A monoclonal antibody 6G2 against DENV NS1 protein, its preparation method and application

Abstract

This invention provides a monoclonal antibody 6G2 against DENV NS1 protein and its application. The CDR amino acid sequence of the heavy chain of the monoclonal antibody 6G2 against DENV NS1 protein is shown in SEQ ID No. 1, and the CDR amino acid sequence of the light chain is shown in SEQ ID No. 3. This invention also provides the application of the above antibody in the preparation of drugs for treating dengue virus infection. The monoclonal antibody 6G2 against DENV NS1 protein of this invention has the ability to competitively bind to DENV1-4NS1 protein, inhibit vascular leakage symptoms, and protect against lethal damage from DENV infection in vivo, thus possessing significant potential value in the treatment of severe dengue fever. The monoclonal antibody 6G2 developed by this invention has important significance and application potential for the prevention and treatment of dengue virus infection, including severe dengue fever.

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 6G2 against NS1 protein that can inhibit vascular leakage symptoms caused by dengue virus infection. Background Technology

[0002] Dengue fever is an acute vector-borne infectious disease caused by the dengue virus, transmitted by mosquitoes. Infection with the dengue virus can lead to asymptomatic infection, dengue fever, or dengue hemorrhagic fever. Typical clinical manifestations of dengue fever include sudden onset, high fever, headache, severe muscle and joint pain, and in some patients, rash, bleeding tendency, swollen lymph nodes, decreased white blood cell count, and decreased platelet count. Dengue fever is mainly prevalent in tropical and subtropical regions and is transmitted by Aedes mosquitoes.

[0003] Early symptoms of severe dengue fever are similar to those of typical dengue fever, but the condition suddenly worsens on days 3-5, with severe headache, nausea, vomiting, altered consciousness, and meningitis signs such as neck stiffness. Clinical features of severe dengue fever may include severe bleeding, shock due to severe plasma leakage, or respiratory distress. Gastrointestinal symptoms and bleeding tendencies are also more pronounced in severe dengue fever. Severe dengue fever can lead to serious organ damage, including severe liver injury, acute respiratory failure, acute cardiac injury, acute renal failure, and encephalopathy.

[0004] Severe bleeding is one of the main differences between severe and ordinary dengue fever. Major precipitating factors include liver and kidney dysfunction, use of antipyretics, insertion of gastric or urinary catheters, intravenous puncture, and intramuscular injection. Severe bleeding often occurs during the acute phase of severe dengue fever and can manifest as subcutaneous hematoma, or bleeding in the gastrointestinal tract, respiratory tract, urinary tract, vagina, and intracranial cavity. Subcutaneous hematoma is the most common, often induced by intramuscular injection or deep arterial / venous puncture, and usually occurs in the trunk and limbs; in severe cases, it can induce hemorrhagic shock. Gastrointestinal bleeding cases are often accompanied by a history of peptic ulcer disease, and bleeding can occur in both the upper and lower gastrointestinal tracts. Intracranial hemorrhage is mostly spontaneous, and the prognosis for patients is poor.

[0005] Key pathogenic mechanisms of severe dengue fever include antibody-dependent enhancement (ADE) and severe vascular leakage damage. ADE targets and enhances viral particle infection, while ADE is associated with the fatal symptoms and damage of severe dengue fever. However, the E and prM proteins that make up the viral particles simultaneously induce both neutralization and ADE effects. Traditional vaccines based on whole-DENV designs, such as attenuated, inactivated, or chimeric vaccines, can mimic natural DENV infection, offering limited protection against DENV infection but potentially leading to ADE and increasing the risk of severe dengue fever. This has resulted in a bottleneck in the development of traditional antiviral drugs and vaccines for dengue virus (DENV).

[0006] Focusing on immune strategies to protect the body from fatal damage caused by vascular leakage is a crucial approach to improving the clinical symptoms of severe dengue fever. Non-structural protein 1 (NS1), which directly participates in the pathogenic mechanism of vascular leakage, exhibits unique antigenic advantages. Targeting the highly conserved NS1 protein can induce a broad-spectrum protective effect against flaviviruses. Antibodies against NS1 protect the body from fatal damage associated with severe dengue fever by blocking vascular leakage caused by endothelial cell dysfunction, and no association with adverse drug reaction (ADE) has been found. However, the screening of broad-spectrum NS1 antibodies is hampered by several factors, including the lack of antigenic stimulation from natural hexamers, low screening efficiency due to immunodeficiency of conserved epitopes, and the limited availability of structural analysis techniques. As a result, none of the reported NS1 antibodies currently available possess the ability to simultaneously resist infection by all four serotypes of dengue fever (DENV). Therefore, targeting the NS1 protein to induce screening and identify broad-spectrum protective antibodies is of great significance for the antiviral treatment or vaccine development of DENV. Summary of the Invention

[0007] To address the shortcomings of existing technologies, this invention provides a monoclonal antibody 6G2 capable of broadly binding to four serotypes of DENV NS1 and inhibiting its vascular leakage effect, its preparation method, and its applications.

[0008] A first aspect of the present invention is to provide a monoclonal antibody 6G2 against DENV NS1 protein, the amino acid sequence of its heavy chain CDR being shown in SEQ ID No. 1 and the amino acid sequence of its light chain CDR being shown in SEQ ID No. 3.

[0009] In some of these embodiments, the monoclonal antibody 6G2 against the DENV NS1 protein is of the I gG2b subtype.

[0010] A second aspect of the invention is the application of the monoclonal antibody 6G2 against the DENV NS1 protein.

[0011] The application of the anti-DENV NS1 protein monoclonal antibody 6G2 in the preparation of drugs or vaccines for the prevention and treatment of dengue virus infection.

[0012] The application of the monoclonal antibody 6G2 against DENV NS1 protein in the preparation of drugs or vaccines to inhibit or treat vascular leakage in severe dengue fever.

[0013] The monoclonal antibody against the DENV NS1 protein 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 embodiments, the dengue virus has serotypes DV-1, DV-2, DV-3, or DV-4, more preferably DV-2, DV-3, or DV-4.

[0014] A third aspect of the present invention is to provide a pharmaceutical composition for treating dengue virus infection, comprising an active ingredient and a pharmaceutically acceptable carrier, said active ingredient being any of the aforementioned monoclonal antibodies against the DENV NS1 protein, 6G2.

[0015] In some of these embodiments, the pharmaceutical composition is a pharmaceutical preparation or a vaccine.

[0016] A fourth aspect of the present invention is to provide a method for neutralizing dengue virus, comprising contacting dengue virus with a monoclonal antibody 6G2 against the DENV NS1 protein.

[0017] A fourth aspect of the present invention is a method for preparing a monoclonal antibody 6G2 against the DENVNS1 protein. The preparation method includes the following steps:

[0018] (1) Animal immunization: A sequential immunization strategy was adopted, and mice were immunized with serum types DV-1, DV-2, DV-3 and DV-4 respectively;

[0019] (2) Culture of mouse myeloma cells: mouse myeloma cells SP2 / 0 were cultured and kept in good growth condition for cell fusion.

[0020] (3) Cell fusion: The spleen lymphocytes of the mouse in step (1) were obtained by polyethylene glycol-mediated cell fusion and mixed with the SP2 / 0 cells in step (2) by centrifugation to mediate cell fusion. The fused cells were cultured under appropriate conditions to obtain the culture.

[0021] (4) Screening of hybridoma cells: The above culture was cultured in HAT selective medium for screening cells with hypoxanthine phosphoribosyltransferase (HPRT) or thymidine kinase (TK) activity deficiency, and positive clones were obtained by screening.

[0022] (5) 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 single-specific hybridoma cell line 6G2 that stably and efficiently expresses the anti-DENV NS1 protein monoclonal antibody 6G2; the amino acid sequences of the heavy chain CDR and light chain CDR of the anti-DENV NS1 protein monoclonal antibody 6G2 are shown in SEQ ID No.1 and SEQ ID No.3, respectively;

[0023] (6) Preparation of monoclonal antibody ascites: 8-10 week old BALB / c mice were selected. One week after intraperitoneal injection of an appropriate amount of Freund's incomplete adjuvant, the mice were inoculated again in PBS buffer containing hybridoma cells 6G2. When the mice's abdomen was significantly distended and the mice were about to die, the ascites was collected, centrifuged, and the antibody titer was determined.

[0024] (7) The monoclonal antibody was purified from mouse ascites fluid.

[0025] A fifth aspect of the present invention is to provide hybridoma cells that secrete the monoclonal antibody 6G2 that inhibits the above-mentioned DENV NS1 protein.

[0026] A sixth aspect of the present invention is to provide a nucleotide in the variable region of the heavy or light chain of a monoclonal antibody 6G2 encoding an anti-DENV NS1 protein.

[0027] In some of these embodiments, the CDR nucleotide sequence of the heavy chain of the anti-DENV NS1 protein monoclonal antibody 6G2 is shown in SEQ ID No. 2, and the CDR nucleotide sequence of the light chain is shown in SEQ ID No. 4.

[0028] The monoclonal antibody 6G2 obtained by screening in this invention has a broad-spectrum binding ability against the DENV1-4 NS1 protein of four serotypes (DV-1, DV-2, DV-3, or DV-4). 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 6G2 can delay the onset and progression of symptoms in AG129 mouse models and protect 25% of mice from lethal doses of DENV2. The monoclonal antibody 6G2 can broadly inhibit the binding of the four serotypes of DENV NS1 protein to target cells, with the best inhibitory effect on DENV1, DENV3, and DENV4 NS1, all reaching over 95%, but the inhibitory effect on DENV2 NS1 is relatively weak. Therefore, the recombinant expression products obtained by antibody mutants or CDR sequence design and modification based on the antibody itself, as described in this invention, have potential antiviral applications in the design of dengue vaccine candidates, screening of therapeutic drugs, or preparation of antiviral therapeutic drugs for DENV1-4 viral infectious diseases.

[0029] The monoclonal antibody 6G2 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 demonstrating significant potential value in the treatment of severe dengue fever. The monoclonal antibody 6G2 developed in this invention has important significance and application potential for the prevention and treatment of dengue virus infection, including severe dengue fever. Attached Figure Description

[0030] Figure 1 This is a graph showing the results of SDS-PAGE analysis of the purity of the monoclonal antibody 6G2.

[0031] Figure 2 This is a graph showing the results of ELISA identification of the binding curves of monoclonal antibody 6G2 to DENV1-4 NS1 protein.

[0032] Figure 3 This is a flow cytometry result verifying the competitive inhibition of NS1 protein binding to target cells by the monoclonal antibody 6G2.

[0033] Figure 4 This is a graph showing the results of validating the protection of the monoclonal antibody 6G2 against fatal vascular leakage caused by DENV infection in the AG129 mouse model. Detailed Implementation

[0034] 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.

[0035] 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.

[0036] 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.

[0037] 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.

[0038] In some embodiments of the present invention, a method is provided to enhance the immune response to conserved epitopes of four serotypes of DENV NS1 protein using a sequential immunization strategy, thereby improving the induction efficiency of broadly cross-reactive antibodies. This method yields a monoclonal antibody 6G2 against DENV NS1 protein. The monoclonal antibody subtype is IgG2b, which specifically and broadly recognizes four serotypes of DENV NS1 protein: DV-1, DV-2, DV-3, and DV-4. The heavy chain CDR amino acid and sequence nucleotides of the anti-DENV NS1 protein monoclonal antibody are shown in SEQ ID No. 1 and SEQ ID No. 2, and the light chain CDR nucleotide and amino acid sequences are shown in SEQ ID No. 3 and SEQ ID No. 4, as detailed below:

[0039] SEQ ID No. 1: Amino acid sequence of heavy chain CDR

[0040] EVKLVESGGALVKPGGSLKLSCAASGFAFSTYDMSWVRQTPEKRLEWVATISSGATYTYYTDSVKGRFTISRDNARNTLYLQMSSLRSEDTAMYYCSRHGPVVATGDYWGQGTTLTVSS

[0041] SEQ ID No. 2: Nucleotide sequence of the heavy chain CDR

[0042] GAAGTGAAGCTGGTGGAGTCTGGGGGAGCCTTAGTGAAGCCTGGAGGGTCCCTGAAACTCTCCTGTGCAGCCTCTGGATTCGCTTTCAGTACCTATGACATGTCTTGGGTTCGCCAGACTCCGGAAAAGAGGCTGGAGTGGGTCGCAACCATTAGTAGTGGTGCTACTTACACCTACTATACAGACAGTGTGAAGGGCCGATTCACCATCTCCAGAGACAATGCCAGGAATACCCTGTACTTGCAAATGAGCAGTCTGCGTTCTGAGGACACGGCCATGTATTACTGTTCAAGACATGGGCCGGTTGTGGCTACAGGAGACTACTGGGGCCAAGGCACCACTCTCACGGTCTCTTCA

[0043] SEQ ID No.3: Amino acid sequence of the light chain CDR

[0044] DIVMSQSPSSLAASVGETVTLNCKSSQSLLYSSDQKNYLAWYQQKPGQSPKLLIYWASTRDSGVPDRFTGSGSGTDFTLTISSVKAEDLAVYYCQQYYNYLTFGAGTKLELK

[0045] SEQ ID No.4: Nucleotide sequence of the light chain CDR

[0046] GACATTGTGATGTCACAGTCTCCATCCTCCCTAGCTGCGTCAGTTGGAGAGACGGTTACTTTGAACTGCAAGTCCAGTCAGAGCCTTTTATATAGTAGCGATCAAAAGAACTACTTGGCCTGGTATCAGCAGAAACCAGGACAGTCTCCTAAACTGCTGATTTACTGGGCATCCACTAGGGATTCTGGGGTCCCTGATCGCTTCACAGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGTGTGAAGGCTGAGGACCTGGCAGTTTATTACTGTCAGCAATATTATAACTATCTCACGTTCGGTGCTGGGACCAAGCTGGAGCTGAAAC。

[0047] In some embodiments of the present invention, the method for preparing the monoclonal antibody against DENV1-4 NS1 protein includes biosynthesis, cell expression, hybridoma cell secretion, etc., for example, by constructing the corresponding expression gene of the antibody and expressing it through an expression vector and host cells.

[0048] The preparation method also includes the hybridoma cell line 6G2 obtained by fusing BALB / c mouse spleen cells and myeloma cells SP2 / 0 that have been sequentially immunized with DENV1-4 NS1 protein, screening, cloning, and stable passage, which secretes the monoclonal antibody 6G2.

[0049] The present invention will be further described in detail below with reference to specific embodiments.

[0050] Example 1: Method for preparing monoclonal antibodies

[0051] 1. Immunization of mice: 6-8 week old BALB / c mice were immunized using a sequential immunization strategy, following a serotype order with a 2-week interval between immunizations. The specific steps are as follows:

[0052] (1) Eight female BALB / c mice aged 4-6 weeks were randomly divided into two groups: a sequential immunization group of 5 mice and a control group of 3 mice.

[0053] (2) Primary immunization: The sequential immunization group was immunized with DENV1 NS1. An equal volume of DENV1 NS1 protein and Freund's complete adjuvant was emulsified. The immunization dose was 50 μg / mouse, and the immunization volume was 0.5 mL / mouse. The antigen emulsion was injected into mice via intraperitoneal injection.

[0054] (3) Booster Immunization: Two weeks after the initial immunization, Freund's complete adjuvant was replaced with Freund's incomplete adjuvant. The immunization dose was halved to 25 μg / mouse, and the immunization volume was 0.5 mL / mouse. The sequentially immunized mice were injected intraperitoneally with emulsified DENV2 NS1, DENV3 NS1, and DENV4 NS1 proteins, with each injection spaced two weeks apart. Seven days after the last immunization, the antibody titer was measured by ELISA in the mouse serum.

[0055] 2. Indirect ELISA assay for serum titer in immunized mice

[0056] Blood was collected from the tail vein and mouse serum was collected by centrifugation. Antibody titers against each serotype of DENV were determined by coating the NS1 protein of each of the four serotypes of DENV in the serum.

[0057] 3. Culture and passage of mouse myeloma cells SP2 / 0: SP2 / 0 myeloma cell lines were cultured and passaged in 1640 medium containing 10% bovine serum and incubated at 37°C in an incubator containing 5% carbon dioxide.

[0058] 4. Cell Fusion: BALB / c mouse peritoneal macrophages were used as feeder cells. One day before fusion, BALB / c mouse peritoneal macrophages were seeded into 96-well plates and cultured in 1×HAT selective medium. Cells showing a titer of 10 against all four serotypes of DENVNS1 were collected. 5 Blood was collected from the orbital venous plexus of the mice to separate serum. Mice were euthanized by cervical dislocation, soaked in 75% alcohol for 15 minutes, and the abdomen was dissected locally. The spleen was aseptically removed. The spleen cells were washed with pre-cooled serum-free 1640 medium and then ground to obtain a suspension.

[0059] The sp2 / 0 cells and spleen cells were respectively brought to a final volume of 30 mL with 1640 medium in centrifuge tubes. After centrifugation at 1000 rpm for 5 min, the supernatant was discarded, and the cells were washed twice with 30 mL of 1640 medium. Finally, they were resuspended in 10 mL of serum-free 1640 medium, and cell counts were performed to determine the concentration and total amount of the two cell types.

[0060] Splenocytes and SP2 / 0 were mixed at a ratio of 5:1 and centrifuged at 1000 rpm for 5 min. The supernatant was discarded. The cell pellet was resuspended and mixed thoroughly by gently tapping the bottom of the tube with a finger. Cell fusion was initiated using 1 mL of preheated 50% PEG solution. After fusion was terminated, the cells were centrifuged at 1000 rpm for 5 min, the supernatant was discarded, and 1×HAT 1640 medium preheated at 37°C was added. The cell suspension was mixed well and then added dropwise (100 μL / well) to 96-well cell culture plates containing feeder cells using a bent-tip dropper. The plates were incubated at 37°C in a 5% CO2 incubator.

[0061] 5. Hybridoma cell screening: After 5 days, the medium in 96-well plates was replaced with half of 1×HAT medium. After 10 days, the medium was changed to 1×HT medium. The fused hybridoma cells were cultured in selective medium for approximately two weeks. When the cell colonies reached an appropriate size, the cell culture supernatant was aspirated for enzyme-linked immunosorbent assay (ELISA) to screen for positive clones. Positive hybridoma clones were also screened using an indirect ELISA method.

[0062] 6. Cloning of Hybridoma Cells: Cloning of hybridoma cells was performed using the limiting dilution method. Cells from hybridoma wells that tested positive for antibody were selected for cell counting. 150-200 cells were used to prepare a cell suspension in 1640 complete medium containing 1×HT and seeded into 96-well plates containing feeder cells (100 μL per well). After 10 days, cell growth was observed, and antibody levels in the supernatant were measured. Wells with the highest antibody titer and showing single-clonal 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-clonal wells were passaged for at least 20 generations to obtain a stable and highly efficient hybridoma cell line, 6G2, expressing a monoclonal antibody against DNEV NS1 protein.

[0063] 7. Preparation of monoclonal antibody ascites: Select 8-10 week old healthy BALB / c mice and inject 0.5 mL of Freund's incomplete adjuvant into each mouse intraperitoneally. After 7-10 days, when the mouse's peritoneum is distended and ascites appears, inject PBS-diluted hybridoma cells (6G2) into the peritoneum. Each mouse's abdomen is inoculated with PBS buffer containing 5 × 10⁶ positive hybridoma cells. After 7-10 days of cell inoculation, the mouse's abdomen is significantly distended. Closely observe the mouse's health status and abdominal signs, and collect the ascites at an appropriate time.

[0064] 8. Purification of monoclonal antibodies: Monoclonal antibody 6G2 was purified from ascites using the Protein A column method.

[0065] 9. Antibody purity was determined by SDS-PAGE electrophoresis. Analysis of Coomassie Brilliant Blue stained monoclonal antibody 6G2 on reducing and non-reducing SDS-PAGE gels: In reducing SDS-PAGE, monoclonal antibody 6G2 showed a single band at both the 55 kDa (heavy chain) and 25 kDa (light chain) positions. Under non-reducing SDS-PAGE conditions, antibody 6G2 showed a band at approximately 150 kDa (intact antibody). See results below. Figure 1 .

[0066] 10. Monoclonal antibody subtype identification: Analysis was performed using the Bio-Rad mouse monoclonal antibody immunoglobulin typing kit. The purified monoclonal antibody was appropriately diluted before detection, and the procedure was strictly performed according to the kit instructions. The ELISA test showed a positive result for the IgG2b subtype, with an OD value of 1.864 measured at 450 nm, indicating that the monoclonal antibody 6G2 secreted by hybridoma cells 6G2 is IgG2b.

[0067] Sequencing of hybridoma cells 6G2 revealed that the amino acids and nucleotides of the heavy chain CDR and light chain CDR of the secreted antibody 6G2 are as shown in SEQ ID No. 1-SEQ ID No. 4.

[0068] The hybridoma cell line 6G2 was cloned four times and cultured for more than four months, and its antibody secretion was stable.

[0069] Example 2: Identification of the binding curve between antibody 6G2 and NS1 protein by indirect ELISA

[0070] DENV1-4 NS1 protein was diluted to 1 μg / mL with PBS, and 100 μL / well was added to each well of a 96-well ELISA plate. The plates were incubated overnight at 4°C. Excess incubation solution was discarded, and the plates were washed three times with PBST and patted dry. 300 μL / well of 5% skim milk powder solution was added, and the plates were blocked at 37°C for 2 h. The blocking solution was discarded, and the plates were washed three times with PBST and patted dry. The test antibody was added to each well at a starting concentration of 100 μg / mL, with eight 10-fold dilutions, and 100 μL / well was added to each well. The plates were incubated at 37°C for 60 min. Negative control was PBS-immunized mouse serum, and blank control was PBS solution. Serum samples were discarded, and the plates were washed three times with PBST and patted dry. 100 μL / well of HRP-labeled goat anti-mouse secondary antibody solution diluted 5000 times with PBS was added, and the plates were incubated at 37°C for 60 min. The secondary antibody solution was discarded, and the plates were washed five times with PBST and patted dry. Add 100 μL of TMB substrate (component A:component B = 1:1) to each well for color development, and incubate at room temperature for 10 min. Stop the color development by adding 50 μL of 2M concentrated sulfuric acid per well. Measure the absorbance (A) of each well at 450 nm using a microplate reader. The calculated KD values ​​of antibody 6G2 indicate strong cross-reactivity with all four serotypes of DENV NS1 protein. The binding affinity to DENV2 NS1 is at the nM level, while the KD values ​​for the other three serotypes of DENV NS1 protein reach the pM level. See the results below. Figure 2 .

[0071] Example 3: Flow cytometry verification of antibody 6G2 competitively inhibiting the binding of NS1 protein to target cells

[0072] HBMECs and HUVECs (human brain microvascular endothelial cells, human umbilical vein endothelial cells) were routinely passaged and cultured to the logarithmic growth phase. Cells were collected after trypsin digestion, centrifuged, and the supernatant discarded. DENV1-4NS1 protein (20 μg / mL) and antibody 6G2 (40 μg / mL) were mixed in PBS solution with 10% FBS and incubated at 37°C for 30 minutes. All reagents were pre-chilled at 4°C, and all operations were performed on ice plates. Cells were resuspended in ice-cold PBS solution containing 10% FBS and 1% sodium azide to approximately 1 × 10⁶ cells / mL. 100 μL of cell suspension was added to each centrifuge tube, and the cells were incubated on ice for 15 minutes. After thorough mixing of the cells with the antibody-NS1 protein complex, the cells were incubated at 4°C for 30 minutes. The cells were centrifuged at 1000 rpm for 5 minutes, resuspended in pre-chilled PBS, and washed three times. Subsequently, the NS1 protein bound to target cells was bound using a FITC-labeled murine His-tagged monoclonal antibody and incubated at 4°C in the dark for 30 min. The cells were then centrifuged at 1000 rpm for 5 min, resuspended in pre-chilled PBS, and washed three times. Finally, the cells were resuspended in ice-cold PBS containing 3% BSA and 1% sodium azide. The cell suspension was immediately transferred to flow cytometry tubes, protected from light, and analyzed as soon as possible. The percentage of target cells bound to NS1 protein was analyzed. Compared with the control group without antibody, the decreased percentage indicated that NS1 protein was competitively bound by antibody 6G2. The results showed that antibody 6G2 broadly inhibited the binding of four serotypes of DENV NS1 protein to target cells, with the best inhibitory effect on DENV1, DENV3, and DENV4 NS1, all exceeding 95%, but the inhibitory effect on DENV2 NS1 was weaker, consistent with the ELISA results in Example 2. (See attached figures for details.) Figure 3 .

[0073] Example 4: Verification of the protective efficacy of NS1 broad-spectrum antibody in an immunodeficient mouse AG129 model.

[0074] Using an AG129 immunodeficient mouse model, mice were pre-immunized with monoclonal antibody 6G2 (300 μg / mouse) and exposed to a lethal dose of DENV2 (5 × 10⁵ PFU / mouse). Changes in mouse signs, disease course, body weight, and survival rate, as well as vascular leakage assays, were continuously observed to evaluate the protective effect of the antibody. Survival curves showed that, in the control group challenged with DENV2 alone, all mice died on day 12, while antibody 6G2 protected mice from a lethal dose of DENV2, with 25% of mice still surviving on day 20 post-infection. Results are shown below. Figure 4 A.

[0075] Meanwhile, the vascular leakage assay based on Evans blue showed that antibody 6G2 significantly improved the degree of vascular leakage in the liver, spleen, and small intestine of mice, especially completely alleviating vascular leakage in the spleen (P > 0.05). See results below. Figure 4 B.

[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 6G2 against DENV NS1 protein, characterized in that, The amino acid sequence of the heavy chain variable region is shown as SEQ ID No. 1, and the amino acid sequence of the light chain variable region is shown as SEQ ID No.

3.

2. The anti-DENV NS1 protein monoclonal antibody 6G2 of claim 1, wherein, The subtype of the anti-DENV NS1 protein monoclonal antibody 6G2 is IgG2b.

3. Use of the anti-DENV NS1 protein monoclonal antibody 6G2 of claim 1 or 2 in the preparation of a medicament for treating dengue virus infection.

4. Use of the anti-DENV NS1 protein monoclonal antibody 6G2 of claim 1 or 2 in the preparation of a medicament for inhibiting or treating vascular leakage in severe dengue.

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

6. A pharmaceutical composition for treating dengue virus infection, comprising an active ingredient which is the anti-DENV NS1 protein monoclonal antibody 6G2 of claim 1 or 2, and a pharmaceutically acceptable carrier.

7. The pharmaceutical composition for treating dengue virus infection according to claim 6, wherein The pharmaceutical composition is a pharmaceutical preparation.

8. A nucleic acid encoding the monoclonal antibody 6G2 against DENV NS1 protein of claim 1, wherein, The nucleotide sequence encoding the heavy chain variable region of the anti-DENV NS1 protein monoclonal antibody 6G2 is shown as SEQ ID No. 2; The nucleotide sequence encoding the light chain variable region of the anti-DENV NS1 protein monoclonal antibody 6G2 is shown as SEQ ID No. 4.

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