Application of IL-37b gene in the preparation of drugs for the prevention and treatment of diseases caused by HSV infection

Abstract

This application discloses the application of the IL-37b gene in the preparation of drugs for the prevention and treatment of diseases caused by HSV infection. In animal model experiments, overexpression of the IL-37bΔ1-45 gene significantly prevented HSK relapse and alleviated corneal function in relapsed mouse models, reduced corneal tissue inflammation and infiltration, reduced corneal stromal fibrosis, and significantly decreased viral load in mouse tears, suggesting that IL-37bΔ1-45 may be a promising new method for preventing HSK relapse and has broad application prospects.

Description

Invention Field

[0001] This application pertains to the fields of infectious disease treatment and gene therapy. Specifically, this application provides the use of the IL-37b gene in the preparation of drugs for the prevention and treatment of diseases caused by HSV infection. Background Technology

[0002] Herpes simplex virus (HSV) belongs to the subfamily Alpha Herpesviridae and is an enveloped spherical virus. It is divided into herpes simplex virus type I (HSV-1) and herpes simplex virus type II (HSV-2). Both HSV-I and HSV-II are pathogenic viruses that can cause herpetic stomatitis, herpetic encephalitis, herpetic keratitis, genital herpes, or neonatal encephalitis. Currently, there is no cure, and no vaccine to prevent infection.

[0003] Humans are the only natural host for this virus, and HSV-1 infection is very common in humans under natural conditions. It is estimated that more than one-third of the world's population has had recurrent herpetic stomatitis, and 30% to 90% of survey subjects have antibodies against herpes simplex virus in their serum, indicating that they have had or are currently experiencing herpes simplex virus infection. Oral herpes simplex is an acute infectious disease of the oral mucosa and perioral skin caused by HSV infection, characterized primarily by herpes vesicles. In traditional Chinese medicine, it is called "heat sores" and is the most common viral infection in oral clinics. Primary herpetic stomatitis is the most common oral lesion caused by herpes simplex virus type 1, which may manifest as a more severe form of gingivostomatitis—acute herpetic gingivostomatitis. Furthermore, recurrent herpetic stomatitis occurs in 30% to 50% of cases after the primary herpes infection has healed, regardless of the extent of the lesions. Recurrent infections typically occur on or near the lips, hence the name recurrent cold sores.

[0004] Herpes simplex keratitis (HSK) is an infectious corneal disease caused by HSV-1, which can lead to blindness in severe cases. It is estimated that 50%-80% of the world's population are HSV-1 carriers, with 1.5 million new cases of keratitis caused by HSV infection each year, of which 40,000 suffer vision loss or blindness. Humans are the only natural host for HSV-1, and infection is very common. Most people are carriers; once infected with HSV-1, the virus remains dormant in neurons for life. When the body's immunity is low, HSV-1 can attack and cause inflammation, specifically in the cornea, causing viral keratitis symptoms. HSV-1-induced HSK is a leading cause of corneal scarring and corneal opacity leading to blindness worldwide.

[0005] Currently, the first-line drugs for treating HSK in clinical practice are broad-spectrum antiviral drugs such as acyclovir (ACV) and ganciclovir. In addition, glucocorticoids, interferon, and cyclosporine are used as adjunctive therapy. New formulations of acyclovir, such as liposomes and micelles, are currently under development (Research Progress in Drug Treatment of Herpes Simplex Virus Matrix Keratitis, Journal of Ophthalmology, 2022, Vol. 37, No. 8). Mild cases show good antiviral response to initial antiviral drug use, but this only temporarily inhibits HSV-1 replication at the affected site and cannot eliminate the virus. Furthermore, it is ineffective against viruses latent in nerves, leading to disease recurrence. In addition, long-term use of broad-spectrum antiviral drugs can easily lead to drug resistance, affecting subsequent treatment outcomes. Severe HSK patients require corneal transplantation to restore vision, but corneal donor resources are scarce, and the transplant needs of all patients cannot be met. Furthermore, there is a possibility of disease recurrence after corneal transplantation. Therefore, there is an urgent need to find new therapeutic drugs and new therapeutic targets to solve the above problems.

[0006] IL-37 is a newly discovered cytokine from 2000, with five isoforms (IL-37a-e). IL-37b protein is known to have negative immunomodulatory effects and plays an important role in controlling various inflammatory diseases. Previous studies have shown that IL-37b protein can alleviate CVB3-induced myocarditis; further research has shown that IL-37b protein has an inhibitory effect on influenza virus replication. However, there are currently no research reports, either domestically or internationally, on IL-37b gene inhibition of HSV-1 or HSV-2, nor are there any reports on the application of the IL-37b gene in the preparation of drugs for the prevention and / or treatment of recurrent herpes simplex virus infection. Summary of the Invention

[0007] Existing treatments for HSV infection have the aforementioned problems. To discover and expand new uses for the IL-37b gene and provide new treatment options for HSV infection, this invention provides the use of AAV9 to deliver the IL-37b gene in the preparation of drugs for treating HSV-related diseases. To address the above problems, the technical solution adopted by this invention is as follows:

[0008] On the one hand, this application provides the use of the IL-37b gene in the preparation of drugs for the prevention and treatment of diseases caused by HSV infection.

[0009] On the other hand, this application provides the use of reagents that increase IL-37b gene expression in the preparation of drugs for the prevention and treatment of HSV infection.

[0010] Furthermore, the reagent for increasing IL-37b gene expression is a vector that overexpresses the IL-37b gene.

[0011] Furthermore, the vector is an adenovirus vector or an adeno-associated virus vector.

[0012] Furthermore, the carrier is an AAV9 carrier.

[0013] Furthermore, the IL-37b gene encodes a protein with the sequence SEQ ID NO.6.

[0014] Furthermore, the IL-37b gene sequence is shown in SEQ ID NO.5.

[0015] The IL-37b gene in this application can be selected from known and undiscovered IL-37b genes and their fragments. These gene sequences can be optimized with codons and truncated as necessary according to the requirements of the vector and the host. Various conventional tags for detection or purification, such as FLAG tags and His tags, can be routinely added or removed from the gene and protein sequences in this application. Those skilled in the art can also add signal peptides, initiating methionine residues, etc., according to the needs of expression.

[0016] Furthermore, the HSV infection is gingivostomatitis, keratitis, encephalitis, reproductive system infection, neonatal herpes, or cervical cancer caused by HSV infection.

[0017] Furthermore, the HSV infection is keratitis.

[0018] Furthermore, the HSV infection is keratitis caused by recurrent HSV infection.

[0019] Furthermore, the drug is an injectable dosage form.

[0020] On the other hand, this application provides a medicament for the prevention or treatment of HSV infection, the medicament comprising an AAV9 vector overexpressing the IL-37b gene.

[0021] Furthermore, the AAV9 vector overexpressing the IL-37b gene is the only effective component in the drug.

[0022] Furthermore, the drug is an injectable form.

[0023] On the other hand, the present invention provides a method for non-therapeutic inhibition of HSV proliferation or inhibition of HSV infection, the method comprising administering an AAV9 vector overexpressing the IL-37b gene.

[0024] Furthermore, the drug or method is applied to mice or humans.

[0025] The drug in this application can be a dosage form known in the art, such as oral administration, injection, or topical administration. The excipients / components therein include, but are not limited to, solvents, cosolvents, suspending agents, dispersants, acidity regulators, isotonic agents, fillers, binders, coating agents, lubricants, capsule shells, flavoring agents, sweeteners, etc., which can be conventionally selected by those skilled in the art based on pharmaceutical knowledge.

[0026] The non-therapeutic methods described in this invention include, but are not limited to, methods used for scientific research and methods used to suppress non-pathogenic latent infections.

[0027] This invention relates to the application of the IL-37bΔ1-45 gene in the preparation of drugs for the prevention and treatment of recurrent HSK, specifically a treatment regimen using AAV9 to deliver the IL-37bΔ1-45 gene for the treatment of HSK and HSK recurrence. In this embodiment, mice with a recurrent HSK mouse model were given a subconjunctival injection of AAV9-IL-37bΔ1-45 adeno-associated virus 30 days before the model was established. Overexpression of IL-37bΔ1-45 significantly prevented corneal function in the HSK recurrence mouse model, reduced corneal tissue inflammation and infiltration, reduced corneal stromal fibrosis, and significantly decreased viral load in mouse tears. These experimental results suggest that IL-37bΔ1-45 holds promise as a novel method for preventing HSK recurrence. This invention experimentally demonstrates that AAV9-IL-37bΔ1-45 overexpression can improve the pathological state of HSK cornea by directly inhibiting viral load in corneal tissue and mouse tears, thereby achieving better therapeutic effects and showing promising application prospects. Attached Figure Description

[0028] Figure 1 Image of GV629 plasmid;

[0029] Figure 2 This is a flowchart of the experimental process of the present invention;

[0030] Figure 3 Fluorescence detection image of successful overexpression of IL-37b gene in corneal tissue;

[0031] Figure 4 Statistical results showing the reversal of weight loss in relapsed HSK model mice with corneal overexpression of the IL-37b gene;

[0032] Figure 5 The statistical results show that the aggravation of disease score in relapsed HSK model mice was reversed after corneal tissue overexpression of IL-37b gene;

[0033] Figure 6 HE staining of corneal tissue sections from relapsed HSK model mice with corneal tissue overexpressing the IL-37b gene;

[0034] Figure 7 The image shows the results of sodium fluorescein assay in mice with corneal tissue overexpression of the IL-37b gene, which showed significant improvement in corneal damage in recurrent HSK model mice.

[0035] Figure 8 The figure shows the results of HSV-1 viral load levels in the tears of relapsed HSK model mice with corneal tissue overexpression of the IL-37b gene. Detailed Implementation

[0036] Example 1: Construction of a corneal tissue-specific AAV vector for mouse Il-37b gene expression

[0037] Firstly, the AAV-293 cell line was provided by Shanghai Jikai Company. The AAV-293 cell line is used to produce adeno-associated virus (AAV). AAV-293 cells are derived from the commonly used HEK293 cell line and can produce higher viral titers. The AAV packaging process is as follows: First, the exogenous gene (Il-37b) is cloned into an adeno-associated virus vector. Then, the recombinant plasmid carrying the exogenous gene, along with helper plasmids AAV Helper Vector and pAAV-rep / capVector, is co-transfected into AAV-293 cells. A large amount of recombinant virus is produced in the cells 72 hours after transfection. The culture medium supernatant and cell pellet are harvested. The virus in the culture medium supernatant is precipitated using PEG8000. The cell pellet is lysed to collect the virus, and the viruses obtained from the cell pellet and supernatant are combined. Finally, the collected virus is purified and concentrated, and its titer and purity are tested before use.

[0038] I. Preparation of Adeno-Associated Virus Overexpression Clones

[0039] 1. Target gene and tool vector information

[0040] Target gene name: Il-37b(AAH20637.1(46-218aa)-3×flag);

[0041] Adeno-associated virus (AAV9-Val46-Asp218) was constructed using the vector GV629. The GV629 vector plasmid contained AAV-pTIE-EGFP-MCS-SV40 polyA, digested with NheI / XhoI enzymes. All components were purchased from Shanghai Jikai Gene Chemical Technology Co., Ltd., and the plasmid was constructed and packaged into AAV9-Val46-Asp218 by this company. The viral element sequence was: TIEp-EGFP-MCS-SV40 PolyA, with cloning sites at NheI / XhoI. Furthermore, TIE (165-1458) is a mouse vascular endothelial cell-specific promoter that promotes the expression of the human Il-37bΔ1-45 gene in mouse vascular endothelial cells. EGFP is a fluorescent protein used to observe the in vivo infection efficiency of AAV9. Multiple cloning sites (MCS) are short DNA sequences containing up to 20 restriction enzyme sites that can serve as insertion sites for foreign genes, including the NheI and XhoI sites used in this study. SV40 polyA acts as a transcription termination signal. Vector map as shown... Figure 1 As shown. AAV9-vector is the empty vector of GV629. AAV9-IL-37b is a GV629 vector packaging the human Il-37bΔ1-45 gene (Accession#AAH20637.1), from Val46 to Asp218 (AAH20637.1(46-218aa)-3×Flag). The following primers were used to amplify the Il-37b gene: forward primer: 5'-ACGAGCTGTACAAGGCTAGCATGGTGCACACTAGCCCTAAG-3', reverse primer: 5'-GCTATCATATGTTACTCGAGTCATTTGTCGTCATCATCCTTATAG-3'. The vector and the human Il-37b gene sequence were digested with restriction enzymes NheI and Xhol, and then cloned using the In-Fusion recombination method. DNA sequencing confirmed the successful construction of the recombinant vector.

[0042] 2. Vector enzyme digestion

[0043] Prepare the enzyme digestion system as follows: 42 μL ddH2O, 5 μL 10x CutSmart Buffer, 2 μL purified plasmid DNA (1 μg / μL), and 1 μL NheI restriction endonuclease (10 U / μL). Gently pipette the digestion system to mix well, centrifuge, and incubate at 37°C for 3 h or overnight. Perform agarose gel electrophoresis on the vector digestion products and recover the target band.

[0044] 3. Obtaining the target gene fragment

[0045] (1) Il-37b gene primers were designed and synthesized, as shown in Table 1 below:

[0046] Table 1 Primers

[0047]

[0048] Primer description: Contains exchangeable base pairs, restriction enzyme sites, and the 5' end sequence of the target gene for PCR extraction of the target gene.

[0049] (2) PCR amplification of the target gene fragment:

[0050] The first step is to clone the foreign gene into a suitable viral vector: Il-37b is cloned into a vector containing an ITR / MCS. The inverted terminal repeat (ITR) sequence in these vectors provides the cis-acting software necessary for all AAV replication and packaging. Following the PrimeSTAR HSDNA polymerase instructions, the reaction mixture is prepared and placed in a PCR instrument for the reaction. After the reaction, the PCR products are recovered by electrophoresis.

[0051] 4. Exchange of PCR products with vector

[0052] Using a homologous recombination kit, the following reaction mixture was prepared in an ice-water bath: 3.5 μL ddH₂O, 2 μL 5×CE II Buffer, 2.52 μL digested vector DNA, 1 μL purified PCR product, and 1 μL Exnase™ II. After preparation, the mixture was gently mixed by pipetting and briefly centrifuged to avoid air bubbles. The reaction was incubated at 37°C for 30 min, followed by cooling in an ice-water bath for 5 min before immediate transformation.

[0053] 5. Transformation

[0054] Add 10 μL of the exchange reaction product to 100 μL of competent cells, gently tap the tube wall a few times to mix, and incubate on ice for 30 min. Heat shock at 42℃ for 90 s, then incubate in an ice-water bath for 2 min. Add 500 μL of antibiotic-free LB medium and incubate at 37℃ with shaking for 1 h. Spread an appropriate amount of the bacterial culture evenly onto a plate containing the appropriate antibiotic and incubate upside down in a constant temperature incubator for 12-16 h.

[0055] 6. Colony PCR identification

[0056] Prepare the identification reaction system: 9 μL ddH2O, 10 μL 2×Taq Plus Master Mix, and 0.5 μL each of forward and reverse primers (10 μM). Vortex to mix and briefly centrifuge. Pick a single colony and add it to 20 μL of the identification system, mix by pipetting, and place in a PCR instrument for reaction.

[0057] Table 2 Primers for PCR identification

[0058]

[0059] The identified positive clones were inoculated into an appropriate amount of LB liquid medium containing the corresponding antibiotics and cultured at 37°C for 12-16 hours. A suitable amount of bacterial culture was then sequenced. The sequencing results were compared and analyzed with the target gene sequence. The sequencing assembly results are shown below, indicating that the sequencing results are completely consistent with the target sequence. The construction of the Il-37b (AAH20637.1(46-218aa)-3×flag) gene sequence involved mouse codon optimization. The Il-37bΔ1-45 gene and protein sequences are as follows:

[0060]

[0061] II. Plasmid Transfection and AAV Virus Harvesting

[0062] 1. 24 hours before transfection, AAV-293 cells in logarithmic growth phase were digested with trypsin, and the cell density was adjusted to approximately 5 × 10⁶ cells / year using culture medium containing 10% serum. 6 Cells / 15mL were reseeded into 10cm cell culture dishes and cultured at 37℃ in a 5% CO2 incubator. Transfection was possible after 24 hours when the cell density reached 70%–80%.

[0063] 2. Replace the culture medium with one containing 2% serum 2 hours before transfection;

[0064] 3. The recombinant expression plasmid, along with pHelper (carrying genes derived from adenovirus) and pAAV-RC (carrying AAV replication and capsid genes), was co-transfected into AAV-293 cells (providing trans-acting factors required for AAV replication and packaging). Add the prepared DNA solutions (5 μg each of GV629-Il-37b recombinant vector plasmid, pHelper vector plasmid, and AAV-RC plasmid) to sterilized centrifuge tubes, mix thoroughly with the corresponding volumes of GIC transfection reagent, adjust the total volume to 1 mL, and incubate at room temperature for 15 min.

[0065] 4. Slowly add the mixture dropwise to the culture medium of AAV-293 cells, mix well, and incubate in a 37°C, 5% CO2 cell culture incubator;

[0066] 5. After culturing for 6 hours, discard the culture medium containing the transfection mixture, add 10 mL of PBS solution to wash once, gently shake the culture dish to wash away the residual transfection mixture, and then discard it.

[0067] 6. Slowly add 10 mL of cell culture medium containing 5% serum, and continue culturing at 37°C in a 5% CO2 incubator for 48 to 72 hours. Recombinant AAV is then assembled in the packaging cells.

[0068] III. AAV9 Virus Concentration and Purification

[0069] 1. Collect AAV virus particles from infected AAV-293 cells. AAV particles typically accumulate in the packaged cells, so collecting the cells and then lysing them to release the AAV particles into the supernatant allows for the recovery of most of the particles. Therefore, depending on the cell state, collect the AAV-293 cell supernatant and cells 72 hours after transfection (0 hours is considered the start of the transfection period).

[0070] 2. Centrifuge at 3000g for 5 minutes at 4℃ to separate the cells from the supernatant;

[0071] 3. Add the Gilead AAV virus concentration kit to the supernatant to obtain the virus from the supernatant. Resuspend the cell pellet in resuspension buffer, and repeatedly freeze and thaw four times in liquid nitrogen at 37°C, followed by centrifugation. Then combine the two virus fractions.

[0072] 4. Balance the samples separately, and place the ultracentrifuge tubes containing the virus supernatant into the Beckman ultracentrifuge one by one. Set the centrifugation parameters to 63,000 rpm, centrifugation time to 2 hours, and centrifugation temperature to 18°C.

[0073] 5. Aspirate the separated layer containing the virus. Place it in an ultrafiltration column for ultrafiltration until a colorless or pale pink clear liquid is obtained;

[0074] 6. Sterilize the obtained virus solution by passing it through a 0.22μm filter membrane and dispense it as required;

[0075] 7. Prepare samples for testing.

[0076] AAV virus titer detection method: The AAV virus titer is detected by qPCR, and the result is the number of virus particles obtained after packaging. The principle is to perform real-time quantitative PCR using adeno-associated virus genome-specific primers. Within the linear range of the quantitative curve, the ratio of the Ct value to the Ct value of a plasmid with a known copy number is the initial copy number of the viral genome.

[0077] AAV virus purity detection method: It can be detected by a sensitive silver staining method. The AAV capsid protein contains three subunits: VP1 (82kDa), VP2 (72kDa), and VP3 (62kDa), and the ratio of VP1:VP2:VP3 is 1:1:10. Therefore, a high-purity AAV will only show three bands in SDS-PAGE analysis.

[0078] Example 2: Mouse HSK relapse model and evaluation indicators

[0079] First, a mouse model of HSK relapse was constructed. Based on a previously published study (Luo, Z., et al., Inhibitory effects of baicalein against herpes simplex virus type 1. ActaPharm Sin B, 2020.10(12):p.2323-2338.), a mouse model of acute HSV-1 corneal infection was established.

[0080] After anesthetizing the mice, the cornea of ​​their right eye was incised in a "#" shape using a 33-gauge needle under a dissecting microscope. Then, 10 μL of HSV-1F strain (1.2 × 10⁻⁶) was injected. 6 PFU (Polydioxanone) strain stock solution was instilled into the injured eye. HSV-1 strain F was a gift from Dr. Hu Kai, Director of the Ophthalmology Department at Nanjing Gulou Hospital. In a recurrent HSK model, 6-week-old male BALB / c mice were primarily inoculated with 1×10⁻⁶ PFU in their right eye. 5 The PFU-1F strain of HSV-1 was used, consistent with an acute HSV-1 corneal infection model. Subsequently, mice were treated with 1 mL of human serum (Sigma, Temecula, CA, USA) 1 day after HSV-1 infection (1 dpi). This serum was anti-HSV reactive, with a 50% virus-neutralizing effective dose of 1:800, to protect mice from HSK infection during the initial infection period. Mice surviving the acute infection were then maintained for 35 dpi, with their right eyes continuously exposed to 302 nm ultraviolet B (UV-B) light for 3 minutes. Levofloxacin eye drops were administered once daily to control bacterial infection, starting 5 hours after HSV-1 infection of the right eye. Corneal images were taken using a stereomicroscope to observe corneal morphology. The condition of the injured cornea was photographed under a microscope using cobalt blue light at a wavelength of 460 nm. Disease severity was assessed using the HSK disease score and fluorescein staining score.

[0081] The HSK disease score (0-5 points) was determined under blinded conditions according to previously reported criteria (Luo, Z., et al., Inhibitory effects of baicalein against herpes simplex virus type 1. Acta Pharm Sin B, 2020. 10(12): p. 2323-2338.), as shown in Table 3. Based on previous research (Marsh, P. and S.C. Pflugfelder, Topical nonpreserved methylprednisolone therapy for keratoconjunctivitis sicca in...), the HSK disease score was determined under blinded conditions. syndrome. Ophthalmology, 1999. 106(4): p. 811-6.), fluorescein staining scores were graded from 0 (none) to 12 (most severe), see Table 4.

[0082] Table 3 HSK Disease Scores

[0083]

[0084] Table 4. Scoring of sodium fluorescein staining

[0085]

[0086] Example 3: AAV9-IL-37bΔ1-45 gene therapy can reduce corneal damage in recurrent HSK mice.

[0087] Mouse experimental procedure as follows Figure 2 As shown.

[0088] First, a mouse model of HSK relapse was established. Then, the relapsed HSK mice were divided into two groups: one group received AAV9-IL-37bΔ1-45, and the other group received AAV9 virus without any gene (AAV9-Vector). On day 7 (dpi) of HSV-1 infection of the mouse eyes, AAV9 virus carrying the Il-37b gene (AAV9-IL-37bΔ1-45) was injected subconjunctivally into the eyes of the experimental group mice, while AAV9 virus without any gene (AAV9-Vector) was injected subconjunctivally into the eyes of the control group mice. The viral load injected was 1.0 × 10⁻⁶ in both groups. 11 vg. In a relapsed HSK mouse model treated with AAV9-IL-37bΔ1-45, 1.0 × 10 11HSV-1 corneal-infected mice were infected with AAV9-Vector or AAV9-IL-37bΔ1-45 of vg and injected subconjunctivally with 10 μL of AAV9-IL-37bΔ1-45 at 5 dpi for 3 weeks. Then, their right eyes were exposed to UV-B at 35 dpi to further induce HSV-1 corneal relapse.

[0089] Figure 3 The results showed that on day 21 after injection of AAV9-IL-37bΔ1-45, corneal tissues from both the control and experimental groups were collected, and the expression of IL-37b in the corneal tissues was detected by immunofluorescence staining. Figure 3 As shown, the control group only showed fluorescent staining of DAPI with cell nuclei, while the experimental group showed red FLAG fluorescent labeling of the entire corneal tissue, indicating that exogenous IL-37b was successfully expressed in the corneal tissue. Figure 4 The results showed that body weight was recorded at 1 dpi, 3 dpi, 5 dpi, 35 dpi, 38 dpi and 40 dpi (n=5). The statistical results showed that AAV9-IL-37bΔ1-45 injection could reverse the weight loss in relapsed HSK mice. Figure 5 The results showed that the disease score of relapsed HSK mice was assessed at 1 dpi, 5 dpi, 35 dpi, 38 dpi and 40 dpi, and the results showed that AAV9-IL-37bΔ1-45 injection could alleviate the ocular surface disease state in relapsed HSK mice. Figure 6 The study showed that corneal damage was indicated by fluorescein staining of corneal tissue at 5 dpi, 35 dpi, 38 dpi, and 40 dpi. Results indicated that AAV9-IL-37bΔ1-45 injection reduced corneal damage in recurrent HSK mice. Representative images of corneal morphology were captured under a stereomicroscope. Fluorescein staining scores (n=5) were also analyzed. Figure 7 Display: Representative H&E images of corneal tissue in relapsed HSK mice after AAV9-IL-37bΔ1-45 treatment at 5 dpi, 35 dpi, 38 dpi and 40 dpi. The results show that AAV9-IL-37bΔ1-45 injection can reduce the level of inflammation and pathological state of corneal tissue in relapsed HSK mice. Figure 8 The results showed that the HSV-1F viral titer in mouse tears was determined by viral plaque detection method, and that AAV9-IL-37bΔ1-45 injection could reduce the viral load in the tears of relapsed HSK mice.

[0090] The above results indicate that AAV9-IL-37bΔ1-45 can improve the corneal pathology of mice with HSK relapse by reducing the viral load in mouse tears, thereby achieving a better effect in preventing HSK relapse. This invention has good application prospects.

Claims

1. The application of a reagent for increasing IL-37b gene expression in the preparation of a drug for preventing recurrence of herpes simplex keratitis; wherein the reagent for increasing IL-37b gene expression is a vector for overexpressing the IL-37b gene; and the IL-37b gene sequence is shown in SEQ ID NO.

5.

2. The application according to claim 1, wherein the vector is an adenovirus vector or an adeno-associated virus vector.

3. The application according to claim 2, wherein the carrier is an AAV9 carrier.

Images