Specific monoclonal antibodies and their application in the detection and neutralization of monkeypox virus

Abstract

The present invention relates to specific monoclonal antibodies and their applications. The amino acid sequence of the light chain CDR1 of the monoclonal antibody is shown in SEQ ID No. 1, the amino acid sequence of CDR2 is shown in SEQ ID No. 2, and the amino acid sequence of CDR3 is shown in SEQ ID No. 3; the amino acid sequence of the heavy chain CDR1 of the monoclonal antibody is shown in SEQ ID No. 4, the amino acid sequence of CDR2 is shown in SEQ ID No. 5, and the amino acid sequence of CDR3 is shown in SEQ ID No. 6. The present invention uses recombinant monkeypox virus surface protein E8L as an antigen. The screened monoclonal antibodies have good affinity for the recombinant monkeypox virus surface protein E8L at the molecular and cellular levels, can significantly inhibit the infection efficiency of VACV replication-deficient strains in VERO cells, and have good in vitro neutralization effect against monkeypox virus.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and in particular relates to a specific monoclonal antibody and its application in the detection and neutralization of monkeypox virus. Background Art

[0002] Monkeypox virus (MPXV) infection primarily damages multiple organs in the human body, including the skin, lungs, and gastrointestinal tract, and can be fatal in severe cases. Currently, the main clinical treatment for monkeypox (MPXV) is anti-smallpox virus drugs, and there is no effective treatment to control monkeypox infection. With the cessation of smallpox vaccination programs worldwide, an increasing number of people lack the adaptive immunity to rapidly respond to monkeypox infection. Therefore, there is an urgent need to develop new therapeutic drugs for monkeypox infection. Neutralizing antibodies have good clinical application prospects in inhibiting viral infection and clearing viruses from the body, such as Ebola hemorrhagic fever, respiratory syncytial infection, and COVID-19. At this stage, screening for high-affinity neutralizing antibodies against monkeypox virus has important clinical value in improving the treatment efficacy of monkeypox-infected patients and preventing the spread of monkeypox.

[0003] E8L is one of the potential protective antigenic proteins of monkeypox virus and is a homologous protein to vaccinia virus (VACV) D8L. E8L is expressed on the surface of IMV viruses and can bind to cell surface chondroitin sulfate and mediate the adsorption of the virus to cells, as reported by Hsiao, JC, et al., "Vaccinia virus envelope D8L protein binds to cells surface chondroitin sulfate and mediates the adsorption of intracellular mature virions to cells." Journal of Virology, vol. 1999, 73(10):8750-61. In addition, studies have shown that six protective antigens of D8L, including A27L, A33R, B5R, H3R and L1R, can induce a strong immune response in mice and induce high levels of neutralizing antibodies, such as Heraud, JM et al., "Subunit recombinant vaccine protects against monkeypox." Journal of immunology, 2006, 177(4): 2552-64.

[0004] In terms of neutralizing antibody development, since homologous proteins have similar biological activities and immunogenicity, most of the antibodies currently used to fight monkeypox virus are induced and screened by VACV-related antigens. For example, Hadas Tamir et al. screened out anti-D8L antibody MV33 and anti-A33R antibody EV42 through phage display technology. Both of them can effectively reduce the VACV titer in mice when used alone and protect them from death when exposed to lethal doses of the virus (Tamir, H. et al., "Synergistic effect of two human-like monoclonal antibodies confers protection against orthopoxvirus infection." Nature communications, 2024, 15(1):3265). At the same time, both have a significant protective effect against MPXV infection. Cross-immunity between members of the orthopoxvirus family suggests the broad-spectrum neutralizing effect of anti-E8L antibodies.

[0005] Currently, there are few reports on directly using MPXV antigens for immunization or screening of monoclonal antibodies. Mengjun Li et al. prepared neutralizing antibodies that bind to MPXV surface proteins A29L and A35R (homologous to VACV A27L and A33R) through hybridoma technology. Among them, anti-A29L antibodies showed good ability to inhibit viral infection both in vivo and in vitro, while anti-A33 antibodies had a weaker neutralizing effect (Li, M. et al., "Three neutralizing mAbs induced by MPXV A29L protein recognizing different epitopes act synergistically against orthopoxvirus." "Emerging microbes & infections", 2023, 12(2): 2223669). Runchu Zhao et al. screened two neutralizing antibodies targeting MPXV surface protein B6R (homologous to VACV B5R) from VACV vaccine recipients. Both antibodies showed broad anti-orthopoxvirus infection effects in a mouse model and have the potential to become antibody drugs for the treatment of monkeypox virus infection (Zhao, R. et al., "Two noncompeting human neutralizing antibodies targeting MPXV B6 show protective effects against orthopoxvirus infections." Nature Communications, 2024, 15(1):4660). However, there are currently no reports of monkeypox neutralizing antibodies produced by immunization or screening against E8L. Summary of the Invention

[0006] Based on the above-mentioned shortcomings and deficiencies in the prior art, one of the objects of the present invention is to at least solve one or more of the above-mentioned problems in the prior art. In other words, one of the objects of the present invention is to provide a specific monoclonal antibody that meets one or more of the above-mentioned needs and its application in the detection and neutralization of monkeypox virus.

[0007] In order to achieve the above-mentioned object of the invention, the present invention adopts the following technical solutions:

[0008] A monoclonal antibody that specifically binds to monkeypox E8L protein, wherein the amino acid sequence of the light chain CDR1 of the monoclonal antibody is shown in SEQ ID No. 1, the amino acid sequence of CDR2 is shown in SEQ ID No. 2, and the amino acid sequence of CDR3 is shown in SEQ ID No. 3;

[0009] The amino acid sequence of the heavy chain CDR1 of the monoclonal antibody is shown in SEQ ID No. 4, the amino acid sequence of CDR2 is shown in SEQ ID No. 5, and the amino acid sequence of CDR3 is shown in SEQ ID No. 6.

[0010] As a preferred embodiment, the amino acid sequence of the light chain variable region of the monoclonal antibody is shown as SEQ ID No. 7, and the amino acid sequence of the heavy chain variable region is shown as SEQ ID No. 8.

[0011] As a preferred embodiment, the amino acid sequence of the light chain variable region of the monoclonal antibody is shown as SEQ ID No. 9, and the amino acid sequence of the heavy chain variable region is shown as SEQ ID No. 10.

[0012] Among them, SEQ ID No. 1: QDVSNT;

[0013] SEQ ID No. 2: WAS;

[0014] SEQ ID No. 3: QHHYNTPYT;

[0015] SEQ ID No. 4: GYSITSDYA;

[0016] SEQ ID No.5: IMYSGNT;

[0017] SEQ ID No. 6: ARSHYGSNFVY;

[0018] SEQ ID No.7:

[0019] DIVMTQSHKFMSTSVGDRVTITCKASQDVSNTVAWYQQRPGQSPKLLI YWASTRHTGVPDRFTGSGSGTDYTLTISSVQAEDLALYYCQHHYNTPYTFG GGTKLEIK;

[0020] SEQ ID No.8:

[0021] DVQLQESGPGLVKPSQSLSLTCTVTGYSITSDYAWNWIRQSPGNKLEW MGYIMYSGNTNYNPSLKSRISITRDTSKNQLFLQLNSVTTEDTATYYCARSH YGSNFVYWGQGTLVTVSA;

[0022] SEQ ID No.9:

[0023] DIQMTQSPSSSLSASVGDRVTITCRASQDVSNTVAWYQQKPGKVPKLLI YWASTRHSGVPSRFSGSGSGTDFTLTISSLQPEDVATYYCQHHYNTPYTFGG GTKLEIK;

[0024] SEQ ID No.10:

[0025] QVQLQESGPGLVKPSETLSLTCTVTGYSITSDYAWNWIRQPPGKGLEW MGYIMYSGNTNYNPSLKSRVTISSVDTSKNQFSLKLSSVTAADTATYYCARS HYGSNFVYWGQGTMVTVSS.

[0026] The present invention also provides use of the monoclonal antibody described in any of the above schemes in the preparation of a medicament for neutralizing monkeypox virus.

[0027] As a preferred embodiment, the drug is a liquid injection or a dry powder.

[0028] The present invention also provides use of the monoclonal antibody described in any of the above schemes in preparing an immunoassay tool for detecting monkeypox virus.

[0029] As a preferred embodiment, the immunoassay tool is a reagent, a kit, a chip or a test paper.

[0030] Compared with the prior art, the present invention has the following beneficial effects:

[0031] The present invention uses the recombinant monkeypox virus surface protein E8L as an antigen and screens against it using an immune library. This method yields specific monoclonal antibodies against the target antigen, which exhibit good affinity for the recombinant monkeypox virus surface protein E8L at both the molecular and cellular levels. Furthermore, the antibodies significantly inhibit the infection efficiency of the replication-deficient VACV strain in VERO cells, demonstrate excellent in vitro orthopoxvirus neutralization efficacy, and can inhibit monkeypox virus infection, demonstrating potential medicinal and detection value. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 This is a flowchart of mouse immunization according to an embodiment of the present invention;

[0033] Figure 2 This is a graph showing the immune effect of recombinant E8L on mice detected by ELISA in an embodiment of the present invention;

[0034] Figure 3 This is an electrophoresis photograph of SDS-PAGE electrophoresis detecting E8-AmAb in ascites according to an embodiment of the present invention;

[0035] Figure 4 This is an affinity curve of E8-AmAb and recombinant E8L detected by ELISA in an embodiment of the present invention;

[0036] Figure 5 This is a graph showing the affinity of E8-AmAb and recombinant E8L detected by ForteBio in an embodiment of the present invention;

[0037] Figure 6 2. This is a graph showing the affinity of E8-AmAb and recombinant E8L on the cell surface detected by flow cytometry in an embodiment of the present invention;

[0038] Figure 7 This is a schematic diagram of the E8-A mAb of an embodiment of the present invention inhibiting the infection of VACV-GFP in VERO cells;

[0039] Figure 8 is a bar graph showing that E8-A mAb according to an embodiment of the present invention inhibits the infection of MPXV in VERO cells;

[0040] Figure 9 1 is an affinity curve of huE8-A mAb and recombinant E8L detected by ELISA in an embodiment of the present invention. DETAILED DESCRIPTION

[0041] The technical solution of the present invention is further explained below through specific embodiments.

[0042] The screening of monoclonal antibodies that specifically bind to the monkeypox E8L protein according to the embodiment of the present invention includes the following steps:

[0043] 1. Mouse Immunization

[0044] Six-week-old male BALB / c mice were immunized with recombinant E8L as the antigen. The first immunization was performed using a mixture emulsified with Freund's complete adjuvant. Seven days later, the second, third, and fourth immunizations were performed using Freund's incomplete adjuvant and the antigen emulsified, with a 7-day interval between immunizations. Three days after the fourth immunization, blood was collected to verify the efficacy of the immunization. Serum anti-E8L antibody titers exceeding 8,000-10,000-fold dilution were used for further experiments.

[0045] 2. Hybridoma Fusion

[0046] After four immunizations, mice were sacrificed after blood sampling, the spleens were removed, and a splenic B cell suspension was prepared. The spleen cells were evenly mixed with mouse myeloma cells Sp2 / 0 and fused under the mediation of polyethylene glycol 4000 (PEG-4000). The fused hybridoma cells were inoculated into 96-well plates and selectively cultured in HAT medium [hypoxanthine H, aminopterin A, and thymidine T]. Unfused cells, BB fusion cells, and Sp2 / 0-Sp2 / 0 fusion cells will not survive.

[0047] 3. Monoclonal Antibody Screening

[0048] After the hybridoma cells have grown to 1 / 3-1 / 2 of the well bottom area, ELISA is used to screen the wells that secrete the most anti-E8L antibodies and the cells in the wells are monocloned by limiting dilution. After the monoclonal cells have grown to 1 / 3-1 / 2 of the well bottom area, ELISA is used to screen the monoclonal hybridoma cells that secrete anti-E8L antibodies and the positive clones are expanded and cultured.

[0049] IV. Antibody variable region gene sequencing

[0050] The positive monoclonal cells were subjected to antibody variable region gene sequencing, and the monoclonal cells were subjected to RNA extraction, 5'RACE reverse transcription, antibody light and heavy chain gene specific amplification, gene cloning and gene sequencing.

[0051] Example 1:

[0052] The screening process of the mouse monoclonal antibody of this embodiment includes:

[0053] 1. Mouse immunization

[0054] like Figure 1 As shown, 6-week-old male BALB / c mice were immunized with recombinant E8L as the antigen. For the first immunization, the antigen was ultrasonically emulsified with an equal volume of Freund's complete adjuvant and administered subcutaneously at multiple sites, with 100 μg of antigen per mouse. Seven days later, the antigen was emulsified with Freund's incomplete adjuvant and administered at the same dose and in the same manner for the second and third immunizations, with a 7-day interval between immunizations. Three days after the third immunization, mouse serum was collected and verified by enzyme-linked immunosorbent assay (ELISA). The fourth immunization was performed after serum anti-E8L antibody titers exceeded 8,000-10,000-fold dilution. The fourth immunization was performed by intraperitoneal injection, with 300 μg of antigen per mouse. Spleens were harvested three days after the fourth immunization for hybridoma fusion experiments.

[0055] 2. Hybridoma fusion

[0056] Before fusion, Sp2 / 0 cells were selected and cultured in a medium containing 8-azaguanine (20 μg / ml) for 14 days. One day before fusion, cells were taken from the peritoneal cavity of ordinary BALB / c mice and inoculated into 96-well plates as feeder cells. On the day of fusion, the mice were bled after 4 immunizations and sacrificed. The spleens were removed and spleen B cell suspensions were prepared. 1×10 8 spleen cell suspension with 2×10 7 Mix the Sp2 / 0 cell suspensions thoroughly, centrifuge at 1000 rpm for 5 minutes, and discard the supernatant. In a 37°C water bath, slowly add 700 μl of 50% PEG-4000 to the mixed cells, followed by the slow addition of 30 ml of serum-free medium to terminate the fusion. After fusion, centrifuge the hybridoma cells at 1200 rpm for 8 minutes, resuspend them in HAT medium, and plate them into a 96-well plate containing feeder cells for selective culture. Unfused cells, BB-fused cells, and Sp2 / 0-Sp2 / 0 fusion cells will not survive.

[0057] 3. Monoclonal Antibody Screening

[0058] After the hybridoma cells have grown to 1 / 3-1 / 2 of the well bottom area, ELISA is used to screen the wells that secrete the most anti-E8L antibodies and the cells in the wells are monocloned by limiting dilution. After the monoclonal cells have grown to 1 / 3-1 / 2 of the well bottom area, ELISA is used to screen the monoclonal hybridoma cells that secrete anti-E8L antibodies and the positive clones are expanded and cultured.

[0059] 4. Antibody variable region gene sequencing

[0060] Positive monoclonal cells were sent to Nanjing Detai Biotechnology for antibody variable region gene sequencing. Technicians performed RNA extraction, 5' RACE reverse transcription, specific amplification of the antibody light and heavy chain genes, gene cloning, and gene sequencing. The results showed that the BCR light and heavy chain sequences of the hybridoma cells tested were identical. These cells were collectively designated E8-A, and the antibodies expressed by them were collectively designated E8-A mAb.

[0061] The amino acid sequence of the light chain variable region (V region) of the monoclonal antibody E8-A mAb is shown in SEQ ID No. 7, and the amino acid sequence of the heavy chain variable region is shown in SEQ ID No. 8. Bioinformatics analysis revealed that the amino acid sequence of the light chain CDR1 is shown in SEQ ID No. 1, the amino acid sequence of CDR2 is shown in SEQ ID No. 2, and the amino acid sequence of CDR3 is shown in SEQ ID No. 3. The amino acid sequence of the heavy chain CDR1 of the monoclonal antibody is shown in SEQ ID No. 4, the amino acid sequence of CDR2 is shown in SEQ ID No. 5, and the amino acid sequence of CDR3 is shown in SEQ ID No. 6.

[0062] Among them, SEQ ID No. 1: QDVSNT;

[0063] SEQ ID No. 2: WAS;

[0064] SEQ ID No. 3: QHHYNTPYT;

[0065] SEQ ID No. 4: GYSITSDYA;

[0066] SEQ ID No.5: IMYSGNT;

[0067] SEQ ID No. 6: ARSHYGSNFVY;

[0068] SEQ ID No.7:

[0069] DIVMTQSHKFMSTSVGDRVTITCKASQDVSNTVAWYQQRPGQSPKLLI YWASTRHTGVPDRFTGSGSGTDYTLTISSVQAEDLALYYCQHHYNTPYTFG GGTKLEIK;

[0070] SEQ ID No.8:

[0071] DVQLQESGPGLVKPSQSLSLTCTVTGYSITSDYAWNWIRQSPGNKLEW MGYIMYSGNTNYNPSLKSRISITRDTSKNQLFLQLNSVTTEDTATYYCARSH YGSNFVYWGQGTLVTVSA.

[0072] Example 2:

[0073] The preparation of mouse ascites and purification of E8-A mAb in this example specifically include the following steps:

[0074] 1. Preparation of Mouse Ascites

[0075] Six-week-old female BALB / c mice were intraperitoneally injected with 0.5 mL of paraffin oil. Fourteen days later, 5 × 10 6 E8-A cells were injected 9-11 days after cell injection. The mice were killed by cervical dislocation when they became sluggish and had swollen abdomens. The abdominal skin was cut open to bluntly expose the peritoneum, and the ascites was aspirated using a syringe. The ascites was allowed to naturally coagulate in paraffin oil after being placed at 4°C for 1 hour. The ascites was then centrifuged at 4000 rpm for 30 minutes at 4°C, and the aqueous phase was collected for purification.

[0076] 2. E8-A mAb purification

[0077] The antibodies in the ascites were purified using a Protein A (MabSelect SuRe LX) column, and the relevant purification operations were performed on an AKTA Avant chromatography system. The column was equilibrated with 10 column volumes of phosphate buffer (pH = 7.4), and the ascites was diluted 10 times with 20mM phosphate buffer and loaded at a rate of 1mL / min. After loading, the column was equilibrated again with 10 column volumes of phosphate buffer. After the post-equilibrium was completed, the target protein was eluted with 0.1M citric acid buffer (pH = 3.0), and the eluted product was immediately adjusted to pH = 7.4 with 1MTris-HCl buffer (pH = 9.0). The eluted target protein was placed in a dialysis bag with a pore size of 30KD and dialyzed in PBS buffer (4°C). Fresh PBS buffer was replaced every 4-6h. After three replacements, the target protein was finally obtained.

[0078] Example 3:

[0079] The affinity test of the above-mentioned E8-A mAb to the antigen includes:

[0080] 1. Enzyme-linked immunosorbent assay (ELISA) to test the affinity of E8-A mAb to antigen

[0081] ELISA experiments were used to detect the specific binding ability of the purified E8-A mAb to recombinant E8L. Recombinant E8L was coated on the ELISA plate at a concentration of 2μg / mL and placed at 4°C overnight. After discarding the coating solution, wash three times with PBST and block with 3% BSA at 37°C for 30 minutes. Discard the liquid in the wells, add three-fold diluted E8-A mAb to the treatment group, and incubate at 37°C for 1 hour. Discard the liquid in the wells and wash three times with BST. Use HRP-labeled rabbit anti-mouse IgG (1:15000) as the secondary antibody for binding reaction, incubate at 37°C for 1 hour, discard the liquid in the wells and wash three times with PBST, add TMB substrate and place in the dark at room temperature. After 5-10 minutes, add ELISA stop solution to stop the color reaction and measure OD 450 .

[0082] 2. FoeteBio method to test the affinity of E8-A mAb to antigen

[0083] The equilibrium dissociation constant (KD) of E8-A mAb and recombinant E8L was determined using the ForteBio assay. Recombinant E8L was biotinylated using a biotin labeling kit (Tongren Chemical) and immobilized on a pre-wetted streptavidin sensor (SA Sensor) at a concentration of 2 μg / mL. After equilibration with PBST, the probe was then bound to a gradient dilution of E8-A mAb (100 nmol / L, 25 nmol / L, 12.5 nmol / L, 6.25 nmol / L, and 3.125 nmol / L). Using PBST as a blank control, the binding time was set to 350 s and the dissociation time to 400 s. After subtracting the background signal from the control sensor, a 1:1 binding model was used to fit the binding and dissociation curves, and the equilibrium dissociation constant (KD) was calculated.

[0084] 3. Flow cytometry determination of the binding affinity between E8-A mAb and E8L on the cell membrane

[0085] E8L stable transfected cells were collected by digestion and washed twice by FACS (PBS + 2% FBS) at 1100 rpm for 5 min. 2×10 5 Cells were resuspended in three-fold serial dilutions of E8-A mAb in each tube and incubated at room temperature for 1 hour. Washed three times by centrifugation using FACS. Diluted FITC-labeled goat anti-mouse IgG (1:20,000) was added and incubated at room temperature for 1 hour in the dark. After incubation, cells were washed three times by centrifugation. Fluorescence intensity was analyzed using flow cytometry and data were analyzed using GraphPad Prism 9 software.

[0086] Example 4:

[0087] The inhibition test of the above-mentioned E8-A mAb against VACV replication-deficient oncolytic virus is as follows:

[0088] An in vitro neutralization activity evaluation system was constructed using VACV (Vaccinia virus) replication-deficient oncolytic virus (VACV-GFP, Wuhan Shumi), and whether E8-A mAb has in vitro neutralization activity was explored. E8-A mAb was diluted to 600 μg / mL with neutralization medium (DMEM+3% FBS+5% complement) and diluted three times, and 50 μL was added to a 96-well plate for each concentration. Three replicate wells were set for each concentration, and a virus group (antibody concentration was 0 mg / mL) and a cell group (no antibody and virus) were set at the same time. VACV-GFP was diluted 5000 times with neutralization medium, 50 μL was added to each well and mixed thoroughly with the antibody dilution solution. The antibody-virus mixture was incubated at 37°C for 1 hour. VERO-E6 cells were digested and collected, and the density was adjusted to 4×10 5cells / mL, add 50 μL of cell suspension to each well, and incubate the 96-well plate at 37°C, 5% CO2 for 24 h.

[0089] The cell culture medium was discarded and the cells were fixed with 4% paraformaldehyde for 20 minutes at room temperature. The liquid in the wells was discarded and the cells were washed twice with PBS. 100 μL of diluted Hoechst stain (1:1000) was added and incubated at room temperature for 30 minutes, followed by two washes with PBS. Cell images were acquired in various fluorescence channels using a high-content microplate imaging system. The green fluorescent area of ​​each well was calculated using Harmony software, and data were analyzed using GraphPad Prism 9.

[0090] Example 5:

[0091] The inhibition test of the above-mentioned E8-A mAb against MPXV is as follows:

[0092] MPXV was used to investigate the in vitro neutralizing activity of E8-A mAb. In a biosafety level 3 laboratory, E8-A mAb was diluted two-fold with neutralization medium (DMEM + 3% FBS + 5% complement), and 50 μL of each concentration was added to a 96-well plate. Three replicates were set for each concentration, and a virus group (antibody concentration was 0 mg / mL) and a cell group (no antibody and virus) were set up at the same time. MPXV was diluted to 100 PFU / mL with neutralization medium, 50 μL was added to each well and mixed thoroughly with the antibody diluent. The antibody-virus mixture was incubated at 37°C for 1 hour. VERO-E6 cells were digested and collected, and the density was adjusted to 2×10 5 cells / mL, add 100 μL of cell suspension to each well. Incubate the 96-well plate at 37°C, 5% CO2. After 24 hours, count the number of plaques in each well under a microscope and calculate the PRNT. 50 .

[0093] Example 6:

[0094] Humanization of the above E8-A mAb:

[0095] Combining bioinformatics and genetic engineering techniques, the laboratory humanized the light and heavy chain sequences of E8-A mAb. The humanized antibody was named huE8-A mAb. The amino acid sequences of its light chain CDR1, CDR2, and CDR3 are the same as SEQ ID No.1, SEQ ID No.2, and SEQ ID No.3, and the amino acid sequences of its heavy chain CDR1, CDR2, and CDR3 are the same as SEQ ID No.4, SEQ ID No.5, and SEQ ID No.6.

[0096] Specifically, the humanized antibody was named huE8-A mAb, the amino acid sequence of the light chain variable region was shown in SEQ ID No. 9, and the amino acid sequence of the heavy chain variable region was shown in SEQ ID No. 10.

[0097] SEQ ID No.9:

[0098] DIQMTQSPSSSLSASVGDRVTITCRASQDVSNTVAWYQQKPGKVPKLLI YWASTRHSGVPSRFSGSGSGTDFTLTISSLQPEDVATYYCQHHYNTPYTFGG GTKLEIK;

[0099] SEQ ID No.10:

[0100] QVQLQESGPGLVKPSETLSLTCTVTGYSITSDYAWNWIRQPPGKGLEW MGYIMYSGNTNYNPSLKSRVTISSVDTSKNQFSLKLSSVTAADTATYYCARS HYGSNFVYWGQGTMVTVSS.

[0101] The following characterizations of the various processes of the E8-A mAb of the above example are as follows:

[0102] like Figure 2 As shown in the figure, after four immunizations, the maximum dilution of mouse serum that produced a positive reaction with the recombinant E8L antibody was 170100 (positive reaction was defined as OD 450 The reading was greater than 2.1 times that of the negative control), indicating that the titer of anti-E8L antibodies in the mice was 170100. After immunization, the mice produced a strong immune response, meeting the requirements for preparing monoclonal antibodies.

[0103] like Figure 3 As shown in the figure, after purification by Protein A affinity chromatography, the eluted fractions contain a single band at 55 kDa and a single band at 25 kDa, representing the heavy and light chains of the antibody, respectively. The eluted fractions contain relatively low levels of contaminants and exhibit high antibody purity, meeting the requirements of subsequent experiments.

[0104] like Figure 4 As shown, the concentration-dependent binding relationship of E8-A mAb to recombinant E8L was analyzed by GraphPad Prism 9 software and a fitting curve was drawn. The EC50 value was calculated to be 16.30 ng / mL, indicating that E8-A mAb has good binding activity to the antigen.

[0105] like Figure 5As shown in Figure 2, the concentration-dependent binding relationship between E8-A mAb and recombinant E8L was analyzed by Fortebio software and a fitting curve was drawn. The equilibrium dissociation constant KD was determined to be 3.22×10 -11 M, belongs to high affinity antibody.

[0106] like Figure 6 As shown, the concentration-dependent binding relationship of E8-A mAb to recombinant E8L on the cell surface was analyzed by GraphPad Prism 9 software and a fitting curve was drawn. The EC50 value was calculated to be 136.5 ng / ml, indicating that E8-A mAb still has strong binding activity when the antigen is expressed on the cell surface.

[0107] like Figure 7 As shown, cells infected with viruses express green fluorescent protein, and the ability of antibodies to inhibit viral infection of cells at different concentrations can be measured by measuring the green fluorescent area. The results show that the E8-A mAb has a concentration-dependent relationship with the GFP area ratio and has a significant inhibitory effect within the tested concentration range.

[0108] like Figure 8 As shown in the figure, the maximum dilution factor at which E8-A mAb achieved 50% inhibition against MPXV was 40, and its PRNT 50 The value was 8.7 μg / mL.

[0109] In addition, the affinity of the above huE8-A mAb to the antigen was tested as follows:

[0110] ELISA experiments were used to detect the specific binding ability of huE8-A mAb to recombinant E8L. Recombinant E8L was coated on the ELISA plate at a concentration of 2μg / mL and placed at 4°C overnight. After discarding the coating solution, wash three times with PBST and block with 3% BSA at 37°C for 30 minutes. Discard the liquid in the wells, add three-fold diluted huE8-A mAb to the treatment group, and incubate at 37°C for 1 hour. Discard the liquid in the wells and wash three times with BST. Use HRP-labeled goat anti-human IgG (1:15000) as the secondary antibody for binding reaction, incubate at 37°C for 1 hour, discard the liquid in the wells and wash three times with PBST, add TMB substrate and place in the dark at room temperature. After 5-10 minutes, add ELISA stop solution to stop the color reaction and measure OD 450 .

[0111] like Figure 9 As shown, the concentration-dependent binding of huE8-A mAb to recombinant E8L was analyzed using GraphPad Prism 9 software, and a fitting curve was drawn. The calculated EC50 value was 31.06 ng / mL, indicating that after humanization, huE8-A mAb still has high binding activity to the antigen.

[0112] Viral adhesion to cell surface glycosaminoglycans is a crucial step in entering cells and initiating viral infection. E8L, a binding molecule for chondroitin sulfate (a glycosaminoglycan), plays a crucial role in monkeypox virus entry. The present invention presents E8-A, a monkeypox neutralizing antibody targeting E8L. This antibody exhibits excellent antigen-binding capacity at both the molecular and cellular levels, inhibiting monkeypox virus infection by blocking the interaction of E8L with the cell surface. This antibody has demonstrated excellent neutralization of VACV in an in vitro neutralization model and holds promise for development as a novel antibody drug for the treatment of monkeypox virus infection.

[0113] Based on this, the present invention also provides the use of the above-mentioned monoclonal antibody in the preparation of a drug for neutralizing monkeypox virus, wherein the drug is a liquid injection or a dry powder.

[0114] The present invention also provides the use of the above monoclonal antibody in the preparation of an immunoassay tool for detecting monkeypox virus, wherein the immunoassay tool is a reagent, a kit, a chip or a test paper.

[0115] The above description is only a detailed description of the preferred embodiments and principles of the present invention. For ordinary technicians in this field, based on the ideas provided by the present invention, there may be changes in the specific implementation methods, and these changes should also be considered as the scope of protection of the present invention.

Claims

1. A monoclonal antibody that specifically binds to monkeypox E8L protein, characterized in that: The amino acid sequence of the light chain CDR1 of the monoclonal antibody is shown in SEQ ID No. 1, the amino acid sequence of CDR2 is shown in SEQ ID No. 2, and the amino acid sequence of CDR3 is shown in SEQ ID No. 3; The amino acid sequence of the heavy chain CDR1 of the monoclonal antibody is shown in SEQ ID No. 4, the amino acid sequence of CDR2 is shown in SEQ ID No. 5, and the amino acid sequence of CDR3 is shown in SEQ ID No.

6.

2. The monoclonal antibody according to claim 1, characterized in that The amino acid sequence of the light chain variable region of the monoclonal antibody is shown in SEQ ID No. 7, and the amino acid sequence of the heavy chain variable region is shown in SEQ ID No.

8.

3. The monoclonal antibody according to claim 1, characterized in that The amino acid sequence of the light chain variable region of the monoclonal antibody is shown in SEQ ID No. 9, and the amino acid sequence of the heavy chain variable region is shown in SEQ ID No.

10.

4. Use of the monoclonal antibody according to any one of claims 1 to 3 in the preparation of a medicament for neutralizing monkeypox virus.

5. The use according to claim 4, characterized in that The medicine is a liquid injection or dry powder.

6. Use of the monoclonal antibody according to any one of claims 1 to 3 in the preparation of an immunoassay tool for detecting monkeypox virus.

7. The use according to claim 6, characterized in that The immunoassay tool is a reagent, a test kit, a chip or a test paper.

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