Antibodies against dengue virus ns1 protein and related products and uses thereof

Abstract

The application discloses an antibody against dengue virus NS1 protein and related products and uses thereof, the antibody has high expression and stable physical and chemical properties, can not only specifically bind to dengue virus 1-4 type NS1 protein, has no cross reaction, has high affinity, solves the technical pain points that existing dengue virus diagnostic means is complex in operation, lacks specificity and lacks a targeted treatment antibody; the antibody can be directly used for developing convenient and efficient dengue virus detection products (such as a kit, a test strip and the like), realizes early rapid screening and accurate typing of infection, can also be used as a core component to prepare a therapeutic drug, provides a novel technical scheme with high specificity, high affinity and wide applicability for prevention and control of dengue virus infection, and has important clinical conversion value and public health significance.

Description

Antibodies against dengue virus NS1 protein and related products and uses Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically, it relates to an antibody against dengue virus NS1 protein, and more specifically, it relates to the antibody against dengue virus NS1 protein and its related products and uses. Background Technology

[0002] Dengue virus (DENV), one of the most widespread arboviruses globally, is classified into 1-4 serotypes (DENV-1, DENV-2, DENV-3, and DENV-4). Infection with DENV can cause a range of diseases, from common dengue fever to severe dengue fever (with fatal symptoms such as vascular leakage and hemorrhage), posing a serious threat to public health in tropical and subtropical regions. Non-structural protein 1 (NS1) is a core protein in dengue virus replication and pathogenesis. It plays a crucial role not only in viral RNA replication and immune evasion but also in being secreted in large quantities into the patient's bloodstream in the early stages of infection (1-9 days after symptom onset), serving as a specific biomarker for early diagnosis of dengue virus infection. Furthermore, NS1 protein induces vascular endothelial damage and triggers inflammatory responses, acting as a key mediator in the pathological process of severe dengue fever. This provides a clear target for intervention strategies targeting NS1 protein and highlights the core value of developing anti-dengue virus NS1 protein antibodies.

[0003] Current dengue virus diagnostic and treatment methods have significant limitations. In terms of diagnosis, traditional RT-PCR (reactive protein-based PCR) is complex, costly, and requires specialized equipment, making it difficult to meet the rapid screening needs of primary healthcare institutions and during outbreaks. Antibody testing is susceptible to cross-reactivity and interference from antibodies from previous infections, failing to effectively differentiate between acute and past infections. Regarding dengue virus treatment, there are currently no specific antiviral drugs, and clinical treatment is primarily symptomatic and supportive. The high mortality rate of severe dengue fever urgently necessitates targeted interventions. Antibodies against the dengue virus NS1 protein can accurately identify conserved antigenic epitopes or serotype-specific epitopes of the NS1 protein. These antibodies can serve as diagnostic probes for early and rapid detection and serotyping of infection, and can also exert therapeutic effects by blocking the pathogenic function of the NS1 protein and mediating viral clearance, potentially overcoming current technological bottlenecks.

[0004] Developing antibodies against dengue virus NS1 protein can provide multi-dimensional technical support for dengue virus prevention and control. In diagnostic applications, these antibodies can be used to develop convenient and efficient antigen detection kits such as colloidal gold and ELISA, enabling rapid screening in grassroots settings and facilitating early detection and control of the epidemic. In therapeutic applications, by neutralizing extracellular NS1 protein and inhibiting its mediated pathological damage, or by using antibody-dependent cell-mediated cytotoxicity (ADCC) to clear infected cells, targeted treatment options can be provided for patients with severe dengue fever, reducing mortality. At the same time, broad-spectrum antibodies targeting conserved epitopes of NS1 protein types 1-4 can also avoid the antibody-dependent enhancement (ADE) effect that may be caused by single serotype antibodies, providing a safer technical option for the prevention and control of multi-serotype dengue virus infection, and have important clinical translational value and public health significance. Summary of the Invention

[0005] In order to solve the above-mentioned technical problems existing in the prior art, the purpose of this invention is to provide antibody 5E3 against dengue virus NS1 protein and related products and uses.

[0006] The present invention achieves the above-mentioned objectives by adopting the following technical solution:

[0007] The first aspect of the present invention provides an antibody against dengue virus NS1 protein or an antigen-binding fragment thereof, wherein the amino acid sequences of the heavy chain variable regions CDR-H1, CDR-H2, and CDR-H3 of the antibody or the antigen-binding fragment are shown in SEQ ID NO:4-6, respectively.

[0008] The amino acid sequences of the light chain variable regions CDR-L1, CDR-L2, and CDR-L3 of the antibody or its antigen-binding fragment are shown in SEQ ID NO:7-9, respectively.

[0009] Furthermore, the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:2.

[0010] Furthermore, the amino acid sequence of the light chain variable region is shown in SEQ ID NO:3.

[0011] In this invention, the dengue virus NS1 protein (Non-structural Protein 1) is a non-structural glycoprotein encoded by the dengue virus (DENV, including serotypes DENV-1 to DENV-5, with DENV-1 to 4 currently being the most prevalent) genome. It is also the only viral protein secreted into the host's bloodstream during viral infection. It has a molecular weight of approximately 45 kDa and can exist in vivo in monomeric, dimeric, or hexamer form. Its amino acid sequence is highly conserved among different serotypes, providing a key target for cross-serotype detection and intervention. This protein not only plays a central role in viral RNA replication, viral particle assembly, and immune escape (such as inhibiting complement activation), but it is also an ideal biomarker for early diagnosis of dengue fever. High concentrations of soluble NS1 protein (peak value up to 600 ng / mL) can be detected in the blood of patients within 72 hours after infection, earlier than the production of IgM / IgG antibodies, and it persists in the blood for 9-18 days, effectively distinguishing acute infection from previous infection. At the same time, NS1 protein can participate in the pathological process of severe dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS) by inducing vascular endothelial cell damage and triggering inflammatory responses, making it a core molecular target for the diagnosis and targeted therapy of dengue virus infection.

[0012] In some embodiments, the present invention does not limit the specific sequences of the amino acid sequences of the heavy chain variable regions CDR-H1, CDR-H2, CDR-H3 and the light chain variable regions CDR-L1, CDR-L2, CDR-L3 of the antibody to the aforementioned specific sequences. For the antibody 5E3 disclosed in this invention, regardless of whether an existing CDR numbering scheme (such as IMGT, Chothia, Kabat, etc.) is used, or a new CDR numbering scheme may emerge in the future, as long as the CDR1, CDR2, and CDR3 regions of the heavy chain variable region or light chain variable region of the antibody are defined according to the corresponding numbering rules, the amino acid sequences corresponding to the heavy chain variable regions CDR1-3 and light chain variable regions CDR1-3 as determined thereby, as well as the nucleotide sequences encoding these amino acid sequences, all fall within the protection scope of this invention.

[0013] In some implementations, the CDR numbering schemes that can be used include, but are not limited to: the IMGT numbering scheme, the Chothia numbering scheme, the AbM numbering scheme, the Kabat numbering scheme, the Martin (enhanced Chothia) numbering scheme, and the Aho numbering scheme. Whether any one of the above numbering schemes is used alone, or two or more of them are used in combination, the corresponding amino acid sequences or their encoding nucleotide sequences obtained after defining the heavy chain variable region CDR1-3 or the light chain variable region CDR1-3 of the antibody as described above based on that numbering scheme fall within the protection scope of this invention.

[0014] A second aspect of the invention provides a nucleic acid molecule encoding an antibody or an antigen-binding fragment thereof as described in the first aspect of the invention.

[0015] In some implementations, based on the degeneracy of codons, the nucleic acid molecule not only contains specific nucleotide sequences that are completely complementary to the above-mentioned amino acid sequences, but also covers all derivative sequences after synonymous codon substitutions. Such substitutions do not change the amino acid composition and spatial conformation of the antibody, but can adapt to the codon preferences of different host cells, significantly improving the translation efficiency of the antibody in the heterologous expression system.

[0016] In some implementations, the protection scope of the nucleic acid molecule is further extended to nucleotide variants of the CDR sequence or complete variable region sequence obtained after defining the heavy chain variable region and light chain variable region CDR1-3 of the antibody described in this invention using various existing or future CDR numbering schemes.

[0017] In some implementations, the nucleic acid molecules can be obtained through mature molecular biology techniques such as artificial synthesis and PCR amplification. They have well-defined sequences and are highly operable, and can be stably inserted into suitable expression vectors. This provides a reliable genetic template for the efficient and stable production of antibodies in host cells, and is the core link between antibody molecule design and practical application transformation.

[0018] A third aspect of the present invention provides an expression vector comprising the nucleic acid molecule described in the second aspect of the present invention.

[0019] In some implementations, the expression vector includes, but is not limited to: prokaryotic expression vectors, eukaryotic expression vectors, shuttle expression vectors, or fusion expression vectors.

[0020] In some implementations, the prokaryotic expression vector is suitable for achieving efficient expression of the target gene in prokaryotic cells (such as *E. coli*). Its core components include a prokaryotic promoter (such as the lac promoter or T7 promoter), a ribosome binding site (SD sequence), a multiple cloning site (for inserting the target nucleic acid molecule), and a prokaryotic replicon (such as the ColE1 replicon, ensuring autonomous replication of the vector in prokaryotic cells). Some prokaryotic expression vectors also carry selection marker genes (such as ampicillin resistance genes or kanamycin resistance genes) to facilitate the screening of successfully transformed host cells. These vectors are suitable for rapidly obtaining large quantities of recombinant proteins without complex post-translational modifications and are often used for preliminary functional verification of the target protein or antigen preparation.

[0021] In some implementations, the eukaryotic expression vector is specifically designed for expressing the target gene in eukaryotic cells. Its core components include a eukaryotic promoter (such as the CMV promoter or SV40 promoter), a transcription termination signal (such as the polyadenylate signal polyA), and a eukaryotic replicon (such as a replicon derived from the SV40 virus or a 2 μm plasmid replicon specific to yeast cells). Depending on the host cell type, it can be further categorized into yeast expression vectors (containing yeast promoters such as the ADH1 promoter, suitable for hosts such as Saccharomyces cerevisiae), insect cell expression vectors (commonly using baculovirus promoters, such as the polyhedrosis protein promoter, adapted to insect cells such as sf9 and sf21), and mammalian cell expression vectors (carrying regulatory elements suitable for cells such as CHO and HEK293, some containing enhancers to improve expression efficiency). Eukaryotic expression vectors typically contain eukaryotic selection markers (such as neomycin resistance genes or hygromycin resistance genes) and support post-translational modifications of the target protein, making them suitable for expressing functional proteins with natural activity.

[0022] In some implementations, the shuttle expression vector combines the core elements of both prokaryotic and eukaryotic expression vectors. It can be constructed and amplified in prokaryotic cells (relying on prokaryotic replicons and selection markers) while simultaneously expressing the target gene in eukaryotic cells (including eukaryotic promoters and termination signals). For example, a vector containing a ColE1 replicon and an ampicillin resistance gene (prokaryotic portion), as well as a CMV promoter, a polyA signal, and a neomycin resistance gene (eukaryotic portion), can be amplified extensively in *E. coli* before being transferred into mammalian cells for protein expression. This greatly simplifies the experimental procedure and is suitable for scenarios requiring stepwise vector preparation and protein expression in different host systems.

[0023] In some implementations, the fusion expression vector carries a tag gene (such as a His tag, GST tag, GFP tag, etc.) upstream or downstream of the target gene insertion site, enabling the fusion expression of the target gene and the tag gene. Such vectors can achieve rapid purification of recombinant proteins (e.g., His tag-bound nickel ion affinity chromatography), intracellular localization observation (e.g., fluorescence tracking with a GFP tag), or protein-protein interaction studies (e.g., pull-down experiments with a GST tag) through the tag. Furthermore, some tag sequences can enhance the solubility of the target protein and improve expression efficiency.

[0024] In this invention, those skilled in the art can conventionally select specific expression vectors according to actual needs (such as host cell selection, protein function research objectives, expression level requirements, etc.).

[0025] A fourth aspect of the present invention provides a recombinant host cell comprising the expression vector described in the third aspect of the present invention;

[0026] Optionally, the host cell is a eukaryotic cell;

[0027] Optionally, the eukaryotic cell is a mammalian cell, a plant cell, or a yeast cell.

[0028] In some embodiments, the present invention preferably uses mammalian cells as host cells. Mammalian cells (such as Chinese hamster ovary cells (CHO), mouse myeloma cells (NS0), and human embryonic kidney cells (HEK293)) are highly consistent with human cells in the type and pattern of post-translational protein modifications, thus maximizing the preservation of the natural structure and function of recombinant proteins. Taking CHO cells as an example, they not only possess stable genetic characteristics and efficient exogenous gene expression capabilities, but also perform complex and uniform glycosylation modifications on antibodies. The resulting recombinant antibodies are extremely similar to natural antibodies in molecular structure, physicochemical properties, and biological activity, making them the most commonly used host cell type for the production of diagnostic antibodies and recombinant therapeutic antibodies. Furthermore, mammalian cells exhibit good growth adaptability and can achieve high-density growth in large-scale suspension culture systems, meeting the industrial production demands for target protein yields and providing a crucial cell expression platform for the industrial application of the antibodies and related detection products mentioned above.

[0029] In some embodiments, the host cells that can be selected by this invention cover a variety of eukaryotic cell types suitable for recombinant antibody expression, specifically including mammalian cells, plant cells, and yeast cells. Among them, mammalian cells are the preferred category, specifically including Chinese hamster ovary cells (CHO, including CHO-K1, CHO-S, and other subtypes), human embryonic kidney cells (HEK293, including engineered cell lines such as HEK293T and HEK293F), mouse myeloma cells (NS0, Sp2 / 0), human cervical cancer cells (HeLa), canine kidney cells (MDCK), mouse mammary epithelial cells (C127), hamster kidney cells (BHK-21), African green monkey kidney cells (Vero, COS-7), mouse hybridoma cells (P3X63Ag8.653), and Cap cells (canine kidney epithelial cells), etc.; plant cells can be selected from plant engineered cell lines such as tobacco cells, Arabidopsis cells, and rice cells; yeast cells can be selected from Pichia pastoris and Saccharomyces cerevisiae. Commonly used yeast expression systems such as *Cerevisiae* and *Hansenula polymorpha* have mature culture processes and expression systems. Those skilled in the art can flexibly choose according to antibody expression needs, modification preferences and industrialization scenarios to ensure efficient antibody production and functional stability.

[0030] The fifth aspect of the invention provides any of the following products:

[0031] (1) A dengue virus detection reagent, wherein the detection reagent comprises the antibody or antigen-binding fragment thereof described in the first aspect of the present invention;

[0032] (2) A dengue virus detection product, wherein the detection product comprises the antibody or antigen-binding fragment thereof described in the first aspect of the present invention, or the detection reagent;

[0033] (3) A diagnostic product for dengue virus infection, wherein the diagnostic product comprises the antibody or its antigen-binding fragment as described in the first aspect of the present invention, or the detection reagent;

[0034] (4) An antibody-drug conjugate for dengue virus detection, the antibody-drug conjugate comprising the antibody or antigen-binding fragment thereof as described in the first aspect of the present invention, and a detectable marker conjugated thereto;

[0035] (5) A pharmaceutical composition comprising the antibody or antigen-binding fragment thereof as described in the first aspect of the present invention;

[0036] (6) A biological agent comprising the antibody or antigen-binding fragment thereof as described in the first aspect of the present invention, or the pharmaceutical composition thereof;

[0037] Optionally, the detection product is a detection kit, a detection chip, or a test strip;

[0038] Optionally, the detectable marker is a fluorescent dye, an enzyme marker, or a radionuclide.

[0039] In some embodiments, the detectable markers include, but are not limited to, fluorescent dyes, avidin, paramagnetic atoms, and radioactive isotopes. In other embodiments, the fluorescent dye is fluorescein, rhodamine, Texas red, phycoerythrin, phycocyanin, allophycocyanin, or polydinoflavin-chlorophyll protein. The avidin is biotin, avidin, streptavidin, vitellavidin, or avidin-like proteins. The radioactive isotopes are radioactive iodine, radioactive cesium, radioactive iridium, or radioactive cobalt.

[0040] In some implementation schemes, to prepare antibody-drug conjugates for dengue virus detection, a mild and efficient conjugation strategy can be selected based on the type of detectable marker. The core is to achieve a stable connection between the two while preserving the antibody's specific binding activity to the NS1 protein and the marker's signaling function. For fluorescent dyes (such as fluorescein, rhodamine, phycoerythrin, etc.) and radioactive isotopes (such as radioactive iodine, cobalt, etc.), covalent coupling is preferred. Taking the most commonly used amino coupling as an example, the amino group (-NH2) of the side chain of the antibody lysine residue is reacted with the label that has been activated by N-hydroxysuccinimide ester (NHS), isothiocyanate, etc., and incubated in PBS buffer at pH 7.2-8.0 at 4°C in the dark for 1-4 hours. Covalent bonds are formed through amidation or addition reactions. The free label is then removed by gel filtration chromatography or dialysis to obtain the purified conjugate. If higher specificity is required, the disulfide bond in the antibody hinge region can be reduced by dithiothreitol (DTT) to expose the cysteine ​​thiol group (-SH), which reacts with the maleimide-activated label to form a thioether bond, reducing the impact on the antibody binding site.

[0041] In some implementations, for avidin-based markers (biotin, streptavidin, ovalbumin, etc.), a specific non-covalent conjugation method can be used to achieve indirect linking by leveraging the ultra-high affinity between biotin and avidin. This method is gentle and does not impair antibody activity. Specifically, activated biotin (such as NHS-biotin) can be first linked to the antibody via amino-linked conjugation to prepare a biotinylated antibody (controlling the biotin to antibody molar ratio to 5-10:1 to avoid over-labeling). This antibody is then mixed with streptavidin-modified fluorescent dyes, enzymes, or other markers and incubated at room temperature for 15-30 minutes to form a stable conjugate through the specific interaction between biotin and avidin. Alternatively, the antibody can be first conjugated to activated avidin, and then combined with a biotinylated marker, adapting to scenarios where direct covalent linking of markers is difficult.

[0042] In some implementations, paramagnetic atoms (such as iron oxide nanoparticles and gadolinium-based contrast agents) are often covalently coupled using surface-active groups. The paramagnetic nanoparticles are first surface-modified to introduce active groups such as carboxyl, amino, or epoxy groups. For carboxyl-modified particles, activation with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) / NHS followed by a condensation reaction with the amino group of the antibody's lysine residue. For amino-modified particles, amide bonds can be formed with the antibody's carboxyl group (C-terminus or aspartic acid / glutamic acid side chain) under EDC catalysis. After the reaction, free antibody is removed by magnetic separation or centrifugation to obtain the antibody-paramagnetic atom conjugate, suitable for applications such as magnetic immunoassay. Regardless of the method used, the molar ratio of antibody to label, reaction pH, and temperature must be optimized to avoid over-labeling leading to antibody polymerization or loss of activity. Furthermore, antibody binding activity must be verified by ELISA after coupling, and the label signal intensity must be detected simultaneously to ensure the conjugate possesses both high specificity and signal effectiveness.

[0043] In some embodiments, the antibody or its antigen-binding fragment contained in the detection product, with its defined heavy chain variable region (SEQ ID NO:2), light chain variable region (SEQ ID NO:3), and CDR region (SEQ ID NO:4-9) sequences, can specifically recognize dengue virus type 1-4 NS1 protein, without cross-reacting with closely related or common pathogens such as Zika virus and influenza virus, and possesses EC 50 With a high affinity of 0.1334 μg / mL, it can efficiently capture NS1 protein in test samples (such as serum, plasma, whole blood, etc.), providing a core guarantee for the accuracy and reliability of the test results.

[0044] In some implementations, the detection kits can be classified into ELISA kits, chemiluminescence kits, etc., according to the detection principle. By optimizing the reaction system (such as antibody coating concentration, incubation conditions, and colorimetric / luminescent substrate ratio), qualitative or quantitative detection of NS1 protein can be achieved. These kits are suitable for accurate diagnosis and batch testing of samples in clinical laboratories and can provide clear numerical results for clinicians to refer to.

[0045] In some implementations, the detection chip is based on microarray technology, which immobilizes antibodies on the surface of the chip carrier to achieve high sensitivity and high throughput detection of NS1 protein in a single sample. It is suitable for large-scale epidemic monitoring or multi-indicator joint detection scenarios and has the characteristics of fast detection speed and small sample volume.

[0046] In some implementation schemes, the test strips (such as colloidal gold immunochromatographic test strips) focus on the need for rapid screening. By combining antibodies with markers such as colloidal gold, a visual detection system is constructed. The system is easy to operate and does not require professional equipment. It can achieve instant detection in scenarios such as primary healthcare institutions and epidemic sites, and positive / negative results can be obtained within 10-20 minutes, which helps in the early detection and isolation of infected cases.

[0047] In some implementation schemes, the detection product can be flexibly integrated with antibody conjugates (such as antibodies conjugated with fluorescent dyes, enzyme labels, or radionuclides) to further improve detection sensitivity and signal recognition. Its overall design follows a standardized production process, with strict control over everything from antibody immobilization and reaction system optimization to product stability verification. This not only solves the pain points of existing detection products that rely on imported antibodies, are costly, and have unstable supply, but also ensures the controllability of product quality and the continuity of supply by relying on core antibody raw materials with independent intellectual property rights. It can be widely used in multiple fields such as clinical diagnosis and treatment, port quarantine, community screening, and epidemiological investigation, providing strong technical support for the precise prevention and control of dengue virus infection.

[0048] A sixth aspect of the present invention provides a method for detecting dengue virus NS1 protein for non-diagnostic and non-therapeutic purposes, the method comprising: contacting a sample to be tested with an antibody or an antigen-binding fragment thereof as described in the first aspect of the present invention, and detecting the formation of an antigen-antibody immune complex.

[0049] A seventh aspect of the present invention provides a method for preparing the antibody or antigen-binding fragment thereof as described in the first aspect of the present invention, the method comprising: culturing the recombinant host cell as described in the fourth aspect of the present invention, inducing antibody expression, separating and purifying the culture product, and obtaining the antibody or antigen-binding fragment thereof.

[0050] Furthermore, the present invention also provides a method for detecting dengue virus NS1 protein in a test sample or a method for diagnosing whether a subject has dengue virus infection or is at risk of dengue virus infection. The method includes the following steps: contacting a test sample from the subject with an antibody or its antigen-binding fragment as described above, a detection reagent, a detection kit, a detection chip, or a diagnostic product; detecting the immune reaction between the sample and the antibody; determining the expression level of dengue virus NS1 protein in the sample; and judging whether the subject has dengue virus infection or is at risk of dengue virus infection based on the result.

[0051] In this invention, the subjects include two main categories: humans and non-human animals. Non-human animals encompass various vertebrates, specifically divided into mammals and non-mammals. Examples of mammals include non-human primates (such as cynomolgus monkeys), sheep, dogs, and cattle, while non-mammals include chickens, amphibians, and reptiles. In some specific embodiments, this invention preferably uses humans as the core subjects to better meet the clinical application needs for the diagnosis and treatment of dengue virus infection-related diseases.

[0052] The eighth aspect of the present invention provides for any of the following applications:

[0053] (1) The use of the antibody or antigen-binding fragment thereof described in the first aspect of the present invention, the nucleic acid molecule described in the second aspect of the present invention, the expression vector described in the third aspect of the present invention, or the recombinant host cell described in the fourth aspect of the present invention in the preparation of diagnostic products for diagnosing dengue virus infection;

[0054] (2) The use of the antibody or antigen-binding fragment thereof described in the first aspect of the present invention, the nucleic acid molecule described in the second aspect of the present invention, the expression vector described in the third aspect of the present invention, or the recombinant host cell described in the fourth aspect of the present invention in the preparation of a detection reagent for detecting dengue virus NS1 protein;

[0055] (3) The use of the antibody or antigen-binding fragment thereof described in the first aspect of the present invention, the nucleic acid molecule described in the second aspect of the present invention, the expression vector described in the third aspect of the present invention, or the recombinant host cell described in the fourth aspect of the present invention in the preparation of a detection product for detecting dengue virus NS1 protein;

[0056] (4) The use of the antibody or antigen-binding fragment thereof described in the first aspect of the present invention, the nucleic acid molecule described in the second aspect of the present invention, the expression vector described in the third aspect of the present invention, or the recombinant host cell described in the fourth aspect of the present invention in the preparation of antibody-drug conjugates for detecting dengue virus NS1 protein;

[0057] (5) The use of the antibody or antigen-binding fragment thereof described in the first aspect of the present invention, the nucleic acid molecule described in the second aspect of the present invention, the expression vector described in the third aspect of the present invention, or the recombinant host cell described in the fourth aspect of the present invention in the preparation of a medicament for the prevention and / or treatment of dengue virus infection.

[0058] In this invention, dengue virus infection refers to the general term for diseases induced by the invasion of the human body by various serotypes of the dengue virus (DENV) family (including the currently widely prevalent DENV-1, DENV-2, DENV-3, and DENV-4 types, as well as other identified or potentially emerging prevalent serotypes). The disease manifestations present a continuous and diverse spectrum, ranging from asymptomatic latent infection to common dengue fever with fever, headache, myalgia, joint pain, and rash as the core symptoms, and then to severe cases that progress rapidly and endanger life. The severe types are specifically dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS). The former is characterized by increased vascular permeability and bleeding tendency caused by abnormal coagulation function, while the latter is accompanied by fatal manifestations such as circulatory failure and multiple organ dysfunction. The occurrence and development of these diseases are closely related to the replication and proliferation of dengue virus in host cells, as well as the pathological processes such as vascular endothelial damage and amplification of inflammatory response mediated by viral NS1 protein.

[0059] Compared with the prior art, the advantages and beneficial effects of the present invention are as follows:

[0060] This invention provides a novel anti-dengue virus NS1 protein antibody, 5E3, which exhibits high expression levels and stable physicochemical properties. It not only specifically binds to dengue virus NS1 proteins of types 1-4 without cross-reactivity but also possesses high affinity (EC). 50 With a concentration of 0.1334 μg / mL, it addresses the technical pain points of existing dengue virus diagnostic methods, such as complex operation, insufficient specificity, and lack of targeted therapeutic antibodies. It can be directly used to develop convenient and efficient dengue virus detection products (such as reagent kits and test strips) to achieve rapid screening and accurate typing in the early stage of infection. It can also be used as a core component to prepare therapeutic drugs, providing a new technical solution for the prevention and control of dengue virus infection that combines high specificity, high affinity, and wide applicability. It has important clinical translational value and public health significance. Attached Figure Description

[0061] Figure 1: Electrophoresis results of anti-dengue virus NS1 protein antibody 5E3;

[0062] Figure 2: HPLC results of anti-dengue virus NS1 protein antibody 5E3;

[0063] Figure 3: Results of ELISA detection of the binding activity of anti-dengue virus NS1 protein antibody 5E3. Detailed Implementation

[0064] The present invention will be further illustrated below with reference to specific embodiments. These embodiments are for illustrative purposes only and should not be construed as limiting the invention. Those skilled in the art will understand that various changes, modifications, substitutions, and variations can be made to these embodiments without departing from the principles and spirit of the invention. The scope of the invention is defined by the claims and their equivalents. The experimental consumables, reagents, and raw materials used in this invention are readily available to those skilled in the art and, unless otherwise specified, can be obtained commercially. Experimental methods not specifying specific conditions are generally performed under conventional conditions or according to the manufacturer's recommendations. In particular, the following embodiments are for illustrative purposes only and should not limit the scope of the invention in any way. It should be noted that the experimental conditions and results described in the following embodiments are for illustrative purposes only and should not, and will not, limit the invention as described in detail in the claims.

[0065] Example 1: Screening for antibodies targeting dengue virus NS1 protein

[0066] 1. Immunogen Recombinant Expression

[0067] The sequence of dengue virus NS1 protein was synthesized and constructed into the pCDNA3.1 vector; plasmid for transfection was extracted; the plasmid was transfected into HEK293 cells and cultured for 7 days; the supernatant was harvested, purified by Ni column, and obtained by concentration and replacement buffer.

[0068] The sequence of the recombinant dengue virus NS1 protein is shown in SEQ ID NO:1, and the sequence information is shown in Table 1 below.

[0069] Table 1. Sequence of recombinant dengue virus NS1 protein

[0070]

[0071] 2. Immunization of mice, SP2 / 0 fusion, selection, and subcloning.

[0072] The first immunization was administered with Freund's complete adjuvant, 100 μg per animal, via intraperitoneal injection, with a total dose of 0.5 mL per animal. A second immunization was administered 3 weeks later. From the second immunization onwards, Freund's incomplete adjuvant was administered at a dose of 50 μg / 0.5 mL per animal, with a third immunization administered 2 weeks later. Cell fusion was prepared 10 days after the third injection.

[0073] To obtain feeder cells, use 10 5 One hole per use, laid up 10 plates the day before fusion. 5 100 μL / well; mouse immune spleen cells and prepared myeloma cells were fused with PEG fusion agent and seeded into 96 cell culture plates containing feeder cells, 100 μL / well.

[0074] Positive wells were screened using ELISA. Recombinant dengue virus NS1 protein was plated overnight. The plates were washed, blocked with skim milk powder, and incubated at 37°C for 1 h. After washing, 100 μL of 96-well culture supernatant was added, and the plates were incubated at 37°C for 1 h. After washing, HRP-labeled goat anti-mouse secondary antibody was added, and the plates were incubated at 37°C for 30 min. After washing, chromogenic buffer was added, and the plates were developed for 10 min. Stop solution was then added, and OD values ​​were read. 450 The numerical value; screening for high expression levels of cell lines for subcloning culture.

[0075] 3. Sequence Fishing

[0076] Cells were collected, and total RNA was extracted using Trizol. cDNA was generated by reverse transcription using oligo(dT)20 primers. Then, the heavy and light chain variable regions were amplified by PCR using specific primers. After electrophoresis purification, the PCR products were transformed into a TA clone insertion vector, and positive clones were selected for sequencing.

[0077] 4. Experimental Results

[0078] Antibody 5E3 targeting the dengue virus NS1 protein was screened out, and its sequence information is shown in Tables 2 and 3.

[0079] Table 2. Sequences of the heavy chain and light chain variable regions of antibody 5E3 targeting dengue virus NS1 protein.

[0080]

[0081] Table 3. CDR sequence of antibody 5E3 targeting dengue virus NS1 protein.

[0082]

[0083] Example 2: Expression and validation of antibodies targeting dengue virus NS1 protein

[0084] 1. Antibody expression

[0085] The amino acid sequence of antibody 5E3, which targets the dengue virus NS1 protein and was screened as in Example 1, was optimized according to the human codon, sent to GenScript for synthesis, and constructed into the pcDNA3.4 vector. Large-scale plasmid preparation was then carried out for later use.

[0086] One day before transfection, healthy 293 cells (70-80% adherent cells and 3×10⁶ suspension cells) were transfected. 5DNA (cells / mL) was seeded into culture dishes. On the day of transfection, endotoxin-free plasmids were prepared at an HC:LC ratio of 1:1-1.2. The amount of PEI was calculated with N / P = 3:1. DNA and PEI were diluted separately with serum-free medium and mixed. The mixture was incubated at room temperature for 15-20 min to form a complex. The complex was added to the cells and cultured at 37°C and 5% CO2. The medium was changed after 6-12 h. The supernatant was collected after 48-96 h of culture. The antibody concentration and activity were detected by UV 280 nm, ELISA, and SDS-PAGE.

[0087] The results showed that the concentration of the 5E3 clone was 389 mg / L.

[0088] 2. Physicochemical properties

[0089] (1) Antibody purity was determined by SDS-PAGE.

[0090] Main instruments: Chemiluminescence imaging system, Tanon-5200, purchased from Tanon; electrophoresis apparatus, Powererpac Basic, purchased from BIO-RAD; electrophoresis tank, DYC-Mini4, purchased from BIO-RAD.

[0091] Main reagents: 1 M Tris-HCl buffer, purchased from Beijing Solarbio Science & Technology Co., Ltd.; 1.5 M Tris-HCl buffer, purchased from Beijing Solarbio Science & Technology Co., Ltd.; 10% SDS, purchased from Beijing Solarbio Science & Technology Co., Ltd.; FastStain, purchased from Gene Universal; 30% gel mixing solution (29:1), purchased from Beijing Solarbio Science & Technology Co., Ltd.; Rainbow 180 broad-spectrum protein marker, purchased from Beijing Solarbio Science & Technology Co., Ltd.

[0092] Sample preparation: Take 20 μL of sample and mix it evenly with 5 μL of 5× reducing buffer, heat it at 95℃ for 5 min, and then cool it; take 20 μL of sample and mix it evenly with 5 μL of 5× non-reducing buffer.

[0093] Electrophoresis: Prepare the gel, add an appropriate amount of electrophoresis buffer, load the sample, and perform electrophoresis.

[0094] Staining and destaining: After electrophoresis, place the gel in an appropriate amount of Coomassie Brilliant Blue staining solution and stain at room temperature for 1 hour or longer; pour out the staining solution, add an appropriate amount of Coomassie Brilliant Blue destaining solution, and destain at room temperature for 4-24 hours. After destaining, soak in ddH2O, compare with the unstained gel using the marker protein as a reference, cut off the gel containing the desired protein component, collect it, and separate the protein to be purified from the gel.

[0095] Figure 1 shows the electrophoresis results of antibody 5E3 targeting dengue virus NS1 protein. From left to right, the bands are the marker and the reduction band, respectively. The results show that the detection purity of 5E3 is greater than 95%.

[0096] (2) The purity of the antibody was determined by HPLC.

[0097] Main instruments: Liquid chromatograph, purchased from Agilent Technologies, model 1100 / 1200 / 1260; Liquid chromatography column, purchased from Tosoh Corporation, model WieTSKgeL G3000SWxl, 7.8 mm I.D. × 30 cm; pH meter, purchased from Sartorius Scientific Instruments Co., Ltd.; Electronic balance, purchased from Sartorius Scientific Instruments Co., Ltd.

[0098] Main reagents: dipotassium hydrogen phosphate trihydrate, potassium dihydrogen phosphate, and potassium chloride were all purchased from Sinopharm Chemical Reagent Co., Ltd.

[0099] Mobile phase preparation: Add potassium dihydrogen phosphate trihydrate, potassium dihydrogen phosphate, and potassium chloride to approximately 900 mL of purified water, stir to dissolve, and bring the volume to 1 L. Measure the pH using a pH meter to ensure it is between 6.2 and 0.1. Filter through a 0.22 μm filter membrane and store at room temperature.

[0100] Sample preparation: System suitability sample: Standard diluted to 2 mg / mL with mobile phase; Test sample: Sample to be tested diluted to 2 mg / mL with mobile phase.

[0101] Chromatographic conditions: injection volume 25 µL, injector temperature 6℃, run time 30 min, flow rate 0.5 mL / min, column temperature 30℃, detection wavelength 280 nm (VWD) or detection wavelength 280 nm with bandwidth 4 nm, reference wavelength 360 nm with bandwidth 80 nm (DAD), peak width 0.025 min (0.5 s).

[0102] Figure 2 shows the HPLC results of antibody 5E3 targeting dengue virus NS1 protein. The results show that the purity of 5E3 is greater than 95%.

[0103] 3. ELISA was used to detect the antibody binding activity.

[0104] Coating: Dilute the antigen (dengue virus NS1 protein) to 2 μg / mL with coating buffer, mix well, add 100 μL / well to a 96-well coated plate, seal with a membrane, and incubate overnight at 4°C. Wash the plate three times with a plate washer, ensuring no liquid residue remains on the plate after the last wash, and pat dry the surface of the plate with absorbent paper.

[0105] Blocking: Add 5% milk powder (0.5 g milk powder dissolved in 10 mL DPBS), 300 μL / well, incubate at 37℃ for 1 h, and wash the plate 3 times as described above. Serially dilute the antibody to 100 μL / well, react at 37℃ for 1 h, and wash the plate 3 times as described above.

[0106] Secondary antibody: Dilute with DPBS at a ratio of 1:2000, add 100 μL / well to a 96-well plate, react at 37°C for 1 h, and wash the plate 3 times according to the above steps.

[0107] Color development: Add TMB, 100 μL / well, and develop color at room temperature in the dark for 10 min.

[0108] Termination: Add 100 μL of 2N H2SO4 per well. Measure OD using a microplate reader. 450 Detection within 10 minutes.

[0109] Figure 3 shows the ELISA results of the binding activity of antibody 5E3, which targets the dengue virus NS1 protein. The results indicate that antibody 5E3 specifically binds to the dengue virus NS1 protein in a concentration-dependent manner. 50 The concentration was 0.1334 μg / mL, indicating high affinity.

[0110] 4. Specific detection of antibodies

[0111] Cultures of dengue virus NS1 protein, Zika virus, SARS-CoV-2, influenza A, influenza B, parainfluenza virus, adenovirus, and respiratory syncytial virus were coated with the same concentration, antibodies and secondary antibodies were added, colorimetric analysis was performed, readings were taken, and data were analyzed.

[0112] The specificity detection results of antibody 5E3 targeting dengue virus NS1 protein are shown in Table 4 below. The results show that only dengue virus NS1 protein (dengue virus type 1-4 NS1 protein) showed normal color development, while the other antigens did not show positive reactions. That is, antibody 5E3 targeting dengue virus NS1 protein can specifically bind to dengue virus NS1 protein and has high specificity.

[0113] Table 4. Specificity detection results of antibody 5E3 targeting dengue virus NS1 protein.

[0114]

Claims

1. An antibody against dengue virus NS1 protein or an antigen-binding fragment thereof, wherein the amino acid sequences of the heavy chain variable regions CDR-H1, CDR-H2, and CDR-H3 of the antibody or the antigen-binding fragment are as shown in SEQ ID NO:4-6, respectively; and the amino acid sequences of the light chain variable regions CDR-L1, CDR-L2, and CDR-L3 of the antibody or the antigen-binding fragment are as shown in SEQ ID NO:7-9, respectively.

2. The antibody or its antigen-binding fragment as described in claim 1, wherein the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:

2.

3. The antibody or antigen-binding fragment thereof as described in claim 1, wherein the amino acid sequence of the light chain variable region is shown in SEQ ID NO:

3.

4. A nucleic acid molecule encoding the antibody or antigen-binding fragment thereof as described in any one of claims 1-3.

5. An expression vector comprising the nucleic acid molecule of claim 4.

6. A recombinant host cell comprising the expression vector of claim 5.

7. The recombinant host cell as described in claim 6, wherein the host cell is a eukaryotic cell.

8. The recombinant host cell of claim 7, wherein the eukaryotic cell is a mammalian cell or a yeast cell.

9. A dengue virus detection reagent, said detection reagent comprising the antibody or antigen-binding fragment thereof as described in any one of claims 1-3.

10. A dengue virus detection product, said detection product comprising the antibody or antigen-binding fragment thereof as described in any one of claims 1-3, or the detection reagent as described in claim 9.

11. The detection product as described in claim 10, wherein the detection product is a detection kit, a detection chip, or a test strip.

12. A diagnostic product for dengue virus infection, the diagnostic product comprising the antibody or antigen-binding fragment thereof as described in any one of claims 1-3, or the detection reagent as described in claim 9.

13. A method for detecting dengue virus NS1 protein for non-diagnostic and non-therapeutic purposes, the method comprising: The sample to be tested is contacted with the antibody or its antigen-binding fragment as described in any one of claims 1-3 to detect the formation of antigen-antibody immune complexes.

14. A method for preparing the antibody or antigen-binding fragment thereof according to any one of claims 1-3, the method comprising: Culturing the recombinant host cell according to any one of claims 6-8, inducing antibody expression, separating and purifying the culture product, and obtaining the antibody or its antigen-binding fragment.

15. The use of the antibody or antigen-binding fragment thereof of any one of claims 1-3, the nucleic acid molecule of claim 4, the expression vector of claim 5, or the recombinant host cell of any one of claims 6-8 in the preparation of diagnostic products for diagnosing dengue virus infection.

16. The use of the antibody or antigen-binding fragment thereof of any one of claims 1-3, the nucleic acid molecule of claim 4, the expression vector of claim 5, or the recombinant host cell of any one of claims 6-8 in the preparation of a detection reagent for detecting dengue virus NS1 protein.

17. The use of the antibody or antigen-binding fragment thereof of any one of claims 1-3, the nucleic acid molecule of claim 4, the expression vector of claim 5, or the recombinant host cell of any one of claims 6-8 in the preparation of a detection product for detecting dengue virus NS1 protein.

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