A monoclonal antibody 6D11 against DENV NS1 protein, its preparation method and application

Abstract

This invention provides a monoclonal antibody 6D11 and its applications. The CDR amino acid sequence of the heavy chain of the anti-monoclonal antibody 6D11 is shown in SEQ ID No. 1, and the CDR amino acid sequence of the light chain is shown in SEQ ID No. 2. This invention also provides the application of monoclonal antibody 6D11 in the preparation of drugs for treating dengue virus infection. The monoclonal antibody 6D11 of this invention has advantages such as competitive binding to DENV1-4NS1 protein, inhibition of vascular leakage symptoms, and protection against lethal damage from DENV infection in vivo, and has significant potential therapeutic value against dengue virus.

Description

Technical Field

[0001] This invention relates to the field of bioengineering technology, and in particular to a method for preparing and applying a broad-spectrum monoclonal antibody D11 against NS1 protein that can inhibit vascular leakage symptoms caused by dengue virus infection. Background Technology

[0002] Dengue virus (DENV) is a mosquito-borne flavivirus, classified into four serotypes (DV-1, DV-2, DV-3, and DV-4). Infection with this virus typically leads to dengue fever and severe dengue fever, the latter of which can be fatal in severe cases. In recent years, cases of severe dengue fever have increased significantly, placing a heavy economic burden on global health systems. Controlling severe dengue fever remains a pressing global public health issue. Clinical features of severe dengue fever include severe bleeding, shock due to severe plasma leakage, or respiratory distress. Gastrointestinal symptoms and bleeding tendencies are also prominent in severe dengue fever cases.

[0003] One of the keys to preventing severe dengue fever is dengue vaccination. Current dengue vaccine development is hampered by antibody-dependent enhancement (ADE), which means that cross-reactive antibodies against one serotype can enhance subsequent infection with a different serotype. Therefore, stimulating stable and durable immunity against all four DENV serotypes and reducing the impact of ADE on the protective efficacy of dengue vaccines is crucial for dengue vaccine development.

[0004] Currently, NS1 protein has become a promising vaccine target for the prevention and treatment of dengue virus (DENV) infection. NS1 is a highly conserved protein in dengue virus, involved in viral replication and immune evasion. Studies have shown that T-cell immune responses to NS1 play a role in preventing dengue infection. Furthermore, inoculation with DENV1, DENV3, or DENV4 NS1 can prevent DENV2 infection, and monoclonal antibodies against DENV2 effectively protect mice from all serotypes of DENV. However, the screening of broad-spectrum NS1 antibodies has been limited by the lack of stimulation from natural hexamer antigens, the low efficiency of screening for conserved epitopes due to immunodeficiency, and the limited availability of structural analysis techniques. Therefore, currently reported NS1 antibodies are insufficient in simultaneously inhibiting vascular leakage caused by four serotypes of dengue infection. To date, no broad-spectrum protective antibodies applicable to the clinical treatment of severe dengue fever have been obtained. Summary of the Invention

[0005] The purpose of this invention is to provide a broad-spectrum protective antibody against dengue virus NS1 protein, 6D11, its preparation method, and its applications. The broad-spectrum protective antibody described in this invention is a monoclonal antibody produced through hybridoma cells. Specifically, this antibody is obtained from the hybridoma cell line 6D11, which is derived from BALB / c mouse spleen cells and mouse myeloma cells SP2 / 0, sequentially immunized with DENV1-4 NS1 protein, through fusion, selection, cloning, and stable passage. This hybridoma cell line stably secretes the monoclonal antibody 6D11.

[0006] In a first aspect, the present invention provides a monoclonal antibody 6D11 against DENV1-4 NS1 protein, wherein the monoclonal antibody isotype is IgG1 and can specifically and broadly recognize DENV1-4 NS1 protein. The CDR amino acid sequence of the heavy chain of the antibody 6D11 is shown in SEQ ID No. 1, and the CDR amino acid sequence of the light chain is shown in SEQ ID No. 2.

[0007] SEQ ID No. 1: Heavy chain CDR amino acid sequence

[0008] QVTLKESGPGILKPSQTLSLTCSFSGFSLSTSGVGVGWIRQPSGKGLEWLAHIWWDDDKFYNPSLKSQLTISKDSSRNQVFLKITSVDTADTATYYCARIYGNFSIFAMDYWGQGTSVTVSS.

[0009] SEQ ID No. 2: Light chain CDR amino acid sequence

[0010] DIQMTQTTSSLSASLGDRVTISCSASQGISNYLNWYQQKPDGTVKLLIYYTSNLHSGVPSRFSGSGSGTDYSLTISNLEPEDIATYYCQQYSKLYTFGGGTKLEIKR.

[0011] SEQ ID No. 3: Heavy chain CDR nucleotide sequence

[0012] CAAGTTACTCTAAAAGAGTCTGGCCCTGGGATATTGAAGCCCTCACAGACCCTCAGTCTGACTTGTTCTTTCTCTGGGTTTTCACTGAGCACTTCTGGTGTGGGTGTAGGCTGGATTCGTCAGCCTTCAGGGAAGGGTCTGGAGTGGCTGGCACACATTTGGTGGGATGATGATAAATTCTAT AACCCATCCCTGAAGAGCCAGCTCACAATCTCCAAGGATTCCTCCAGAAACCAGGTTTTCCTCAAGATCACCAGTGTGGACACTGCAGATACTGCCACTTACTACTGTGCTCGAATATATGGTAACTTTTCTATCTTTGCTATGGACTACTGGGGTCAAGGCACCTCAGTCACCGTCTCCTCA.

[0013] SEQ ID No. 4: Light chain CDR nucleotide sequence

[0014] GATATCCAGATGACACAAACTACATCCTCCCTGTCTGCCTCTCTGGGAGACAGAGTCACCATCAGTTGCAGTGCAAGTCAGGGCATTAGCAATTATTTAAACTGGTATCAGCAGAAACCAGATGGAACTGTTAAACTCCTGATCTATTACACATCAAATTT ACACTCAGGAGTCCCATCAAGGTTCAGTGGCAGTGGGTCTGGGACAGATTATTCTCTCACCATCAGCAACCTGGAACCTGAAGATATTGCCACTTACTATTGTCAGCAGTATAGTAAGTTGTACACGTTCGGAGGGGGGACCAAGCTGGAAATAAAACGG.

[0015] In some embodiments, the monoclonal antibody 6D11 is a murine antibody.

[0016] In some embodiments, the monoclonal antibody 6D11 is of the IgG1 subtype.

[0017] Secondly, this invention proposes a method for screening monoclonal antibodies, 6D11, against DENV1-4 NS1 proteins based on a sequential immunization strategy. This method induces an immune response to conserved epitopes by sequentially immunizing four serotypes of DENV NS1 proteins and improves the induction efficiency of broadly cross-reactive antibodies. Using this method, a monoclonal antibody, 6D11, capable of broadly binding to four serotypes of DENV NS1, was identified.

[0018] A method for preparing the above-mentioned monoclonal antibody 6D11 against DENV1-4 NS1 protein, the method comprising the following steps:

[0019] (1) Immunization of animals: BALB / c mice were immunized with serum types DV-1, DV-2, DV-3 and DV-4 using a sequential immunization strategy.

[0020] (2) Preparation of hybrid cells: Culture mouse myeloma cells SP2 / 0 and maintain them in a good growth state;

[0021] The spleen lymphocytes of the mouse in step (1) were obtained by polyethylene glycol-mediated cell fusion. They were mixed with the above SP2 / 0 cells and centrifuged to mediate cell fusion. The fused cells were cultured under appropriate conditions to obtain hybrid cells.

[0022] (3) Screening of hybridoma cells: The hybrid cells were cultured in HAT selective medium, which is used to screen for cells with hypoxanthine phosphoribosyltransferase (HPRT) or thymidine kinase (TK) activity deficiency, and positive clones were obtained by screening.

[0023] (4) Cloning of hybridoma cells: Select the culture wells with the highest antibody titer and showing single-clonal cell growth, perform limiting dilution again, perform limiting dilution more than 4 times, and passage more than 20 times to obtain a stable and efficient single-specific hybridoma cell line 6D11 expressing the monoclonal antibody 6D11; the amino acid sequences of the heavy chain CDR and light chain CDR of the monoclonal antibody 6D11 are shown in SEQ ID No.1 and SEQ ID No.2, respectively;

[0024] (5) Production of monoclonal antibodies using BALB / c mice: Select 8-10 week old BALB / c mice, inject an appropriate amount of Freund's incomplete adjuvant into the peritoneum one week later, and then inoculate the abdomen again with PBS buffer containing hybridoma cells 6D11. When the mouse abdomen is obviously distended and the mouse is about to die, collect the ascites fluid, centrifuge it, and determine the antibody titer.

[0025] (6) The monoclonal antibody was purified from mouse ascites fluid.

[0026] Thirdly, this invention provides a monoclonal antibody hybridoma line targeting the DENV1-4 NS1 protein, namely 6D11. This cell line, after being cryopreserved in liquid nitrogen, grows well upon thawing, and antibody secretion shows no decline after more than 20 generations of continuous culture.

[0027] Fourthly, the present invention provides the potential antiviral therapeutic value and application of the monoclonal antibody 6D11.

[0028] The use of any of the above-mentioned monoclonal antibodies 6D11 in the preparation of drugs or vaccines for the prevention or treatment of dengue virus infection.

[0029] The use of any of the above-mentioned monoclonal antibodies 6D11 in the preparation of drugs or vaccines that inhibit or treat vascular leakage caused by dengue virus infection.

[0030] The use of any of the above-mentioned monoclonal antibodies 6D11 in the preparation of drugs or vaccines to inhibit or treat dengue fever.

[0031] In some of these embodiments, the dengue fever is severe dengue fever.

[0032] The monoclonal antibody 6D11 described in this invention has the ability to competitively bind to the DENV1-4 NS1 protein and can inhibit vascular leakage symptoms, while providing protection against fatal damage caused by DENV infection in vivo.

[0033] The anti-DENV NS1 protein monoclonal antibody described in this invention is capable of treating at least two, three, or four dengue virus serotypes. Therefore, in some embodiments, the antibody molecule is administered to a patient infected with dengue virus or at risk of dengue virus infection, when no test is performed to determine the dengue virus serotype, for example, the dengue virus serotype may be unknown. In some of these embodiments, the dengue virus has serotypes DV-1, DV-2, DV-3, or DV-4.

[0034] Fifthly, the present invention provides a pharmaceutical composition for treating dengue virus infection, comprising any of the above-mentioned monoclonal antibodies 6D11 and a pharmaceutically acceptable carrier.

[0035] In some of these embodiments, the drug is used to treat severe dengue fever caused by dengue virus infection.

[0036] In some of these embodiments, the pharmaceutical composition is a pharmaceutical preparation or a vaccine.

[0037] In a sixth aspect, the present invention provides a method for neutralizing dengue virus, comprising contacting dengue virus with monoclonal antibody 6D11.

[0038] In a seventh aspect, the present invention provides a nucleotide encoding a variable region of the antibody heavy chain and / or light chain of an anti-monoclonal antibody 6D11.

[0039] In some of these embodiments, the CDR nucleotide sequence of the heavy chain of the monoclonal antibody 6D11 is shown in SEQ ID No. 3, and the CDR nucleotide sequence of the light chain is shown in SEQ ID No. 4.

[0040] The monoclonal antibody 6D11 obtained by screening in this invention has a broad-spectrum binding ability against four serotypes (DV-1, DV-2, DV-3, or DV-4) of DENV1-4 NS1 protein (especially with better inhibitory effects on DENV2 and DENV3 NS1 proteins). In in vitro cell models, it competitively inhibits the binding of NS1 protein to target cells and effectively inhibits the biological functions of NS1 protein. The antibody 6D11 can delay the onset and progression of symptoms in AG129 mouse models and protect 25% of mice from lethal doses of DENV2. Therefore, the monoclonal antibody 6D11 of this invention has potential antiviral applications in the design of dengue vaccine candidates, screening of therapeutic drugs, or preparation of antiviral therapeutics for DENV1-4 viral infectious diseases.

[0041] The monoclonal antibody 6D11 against DENV NS1 protein described in this invention possesses the ability to competitively bind to DENV1-4 NS1 protein, inhibit vascular leakage symptoms, and protect against lethal damage from DENV infection in vivo, thus exhibiting potential antiviral therapeutic value. The monoclonal antibody 6D11 developed in this invention is of significant importance and has application potential for the prevention and treatment of dengue fever infection (including severe dengue fever). Attached Figure Description

[0042] Figure 1 This is a technical roadmap for the preparation of the broad-spectrum protective monoclonal antibody 6D11.

[0043] Figure 2 This is a graph showing the results of SDS-PAGE assay for antibody purity.

[0044] Figure 3 This is a graph showing the results of ELISA identification of the binding curves of the monoclonal antibody 6D11 to the DENV1-4 NS1 protein.

[0045] Figure 4 This is a flow cytometry diagram showing the results of the monoclonal antibody 6D11 competitively inhibiting the binding of NS1 protein to target cells.

[0046] Figure 5 This is a graph showing the results of validating the protection of the monoclonal antibody 6D11 against fatal vascular leakage caused by DENV infection in the AG129 mouse model. Detailed Implementation

[0047] To facilitate understanding of the present invention, a more complete description will be provided below. The present invention can be implemented in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided to provide a thorough and complete understanding of the disclosure of the present invention.

[0048] Unless otherwise specified, experimental methods in the following examples were performed under standard conditions, such as those described in the fourth edition of *Molecular Cloning: A Laboratory Manual*, edited by Green and Sambrook, published in 2013, or according to the manufacturer's recommendations. All commonly used chemical reagents used in the examples are commercially available products.

[0049] Unless otherwise defined, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. The terminology used in this specification is for the purpose of describing particular embodiments only and is not intended to limit the invention. The term "and / or" as used in this invention includes any and all combinations of one or more of the associated listed items.

[0050] CDR: Ig, complementarity-determining region. The hypervariable region within the variable regions of the heavy and light chains of an antibody (Ab) constitutes the antigen (Ag) binding site of the antibody molecule. Because the antigen binding site is complementary to the antigen epitope structure, the hypervariable region is also called the complementarity-determining region of the antibody molecule.

[0051] The present invention will be further described in detail below with reference to specific embodiments.

[0052] Example 1: Method for preparing monoclonal antibodies

[0053] Please refer to the technical roadmap for the preparation of monoclonal antibody 6D11. Figure 1 .

[0054] 1.1 Immunization of Mice: Mice were immunized using a sequential immunization strategy. DENV1 NS1 protein was emulsified with Freund's complete adjuvant for primary immunization, followed by immunization with DENV2 NS1 protein, DENV3 NS1 protein, and DENV4 NS1 protein emulsified with Freund's incomplete adjuvant, in serotype order, with an interval of 2 weeks. The control group received the same immunization procedure using PBS instead of the antigen. In this experiment, 10 female BALB / c mice aged 4-6 weeks were randomly divided into two groups: a sequential immunization group and a blank control group. Primary immunization was performed first, followed by booster immunization 2 weeks later. The booster immunization interval was 2 weeks.

[0055] 1.2 Serum titer determination

[0056] Seven days after the last immunization of mice, serum was collected and the titer of the immunized mouse serum was determined by indirect ELISA. Blood was collected from the tail vein, allowed to stand at room temperature for 2 hours, and then centrifuged (10,000 rpm, 10 min). The clear supernatant was the mouse serum. DENV1-4 NS1 protein was diluted to 1 μg / mL with PBS, and 100 μL / well was added to a 96-well ELISA plate and incubated overnight at 4°C. After washing the plate three times with PBST, 300 μL / well of 5% skim milk powder solution was added, and the plate was blocked at 37°C for 2 h, followed by washing three times with PBST. 1 μL of mouse serum was taken, diluted to 1 mL with PBS, and 12 2-fold dilutions were performed. Then, 100 μL / well of the solution was added to the corresponding well of the ELISA plate and incubated at 37°C for 60 min. The negative control was PBS-immunized mouse serum, and the PBS solution served as a blank control. The serum sample was then discarded, and the plate was washed three times as described above. Add 100 μL / well of PBS diluted 5000-fold with HRP-labeled goat anti-mouse secondary antibody solution and incubate at 37°C for 60 min. Discard the solution and wash 5 times with PBST. Then add 100 μL / well of TMB substrate for color development and incubate at room temperature for 10 min. Add 50 μL / well of 2M concentrated sulfuric acid to stop the color development and read the absorbance of each well at OD450 using a microplate reader.

[0057] Example 2: Cell fusion and hybridoma cell screening

[0058] 2.1 Fusion of mouse spleen cells with myeloma cells SP2 / 0

[0059] A sterile spleen cell suspension (10) was collected from the spleen of immunized BALB / c mice. 8 (cells) and myeloma cells SP2 / 0 (2×10) 7Add 100 cells to a new 50 mL centrifuge tube, mix well, and bring the volume up to 30 mL. Centrifuge at 1000 rpm for 5 min and discard the supernatant. At 37°C, slowly add 1 mL of preheated 50% PEG solution over 1 min and allow it to react for 90 s. Immediately afterwards, add 15 mL of preheated 1640 medium at 37°C, specifically 1 mL over the first 30 s, 3 mL over the next 30 s, and the remaining 11 mL over the last min to dilute the PEG and terminate the fusion process. Add 50 mL of preheated 1×HAT 1640 medium at 37°C, mix well, and then use a bent-tip dropper to add 100 μL / well to a 96-well cell culture plate that has been pre-coated with feeder cells the previous day. Incubate at 37°C in a 5% CO2 incubator.

[0060] 2.2 Screening of hybridoma cells

[0061] Specific antibody titers were detected using indirect ELISA, and fusion cell lines exhibiting high titers and single-clone growth were selected for cloning. Cloning of hybridoma cells was performed using a limiting dilution method, and cell counts were performed on wells showing positive antibody detection. 150-200 cells were cultured in 1640 complete medium to prepare a cell suspension and seeded into 96-well plates containing feeder cells (100 μL per well). Cell growth was observed, and antibody levels in the supernatant were measured. Wells with the highest antibody titers and exhibiting single-clone cell growth were selected for further limiting dilution. This process was repeated at least four times until all diluted wells were positive. Cells from single-clone wells were passaged for at least 20 generations to obtain the stable and highly efficient hybridoma cell line 6D11 expressing the anti-DNEV NS1 protein monoclonal antibody.

[0062] Sequencing of hybridoma cells 6D11 revealed that the amino acids and nucleotides of the heavy chain CDR and light chain CDR of the secreted antibody 6D11 are as shown in SEQ ID No. 1-SEQ ID No. 4.

[0063] Example 3: Preparation of ascites fluid and purification and identification of monoclonal antibodies

[0064] One week after intraperitoneal injection of 0.5 ml / mouse Freund's incomplete adjuvant, 6-8 week old female BALB / c mice were injected with hybridoma cells 6D11 at a rate of 5 × 10⁻⁶. 6 Mice were immunized via intraperitoneal injection. Ascites production was observed daily. After approximately 7-10 days, when the mice exhibited significant abdominal distension and a feeling of tightness upon palpation, ascites fluid could be extracted. Monoclonal antibodies were purified from the ascites fluid using the Protein A column method. The antibody purification process consisted of the following steps: ascites fluid treatment, equilibration, sample loading, washing, and elution.

[0065] Subsequently, antibody purity was identified by SDS-PAGE electrophoresis, and the reducing and non-reducing SDS-PAGE gels of the monoclonal antibody stained with Coomassie Brilliant Blue were analyzed: In reducing SDS-PAGE, antibody 6D11 showed a single band at the 55 kDa (heavy chain) and 25 kDa (light chain) positions. Under non-reducing SDS-PAGE conditions, antibody 6D11 showed a band at approximately 150 kDa (intact antibody). Results are shown below. Figure 2 .

[0066] Example 4: Biological identification of monoclonal antibody 6D11

[0067] 4.1 Antibody subtype identification

[0068] Antibody subtypes were analyzed using a mouse monoclonal antibody immunoglobulin typing kit (Bio-Rad). Following the kit instructions, purified antibodies were appropriately diluted before subtype detection. The ELISA test showed a positive result for the IgG1 subtype, with an OD value of 2.497 measured at 450 nm, indicating that the monoclonal antibody 6D11 secreted by hybridoma cells 6D11 is IgG1.

[0069] 4.2 Identification of antibody binding curves

[0070] The binding curves of antibody 6D11 to DENV1-4 NS1 protein were determined using an indirect ELISA method. The procedure for the indirect ELISA method can be found in section 1.2 of Example 1. 1 μg / ml DENV1-4 NS1 protein was coated with 6D11. Primary antibody was prepared at a starting concentration of 100 μg / ml, serially diluted 10-fold. Secondary antibody was HRP-labeled goat anti-mouse IgG diluted 5000-fold. Absorbance at 450 nm was measured. Binding curves of antibody 6D11 to DENV NS1 were fitted, and the KD value was calculated. The results showed that antibody 6D11 exhibited strong cross-reactivity with all four serotypes of DENV NS1 protein. The binding ability with DENV1 and DENV4 NS1 was at the nM level, while the KD values ​​for the other two serotypes of DENV NS1 protein reached the pM level. (See attached table). Figure 3 .

[0071] 4.3 Identification of antibody competitive inhibition of NS1 protein binding to target cells

[0072] DENV NS1 protein of four serotypes (20 μg / mL each) was mixed with antibody 6D11 (40 μg / mL) in PBS solution containing 10% FBS and incubated at 37°C for 30 min. Cells (HBMEC human brain microvascular endothelial cells, human umbilical vein endothelial cells (HUVECs)) were resuspended in ice-cold PBS solution containing 10% FBS and 1% sodium azide to approximately 1 × 10⁻⁶ cells / mL. 6 Cells / mL. Add 100 μL of cell suspension to each centrifuge tube and incubate on ice for 15 min. After thoroughly mixing the cells with the antibody-NS1 protein complex, incubate at 4°C for 30 min. Centrifuge at 1000 rpm for 5 min, resuspend in pre-chilled PBS, and wash the cells three times. Then, use a FITC-labeled murine His-tagged monoclonal antibody to bind the NS1 protein bound to the target cells, and incubate at 4°C in the dark for 30 min. Centrifuge at 1000 rpm for 5 min, resuspend in pre-chilled PBS, and wash the cells three times. Finally, resuspend the cells in ice-cold PBS containing 3% BSA and 1% sodium azide. Immediately transfer the cell suspension to flow cytometry tubes for analysis. The percentage of target cells binding to NS1 protein was analyzed. Compared with the control group without antibody, the decreased percentage indicates that NS1 protein is competitively bound by antibody 6D11. This result is consistent with the ELISA experiment in section 4.2. Antibody 6D11 can broadly inhibit the binding of four serotypes of DENV NS1 protein to target cells, but the inhibitory effect on DENV1 and DENV4 NS1 proteins is weaker than that on DENV2 and DENV3 NS1 proteins. (See attached results). Figure 4 .

[0073] 4.4 Identification of antibody protective efficacy in vivo

[0074] Using the AG129 immunodeficient mouse model, under pre-immunization with monoclonal antibody 6D11 (300 μg / mouse), the mice were exposed to a lethal dose of DENV2 (5 × 10⁻⁶). 5 (PFU / mouse). Changes in mouse signs, disease course, body weight, and survival rate were continuously observed, and the vascular leakage test was used to evaluate the protective effect of the antibody. The survival curves showed that within 15 days after DENV2 challenge, all mice in the challenged control group died on day 12, while 25% of mice in the antibody 6D11 pre-immunized group were still alive on day 15, demonstrating better efficacy in protecting mice from lethal doses of DENV2. Simultaneously, the vascular leakage test results showed that vascular leakage in the liver, spleen, and small intestine of mice in the antibody 6D11 pre-immunized group was effectively alleviated, especially showing complete inhibition of splenic vascular leakage. See results below. Figure 5 .

[0075] It is evident that the monoclonal antibody 6D11 has significant preventive and therapeutic value against dengue fever, including severe dengue fever.

[0076] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the protection scope of the present invention. Therefore, the protection scope of this invention patent should be determined by the appended claims.

Claims

1. A monoclonal antibody 6D11 against DENV1-4 NS1 protein, characterized in that, The amino acid sequence of the heavy chain variable region of the monoclonal antibody 6D11 is shown in SEQ ID No. 1, and the amino acid sequence of the light chain variable region is shown in SEQ ID No.

2.

2. The monoclonal antibody 6D11 against DENV1-4 NS1 protein according to claim 1, characterized in that, The nucleotide sequence of the heavy chain variable region of the monoclonal antibody 6D11 is shown in SEQ ID No. 3, and the nucleotide sequence of the light chain variable region is shown in SEQ ID No.

4.

3. The monoclonal antibody 6D11 against DENV1-4 NS1 protein according to claim 1, characterized in that, The monoclonal antibody 6D11 is of the IgG1 subtype.

4. The use of the monoclonal antibody 6D11 against DENV1-4 NS1 protein as described in any one of claims 1-3 in the preparation of a medicament for treating dengue virus infection.

5. The use of the monoclonal antibody 6D11 against DENV1-4 NS1 protein as described in any one of claims 1-3 in the preparation of a medicament for inhibiting or treating vascular leakage caused by dengue virus infection.

6. The application according to claim 4 or 5, characterized in that, The dengue virus has serotypes DV-1, DV-2, DV-3, or DV-4.

7. A pharmaceutical composition for treating dengue virus infection, characterized in that, The pharmaceutical composition comprises the monoclonal antibody 6D11 against the DENV1-4 NS1 protein as described in any one of claims 1-3, and a pharmaceutically acceptable carrier.

8. A nucleic acid encoding a monoclonal antibody 6D11 against the DENV1-4 NS1 protein as described in any one of claims 1-3, characterized in that, The nucleotide sequence of the heavy chain variable region of the monoclonal antibody 6D11 against DENV1-4 NS1 protein is shown in SEQ ID No. 3, and the nucleotide sequence of the light chain variable region is shown in SEQ ID No. 4.

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