West nile virus monoclonal antibody with neutralizing activity and preparation method and application thereof

Abstract

This invention provides a neutralizing West Nile virus monoclonal antibody, its preparation method, and its application. The invention involves immunizing mice with inactivated West Nile virus as an antigen, monitoring the neutralizing titer of the mouse serum during the immunization process, and screening for mice with the highest neutralizing titer. Then, myeloma cells SP2 / 0 are fused with immune spleen cells from the selected mice with the highest neutralizing titer. Positive hybridoma cells are screened, subcloned, and further screened through neutralization tests to obtain the hybridoma cell line C9-G11-F3, which secretes a neutralizing West Nile virus monoclonal antibody. The monoclonal antibody secreted by this hybridoma cell line C9-G11-F3 not only has the ability to neutralize West Nile virus but also exhibits cross-protective ability against Japanese encephalitis virus, demonstrating significant research and application value.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to a neutralizing West Nile virus monoclonal antibody, its preparation method, and its application. Background Technology

[0002] West Nile virus (WNV) was first discovered in 1937 from the blood of a woman with a fever in the West Nile region of Uganda, hence its name. It is an enveloped positive-sense RNA virus belonging to the Flaviviridae family and Flavivirus genus. It is also a mosquito-borne virus that can cause fatal neurogenic encephalitis in humans and horses, and can cause death in birds, chickens, and other animals.

[0003] In 2022, cases of human WNV infection were reported in Antelope Canyon, San Fernando Valley, and San Gabriel Valley, with a total of 6 cases identified, most of which developed symptoms in late July and early August. According to statistics released by the Hellenic Centre for Disease Control, Greece confirmed 35 cases of WNV infection in 2019, including 2 deaths reported in the week of August 2-8 alone. This has had a significant impact on the local healthcare system and public health, and has also posed a major challenge to global public health.

[0004] Although there are currently no confirmed cases of WNV in my country, it has already ravaged temperate regions of Europe and North America. With the development of global economic and trade integration, invasive diseases pose a threat to global health, and their prevention and control hold a crucial strategic position in safeguarding national health and the stability of the healthcare system. However, there are currently no specific drugs or commercially available vaccines against WNV, while neutralizing antibodies are well-known for their effectiveness in disease prevention and control. Therefore, screening and preparing WNV neutralizing antibodies is of great significance for further research into the pathogenic mechanism of WNV and the prevention and control of invasive diseases.

[0005] In the prior art, patent CN104498438A discloses a West Nile virus monoclonal antibody and kit. This patent involves purifying recombinantly expressed West Nile virus envelope protein E and immunizing mice with it. Spleen cells from these immunized mice are then fused with SP2 / 0 mouse myeloma cells, and the cells are cultured, further subcloned, and screened for a monoclonal antibody against West Nile virus and hybridoma cells that secrete these antibodies. However, this method, which uses E protein to immunize mice, only yields antibodies with high binding titers, which have low neutralizing activity. Furthermore, the antibodies obtained by this method are only specifically targeting West Nile virus and do not provide cross-protection against other flavivirus species, thus limiting their applicability as therapeutic antibody drugs.

[0006] In view of this, it is necessary to design an improved West Nile virus monoclonal antibody with neutralizing activity, as well as its preparation method and application, to solve the above problems. Summary of the Invention

[0007] In view of the shortcomings of the prior art, the purpose of this invention is to provide a monoclonal antibody that has a good neutralizing effect on West Nile virus and cross-protective ability against Japanese encephalitis virus.

[0008] To achieve the above objectives, the present invention provides a West Nile virus monoclonal antibody with neutralizing activity, secreted by the hybridoma cell line C9-G11-F3, which has the ability to neutralize West Nile virus and also has cross-protective ability against Japanese encephalitis virus; the hybridoma cell line C9-G11-F3 is deposited at the China Center for Type Culture Collection, with accession number CCTCC NO:C202391.

[0009] As a further improvement of the present invention, the monoclonal antibody includes a heavy chain variable region and a light chain variable region; the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:1, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:2.

[0010] As a further improvement of the present invention, the nucleotide sequence encoding the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:3, and the nucleotide sequence encoding the amino acid sequence of the light chain variable region is shown in SEQ ID NO:4.

[0011] To achieve the above objectives, the present invention provides a method for preparing the above-mentioned neutralizing West Nile virus monoclonal antibody, comprising the following steps:

[0012] S1. Prepare West Nile virus solution, and after sequentially inactivating and concentrating it, store it as an antigen for later use.

[0013] S2. The antigen prepared in step S1 is used to perform an immunization test on mice, and the serum neutralization titer of the mice in the immunization test is monitored. Mice with the highest neutralization titer are selected for shock immunization.

[0014] S3. Mouse myeloma cells SP2 / 0 were fused with immune spleen cells from mice after the shock immunization in step S2. Positive hybridoma cells were screened for subcloning, and the subcloned positive cell lines were screened by neutralization test to obtain hybridoma cell line C9-G11-F3 that can secrete West Nile virus monoclonal antibodies with neutralizing activity.

[0015] As a further improvement of the present invention, in step S2, after monitoring the neutralizing titer of the antiserum of the test animals, the immunization procedure of the immunization test is adjusted according to the monitored neutralizing titer.

[0016] As a further improvement of the present invention, in step S2, the serum of the mouse with the highest neutralizing titer selected has a neutralizing titer of 1:3200 against West Nile virus.

[0017] As a further improvement of the present invention, in step S3, the monoclonal antibody secreted by the obtained hybridoma cell line C9-G11-F3 has a neutralizing titer of 1:50 against West Nile virus.

[0018] As a further improvement of the present invention, in step S3, the monoclonal antibody secreted by the obtained hybridoma cell line C9-G11-F3 has a protective efficacy of 40% against West Nile virus infection and a protective efficacy of 20% against Japanese encephalitis virus infection.

[0019] This invention also provides the application of the above-mentioned neutralizing West Nile virus monoclonal antibody in the preparation of humanized antibodies.

[0020] The present invention also provides the application of the above-mentioned neutralizing West Nile virus monoclonal antibody in the preparation of a drug; the drug is a drug for the prevention or treatment of diseases caused by at least one of West Nile virus and Japanese encephalitis virus.

[0021] The beneficial effects of this invention are:

[0022] This invention provides a method for preparing a neutralizing West Nile virus monoclonal antibody. The method involves immunizing mice with inactivated West Nile virus as the antigen, monitoring the neutralizing titer of the mouse serum during the immunization process, and adjusting the immunization program based on the monitoring results to optimize the most suitable immunization program for screening mice with high serum neutralizing titers. Furthermore, this invention fuses immune spleen cells from the selected mice with high serum neutralizing titers with myeloma cells SP2 / 0. Positive hybridoma cells are screened, subcloned, and then further screened through a neutralization test. The resulting hybridoma cell line, C9-G11-F3, secretes a neutralizing West Nile virus monoclonal antibody. This monoclonal antibody is a neutralizing antibody that not only has a high neutralizing titer against West Nile virus but also exhibits cross-protective ability against Japanese encephalitis virus. This provides new raw materials for exploring the pathogenic mechanism of West Nile virus and also offers new ideas for the research and design of therapeutic antibodies or drugs targeting West Nile virus and Japanese encephalitis virus. Attached Figure Description

[0023] Figure 1 This is a diagram verifying the inactivation effect of West Nile virus.

[0024] Figure 2 The results show the neutralizing titer of mouse serum immunized with inactivated West Nile virus as an antigen on day 90.

[0025] Figure 3 A comparison of neutralizing titers in mouse serum on day 43 after immunization with different antigens.

[0026] Figure 4 The neutralizing titer of monoclonal antibodies secreted by hybridoma cell line C9-G11-F3 against West Nile virus and Japanese encephalitis virus was determined.

[0027] Figure 5 The graph shows the binding and neutralization effects of JEV on cell lines screened using the conventional ELISA method.

[0028] Figure 6 Comparison of neutralization effects of hybridoma cell supernatants obtained using different screening methods.

[0029] Figure 7 This is the result of testing the genetic stability of hybridoma cells.

[0030] Figure 8 The results of SDS-PAGE analysis show the purification effect of ascites fluid.

[0031] Figure 9 Analysis of the protective rate of the monoclonal antibody provided by this invention in mice.

[0032] Figure 10 The results show the viral load in the mouse brain.

[0033] Figure 11 The results show the detection of relevant cytokines in mouse brain tissue.

[0034] Figure 12 The results of IHC analysis of mouse brain tissue sections.

[0035] Figure 13 HE staining results of mouse brain tissue sections.

[0036] Figure 14 It is a humanized heavy chain plasmid for monoclonal antibodies.

[0037] Figure 15 It is a humanized light chain plasmid for monoclonal antibodies.

[0038] Figure 16 This is a diagram illustrating the neutralization effect of humanized monoclonal antibodies. Detailed Implementation

[0039] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be described in detail below with reference to the accompanying drawings and specific embodiments.

[0040] It should also be noted that, in order to avoid obscuring the present invention with unnecessary details, only the structures and / or processing steps closely related to the present invention are shown in the accompanying drawings, while other details that are not closely related to the present invention are omitted.

[0041] Additionally, it should be noted that the terms “comprising,” “including,” or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such process, method, article, or apparatus.

[0042] This invention provides a neutralizing West Nile virus monoclonal antibody secreted by hybridoma cell line C9-G11-F3, which has the ability to neutralize West Nile virus and also has cross-protective ability against Japanese encephalitis virus; the hybridoma cell line C9-G11-F3 is deposited at the China Center for Type Culture Collection, with accession number CCTCC NO:C202391.

[0043] The monoclonal antibody includes a heavy chain variable region and a light chain variable region; the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:1, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:2; the nucleotide sequence encoding the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:3, and the nucleotide sequence encoding the amino acid sequence of the light chain variable region is shown in SEQ ID NO:4.

[0044] The present invention also provides a method for preparing the above-mentioned neutralizing West Nile virus monoclonal antibody, comprising the following steps:

[0045] S1. Prepare West Nile virus solution, and after sequentially inactivating and concentrating it, store it as an antigen for later use.

[0046] S2. The antigen prepared in step S1 is used to perform an immunization test on mice, and the serum neutralization titer of the mice in the immunization test is monitored. Mice with the highest neutralization titer are selected for shock immunization.

[0047] S3. Mouse myeloma cells SP2 / 0 were fused with immune spleen cells from mice after the shock immunization in step S2. Positive hybridoma cells were screened for subcloning, and the subcloned positive cell lines were screened by neutralization test to obtain hybridoma cell line C9-G11-F3 that can secrete West Nile virus monoclonal antibodies with neutralizing activity.

[0048] Through the above methods, this invention selects inactivated West Nile virus as the antigen and uses the results of the neutralization test as the main screening basis in both the immunization test and the screening of hybridoma cell lines. Compared with the existing technology that uses protein as the antigen for immunization and uses the binding titer detected by indirect ELISA as the main screening basis, this invention effectively solves the problems of low antibody neutralization activity and lack of cross-protection against other species of flaviviruses. The West Nile virus monoclonal antibody secreted by the hybridoma cell line C9-G11-F3 obtained by this invention is a neutralizing antibody that not only has a high neutralizing titer against West Nile virus, but also has cross-protection against Japanese encephalitis virus, and has high research significance and application value.

[0049] Preferably, in step S2, after monitoring the neutralizing titer of the antiserum of the test animals, the immunization procedure for the immunization test is adjusted according to the monitored neutralizing titer. This operation allows for the selection of the most suitable immunization procedure to improve the neutralizing titer of mouse serum. The resulting selected mice exhibit a neutralizing titer of 1:3200 against West Nile virus, significantly superior to the conventional neutralizing titer levels in existing technologies.

[0050] Preferably, in step S3, after screening the subcloned positive cell lines through a neutralization test, a hybridoma cell line with high neutralization titer is obtained; the monoclonal antibody secreted by the hybridoma cell line C9-G11-F3 obtained by the final screening has a neutralization titer of 1:50 against West Nile virus; the monoclonal antibody has a 40% protective efficacy against West Nile virus infection and a 20% protective efficacy against Japanese encephalitis virus infection, thus achieving cross-protection against West Nile virus and Japanese encephalitis virus.

[0051] This invention also provides the application of the aforementioned neutralizing West Nile virus monoclonal antibody in the preparation of humanized antibodies. Humanized antibodies can be obtained by sequencing and humanizing a mouse-derived monoclonal antibody. Furthermore, this invention also provides the application of the aforementioned neutralizing West Nile virus monoclonal antibody in the preparation of a drug; said drug is a remedy for the prevention or treatment of diseases caused by at least one of West Nile virus and Japanese encephalitis virus.

[0052] The following detailed description, with reference to specific embodiments, illustrates the neutralizing West Nile virus monoclonal antibody, its preparation method, and its application provided by the present invention.

[0053] Example 1

[0054] This embodiment provides a method for preparing a West Nile virus monoclonal antibody with neutralizing activity, specifically including the following steps:

[0055] S1. Preparation of antigen

[0056] 1.1. Large-scale culture of West Nile virus

[0057] BHK-21 cells were revived and passaged at least three times until the cells were stable. The cells were then inoculated into T175 mm cells. 2 When the cell culture flasks are approximately 80% full, WNV NY99 cells are inoculated at a dose of 0.01 MOI and incubated at 37°C in a 5% CO2 incubator for 1 hour. The supernatant is discarded, and DMEM maintenance medium is added. The cells are then cultured at 37°C in a 5% CO2 incubator for 36-72 hours, with continuous monitoring of cell status. When cells exhibit pathological morphology such as rounding, bubbling, or detachment, they are placed at -80°C. This freeze-thaw cycle is repeated three times. Cells are collected in 50 mL sterile centrifuge tubes and centrifuged at 1000 rpm for 10 minutes. Cell debris is filtered through a 0.22 μm filter membrane, and the resulting supernatant is the West Nile virus solution. The West Nile virus solution is aliquoted into 1.5 mL sterile centrifuge tubes, 500 μL per tube, and stored at -80°C for later use.

[0058] 1.2 Virus titer determination

[0059] BHK-21 cells in good growth condition were seeded into 12-well plates and cultured until the cells covered 90% of the wells. The cells were then washed twice with serum-free DMEM medium. The West Nile virus solution to be measured was serially diluted 10-fold, thoroughly mixed, and added to each well of the 12-well plate. 200 μL of the DMEM-diluted virus solution was added to each well, with two replicates for each dilution. The plates were incubated at 37°C in a 5% CO2 incubator for 1 hour, followed by washing twice with DMEM basal medium. 1 mL of 1.5% sodium carboxymethyl cellulose was added to each well, and the plates were incubated at 37°C without disturbing the cells. On the fifth day, the cells were fixed with plaque fixative for 12 hours. The plaque fixative was discarded, and the cells were gently rinsed with tap water and air-dried. Then, the cells were stained with plaque staining solution for 4 hours, gently rinsed with tap water, patted dry, and the plaques were counted. The virus titer was calculated using the formula: Plaque Forming Units (PFU / mL) = X1 + X2 + X3 + ... + Xn / n * V × d. Where X1, X2, X3, ..., Xn represent the number of empty plaques obtained in different well plates with the same virus dilution, n represents the number of wells in the culture plate used for calculation, V represents the volume of virus diluent added, and d represents the dilution factor of the virus diluent. In this embodiment, the calculated virus titer is 2 × 10⁻⁶. 7 PFU / mL.

[0060] 1.3. Inactivation of West Nile virus and verification of inactivation effect

[0061] West Nile virus solution was inactivated using β-propiolactone: β-propiolactone at a final concentration of 0.05% was added to the West Nile virus solution to be inactivated. After incubation at 4°C for 24 hours, the solution was placed at 37°C for 2 hours to hydrolyze the β-propiolactone, yielding the inactivated West Nile virus solution. The inactivated West Nile virus solution was blindly passaged into BHK-21 cells for 3 passages to verify its inactivation effect. The results are as follows: Figure 1 As shown.

[0062] exist Figure 1 In the image, A shows a comparison of cell states after three blind passages in BHK-21 cells: the negative control group (con), uninactivated West Nile virus (WNV) solution, and inactivated West Nile virus solution (WNV + β-propiolactone). B shows a comparison of plaque results between inactivated West Nile virus solution (WNV + β-propiolactone) and uninactivated West Nile virus solution (WNV) after three blind passages. Figure 1 It can be confirmed that the West Nile virus in the inactivated West Nile virus solution obtained by the above method has been inactivated.

[0063] 1.4 Concentration of inactivated virus solution

[0064] The inactivated West Nile virus solution was centrifuged at 4°C and 30,000 r / min for 2 h using a preparative high-speed centrifuge. The supernatant was carefully discarded, and the inactivated West Nile virus particles were resuspended in 1 mL of sterile PBS and stored as antigens for later use. The protein concentration was determined to be 0.9 mg / mL by the BCA method.

[0065] S2, Animal Immunity

[0066] The antigen prepared in step S1 was used to perform an immunization test on mice. In this example, the mice used for the immunization test were 6-week-old female BALB / c mice (purchased from the Veterinary Hospital of Huazhong Agricultural University, referred to as mice).

[0067] After each immunization, blood was collected from the mandibular vein of mice. The serum titer was first determined using an indirect ELISA method to confirm successful immunization, followed by a neutralization test. The neutralization titer of the mouse serum was monitored in real time. The specific steps for determining the neutralization titer using the neutralization test are as follows:

[0068] (1) Place the serum in a water bath at 56°C for 30 minutes to inactivate complement;

[0069] (2) Dilute the serum using a serial dilution method;

[0070] (3) Mix the diluted serum with an equal volume of virus thoroughly and place in a 37°C incubator for 1.5 hours;

[0071] (4) Inoculate the virus-serum mixture onto a 24-well cell plate covered with a monolayer of BHK-21 cells;

[0072] (5) After incubating the cell plate at 37°C for 1 hour, wash it twice with serum-free DMEM medium.

[0073] (6) Add the prepared 1.5% sodium carboxymethyl cellulose to the cell plate and incubate at 37°C without moving it. On the fifth day, fix with 10% formaldehyde fixative for 12 hours, then stain with crystal violet staining solution for 4 hours. After recovering the staining solution, gently rinse under tap water, pat dry, and count the empty spots.

[0074] (7) Calculation: Neutralizing antibody is calculated using the PRNT50 method, which is the serum dilution with half the number of plaques as the positive control.

[0075] After determining the neutralizing titer of mouse serum using the method described above, the immunization procedure for the immunization test was adjusted based on this neutralizing titer. The principle of adjustment was to obtain mice with the highest possible neutralizing titer. After numerous experimental adjustments, the optimal mouse immunization procedure is shown in Table 1.

[0076] Table 1 shows the preferred mouse immunization program.

[0077]

[0078] Following the primary immunization and four booster immunizations according to the above immunization schedule, the serum neutralization titer of mice was measured on day 90 post-immunization as follows: Figure 2 As shown in the figure. A represents the mouse serum neutralization curve, B represents the mouse serum neutralization titer statistics, C represents the mouse serum titer against West Nile virus (WNV) and Japanese encephalitis virus (JEV), and 8-22 in the figure represents the mouse number. Figure 2 It can be seen that the serum of mouse No. 21, which has the highest neutralizing titer against West Nile virus, also has the highest neutralizing titer against Japanese encephalitis virus. Its neutralizing titer against West Nile virus can reach 1:3200, and its neutralizing titer against Japanese encephalitis virus can reach 1:400, which is significantly better than the conventional neutralizing titer level in the existing technology.

[0079] To verify the effect of the choice of immunogenic antigen on the neutralizing titer of mouse serum, the conventional envelope protein (E protein) was used as the antigen, and the immunization experiment was performed as described above. Mouse serum was collected on day 43 post-immunization, and the neutralizing titer was determined using the PRNT method. This result was compared with the neutralizing titer of mouse serum immunized with inactivated West Nile virus (WNV) 43 days post-immunization in this example. The results are as follows: Figure 3 As shown. By Figure 3 It can be seen that the conventional method of using E protein as an antigen not only has a low neutralizing titer, but also does not provide cross-protection against JEV in mouse serum. However, the present invention selects inactivated West Nile virus as the antigen, which can significantly improve the neutralizing titer against WNV and also enable some mice to show cross-protection against JEV, so as to obtain monoclonal antibodies with high neutralizing titers and cross-protection against JEV in subsequent screening. At the same time, the neutralizing titer of mouse serum on day 43 after immunization with inactivated West Nile virus as the antigen is significantly lower than that on day 90 after immunization, indicating that immunization according to the above immunization procedure can also effectively improve the neutralizing titer of mouse serum.

[0080] Three days before cell fusion, the selected 21 mice were given a shock immunization (0.2 mg of antigen was injected intraperitoneally).

[0081] S3, Cell Fusion and Hybridoma Cell Screening

[0082] 3.1 Preparation of SP2 / 0 tumor cells

[0083] SP2 / 0 cells were prepared from mouse solid tumors. The specific steps are as follows:

[0084] (1) Resuscitate SP2 / 0 cells frozen in the laboratory and culture them in six-well plates. When the cells are in good condition, resuspend them in 1640 basal culture medium (purchased from Wuhan Saiwei Biotechnology Co., Ltd.) and inject them subcutaneously into BALB / c mice (without immunization). About 14 days later, tumors grow on the backs of the mice.

[0085] (2) Kill the mice by pulling their necks and soaking them in 75% alcohol for 5 minutes;

[0086] (3) Under sterile conditions on a clean bench, take the tumor and place it in a sterile homogenizer. Add 5 mL of 1640 basic culture medium and grind it thoroughly. Then add 10 mL of 1640 basic culture medium, mix well, and let it stand for 5 min. After the larger tissue blocks settle to the bottom of the tube, take the upper cell suspension into a centrifuge tube for later use. Add 10 mL of 1640 basic culture medium to resuspend the tissue. Repeat this washing process twice. Centrifuge the cell suspension at 1000 r / min for 10 min, and take the precipitate to resuspend it in 15 mL of 1640 basic culture medium.

[0087] (4) Add 20 mL of lymphocyte separation medium to another 50 mL centrifuge tube, gently add the cell suspension on top of the lymphocyte separation medium, centrifuge at 1000 r / min for 10 min, use a pipette to aspirate the dense white cell layer at the interface, wash once with 10 mL of 1640 basal culture medium, count and set aside.

[0088] 3.2 Preparation of immune spleen cells

[0089] Take one mouse that has undergone shock immunization in step S2, euthanize it by exsanguination through the eye socket, collect the blood and separate the serum, which is the positive serum.

[0090] After immersing the mice in 75% ethanol for 5 minutes, they were transferred to a clean bench and placed on a dissecting board. The forelimbs were fixed, and the hindlimbs were crossed (left hindlimb crossed). The skin was first cut and torn to expose the peritoneum. A new set of scissors and forceps were used to cut open the peritoneum to expose the spleen. Under aseptic conditions, the spleen was removed and placed in a homogenizer. 5 mL of 1640 basal medium was added and the cells were homogenized. Then, 10 mL of 1640 basal medium was added, and the mixture was allowed to stand for 5 minutes. The supernatant was gently aspirated into a centrifuge tube. 10 mL of 1640 basal medium was added to the homogenizer, and the mixture was allowed to stand for 5 minutes. This washing process was repeated twice. The cells were centrifuged at 1000 rpm for 10 minutes, and the supernatant was discarded. The spleen cells were resuspended in an appropriate amount of 1640 basal medium for later use.

[0091] 3.3 Preparation of feeder cells

[0092] An unimmunized BALB / c mouse was euthanized by exsanguination through the orbital rim, and the blood was collected and the serum was separated to obtain negative serum.

[0093] After immersing mice in 75% alcohol for 5 minutes, feeder spleen cells were prepared according to the method for preparing immune spleen cells in step 3.2 above. The resulting feeder spleen cells were resuspended in an appropriate amount of HAT medium (purchased from Sigma) and evenly spread in a 96-well cell culture plate for later use, 100 μL / well.

[0094] 3.4 In vitro fusion of myeloma cells and immune spleen cells

[0095] (1) Mix the SP2 / 0 myeloma cell suspension (1×107 cells) obtained in step 3.1 with the immune spleen cell suspension (1×108 cells) obtained in step 3.2 in a 50 mL centrifuge tube and centrifuge at 1000 r / min for 10 min.

[0096] (2) Empty the supernatant (you can use sterile filter paper to absorb the water), and gently tap the bottom of the centrifuge tube to loosen the cells;

[0097] (3) Place the centrifuge tube containing the cell mixture in a 37°C water bath and slowly add 0.8 mL of 50% polyethylene glycol (PEG) preheated to 37°C within 1 min, while gently stirring with the tip of a pipette.

[0098] (4) Continue stirring for 1 minute;

[0099] (5) Slowly add 10 mL of 1640 basic culture medium preheated to 37°C within 5 min. When adding, it should be slow and constantly and gently stirred. Finally, slowly add 30 mL of 1640 basic culture medium preheated to 37°C.

[0100] (6) Centrifuge at 1000 r / min for 10 min, discard the supernatant, and place at 37℃ for 7 min;

[0101] (7) Resuspend the cells in an appropriate amount of HAT medium, spread them evenly on a 96-well cell culture plate containing feeder cells, 100 μL / well, and incubate at 37°C in a 5% CO2 incubator.

[0102] 3.5 Screening of positive hybridoma cells

[0103] On day 4 after fusion, 50 μL of fresh HAT medium was added. On days 8-10, all medium was discarded and replaced with HT medium (purchased from Sigma) (i.e., 1640 basal medium + 20% fetal bovine serum + 1% penicillin / streptomycin + 2% HT (hypoxanthine + thymidine)). When the fused cell colonies reached 1 / 4 the size of the culture wells and the cell supernatant turned yellow, the hybridoma cell supernatant was detected using a standard indirect ELISA method. The specific steps were as follows:

[0104] Concentrated inactivated West Nile virus solution and concentrated supernatant of uninfected BHK-21 cells (as negative controls) were coated onto 96-well ELISA plates and incubated overnight at 4°C. After coating, the plates were washed three times with PBST buffer (PBS buffer + 0.5% Tween), and incubated at 37°C for 1 hour with 1% blocking buffer (1 g bovine serum albumin dissolved in 100 mL PBST buffer). The plates were then washed three times with PBST, and hybridoma cell culture supernatant was added and incubated at 37°C for 1 hour. After washing three times with PBST, goat anti-mouse IgG-HRP (Wuhan Boster Biological Engineering Co., Ltd.) was added and incubated at 37°C for 1 hour. After washing three times with PBST, substrate solution (purchased from Wuhan Keqian Animal Biological Products Co., Ltd.) and chromogenic solution (purchased from Wuhan Keqian Animal Biological Products Co., Ltd.) were added. After 10 minutes, the colorimetric reaction was observed, and stop solution (purchased from Wuhan Keqian Biotechnology Co., Ltd.) was added. The OD value was measured at 630 nm. Cell growth wells that reacted positively with the antigen-coated plate but negatively with the negative control plate, and showed vigorous cell growth and good morphology, were selected for further subcloning.

[0105] 3.6 Subcloning of positive hybridoma cells

[0106] Before cloning, prepare feeder cells (the specific steps are the same as in step 3.3). Limit dilution of hybridoma cells that tested positive for ELISA is performed so that each well contains 1-2 hybridoma cells. Cell growth in the wells is then observed and recorded periodically. After 8-10 days of cloning, when the cells have grown to approximately 1 / 3-1 / 2 the size of the culture well, they are detected using an indirect ELISA method (the specific steps are the same as in step 3.5). Wells with single colonies that test positive are selected and cloned again using the same method to further purify the cell line.

[0107] 3.7 Screening of hybridoma cell lines

[0108] Positive cell lines that underwent two subcloning processes were screened using a neutralization assay (the specific steps are the same as in step S2). Cell lines with the highest neutralizing activity were then subjected to a third subcloning process to obtain hybridoma cell lines capable of secreting monoclonal antibodies against West Nile virus with high neutralizing activity. The neutralizing titers of these hybridoma cell lines against other species of flaviviruses, including Japanese encephalitis virus (JEV), dengue virus (DENV), and Zika virus (ZIKV), were then determined. The results showed that the monoclonal antibodies secreted by the selected hybridoma cell line C9-G11-F3 had neutralizing effects against both West Nile virus (WNV) and Japanese encephalitis virus (JEV). The neutralizing titer results are as follows: Figure 4 As shown.

[0109] Depend on Figure 4It can be seen that the monoclonal antibody secreted by the hybridoma cell line C9-G11-F3 has a neutralizing titer of 1:50 against West Nile virus, indicating that it has a good neutralizing effect against West Nile virus; at the same time, the monoclonal antibody also has a certain neutralizing effect against Japanese encephalitis virus, and has cross-protective ability against Japanese encephalitis virus.

[0110] To verify the impact of hybridoma cell line screening methods on neutralization efficacy, hybridoma cell lines were screened using only the standard ELISA method without additional neutralization assays. Hybridoma cell lines exhibiting strong WNV binding reactions (OD630 > 2.0) in their supernatants were selected, and their JEV binding capacity was measured. The results are as follows: Figure 5 As shown in Figure A, blue wells indicate cross-reactivity with JEV, meaning they have the ability to bind JEV, while gray wells indicate negative reactions, meaning they do not have the ability to bind JEV. This figure shows that the supernatant of 28 cell lines had the ability to bind JEV. Further testing of the JEV neutralization effect on these 28 cell lines yielded the following results: Figure 2 As shown in Figure B, only one of the 28 strains exhibited a low (<10%) neutralizing effect at the cellular level. This extremely low neutralizing effect at the cellular level is insufficient to provide actual protection in animal experiments and offers no cross-protection against Japanese encephalitis virus. Further comparison of the neutralizing effect of this cell line against West Nile virus with the neutralizing effect of the hybridoma cell supernatant screened using the neutralization test in this embodiment yielded the following results: Figure 6 As shown. In Figure 6 In the diagram, A represents the plaque assay results of the inhibitory effect of hybridoma cell supernatants selected under two screening methods on West Nile virus, and B represents the statistical data on the inhibitory effect of hybridoma cell supernatants selected under two screening methods on West Nile virus. Figure 6 It can be seen that the neutralization effect of the hybridoma cell supernatant selected by the neutralization assay in this embodiment is significantly better than that of the hybridoma cell supernatant selected by the conventional ELISA method alone.

[0111] Therefore, compared with conventional ELISA screening methods, this invention obtains mice with high serum neutralizing titers based on specific immune conditions, and then combines neutralization tests as screening conditions. This not only allows for more accurate and efficient screening of hybridoma cell lines that have cross-protective effects against Japanese encephalitis virus, but also provides high neutralizing titers against both West Nile virus and Japanese encephalitis virus.

[0112] To test the genetic stability of the hybridoma cell line C9-G11-F3 obtained in this embodiment, the hybridoma cells were treated with colchicine, and chromosome counting was performed under an oil immersion microscope using Giemsa staining. SP2 / 0 cells were treated in the same way as a negative control. The results are as follows: Figure 7 As shown in the figure, counting revealed that the hybridoma cells had far more chromosomes (approximately 100) than the control group myeloma SP2 / 0 cells, approximately the sum of the chromosomes in myeloma cells (approximately 60) and mouse spleen cells (approximately 40). Therefore, it can be inferred that the harvested hybridoma cells were indeed derived from the fusion of myeloma cells and mouse spleen cells, and that they were capable of stable inheritance.

[0113] In this embodiment, the obtained monoclonal antibody was also prepared and purified in large quantities, and the specific operations are as follows:

[0114] Four 10-week-old BALB / c mice were intraperitoneally injected with 0.5 mL of incomplete Fluor adjuvant per mouse. Seven to ten days later, the expanded hybridoma cell lines were collected and injected into mice intraperitoneally (10⁵ to 10⁶ cells per mouse). Seven to ten days later, ascites fluid was collected, centrifuged at 2000 r / min for 10 min, and the supernatant was used for later use.

[0115] The ascites fluid obtained by centrifugation was purified according to the instructions of the IgG antibody purification kit (purchased from Thermo Biotechnology). The specific steps are as follows:

[0116] 1) Sample preparation: The mouse ascites fluid was thawed on ice. The thawed ascites fluid was diluted 80 times with equilibration buffer (20mM PB pH=7.0). First, all the thawed ascites fluid was transferred into a beaker. The equilibration buffer was slowly added to the wall in small amounts. The diluted sample was filtered through a 0.45μm filter membrane.

[0117] 2) Clean the AKTA instrument and purification column according to the operating requirements of the AKTA instrument and antibody purification kit, and complete the operations such as equilibration and sample loading.

[0118] 3) Elution: Pause the AKTA instrument and elute the antibody with citric acid (0.1M citric acid pH=3.0). Adjust the flow rate to 1mL / min. After the peak is eluted, collect the eluent in a clean centrifuge tube.

[0119] 4) Refolding: Pause the AKTA instrument and use acetic acid (1M) to refold the antibody. Adjust the flow rate to 2.5 mL / min, approximately 3 column volumes.

[0120] 5) Pause the instrument and use equilibration buffer to wash away the strong acid solution used in the first two steps. Elute at a flow rate of 3 mL / min for about 3 column volumes.

[0121] 6) Washing: Stop the instrument and use NaOH (0.1M) solution to wash for about 3 column volumes at a flow rate of 2.5 mL / min to remove impurities.

[0122] 7) Instrument cleaning: After purification, clean the instrument with ddH2O and 20% ethanol respectively.

[0123] 8) Take a portion of the antibody eluted in step 3) and perform SDS-PAGE gel verification. The analysis results are as follows: Figure 8 As shown, the remaining antibody was aliquoted into 1.5 mL EP tubes and stored at -20°C for later use.

[0124] exist Figure 8 In the diagram, M represents the protein marker, and 1 represents the purified monoclonal antibody sample. Figure 8 It can be seen that the purified monoclonal antibody samples all showed antibody heavy chain bands at 55 kDa and antibody light chain bands at 25 kDa, indicating good antibody purification effect.

[0125] To verify the protective effect of the monoclonal antibody obtained in this embodiment in vivo, the following animal challenge protection experiment was conducted:

[0126] The animal experiments were divided into five groups as follows: monoclonal antibody treatment group (WNV+C9-G11-F3), cross-protection group (JEV+C9-G11-F3), WNV negative control group (WNV+Negative serum), JEV negative control group (JEV), and positive control group (WNV+Positive serum). The WNV infection dose was 10... 6 PFU, JEV infection dose is 10 5 PFU was administered via intraperitoneal injection in all cases. Monoclonal antibodies were administered intraperitoneally at a dose of 20 mg / kg on day 1 post-challenge, and at a dose of 5 mg / kg on days 2 and 3 post-challenge. Clinical manifestations, weight changes, and mortality were observed and recorded at the same time each day from day 1 to 20 post-challenge. Results are as follows: Figure 9 As shown. By Figure 9 It can be seen that the monoclonal antibody provided in this embodiment has a 40% protective efficacy against West Nile virus infection in mice and a 20% protective efficacy against Japanese encephalitis virus infection in mice; furthermore, treatment with the monoclonal antibody prolongs the time to onset of the disease in mice after challenge.

[0127] To investigate the effects of the monoclonal antibody prepared in this embodiment on mice infected with West Nile virus or Japanese encephalitis virus, the mice were grouped and challenged as described above. On day 7 post-challenge, the mice were euthanized and immersed in 75% ethanol for 5 minutes. The skin on the back of the neck of the mice was cut open using sterile surgical instruments to expose the brain tissue. The brain tissue was aseptically removed, washed in sterile PBS, and stored for later use. A portion of the stored mouse brain tissue was used to determine the viral titer using a plaque assay. Another portion was used to extract RNA from the mouse brain tissue using the Trizol method and reverse transcribed into cDNA to detect the expression of relevant inflammatory factors and viral load in the mouse brain tissue. The remaining portion was prepared into brain tissue sections for IHC and HE staining analysis. The experimental results are as follows: Figure 10-13 As shown.

[0128] exist Figure 10 In the table, A represents the West Nile virus titer in mouse brain tissue determined by plaque assay; B represents the West Nile virus load in mouse brain tissue determined by quantitative real-time fluorescence assay; C represents the Japanese encephalitis virus titer in mouse brain tissue determined by plaque assay; and D represents the Japanese encephalitis virus load in mouse brain tissue determined by quantitative real-time fluorescence assay. Figure 10 and Figure 11 As can be seen, compared with the negative control group, the viral load in the brain tissue of mice treated with monoclonal antibodies and the expression levels of cytokines such as IL-1β, CCL-5, IFN-β, and IL-6 were significantly reduced, proving that the monoclonal antibody provided in this embodiment can protect mice from West Nile virus and Japanese encephalitis virus infection in vivo.

[0129] Microglia and astrocytes in brain tissue, when activated, can secrete various chemokines and cytokines to defend against the invasion of pathogenic microorganisms. Therefore, the number and state of these two cell types are often used to evaluate the intensity of the inflammatory response in brain tissue. IBA-1 is a specific marker for microglia; when microglia are activated, their cell bodies enlarge, their spikes increase, and they appear deeply stained brown in IHC staining. GFAP is a specific marker for astrocyte maturation; after activation, it appears as irregular "star-shaped" cells deeply stained brown in IHC staining. Figure 12 It can be seen that astrocytes and microglia were significantly activated in the negative serum treatment group mice. The average optical density statistics also showed that the number of IBA-1 and GFAP cells was significantly higher in the group mice than in the antibody treatment group mice, indicating that the monoclonal antibody prepared in this invention has a protective effect against West Nile virus infection in mice. Similarly, dark brown microglia and astrocytes were observed in the brain tissue of mice infected with Japanese encephalitis virus, while the activation and proliferation of these two cell types were significantly reduced in the brain tissue of mice treated with monoclonal antibody, indicating that the monoclonal antibody has a therapeutic effect against Japanese encephalitis virus infection in mice.

[0130] Combination Figure 13 HE staining results also showed that on day 7 post-infection, mice in the negative serum treatment group exhibited typical pathological features of encephalitis, with extensive inflammatory cell infiltration in the brain tissue, a large number of inflammatory cells accumulating around blood vessels in the cerebral cortex forming a vascular "cuff" phenomenon, and extensive inflammatory cell infiltration, necrosis, and neuronal atrophy in the striatum, thalamus, and midbrain. In contrast, the monoclonal antibody treatment group did not show these pathological features of encephalitis. These results further demonstrate that monoclonal antibodies have a therapeutic effect against West Nile virus. Similarly, the brain tissue of mice infected with Japanese encephalitis virus also showed pathological features of JEV encephalitis, with extensive inflammatory cell infiltration, while the brain tissue of mice in the monoclonal antibody treatment group did not show similar pathological changes. These results demonstrate that the monoclonal antibody C9-G11-F3 also has a certain therapeutic effect on Japanese encephalitis virus infection.

[0131] Example 2

[0132] This embodiment provides the application of the neutralizing West Nile virus monoclonal antibody prepared in Example 1 in the preparation of humanized antibodies.

[0133] 1. Amplification of VH and VL sequences of murine monoclonal antibodies and construction of recombinant humanized antibody plasmids

[0134] After PCR amplification of the C9-G11-F3 variable region gene of the monoclonal antibody prepared in Example 1, agarose gel electrophoresis was performed. A single, clear band appeared at approximately 500 bp under a gel imaging system. The target band was recovered and mixed with the pMD vector. TM After 19-T ligation, sequencing was performed, and the structures with antibody variable regions were predicted using antibody structure prediction software (http: / / www.vbase2.org / ).

[0135] The amino acid sequence of the heavy chain variable region of monoclonal antibody C9-G11-F3 is shown in SEQ ID NO:1, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:2; the nucleotide sequence encoding the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:3, and the nucleotide sequence encoding the amino acid sequence of the light chain variable region is shown in SEQ ID NO:4. The specific sequence listing is as follows:

[0136] SEQ ID NO:1

[0137] DVQPQQSGVELMKPGASVKMSCKASGYRFTSYLIEWVKQRPGHGLEWIGEILPGSGSTNYNEKFQGKATFTADTSSNTAYLQLSTLTSEDSAVYYCGREKGGYAMDYWGQGTSVIVSS

[0138] SEQ ID NO:2

[0139] DIVMTQTPLSLPVSLGDQASISCKSSHSLTHINGNTYLHWYLQKPGQSPQLLIYKVSNRFSGVPDRFIGSGSGTDFTLRITRVEAEDLGVYFCSQSTHVPPWSFGGGTKLEIK

[0140] SEQ ID NO:3

[0141] GACGTCCAACCGCAGCAATCTGGAGTTGAATTGATGAAGCCTGGGGCCTCAGTGAAGATGTCCTGCAAGGCTTCTGGCTACAGATTCACTAGTTACTTGATAGAGTGGGTAAAACAGAGGCCTGGACATGGCCTTGAGTGGATTGGAGAGATTTTACCTGGAAGTGGTAGTACTAATTATAATGAGAAGTTTCAGGGCAAGGCCACATTCACTGCAGATACATCCTCCAATACAGCCTACTTGCAGCTCAGTACCCTGACATCTGAGGACTCTGCCGTCTATTACTGTGGAAGAGAGAAGGGGGGCTATGCTATGGACTACTGGGGTCAAGGAACTTCAGTCATCGTCTCCTCA

[0142] SEQ ID NO:4

[0143] GATATTGTGATGACCCAAACTCCACTCTCCCTGCCTGTCAGTCTTGGAGATCAAGCCTCCATCTCTTGCAAATCTAGTCACAGCCTTACACACATTAATGGCAACACCTATTTACATTGGTACCTGCAGAAGCCAGGCCAGTCTCCACAGCTCCTAATCTACAAAGTTTC CAACCGATTTTCTGGGGTCCCAGACAGGTTCATTGGCAGTGGATCAGGGACAGATTTCACACTCAGGATCACCAGAGTGGAGGCTGAGGATCTGGGAGTTTATTTCTGCTCTCAAAGTACACATGTTCCTCCGTGGTCGTTCGGTGGAGGCACCAAGCTGGAAATCAAGC

[0144] The PCR products of the VH and VL genes of the monoclonal antibody C9-G11-F3 were ligated to the antibody heavy chain vector backbone pFUSE2ss-CHIg-hG1 (double-digested with restriction endonucleases EcoRI and BamHI) and the antibody light chain vector backbone pFUSE2ss-CLIg-hK (double-digested with restriction endonucleases EcoRI and KpnI), respectively, via homologous recombination. The ligation products were transformed into *E. coli* Trans5α, and single colonies were selected for colony PCR identification. Colonies showing bands of the expected size were sequenced to obtain recombinant plasmids with completely correct nucleotide sequences. The successfully constructed recombinant plasmids of the monoclonal antibody C9-G11-F3 were named pFUSE2ss-CHIg-hG1-C9(…). Figure 14 ) and pFUSE2ss-CLIg-hK-C9( Figure 15 ).

[0145] 2. Acquisition of recombinant antibodies and verification of their neutralizing effect

[0146] HEK-293T cells were transfected with the successfully constructed monoclonal antibody C9-G11-F3 humanized heavy and light chain plasmids pFUSE2ss-CHIg-hG1-C9 and pFUSE2ss-CLIg-hK-C9. After 48 hours, the culture supernatant was collected, and the neutralization effect was measured. The results are as follows: Figure 16 As shown. By Figure 16 It can be seen that the recombinant humanized antibody (C9-G11-F3[human]) can still neutralize WNV infection.

[0147] In summary, this invention provides a neutralizing West Nile virus monoclonal antibody, its preparation method, and its application. This invention involves immunizing mice with inactivated West Nile virus as an antigen, monitoring the neutralizing titer of the mouse serum during the immunization process, and screening for mice with the highest neutralizing titer. Then, myeloma cells SP2 / 0 are fused with immune spleen cells from the selected mice with the highest neutralizing titer. Positive hybridoma cells are screened, subcloned, and further screened through neutralization tests to obtain the hybridoma cell line C9-G11-F3, which secretes a neutralizing West Nile virus monoclonal antibody. The monoclonal antibody secreted by this hybridoma cell line C9-G11-F3 not only has the ability to neutralize West Nile virus but also exhibits cross-protective ability against Japanese encephalitis virus, demonstrating significant research and application value.

[0148] The above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention.

Claims

1. A monoclonal antibody against West Nile virus having neutralizing activity, characterized in that: It is secreted by hybridoma cell line C9-G11-F3 and has the ability to neutralize West Nile virus, while also having cross-protective ability against Japanese encephalitis virus; the hybridoma cell line C9-G11-F3 is deposited at China Center for Type Culture Collection, with accession number CCTCC NO:C202391.

2. The West Nile virus monoclonal antibody having neutralizing activity according to claim 1, characterized in that: The monoclonal antibody includes a heavy chain variable region and a light chain variable region; the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:1, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:

2.

3. The West Nile virus monoclonal antibody having neutralizing activity according to claim 2, characterized in that: The nucleotide sequence encoding the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:3, and the nucleotide sequence encoding the amino acid sequence of the light chain variable region is shown in SEQ ID NO:

4.

4. The use of a West Nile virus monoclonal antibody with neutralizing activity as described in any one of claims 1-3 in the preparation of humanized antibodies.

5. Use of a West Nile virus monoclonal antibody having neutralizing activity according to any one of claims 1 to 3 for the manufacture of a medicament. 5 The drug is a drug for the prevention or treatment of diseases caused by at least one of West Nile virus and Japanese encephalitis virus.

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