing for 48 hours until cytopathic effects are observed. Fixation: Fix cells with 4% paraformaldehyde for 5 minutes at room temperature. After fixation, wash cells three times with pre-warmed PBS at 37°C, and observe under a microscope to ensure cell morphology remains intact. Blocking: Block cells with 1% BSA at room temperature for 1 hour. Primary antibody incubation: Dilute the primary antibody solution to an appropriate concentration and incubate at room temperature for 2 hours, followed by three washes with PBS. Secondary antibody incubation: Add CY3-labeled anti-rabbit IgG fluorescent secondary antibody (1:200 dilution) or 488-labeled anti-mouse IgG fluorescent secondary antibody and incubate at room temperature for 40 minutes, followed by three washes with PBS. Protect from light during secondary antibody incubation to prevent fluorescence quenching. Nucleus labeling: Label nuclei with Hoechst (1:1000), incubate at room temperature for 10 minutes, and wash three times with PBS. Mounting: Use Beyotime's immunofluorescence anti-quenching mounting solution to mount the slides, avoiding air bubbles during the mounting process. Photography: Take photos under a fluorescence microscope.

[0068] In the following example, the plaque analysis method for recombinant viruses is as follows: Recombinant virus rCVB3(mu)-TGFβ1 is taken and serially diluted 10-fold: [The text abruptly ends here, likely due to an incomplete sentence or missing information.] -3 10 -4 10 -5 10 -6 10 -7 10 -8 The cells were seeded into 100% monolayer Vero cells; after adsorption in a 37°C, 5% CO2 incubator for 1 h, they were washed three times with PBS; 2 ml of 1X DMEM maintenance medium containing 0.9% low-melting-point agarose was added; after 2.5 days, 4% paraformaldehyde diluted with PBS was added and fixed at room temperature for 1 h; the low-melting-point agarose was removed by running water, crystal violet staining solution was added, and the solution was incubated at room temperature for 30 min; residual staining solution was washed away by running water, and the morphology and diameter of the empty spots were observed.

[0069] In the following example, the method for detecting recombinant viral protein expression by immunoblotting is as follows: rCVB3(mu)-TGFβ1 recombinant virus is injected at 10... 2 TCID50 / 100 μL was seeded into 6-well cell culture plates containing 80% confluence of Vero cells. After incubation at 37°C and 5% CO2 for 1 h, the cells were washed three times with PBS, and 2 ml of DMEM maintenance medium containing 10% FBS was added. The cells were then incubated at 37°C and 5% CO2 until complete cytopathic effect was achieved. The supernatant was removed, RIPA was added, and the cells were placed on ice to lyse the proteins. Then, 50 μL of 2×SDS-PAGE protein loading buffer was added. The lysate was collected and boiled in water for 10 min for complete denaturation. 20 μL of the sample was loaded onto an SDS-PAGE gel for electrophoresis to separate the proteins. Transfer: Immerse the PVDF membrane in methanol to activate it until transparent; immerse the filter paper in transfer buffer; remove the PVDF and filter paper, and transfer the membrane using a transfer apparatus at a constant current of 220 mA for 1 hour. Then, block the membrane in a shaker with 10% skim milk at room temperature for 1 hour; wash three times with TBST for 5 minutes each time, add 1:1000 diluted anti-flag mouse monoclonal antibody, and incubate overnight at 4°C; wash three times with TBST for 15 minutes each time, add 1:1000 diluted HRP-labeled rabbit anti-mouse secondary antibody, incubate in a shaker at room temperature for 1 hour, wash three times with TBST for 15 minutes each time, and finally add exposure solution to the PVDF membrane for color development; photograph using a general-purpose chemiluminescence system.

[0070] In the following examples, statistical analysis of the experimental data was performed using GraphPad Prism 8 software. Normally distributed data were analyzed using ANOVA and LSD multiple comparisons. Normally distributed data with heterogeneous variance were analyzed using Brown-Forsythe and Weich ANOVA tests. Non-normally distributed data were analyzed using non-parametric Kruskal-Wallis rank ANOVA and the Dunn test. Experimental data are expressed as mean ± SEM. P < 0.05 was considered statistically significant.

[0071] I. 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) TGF-β1 expression gene was obtained by PCR.

[0074] The TGF-β1 monomer required for constructing the rCVB3(mu)-TGFβ1 recombinant virus was a 12.5 kDa protein, with an expressed gene size of 336 bp. High-fidelity DNA polymerase was used for PCR segmental 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℃ for 3 min; cycling parameters: 95℃ for 15 s, 58℃ for 15 s, 72℃ for 1 min, for a total of 35 cycles; finally, extension at 72℃ for 5 min to complete the PCR reaction.

[0078] Agarose gel electrophoresis and gel recovery of amplified nucleic acid products were performed as follows: A 1% agarose gel was prepared using 1X TAE electrophoresis buffer pre-laced with SYBR safe nucleic acid dye. After sample loading, electrophoresis was performed at 110V for 30 min. After the gel completely melted, it was equilibrated to room temperature. 1 / 3 volume of isopropanol was added, followed by centrifugation at 12000 rpm for 1 min. The centrifuged liquid was discarded, and 500 μL of membrane wash buffer was added, followed by centrifugation at 12000 rpm for 1 min. The washing procedure was repeated once, discarding the wash buffer. The gel was centrifuged at 12000 rpm for 2 min, and the liquid was discarded. The gel was incubated at room temperature for 6 min to allow complete ethanol evaporation. Then, 30 μL of RNase-free / DNase-free TB buffer was added, and elution was performed at 12000 rpm for 2 min.

[0079] (2) Enzyme digestion

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

[0081] Table 2 Enzyme digestion system

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

[0083] The enzyme digestion system was incubated overnight at 37°C for complete digestion. The digested products were then subjected to agarose gel electrophoresis again. The nucleic acids in the gel were recovered to purify the digested products, and the concentration and purity of the final products were recorded using a microplate reader.

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

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

[0086] Table 3. Reaction system for constructing full-length cDNA cloning plasmids

[0087] pCV 50ng TGF-β1 DNA 150ng T4 ligase 0.5 μl (one unit) 10X buffer 1μl <![CDATA[ddH2O]]> up to 10μl Total 10μl

[0088] Transformation was performed using *E. coli* TOP10 competent cells after overnight incubation at 4°C with the ligation product: 5 μl of ligation product was added to 50 μl of TOP10 competent cells (competent cells were freeze-thawed on ice), and incubated on ice for 30 min; heat shock was performed in a 42°C water bath for 90 s, followed by incubation on ice for 2 min; 600 μl of antibiotic-free LB medium was added, and the cells were cultured with shaking at 37°C for 1 h; the bacterial solution was then spread onto a substrate containing AMP. + On LB agar plates containing antibiotics, incubate upside down in a 37°C incubator for about 24 hours. After individual colonies have formed, select colonies and identify positive clones by colony PCR.

[0089] (4) Cloning identification and plasmid extraction

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

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

[0092] The plasmids were identified using restriction endonuclease digestion, and the identification system is shown in Table 4 below.

[0093] Table 4 Identification System

[0094] plasmid 5μl (2ug) Cla I 1μl Stu I 1μl 10×NEB buffer 1μl Replenish water to 10μl Total 10μl

[0095] The enzyme digestion fragments were detected by 1% agarose gel electrophoresis after being reacted in a 37℃ water bath for 2 hours. 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) Rescue of recombinant viruses by transfection with Vero cells

[0097] The pCVB3(mu)-TGFβ1 plasmid was introduced into Vero cells using the lipo2000 liposome transfection method. The transfection steps were as follows: First, Vero cells were seeded in 6-well cell culture plates and cultured to 90% confluence using DMEM complete medium containing 10% FBS fetal bovine serum. 10 μl of Lipofectamine 2000 was thoroughly mixed with 125 μl of Opti-MEM and incubated at room temperature for 5 min. Simultaneously, 3 μg of plasmid was mixed with 125 μl of Opti-MEM. The two mixtures were thoroughly mixed and incubated at room temperature for 20 min. The cell culture medium was discarded, and 1.5 ml of Opti-MEM was added. The plasmid and liposome mixture was added to the cells and incubated at 37°C in a 5% CO2 incubator. Cytopathic effects were observed during incubation. Once cytopathic effects appeared, the cell suspension was collected. The cells were centrifuged at 12000 rpm at 4°C for 30 min, and the supernatant containing the virus was collected.

[0098] (7) Recombinant virus amplification and passage

[0099] The collected viral supernatant was amplified as follows: 10 μl of the primary viral solution supernatant was inoculated into a 25 cm² culture chamber containing Vero cells at 90% density. 2 Cells were cultured in cell culture flasks and then incubated at 37°C for 1 hour. After washing three times with pre-warmed PBS, 5 ml of DMEM medium containing 2% FBS was added, and the cells were cultured for 48 hours. During this period, cytopathological effects were observed. When 90% of the cells reached confluence, the supernatant containing the virus was collected at 12,000 rpm for 30 minutes at 4°C as the first-generation virus (P1) and frozen at -80°C. The TCID50 of the virus was determined using the same method, i.e., 10-1 viruses per passage. 2 TCID50 / 100ul and frozen at -80°C. Virus samples were frozen in a freezer at -80°C.

[0100] (8) Virus purification and concentration

[0101] Vero cells were cultured by seeding them into 25T cell culture dishes until 100% coverage. rCVB3(mu)-TGFβ1 was then inoculated into Vero cells until complete viral transformation. The cells were then frozen at -80°C and subjected to three freeze-thaw cycles to completely release the virus. Approximately 15 ml of viral lysis supernatant was collected using the same procedure. The virus was then collected by transferring the viral solution to a centrifuge tube and centrifuging at 4°C for 30 min at the highest speed. The supernatant was collected and filtered once through a 0.45 μm filter to remove cell debris. The virus was then purified and concentrated by ultrafiltration centrifugation. 15 ml of the viral supernatant was added to an ultrafiltration centrifuge tube (PALL Life Sciences Macrosep Centrifugal Devices, 1000K, purple 6 / pkg). The specific steps for using the sample collector (OD990C36) are as follows: 1) Firmly insert the filter membrane paddle 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 aspirate 15 mL of viral supernatant onto the non-membrane side of the sample tube. Place the cap on the sample tube. Place the ultrafiltration device in a cradle centrifuge; 4) Centrifuge at 3220 g at 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-fold (e.g., 15 mL to 1.5 mL); 7) Exchange the buffer with physiological saline and concentrate again 10-fold; 8) Repeat this procedure 3 to 5 times to remove 95 to 99% of the salt or buffer, and finally determine the viral titer using TCID50.

[0102] (9) TCID50 assay for viral titer

[0103] Cell preparation: Vero cells were seeded in 96-well plates and cultured overnight. The virus was serially diluted 10-fold, resulting in 11 dilutions. The virus was then seeded into 8 replicates per well of the 96-well plates containing the cultured cells. The virus-inoculated cells were placed in a cell culture incubator and cultured for 48 hours. Cytopathic effects were observed under a microscope and recorded. TCID50 was calculated using the Reed-Muench method.

[0104] (II) rCVB3(mu)-TGFβ1 test

[0105] The construction diagram of pCVB3(mu)-TGFβ1 is shown below. Figure 1A As shown, the constructed pCVB3(mu)-TGFβ1 is as follows Figure 1B As shown, its protein immunoblotting and immunofluorescence are as follows: Figure 1C and 1D As shown, the plaque-attracting characteristic is as follows Figure 1E As shown.

[0106] TBI mice were constructed using the above method. Mice were administered pCVB3(mu)-TGFβ1 via gavage, with physiological saline as a control. A viral titer of 1 x 10⁻⁶ was used. 5 TCID50 / 100ul; no live virus was detected in the pancreas, heart, large intestine, small intestine, spleen, lungs, kidneys, muscles, serum and other tissues on day 5 post-infection (Table 5), so it is believed that this strain will only exist in mice for no more than 5 days.

[0107] Table 5. Dynamic distribution of rCVB3(mu)-TGFβ1 virus in mouse tissues after infection.

[0108]

[0109] Note: "+" indicates live virus detected; "-" indicates live virus not detected.

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

[0111] II. Experimental Design

[0112] TBI mice were constructed using the above method. Twenty-four hours after TBI, the mice were randomly assigned to one of the following four groups: sham + saline group; TBI + saline group; TBI + CVB3 (mu) group; and TBI + rCVB3 (mu)-TGFβ1 group; with six mice in each group. A viral titer of 1 x 10⁻⁶ was used. 5 TCID50 / 100ul; perform the following tests.

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

[0114] A mouse model of severe traumatic brain injury (TBI) was established by striking the right cerebral cortex with a controlled impactor to a depth of 2 mm. Body weight changes in mice were recorded from day 1 to 14 post-TBI. Results showed that the mice maintained good vital signs and overall condition. Compared to the sham-operated control group (sham + saline group), the experimental group mice experienced a decrease in body weight after TBI, reaching its lowest point on the second day, followed by recovery. The rate of weight gain in the TBI and treatment groups was greater than that in the sham-operated control group. After day 8, the weight gain in the TBI and rCVB3(mu)-TGFβ1 administration groups began to be higher than that in the sham-operated control group. Figure 2A ).

[0115] To assess the safety of attenuated Coxsackievirus B3 and its vector in TBI mice, histopathological sections of the heart and pancreas were examined on day 15. H&E tissue section results showed that rCVB3(mu)-TGFβ1 did not cause cardiac pathological damage in either normal or TBI mice, and did not induce myocarditis, pancreatitis, or acinar atrophy. A small amount of fatty pancreas was observed in the rCVB3(mu)-TGFβ1 treatment group of TBI mice. Figure 2B ).

[0116] To further clarify the effects of CVB3(mu) and rCVB3(mu)-TGFβ1 on cardiac and pancreatic function in mice, the blood glucose levels in fasting serum of mice were measured (using an ARCHITECT c16000 clinical chemistry analyzer). Specifically, blood glucose levels in TBI mice were monitored on days 3 and 10. The results showed that blood glucose levels in the TBI group mice continued to rise after TBI, with a significant difference on day 10. In contrast, blood glucose levels in the CVB3(mu) and rCVB3(mu)-TGFβ1 treatment groups remained stable, similar to those in the sham-operated control group. These results suggest that TBI mice have impaired glycemic control due to traumatic brain injury, while the CVB3(mu) and rCVB3(mu)-TGFβ1 treatment groups maintained normal glycemic regulation. Figure 2C ).

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

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

[0119] IV. Experiments on the reduction of cerebral edema lesion volume and apoptosis of neuronal cells in the affected side of a severe TBI mouse model using recombinant Coxsackievirus CVB3(mu) and rCVB3(mu)-TGFβ1.

[0120] To clarify the therapeutic effect of rCVB3(mu)-TGFβ1 on mice with severe TBI, MRI of the mouse brain was performed. The results showed that rCVB3(mu)-TGFβ1 significantly promoted the absorption rate of cerebral hematoma. Figure 3A and Figure 3B ).

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

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

[0123] V. Experiment on the regulation of the proportion of M2 microglia and astrocytes by recombinant Coxsackievirus CVB3(mu) and rCVB3(mu)-TGFβ1 in a severe TBI mouse model

[0124] Microglia play a crucial role in the occurrence and development of traumatic brain injury (TBI)-induced neuroinflammation. This study examined the distribution characteristics of M1 and M2 microglia in the periphery of traumatic brain injury in mice on day 14. The results showed no significant difference in the proportion of M1 microglia after TBI compared to the sham-operated control group. However, the proportion of M2 microglia was significantly increased in the CVB3(mu) and rCVB3(mu)-TGFβ1 treatment groups. Specifically, the rCVB3(mu)-TGFβ1 treatment group had a significantly higher proportion of M2 microglia than the CVB3(mu) treatment group, indicating the important role of TGF-β1 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] Behavioral changes in mice were observed on day 50, and cognitive function was assessed using the water maze test. Results showed that TBI caused significant neurological damage compared to the sham-operated group, manifested as reduced dwell time on the platform, with almost no ability to find it (P<0.01). Mice treated with low and high doses of rCVB3(mu)-TGFβ1 maintained neurological function; the time spent finding and remaining on the platform was not significantly different from the Sham group, although there was greater individual variation in the high-dose group. 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 treatment group showed better performance. Figure 5A , Figure 5B , Figure 5C and Figure 5D As expected, mice treated with low and high doses of rCVB3(mu)-TGFβ1 exhibited better behavioral performance in the MWM test after TBI than mice in the TBI group (P<0.05). These findings suggest that low and high doses of rCVB3(mu)-TGFβ1 treatment alleviated neurological damage and improved neurological functional outcomes after TBI. The treatment groups showed improvements in memory and motor skills.

[0127] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, and all such modifications or substitutions should be covered within the scope of the claims of the present invention.

Claims

1. The use of a recombinant attenuated Coxsackie B3 virus loaded with an anti-inflammatory factor or a composition comprising a recombinant attenuated Coxsackie B3 virus loaded with an anti-inflammatory factor in the preparation of a medicament for treating traumatic brain injury, wherein the recombinant attenuated Coxsackie B3 virus loaded with an anti-inflammatory factor comprises the sequence shown in SEQ ID NO:

1.

2. The application according to claim 1, characterized in that, (a) The drug is used to increase the rate of absorption of cerebral edema on the affected side; or (b) The drug is used to reduce apoptosis of brain cells on the affected side; or (c) The drug is used to improve neurological function.

3. The application according to claim 2, characterized in that, Neurological functions include memory and / or motor abilities.

4. The application according to any one of claims 1 to 3, characterized in that, Drug dosage forms include injections, tablets, powders, or patches.

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