Use of an engineered attenuated Coxsackievirus B3 virus vector in the preparation of a medicament for treating traumatic brain injury

Abstract

The present invention belongs to the technical field of disease drugs, and particularly relates to the application of an engineered attenuated coxsackievirus B3 vector (CVB3(mu)) in the preparation of a drug for treating traumatic brain injury. The present invention discovers that after severe traumatic brain injury in mice, single intraperitoneal injection treatment with CVB3(mu) can reshape the host immune system, can significantly increase the absorption rate of ipsilateral brain edema, and achieve the effect of treating traumatic brain injury; in addition, CVB3(mu) has remarkable safety.

Description

Technical Field

[0001] The present invention belongs to the technical field of disease drugs, and particularly relates to the application of an engineered attenuated coxsackievirus B3 virus vector in the preparation of a drug for treating traumatic brain injury. Background Art

[0002] Central nervous system injury triggers a dynamic neuroinflammatory response, which is mediated by the activation of the neuroinflammatory cascade by resident and infiltrating immune cells. After primary injury, the dysregulated inflammatory cascade is associated with the maintenance of a pro-inflammatory microenvironment, leading to secondary neurodegeneration and the development of persistent neurological deficits. Due to the multifaceted nature of central nervous system injury, clinically effective therapies for diseases such as traumatic brain injury (TBI), spinal cord injury (SCI), and stroke have proven difficult to develop. There is currently no treatment method that adequately addresses the chronic inflammatory component of secondary central nervous system injury. Existing research has developed selective immunomodulatory methods, such as promoting anti-inflammatory M2-like positive tissue remodeling by improving pro-inflammatory M1-like responses, which has played a neuroprotective role, suggesting the feasibility of treating TBI through immunological intervention.

[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 variant forms have different abilities to shape the immune system, having beneficial or harmful effects on the host; for example, HIV infection can lead to severe damage to the host immune system, especially the reduction of CD4+ T cells. This change in the immune environment makes the host more vulnerable to other infections; different genotypes and mutant forms of the hepatitis B virus can lead to different immune responses; some variant forms may lead to chronic infection, while other forms 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 property of pathogens, existing research has used 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 has the potential to have specific therapeutic effects on some diseases unrelated to the pathogens, such as oncolytic viruses promoting tumor clearance by regulating the tumor immune microenvironment. Adenoviruses can infect tumor cells and trigger cell lysis, releasing tumor antigens and activating the host immune system to clear tumor cells. Adeno-associated virus (AAV) can also be genetically engineered to express cytokines or other immunomodulatory molecules, thereby enhancing the anti-tumor immune response, and is widely used in gene therapy, especially for the treatment of genetic diseases. These viruses and virus-related vectors 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 discovers that the engineered attenuated coxsackievirus B3 vector (CVB3(mu)) can significantly treat traumatic brain injury with high safety.

[0006] 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 coxsackievirus B3 vector in the preparation of a drug for treating traumatic craniocerebral injury.

[0008] Preferably, the above application includes one or more combinations of the following applications: the application of the engineered attenuated coxsackievirus B3 vector in the preparation of a drug for increasing the absorption rate of ipsilateral brain 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 at least include: The present invention discovers that after severe craniocerebral injury in mice, single intraperitoneal injection treatment with the engineered attenuated coxsackievirus B3 vector (CVB3(mu)) can reshape the host immune system, reduce the volume of ipsilateral brain edema lesions, and achieve the effect of treating traumatic brain injury; in addition, CVB3 has significant safety. Brief Description of the Drawings

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

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

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

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

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

[0016] Figure 6 Effect of right ipsilateral brain impact in a mouse model of severe traumatic brain injury, damage to the cerebral cortex and hippocampus of mice caused by a 2-mm impact depth;

[0017] Figure 7 Curves of monitored changes in body weight of mice in each group (n = 6 / group);

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

[0019] Figure 9 MRI scanning of mice using a TMRI scanner;

[0020] Figure 10 Degree of absorption of ipsilateral cerebral edema in different groups of mice. Detailed implementation manners

[0021] The examples given are for better illustration of the present invention, but the content of the present invention is not limited only to the examples given. Therefore, those skilled in the art who make non-essential improvements and adjustments to the implementation manners based on the above-mentioned inventive content still fall within the protection scope of the present invention.

[0022] The terms used herein are only for describing specific embodiments and are not intended to limit the present disclosure. Unless having significantly different meanings in the context, the expressions in the singular form include those in the plural form. As used herein, it should be understood that terms such as "including", "having", "containing" 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 of the existence or addition of one or more other features, numbers, operations, components, parts, elements, materials or their combinations. As used herein, depending on the circumstances, " / " can be interpreted as "and" or "or".

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

[0024] In some specific examples, the above application includes: the application of an engineered attenuated Coxsackievirus B3 vector in the preparation of a medicament for increasing the absorption rate of ipsilateral cerebral edema.

[0025] In some specific examples, the dosage forms of the medicament in the above application include injections, tablets, powders or patches.

[0026] It should be noted that the engineered attenuated Coxsackievirus B3 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 engineered oncolytic virus (OV) has been developed as an immunotherapy. Among them, the CVB3 virus has also been developed as an oncolytic virus, and its damage-associated molecular patterns can effectively induce host immune regulatory responses. In addition, CVB3 can also be used as a vector for delivering immunomodulators, such as carrying cytokines, to obtain a cytokine-equipped CVB3, which further modifies the host immune microenvironment. In the early stage of this invention, the safety and immune system regulatory characteristics of CVB3(mu) were clarified in a normal mouse animal model. Among them, CVB3(mu) upregulates TGF-β1 and can regulate the expression of CCR5 in mouse brain tissue. This vector does not significantly induce a pro-inflammatory response, but instead has a positive regulation on the regulation of anti-inflammatory effects.

[0028] It should also be noted that neuroinflammatory response is a multi-factorial and dynamically developing process in response to acute central nervous system (CNS) injury, including acute and chronic phases mediated by resident and infiltrating immune cells. Any effective treatment for diseases such as traumatic brain injury (TBI), spinal cord injury (SCI), and stroke must address this complex situation. Single chemical immunomodulatory drugs often exhibit dual effects of neuroprotection and neurotoxicity in the CNS injury microenvironment, which explains why several immunosuppressive therapeutic drugs, including methylprednisolone and erythropoietin, have failed to significantly improve outcomes in clinical trials of TBI and SCI. Inhibiting the early inflammatory response may even exacerbate the injury, as shown by increased edema in rats treated with methylprednisolone after SCI. After CNS injury, the acute inflammatory process plays a crucial role in establishing a microenvironment conducive to nerve repair and regeneration. In addition, acute immunosuppression fails to address the chronic inflammatory cascade leading to secondary neurodegeneration and the development of long-term neurological dysfunction. Neurocentric therapeutic approaches such as free radical scavengers and N-methyl-D-aspartic acid antagonists have also been studied in CNS injury but with poor results, indicating 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 modify the secondary neuroinflammation and 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 of cytokines and chemokines, etc. The present invention uses an attenuated Coxsackievirus, a strong immunogen, 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 by single intraperitoneal injection of CVB3(mu) was demonstrated. An anti-inflammatory immune microenvironment phenotype in the injury area was obtained through immune system regulation, which alleviated the progression of edema and exerted a neuroprotective effect. The proportion of M2 microglia increased around the craniocerebral injury site on the 14th day after TBI. There were also obvious changes in the activation and distribution of astrocytes. Apoptosis and autophagy decreased around the injury site. The cognitive ability of mice was improved on the 50th day, and the memory and motor abilities of mice were improved. When observed up to the 40th week, the mice still survived healthily without affecting their normal lifespan. Different time points of TBI represent different inflammatory development periods. Several different time points were concerned, including the acute, middle, and late stages of TBI, so the observations of acute and chronic inflammation were involved. The results showed that single administration of CVB3(mu) seemed to achieve good efficacy at different periods. When we were in the late stage of TBI, we did not observe the phenomenon of chronic inflammation of TBI.CVB3(mu)-based immunotherapy is different from current single chemical anti-inflammatory drugs. Instead of directly and non-specifically inhibiting inflammation, it 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 after 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-response and bring about significant and lasting clinical recovery.

[0029] To better understand the present invention, the content of the present invention will be further clarified 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 re-rescued based on the pCVB3(mu) cloning vector prepared in the early stage of the laboratory; the cell culture medium used in the cell transfection experiment was DMEM medium, and the plasmid vector ligation transformation experiment was used for engineering Escherichia coli TOP10. The DH5α competent strain was purchased from Tiangen Biochemical Co., Ltd. (Beijing); the obtained virus strain was identified for plaque characteristics and virus titer by plaque assay, and was aliquoted and frozen in an -80°C refrigerator.

[0031] In the following examples, male Balb / c mice, 8 - 10 weeks old, were purchased from Vital River Laboratories Inc. (Beijing, China). The experimental protocols in the present invention were approved by the Animal Care and Use 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-hour light / dark cycle housing facility with free access to food and water.

[0032] In the following examples, the traumatic brain injury experimental model was constructed as follows: A traumatic brain injury (TBI) mouse animal model was constructed using 9 - 10-week-old male Balb / c mice. The mice were anesthetized with 1% sodium pentobarbital (35 mg / kg). A craniotomy with a diameter of 4 mm was performed in the left parietal bone behind the coronal suture and next to the sagittal suture using a high-speed electric drill. A 2-mm deep cortical contusion was caused on the 4-mm diameter cranial wound using an electric craniocerebral spinal cord injury impactor.

[0033] In the following examples, the MRI experimental method was as follows: A 7.0T MRI scanner (Agilent Technologies Co., Ltd., CA,

[0034] MRI scans of mice were performed in the USA. After anesthesia with isoflurane, the mice were fixed on the fixation system of the MRI scanner. MRI was performed on the 5th and 15th days after TBI. During the MRI data acquisition, the body temperature and respiratory rate of the mice were observed with the aid of an MRI-compatible monitoring device (Small Animal Instruments, Inc., USA). The 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, average value = 3. The experimental data were analyzed using MATLAB software.

[0035] In the following example, the H&E staining method was as follows: After fixing the tissue samples, dehydration was carried out, followed by embedding in paraffin and sectioning; after sectioning, first stained with hematoxylin dye to make the cell nuclei blue-violet, then rinsed with tap water to differentiate the staining; then briefly differentiated with 1% hydrochloric acid alcohol, and then thoroughly washed with tap water to remove the acidic reaction; next stained with eosin dye to make the cytoplasm and extracellular matrix pink, then dehydrated successively with 70%, 95% and 100% ethanol, and finally cleared with xylene and sealed with neutral gum. The prepared sections were observed under a microscope.

[0036] In the following example, the statistical analysis of the experimental data was performed using GraphPad Prism 8 software. ANOVA variance analysis and LSD multiple comparison analysis methods were used for data conforming to the normal distribution. For data conforming to the normal distribution and non-homogeneous variance, Brown-Forsythe and Welch variance analysis tests were used. For data not conforming to the normal distribution, non-parametric Kruskal-Wallis rank variance analysis and Dunn test methods were used. The experimental data were expressed as mean ± SEM. P < 0.05 was considered statistically significant.

[0037] I. Obtaining the CVB3(mu) virus

[0038] The experiment used lipo2000 liposome transfection method to introduce pCVB3(mu) plasmid into Vero cells. The transfection steps were as follows: First, inoculate Vero cells into a 6-well cell culture plate and culture the cells to 90% density using DMEM complete medium containing 10% FBS fetal bovine serum. After mixing 10 μl of Lipofectamine2000 with 125 μl of Opti-MEM thoroughly, let it stand at room temperature for 5 min. Meanwhile, mix 3 μg of plasmid with 125 μl of Opti-MEM. After thoroughly mixing the two tubes of mixed solution, let it stand at room temperature for 20 min. Aspirate the cell culture medium, and add 1.5 ml of Opti-MEM blood medium. Add the plasmid and liposome mixed solution to the cells, and place it in an incubator at 37°C and 5% CO2. Observe the cytopathic effect during this period. When cytopathic changes occur in the cells, collect the cell suspension. Centrifuge at 12,000 rpm at 4°C for 30 min, and collect the supernatant containing the virus.

[0039] (I) Amplification and passage of recombinant virus

[0040] The collected virus supernatant was amplified by the following method. Inoculate 10 μl of the supernatant of the primary virus solution into a 25 cm 2 cell culture flask cultured with Vero cells at 90% density, then adsorb it in a cell incubator at 37°C for 1 h. After washing 3 times with pre-warmed PBS, add 5 ml of DMEM medium containing 2% FBS and culture for 48 h. During this period, observe the cytopathological effect. When 90% of the cells 2 are centrifuged at 12,000 rpm at 4°C for 30 min, collect the supernatant containing the virus as the first-generation virus (P1) and freeze it at -80°C. The TCID50 of the virus was determined according to the same method, that is, the virus for each passage was 10

[0041] (II) Virus purification and concentration

[0042] Cultivation of Vero cells: Inoculate Vero cells into a 25T cell culture dish until the confluence reaches 100%; inoculate CV-B3 (WT) and rCV-TGFβ1 into Vero cells until complete viral transformation, freeze them in an -80°C refrigerator, and perform three cycles of freeze-thawing to completely release the virus in the cells. Follow the same steps to collect approximately 15 ml of virus lysate supernatant; harvest the virus, transfer the virus solution to a centrifuge tube, centrifuge at the maximum speed at 4°C for 30 min, collect the supernatant, and filter it once through a 0.45 μm filter to remove cell debris; ultrafiltration centrifugation is used to purify and concentrate the virus. Add 15 ml of virus supernatant to the sample collector (Sample Reservoir) of an ultrafiltration centrifugation tube (PALL life Sciences Macrosep Centrifugal Devices, 1000K, purple 6 / pkg OD990C36). The specific steps 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 the virus supernatant sample into the non-membrane side of the sample tube. Place the lid on the sample tube. Place the ultrafiltration device into a swing-out rotor centrifuge; (4) Centrifuge at 3220g at 4°C for 90 min to achieve the required concentrated volume; (5) Remove the filtrate collection tube and aspirate and discard the filtrate; (6) Concentrate the sample by at least 10 times (for example, concentrate 15 ml to 1.5 ml); (7) Exchange the buffer with normal saline and concentrate it by 10 times again; (8) Repeat this procedure 3 to 5 times to remove 95 to 99% of the salt or buffer, and finally measure the virus titer using TCID50.

[0043] (III) Measuring the virus titer using TCID50

[0044] Cell preparation: Inoculate Vero cells into a 96-well plate and culture overnight. Perform serial 10-fold dilutions of the virus, with a total of 11 dilution factors; inoculate the virus into the cultured cells in the 96-well plate, with 8 replicates for each dilution factor; place the cells inoculated with the virus in a cell culture incubator and continue culturing. Observe the cytopathic effect under a microscope after 48 hours and record it; calculate the TCID50 using the Reed-Muench method.

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

[0046] Use the above method to identify the plaque characteristics of CVB3(WT) and CVB3(mu) by plaque assay respectively. The results are shown in Figure 1 as follows. The results show that CVB3(mu) has a smaller plaque-forming ability compared to CVB3(WT), indicating that CVB3(mu) has the attenuation characteristic.

[0047] Using the above method to construct TBI mice, 24 hours after TBI, the mice were randomly assigned to the following 3 groups: normal saline group, CVB3 (WT) group, and CVB3 (mu) group; using a virus titer of 1X10 5 TCID50 / 100ul; and the following experiments were carried out according to the Figure 2 flow chart shown.

[0048] The body weight was measured every one to two days until the end and compared among groups. The results Figure 3 showed that the animals in the CVB3 (mu) inoculation group did not lose weight throughout the study, which is typical of healthy mice. In contrast, the mice infected with the same dose of wild-type CVB3 (WT) showed a continuous decrease in body weight; and the survival rates of the two groups of mice at the end were calculated. The results were as Figure 4 shown. The animals in the CVB3 (mu) group and the normal saline group were clinically healthy and no deaths were observed. Deaths of mice were recorded on the 10th, 16th, and 28th days after infection with CVB3 (WT).

[0049] In addition, for the histopathology induced by CVB3 (WT) and CVB3 (mu) strains, the heart and pancreas collected at the end on the 21st day were subjected to standard histological processing to evaluate inflammatory changes. The results were as Figure 5 shown. The pathological changes in the heart and pancreas tissues were examined. Myocarditis or pancreatitis was not detected in the control group. The pancreatic tissue in 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 infiltration of inflammatory cells in the interstitial tissue and most of the acini were replaced by adipose tissue; the pancreatic tissue in the CVB3 (mu) P1 group was dense and the acinar structure remained intact. No damage was observed in the heart tissue in CVB3 (mu).

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

[0051] Using the above method to construct TBI mice, 24 hours after TBI, the mice were randomly assigned to the following 4 groups: CVB3 (mu) group, sham + normal saline group; TBI + normal saline group (hereinafter also referred to as the TBI group); TBI + CVB3 (mu) group; 6 mice in each group; by intraperitoneal injection of 100ul / mouse of virus, using a virus titer of 1X10 5 TCID50 / 100ul; the following experiments were carried out.

[0052] A controlled impactor was used to strike the right cerebral cortex tissue at a depth of 2 mm to establish a severe traumatic brain injury TBI model in mice (seeFigure 6 );Data on the body weight changes of mice from 1 to 10 days after TBI in mice were recorded. The results showed that the vital signs and status of the mice were good. Compared with the sham operation control group (sham + normal saline group), the body weight of the mice in the experimental group decreased after TBI, reaching the lowest point on the second day and then starting 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] To evaluate the safety of attenuated coxsackievirus B3 and its vector in TBI mice, pathological sections of the heart and pancreas tissues of mice on the 15th day were examined. The results of H&E tissue sections showed that CVB3(mu) did not cause pathological damage to the hearts of normal and TBI mice, and did not cause myocarditis, pancreatitis, and islet atrophy ( Figure 8 ).

[0054] These results together suggest that CVB3(mu) has no obvious toxic and side effects in normal and TBI mice, showing safety characteristics whether in the early stage of infection or for a relatively long time.

[0055] IV. Recombinant coxsackievirus CVB3(mu) significantly increases the absorption rate of ipsilateral brain edema in a severe TBI mouse model

[0056] To clarify the therapeutic effect of CVB3(mu) on severe TBI mice, magnetic resonance imaging MRI (Agilent Technologies Co., Ltd., CA, USA) of the mouse brain was performed, T2-weighted (T2w) imaging was carried out, and statistical analysis was performed. The results showed that CVB3(mu) could significantly promote the absorption of cerebral hematomas ( Figure 9 and Figure 10 ). The above results illustrate that recombinant coxsackievirus CVB3(mu) significantly increases the absorption rate of ipsilateral brain edema in a severe TBI mouse model and has a significant therapeutic effect on TBI.

[0057] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not restrictive. Although the present invention has been described in detail with reference to the preferred embodiments, those of ordinary skill in the art should understand that the technical solutions of the present invention can be modified or equivalently replaced without departing from the purpose and scope of the technical solutions of the present invention, and they should all be covered by the scope of the claims of the present invention.

Claims

1. An engineered attenuated Coxsackie B3 virus vector for use in the preparation of a drug for increasing the absorption rate of lateral cerebral edema in patients, wherein the engineered attenuated Coxsackie B3 virus vector comprises a nucleic acid sequence as shown in SEQ ID NO:

1.

2. The application according to claim 1, characterized in that: The medicine comes in the form of an injection, tablet, powder, or patch.

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