Monoclonal antibody V6-M15 against tick-borne encephalitis virus and preparation method and application thereof

Abstract

The application provides a hybridoma cell strain, the preservation number of the hybridoma cell strain is CCTCC NO: C2024394, and the hybridoma cell strain is prepared from spleen lymphocytes of mice immunized by a vaccine containing a recombinant protein with an amino acid sequence as shown in SEQ ID NO. 1. Further, the hybridoma cell is used to prepare a monoclonal antibody V6-M15 which can be highly specific to bind to a trimer conformation in a membrane fusion process of EDIII, and the monoclonal antibody V6-M15 can effectively inhibit the amplification of three TBEV, and can provide broad-spectrum protection against three TBEV subtype strains. The application provides a new strategy for developing more effective TBEV specific monoclonal antibodies and prevention and treatment of TBEV.

Description

Technical Field

[0001] This invention belongs to the field of monoclonal antibody preparation technology, specifically relating to a monoclonal antibody V6-M15 against tick-borne encephalitis virus, its preparation method, and its application. Background Technology

[0002] Tick-borne encephalitis virus (TBEV) belongs to the genus Flaviviridae in the family Flaviviridae. It is an enveloped virus with a single-stranded positive-sense RNA genome approximately 11 kb long, encoding a polyprotein that later produces three structural proteins and seven non-structural proteins through protease cleavage. TBEV is an arthropod-borne virus (arbovirus), primarily transmitted by the castor beetle and the persulcatus tick; there are also cases of transmission through unpasteurized dairy products. TBEV is mainly distributed in forests and grasslands of Europe and Asia. With climate change, the spread of TBEV has gradually expanded, and it is now widely distributed worldwide. It can infect the central nervous system, leading to severe neurological diseases such as encephalitis and meningitis, and in severe cases, even death. Many patients develop serious long-term neurological complications. In recent years, the incidence of tick-borne encephalitis has been rising, and TBEV has become the third leading cause of infectious encephalitis globally, causing a significant clinical and economic burden worldwide. Currently, there are no specific antiviral drugs for TBEV; clinical treatment is mainly supportive, including analgesia, antipyretics, and maintenance of vital signs.

[0003] For enveloped viruses, protein-mediated membrane fusion is a dynamic process in which specialized protein mechanisms undergo significant conformational changes that drive the two membrane bilayers together, resulting in lipid mixing and opening fusion pores between previously separated membrane-binding compartments. Membrane fusion is a critical step in enveloped viral entry, facilitating the delivery of the viral genome into the host cell. The low pH environment of the endosomal compartment triggers the fusion of the viral particle with the host cell membrane, leading to the release of the viral RNA genome into the host cell cytoplasm. The fusion process is a key step in viral infection and is essential for the release and replication of the viral genome. For TBEVs, external structural proteins primarily facilitate the recognition and binding of host cell receptors or membrane interactions to enable cell entry. Surface structural proteins typically contain antigenic determinants capable of inducing neutralizing antibodies. Of the three structural proteins of flaviviruses (capsid, membrane, and envelope), envelope glycoprotein (E) plays a crucial role in various viral biological processes, including viral entry, genome release, assembly, viral particle maturation, and expulsion. Structural protein E is considered the major viral antigen responsible for inducing neutralizing antibodies in vivo. During endosome fusion, the E protein undergoes a major low-pH-induced rearrangement, transforming from a dimer to a trimer. At pH 5.5, the E protein forms a fusion-open trimer with its fusion loop exposed, inserting into the endosome membrane. The three domains of the E protein rotate, bringing the viral membrane closer to the endosome membrane. The virus then fuses with the endosome membrane, creating a pathway for the release of the viral genome. Domain III (EDIII) plays a crucial role in this pH-induced rearrangement, which is essential for the subsequent release of the viral genome and for combating viral infection.

[0004] Monoclonal antibodies (mAbs) are antibodies produced by a single type of B cell clone, exhibiting high specificity and homogeneity. Their research and application have seen significant advancements in recent decades. Currently, monoclonal antibodies are widely used in the diagnosis and treatment of various diseases, particularly cancer, autoimmune diseases, and infectious diseases. With the development of molecular biology and genomics, researchers are constantly uncovering new disease targets, driving the development of new monoclonal antibodies. Monoclonal antibodies have shown promising efficacy in the antiviral field. Screening and isolating monoclonal antibodies from the serum of recovered patients or post-vaccination serum is an effective method for obtaining monoclonal antibodies. Previous studies have shown that monoclonal antibodies are mainly obtained from the serum of infected individuals, making them difficult to obtain and costly. During flavivirus infection, monoclonal antibodies can protect the host through various mechanisms, including blocking viral attachment to the cell surface, disrupting viral membrane fusion, mediating viral clearance through Fc-dependent effector function, or directly neutralizing the virus. Currently, several monoclonal antibodies that can effectively neutralize West Nile virus, dengue virus, and Zika virus have been reported. Monoclonal antibodies inhibit various stages of the viral life cycle. Most of these antibodies target the lateral crest of EDIII, a highly conserved epitope in flaviviruses. This indicates that EDIII domain-induced monoclonal antibodies play a crucial role in the prevention and treatment of flaviviruses. However, research on monoclonal antibodies against TBEV is currently lacking. Therefore, there is an urgent need to develop more effective TBEV-specific monoclonal antibodies to provide new strategies for the prevention and treatment of TBEV. Summary of the Invention

[0005] In view of this, the present invention utilizes the vaccine TBEV-EDIII-Fd-F containing a recombinant protein with an amino acid sequence as shown in SEQ ID NO.1 (this vaccine has been described in detail in another patent application filed by the applicant on the same day, entitled "A recombinant protein and its construction method and its application in a tick-borne encephalitis virus vaccine") to immunize the spleen lymphocytes of mice to obtain hybridoma cells, and finally obtains the monoclonal antibody V6-M15 from the hybridoma cells.

[0006] The SEQ ID NO.1 is as follows: KFTWKRIPTDSGHDTVVMEVAFSGTKPCRIPVRAVAHGSPDVNVAMLITPNPTIETNGGGFIEMQLPPGDNIIYVGELSHQWFQKGSGSGSGSGYIPEAPRDGQAYVRKDGEWVLLSTFLGGSGSGSMTTASTSQVRQNYHQDSEAAINRQ INLELYASYVYLSMSYYFDDRDDVALKNFAKYFLHQSHEEREHAEKLMKLQNQRGGRIFLQDIKKPDCDWESGLNAMECALHLEKNVNQSLLELHKLATDKNDPHLCDFIETHYLNEQVKAIKELGDHVTNLRKMGAPESGLAEYLFDKHTLGDSDNES.

[0007] One of the objectives of this invention is to provide a hybridoma cell line, the accession number of which is CCTCC NO: C2024394.

[0008] The second objective of this invention is to provide a clonal antibody V6-M15 produced from the above-mentioned hybridoma cell line.

[0009] The third objective of this invention is to provide the application of the above-mentioned monoclonal antibody V6-M15 in the preparation of a kit for detecting or assisting in the detection of tick-borne encephalitis virus.

[0010] The fourth objective of this invention is to provide the application of the above-mentioned monoclonal antibody V6-M15 in the prevention and treatment of tick-borne encephalitis virus.

[0011] Compared with the prior art, the beneficial effects of the present invention are as follows: This invention utilizes hybridoma cells with accession number CCTCC NO: C2024394 to prepare the monoclonal antibody V6-M15, which can specifically bind to the trimer conformation during EDIII membrane fusion. Monoclonal antibody V6-M15 exhibits good neutralizing effects against three tick-borne encephalitis viruses (Far Eastern subtype TBEV-FE, European subtype TBEV-Nd, and Siberian subtype TBEV-SiB), with a concentration reaching 10... 2 The infection rate was almost 0 at µg / mL; in mouse infection experiments, the monoclonal antibody V6-M15 showed good protective effects, with the survival rate of mice in the TBEV-Nd and TBEV-SiB groups reaching 100% and the survival rate of mice in the TBEV-FE group reaching 80%. Attached Figure Description

[0012] Figure 1The image shows the results of highly specific binding of the monoclonal antibody V6-M15 to the EDIII-Fd-F protein.

[0013] Figure 2 Figure 1 shows the results of neutralization assays of monoclonal antibody V6-M15 against tick-borne encephalitis virus; Figure A is the WH2012-Far East subtype GenBank: KJ755186.1; Figure B is the European subtype GenBank: U27495.1; Figure C is the IM-Sib-2020-Siberian subtype GenBank: OR827302.

[0014] Figure 3 Figure 1 shows the results of the protective experiment of monoclonal antibody V6-M15 against tick-borne encephalitis virus (WH2012-Far East subtype GenBank: KJ755186.1); Figure A shows viremia 3 days after infection; Figure B shows viremia 7 days after infection; Figure C shows the viral titer of brain tissue after infection; Figure D shows the survival rate of mice after infection; Figure E shows the pathological section of the brain of mice after infection.

[0015] Figure 4 Figure 1 shows the results of a protective experiment against tick-borne encephalitis virus (European subtype GenBank: U27495.1) with monoclonal antibody V6-M15. Among them, A is viremia 3 days after infection; B is viremia 7 days after infection; C is a statistical graph of viral titer in brain tissue after infection; D is a statistical graph of mouse survival rate after infection; and E is a pathological section of mouse brain after infection.

[0016] Figure 5 Figure 1 shows the results of a protective assay against tick-borne encephalitis virus (IM-Sib-2020-Siberian subtype GenBank: OR827302) with monoclonal antibody V6-M15. In the figure, A represents viremia 3 days after infection; B represents viremia 7 days after infection; C represents the viral titer in brain tissue after infection; D represents the survival rate of mice after infection; and E represents a pathological section of the brain of mice after infection. Detailed Implementation

[0017] The present invention will now be described in further detail with reference to specific embodiments, so that those skilled in the art can more clearly understand the present invention.

[0018] Example 1 This embodiment provides a method for preparing the monoclonal antibody V6-M15, as detailed below: (1) Animal immunization Five Balb / c mice were immunized, each with an immunization dose of 20 μg. For the first immunization, the immunogen EDIII-Fd-F was prepared as an emulsion with an equal volume of complete Freund's adjuvant and injected subcutaneously at multiple sites in the abdomen. Two weeks later, the same dose of immunogen was prepared as an emulsion with an equal volume of incomplete Freund's adjuvant and injected subcutaneously at multiple sites in the abdomen. Blood was collected one week after the third immunization to determine serum titers. A booster immunization was performed 39 days after the third immunization, and blood was collected one week after that to determine serum titers. Mice were selected for booster immunization, and the spleens of the mice were harvested three days after the booster immunization for hybridoma fusion.

[0019] (2) Blood collection and titer testing One week after the last immunization, 50-60 μL of blood was collected from the orbital venous plexus of mice. After standing overnight at 4°C, the supernatant serum was separated by centrifugation for testing. Appropriate amounts of the target protein EDIII-Fd-F and the tag protein F were taken for detection. The proteins were diluted to 5 μg / mL with coating buffer, and then 100 μL was added to each well of a 96-well plate using a single-channel pipette. The plate was gently tapped to mix the sample, sealed tightly with plastic wrap, and coated overnight at 4°C. The plate was washed once with 200 μL of washing buffer per well, and the plate was shaken dry. The plate was then blocked with 300 μL of blocking buffer per well at room temperature for 1 hour. After washing the plate twice with 400 μL / well of the solution, add the serially diluted sample and sample diluent at 100 μL / well. Simultaneously add the detection antibody at 100 μL / well to each 96-well plate and incubate at room temperature for 2 hours. Wash the plate five times with 400 μL / well of the washing buffer, add 200 μL / well of the chromogenic solution, and incubate at room temperature for 12 minutes. Stop the reaction by adding 50 μL / well of the stop solution. Detect the reaction using a microplate reader at a wavelength of 450 nm. Spleen cells were extracted from mice with high EDIII-Fd-F serum titers and low F serum titers.

[0020] (3) Mouse monoclonal antibody fusion and screening All spleen cells from immunized mice were collected and mixed with mouse myeloma cells at a 1:1 ratio. Hybridoma cells were obtained by electrofusion. The cells were coated with the antigen protein EDIII-Fd-F and the tag protein F. The cell supernatant was analyzed by ELISA to obtain positive clones that bound the antigen protein but not the tag protein. Positive clones were selected and subjected to two limiting dilutions. The subclonal supernatants obtained after limiting dilution were screened by ELISA to obtain monoclonal cells for subsequent production.

[0021] (4) Hybridoma cell culture Transfer 1 mL of hybridoma cells into a 100 mL culture flask. Add 10 mL of fresh culture medium every 48 hours to expand the cells to the required production volume. Then add hybridoma feeding solution every other day and culture for 6-8 days.

[0022] (5) Antibody purification Centrifugation: Centrifuge using a benchtop centrifuge at 1000g for 20 minutes, collect the supernatant, and select different filtration methods according to the volume of the liquid, using a 0.45μm filter membrane.

[0023] Connection system: Connect protein column A to the purification system; Water balance: Rinse the 3CV with ultrapure water to replace the 25% ethanol preservation solution; Equilibration column: AC binding + 1 / 5 stock buffer equilibrate 3CV until UV baseline is stable; Sample loading: Adjust the sample loading flow rate to 4CV; Rinse: Rinse with eluent for 5-10 CV until the UV baseline is stable, at the same flow rate as the sample loading flow rate; Elution: elute with AC elution and collect based on UV peak; Neutralization: Add 2M tris and neutralize the eluted antibody at pH 8.0; Balance: AC binding balances 3CV to neutral; CIP cleaning: CIP cleaning 5CV or higher; Rinse with alkali: Rinse with AC Binding until the pH at the outlet is neutral; Storage: Equilibrate to 2CV with 25% ethanol and store the column.

[0024] (6) ELISA identification Coating: Coating of the project antigen EDIII-Fd-F and tag protein F at concentrations of 0.1 μg / mL, 1 μg / mL, 5 μg / mL, 100 μL / well, overnight at 4°C; Sealing: Shake off the liquid inside the plate and pat dry. Add 2% BSA, 300 μL / well, seal, and incubate at room temperature for 1 hour. Washing: 300 μL / well washing solution, wash twice, pat dry after the last wash; Antibody dilution: Dilute the antibody to 1 μg / mL, add 100 μL to each well of the corresponding plate, mix well, and react at room temperature for 2 h; Wash the plate: 300 μL / well washing solution, wash the plate 3 times, and pat dry after the last wash; Add secondary antibody: Dilute Rabbit Anti-Mouse IgG F(ab)2 / HRP secondary antibody to 0.09 μg / mL, 100 μL / well, mix well, and incubate at room temperature for 1 h; Wash the plate: 300 μL / well washing solution, wash the plate 3 times, and pat dry after the last wash; Color development: Mix solution A and solution B at a 1:1 ratio, add 200 μL to each well, incubate at room temperature in the dark for 20 min to terminate, add 50 μL of stop solution to each well, and immediately measure the OD value at a wavelength of 450 nm. The specific results are shown in Table 1.

[0025] Table 1 ELISA Identification Results

[0026] As shown in Table 1, the V6-M15 monoclonal antibody can specifically recognize the antigen EDIII-Fd-F, but does not bind to the tag protein F.

[0027] (7) Identification of BLI biomolecular interactions Biomembrane interference experiments were performed using an Octet RED96e (Sartorius) instrument. V6-M15 antibody was immobilized at a concentration of 200 nM on an anti-mouse Fc Capture (AMC) biosensor (Sartorius 18-5088). EDIII-Fd-F and 3EDIII-F (representing the EDIII region of TBEV in ferritin, with three tandemly expressed EDIII epitopes lacking conformational structure) were bound and dissociated at concentrations of 1000 nM, 100 nM, and 10 nM. The instrument was operated according to the manual, with the experimental steps as follows: baseline time 120 s, loading time 600 s, baseline 2 time 120 s, association time 900 s, and dissociation time 900 s. Data were collected and analyzed; results are shown below. Figure 1 .

[0028] Depend on Figure 1 It was found that by treating mice with TBEV-EDIII-Fd-F nanoparticles and preparing monoclonal antibody cells using hybridoma technology, the monoclonal antibody V6-M15 was successfully screened and obtained after ELISA and BLI screening. V6-M15 showed high affinity for EDIII-Fd-F protein, but weak affinity for 3EDIII-F protein (which recognizes linear epitopes). This invention successfully screened out the monoclonal antibody V6-M15, which specifically binds to the trimer conformation during EDIII membrane fusion.

[0029] Example 2 This embodiment provides the neutralizing effect of the prepared monoclonal antibody V6-M15 and its in vivo validation experiment in mice. The specific steps are as follows: (1) Neutralization test of monoclonal antibody V6-M15 Mix the culture medium and V6-M15 (30 µg monoclonal antibody) with 500 PFU of TBEV, incubate at 4°C for 1 hour, and then add 5 × 10⁻⁶ PFU to each well of a 24-well plate. 4 In Vero, after 1 hour, the cells were washed four times with PBS to remove free virus and monoclonal antibodies. DMEM containing 10% FBS was added, and the cells were incubated for another 24 hours. The supernatant was then collected for qRT-PCR to determine viral genome content. The supernatant was collected by centrifugation at 5000 rpm for 5 min, and total RNA was extracted using an RNA extraction kit (Magen Cat: R4173-03). Virus-specific universal primers were used, as follows: TBEV-Far East Asian type sense primer: 5'-TAGGCGTGGTGGTTTTGAGG-3' and reverse primer: 5'-GTTTAGCCGTGCCCCGTGAC-3'; TBEV-European subtype sense primer: 5′-GGGCGGTTCTTGTTCTCC-3′ and reverse primer: 5′-ACACATCACCTCCTTGTCAGACT-3′; TBEV-Probe-WT, 5′-TGAGCCACCATCACCCAGACACA-3′, 5′-6-FAM, 3′-TAMRA-N; TBEV-Siberian subtype sense primer: 5′-GTTGTACGCATGGCTATGACTG-3′ and reverse primer: 5′-CAGGATCCAGTCATTTACCGCTC-3′.

[0030] Three different TBEV RNA subtypes were amplified. All real-time quantitative PCR was performed using the One Step TB GreenPrimeScript™ PLUS RT-PCR Kit (TaKaRa Code No. RR096A). RT-PCR assays were performed on a CFX96 Touch real-time PCR system (Bio-Rad). Specific results can be found in [link to results]. Figure 2 .

[0031] Depend on Figure 2 It can be seen that monoclonal antibody V6-M15 effectively inhibited the amplification of the three TBEV strains. After the addition of monoclonal antibody V6-M15, the viral titer in the cell culture supernatant was reduced by nearly 100% compared with the PBS group.

[0032] (2) Monoclonal antibody V6-M15 protection experiment Mice were infected with tick-borne encephalitis virus via intraperitoneal injection and treated with monoclonal antibodies 24 hours post-infection. Serum samples were collected from five mice on days 3 and 7 post-infection, and brain tissue samples were collected from the mice before death for viral titer detection. Tissue viral titer was determined using quantitative real-time PCR (qRT-PCR). After homogenization, centrifugation to remove the supernatant, viral RNA extraction and RT-PCR quantification were performed as described above.

[0033] For practical application, monoclonal antibody V6-M15 was used to treat animals infected with TBEV. Blank background mice were infected with a lethal dose of TBEV (TBEV-FE, 200 TCID50, 100 µL). Twenty-four hours later, the mice were inoculated with monoclonal antibody V6-M15 via intraperitoneal injection. Following the experimental method described above, the survival rate of the mice over the next 14 days was calculated. Specific results are shown below. Figure 3 .

[0034] Depend on Figure 3 It can be seen that, compared with the PBS group, the viral RNA levels in the serum and brain tissue of mice treated with monoclonal antibody V6-M15 were significantly reduced. Figure 3 AC); mice experienced less weight loss, most survived to the end of the experimental period, and survival time was significantly prolonged ( Figure 3 D); In addition, V6-M15 also inhibited the development of encephalitis symptoms (D); Figure 3 E).

[0035] Mice were infected with live TBEV-SiB (200 TCID50, 100 µL) or TBEV-Nd (200 TCID50, 100 µL). Data, including changes in body weight and survival rate, were collected according to the experimental methods described above. Results are shown in the table below. Figure 4 , 5 .

[0036] Depend on Figure 4 , 5 It can be seen that compared with the PBS group, the viral RNA level in mouse serum was reduced and the survival time was prolonged. The monoclonal antibody V6-M15 also inhibited the development of encephalitis symptoms. The monoclonal antibody V6-M15 successfully provided protection against TBEV-Nd and TBEV-SiB strains, and all infected mice survived during the experiment.

[0037] In summary, this invention successfully prepared a monoclonal antibody, V6-M15, that specifically binds to the trimer conformation during EDIII membrane fusion. Furthermore, the monoclonal antibody V6-M15 effectively inhibits the amplification of three TBEV strains and can provide broad-spectrum protection against three TBEV subtypes.

[0038] Unless otherwise specified, all raw materials used in this invention are existing substances that can be purchased directly from the market.

[0039] The above are merely preferred embodiments of the present invention and are not intended to limit the scope of protection of the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A hybridoma cell line, characterized in that, The accession number of the hybridoma cell line is CCTCC NO: C2024394.

2. The hybridoma cell line according to claim 1, characterized in that, The hybridoma cell line was prepared from spleen lymphocytes of mice immunized with a vaccine containing a recombinant protein with an amino acid sequence as shown in SEQ ID NO.

1.

3. A monoclonal antibody V6-M15 produced from the hybridoma cell line of claim 1.

4. The use of the monoclonal antibody V6-M15 according to claim 3 in the preparation of a kit for detecting or assisting in the detection of tick-borne encephalitis virus.

5. The use of the monoclonal antibody V6-M15 according to claim 3 in the prevention and treatment of tick-borne encephalitis virus.

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