Application of engineered attenuated coxsackie B3 virus vector 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 an engineered attenuated coxsackie B3 virus vector (CVB3 (mu)) in preparation of drugs for treating traumatic craniocerebral injury. According to the application disclosed by the invention, after severe craniocerebral injury of a mouse, CVB3 (mu) can remodel a host immune system through single intraperitoneal injection treatment, so that the absorption speed of encephaledema on an affected side can be remarkably increased, and the effect of treating traumatic craniocerebral injury is achieved; in addition, the CVB3 (mu) has remarkable safety.

Description

Technical Field

[0001] The present invention belongs to the technical field of disease medicines, and specifically relates to the use of an engineered attenuated Coxsackie B3 virus vector 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 the engineered attenuated Coxsackie B3 virus vector (CVB3(mu)) 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] On the one hand, the present invention provides an application of an engineered attenuated Coxsackie B3 virus vector in the preparation of a drug for treating traumatic brain injury.

[0008] Preferably, the above application includes one or more combinations of the following applications: application of an engineered attenuated Coxsackie B3 virus vector in the preparation of a drug for increasing the absorption rate of affected-side cerebral edema.

[0009] Preferably, the dosage form of the drug in the above application includes injection, tablet, powder or patch.

[0010] The beneficial effects of the present invention include at least: the present invention found that a single intraperitoneal injection of an engineered attenuated Coxsackie B3 virus vector (CVB3(mu)) after severe craniocerebral injury in mice can reshape the host immune system and reduce the volume of lateral cerebral edema lesions, thereby achieving the effect of treating traumatic craniocerebral injury; in addition, CVB3 has significant safety. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] Figure 1 Comparison of CVB3(mu) and CVB3(WT) plaques;

[0012] Figure 2 Schematic diagram of the experimental process for evaluating the safety of CVB3(mu) in mouse animal models;

[0013] Figure 3 The body weight changes of mice in the CVB3(mu) and CVB3(WT) groups;

[0014] Figure 4 The survival rates of mice in the CVB3(mu) and CVB3(WT) groups;

[0015] Figure 5 Representative sections of heart and pancreatic tissues of mice in the CVB3(mu) and CVB3(WT) groups;

[0016] Figure 6 This is the effect of the right side of the brain being struck in a mouse model of severe traumatic brain injury. The damage to the cerebral cortex and hippocampus of the mouse was caused by a 2mm strike depth.

[0017] Figure 7 The weight monitoring change curve of mice in each group (n=6 / group);

[0018] Figure 8 Representative results of H&E staining of heart and pancreatic tissue sections of mice in each group;

[0019] Fig. 9 MRI scans of mice in a TMRI scanner;

[0020] Fig.10 The figures show the degree of cerebral edema in different groups of mice. DETAILED DESCRIPTION

[0021] 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.

[0022] 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.

[0023] The embodiment of the present invention provides an application of an engineered attenuated Coxsackie B3 virus vector (CVB3(mu)) in the preparation of a drug for treating traumatic brain injury.

[0024] In some specific examples, the above applications include: use of an engineered attenuated Coxsackie B3 virus vector in the preparation of a drug for increasing the absorption rate of affected-side cerebral edema.

[0025] In some specific examples, the dosage form of the drug in the above application includes injection, tablet, powder or patch.

[0026] It should be noted that the engineered attenuated Coxsackie B3 virus vector (CVB3(mu)) in the present invention is well known in the art, and includes the nucleic acid sequence shown in SEQ ID NO:1.

[0027] It should also be noted that in the treatment of tumors, a naturally occurring or genetically modified oncolytic virus (OV) has been developed as an immunotherapy. Among them, the CVB3 virus has also been developed as a tumor-melting virus, and its damage-related molecular pattern can effectively induce the host immune regulatory response. In addition, CVB3 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. In the early stage of the present invention, the safety and immune system regulation properties of CVB3 (mu) were clarified in a normal mouse animal model, in which CVB3 (mu) upregulated TGF-β1 and could 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.

[0028] 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 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 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.

[0029] 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.

[0030] In the following examples, the CVB3(mu) used in the experiment was rescued based on the pCVB3(mu) cloning vector prepared in the laboratory earlier; 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 strains were identified by plaque identification of the virus's plaque characteristics and virus titer, and were packaged and frozen in a -80℃ refrigerator.

[0031] In the following examples, male Balb / c mice, 8-10 weeks old, were purchased from Beijing Weitong Lihua Biological Co., Ltd. (Beijing, China). The experimental protocol in the present invention 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 conducted 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 feeding box with free access to food and water.

[0032] In the following example, the experimental model of traumatic brain injury was constructed as follows: a 9-10 week old Balb / c male mouse was used to construct a mouse animal model of traumatic brain injury (TBI), and the mouse was 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.

[0033] In the following example, the MRI experimental method is as follows: a 7.0T MRI scanner (Agilent Technologies Co. Ltd., CA,

[0034] 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.

[0035] 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.

[0036] 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.

[0037] 1. Acquisition of CVB3(mu) virus

[0038] The experiment used lipo2000 liposome transfection method to introduce pCVB3 (mu) plasmid into Vero cells. The transfection steps are as follows: First, Vero cells were inoculated in 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 the period, observe the cytopathic effect, and collect the cell suspension when the cells show cytopathic effect; centrifuge at 12000rpm4℃ for 30 minutes, and collect the supernatant containing the virus.

[0039] (I) Recombinant virus amplification and passaging

[0040] 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.

[0041] (II) Virus purification and concentration

[0042] Vero cell culture, Vero cells were inoculated in 25T cell culture dishes to a coverage of 100%; CV-B3 (WT) and rCV-TGFβ1 were inoculated in Vero cells until complete virus transformation, frozen in a -80°C refrigerator, and repeatedly frozen and thawed three times to completely release the virus in the cells. In the same steps, about 15 ml of virus lysis supernatant was collected; the virus was collected, the virus solution was transferred to a centrifuge tube and centrifuged at a maximum speed of 4°C for 30 minutes, the supernatant was collected, and filtered once with a 0.45 μm filter to remove cell debris; ultrafiltration centrifugation was used to purify and concentrate the virus, and 15 ml of the virus supernatant was added to the sample collector of an ultrafiltration centrifuge tube (PALL life Sciences Macrosep Centrifugal Devices, 1000K, purple 6 / pkg OD990C36) (Sample The specific steps are as follows: (1) firmly insert the 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 draw 15 ml of the virus supernatant sample to the non-membrane side of the sample tube. Place the lid on the sample tube. Place the ultrafiltration device in a cradle rotor centrifuge; (4) centrifuge at 3220g 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 times (for example, 15 ml is concentrated to 1.5 ml); (7) exchange the buffer with saline and concentrate it again 10 times; (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.

[0043] (III) TCID50 determination of virus titer

[0044] 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 further culture, and the cell lesions were observed and recorded under a microscope after 48 hours; TCID50 was calculated using the Reed-Muench method.

[0045] 2. Verification of the attenuation and safety characteristics of CVB3(mu)

[0046] The above method was used to identify the plaque characteristics of CVB3 (WT) and CVB3 (mu) by plaque, and the results are shown in the figure. Figure 1 The results showed that CVB3(mu) had less plaque phagocytosis than CVB3(WT), suggesting that CVB3(mu) had attenuated characteristics.

[0047] TBI mice were constructed using the above method. 24 hours after TBI, mice were randomly divided into the following three groups: saline group, CVB3 (WT) group, and CVB3 (mu) group. The virus titer was 1X10 5 TCID50 / 100ul; and according to Figure 2 The following experiments were performed according to the flow chart shown.

[0048] Body weight was measured every one to two days until termination and compared between groups. Figure 3 As shown in the figure, animals in the CVB3(mu)-vaccinated group did not lose weight throughout the study, which is typical of healthy mice. In contrast, mice infected with the same dose of wild-type CVB3(WT) continued to lose weight; and the survival rates of the two groups of mice at the end of the study were calculated, as shown in the figure. Figure 4 As shown, animals in both the CVB3(mu) and saline groups were clinically healthy and no mortality was observed. Deaths of mice were recorded in CVB3(WT) mice on days 10, 16, and 28 of infection.

[0049] In addition, the hearts and pancreas collected at day 21 were subjected to standard histological processing to evaluate inflammatory changes induced by CVB3(WT) and CVB3(mu) strains. Figure 5 As shown, pathological changes in heart and pancreatic tissues were examined. No myocarditis or pancreatitis was detected in the control group. The pancreatic tissue of the control group was dense, and the acinar structure was intact. In contrast, in the pancreatic tissue of the CVB3(WT) group, we observed pancreatic acinar atrophy, reduced acinar volume, reduced acinar number, and partial necrosis of acinar cells. There was a large amount of inflammatory cell infiltration in the interstitial tissue, and most of the acini were replaced by adipose tissue; the pancreatic tissue of the CVB3(mu)P1 group was dense, and the acinar structure remained intact. In CVB3(mu), no damage was observed in the heart tissue.

[0050] 3. Recombinant Coxsackievirus CVB3(mu) has no toxic side effects and has safety characteristics in a severe TBI mouse model

[0051] TBI mice were constructed using the above method. 24 hours after TBI, mice were randomly divided into the following 4 groups: CVB3(mu) group, sham+saline group, TBI+saline group (hereinafter referred to as TBI group), and TBI+CVB3(mu) group. There were 6 mice in each group. 100ul / mouse virus was injected intraperitoneally with a virus titer of 1X10 5 TCID50 / 100ul; perform the following test.

[0052] The mouse model of severe craniocerebral injury (TBI) was established by using a controlled impactor to strike the right cerebral cortex at a depth of 2 mm (see Figure 6 ); recorded the weight change data of mice from 1 to 10 days after TBI. The results showed that the mice had good vital signs and condition. 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 ( Figure 7 ).

[0053] 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 day 15 were detected. The results of H&E tissue sections showed that CVB3(mu) did not cause cardiac pathological damage to normal and TBI mice, nor did it cause myocarditis, pancreatitis, and pancreatic islet atrophy ( Figure 8 ).

[0054] These results collectively suggest that CVB3(mu) has no obvious toxic side effects in normal and TBI mice, either in the early stages of infection or over a longer period of time, showing a safe profile.

[0055] IV. Recombinant Coxsackievirus CVB3(mu) significantly improves the absorption rate of brain edema on the affected side in a severe TBI mouse model

[0056] In order to clarify the therapeutic effect of CVB3(mu) on severe TBI mice, the mouse brain was examined by magnetic resonance imaging (MRI) (Agilent Technologies Co. Ltd., CA, USA), T2-weighted (T2w) imaging, and statistical analysis. The results showed that CVB3(mu) could significantly promote the absorption of cerebral hematoma ( Fig. 9 and Fig.10 ). The above results show that recombinant Coxsackievirus CVB3(mu) significantly increases the absorption rate of brain edema on the affected side in a severe TBI mouse model and has a significant TBI therapeutic effect.

[0057] 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. Use of an engineered attenuated Coxsackie B3 virus vector in the preparation of a drug for treating traumatic brain injury.

2. The use according to claim 1, characterized in that: Applications include: Application of engineered attenuated Coxsackie B3 virus vector in the preparation of drugs for increasing the absorption rate of affected-side brain edema.

3. The use according to any one of claims 1 to 2, characterized in that: The medicine comes in the form of an injection, tablet, powder, or patch.

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