Specific monoclonal antibody and application thereof in detection and neutralization of monkey pox virus

Abstract

The invention relates to a specific monoclonal antibody and application thereof in detection and neutralization of monkey pox virus. The amino acid sequence of a light chain CDR1 of the monoclonal antibody is as shown in SEQ ID No.1, the amino acid sequence of CDR2 of the monoclonal antibody is as shown in SEQ ID No.2, and the amino acid sequence of CDR3 of the monoclonal antibody is as shown in SEQ ID No.3; the amino acid sequence of a heavy chain CDR1 of the monoclonal antibody is as shown in SEQ ID No.4, the amino acid sequence of CDR2 of the monoclonal antibody is as shown in SEQ ID No.5, and the amino acid sequence of CDR3 of the monoclonal antibody is as shown in SEQ ID No.6. The recombinant monkey pox virus surface protein B6R is used as an antigen, the monoclonal antibody obtained through screening has good affinity with the recombinant monkey pox virus surface protein B6R, the infection efficiency of VACV replication defective strains and MPXV in VERO cells can be inhibited, and a good MPXV in-vitro neutralization effect is shown in a mouse body.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a specific monoclonal antibody and its application in the detection and neutralization of monkeypox virus. Background Technology

[0002] Monkeypox virus (MPXV) infection primarily damages multiple organs in the human body, including the skin, lungs, and gastrointestinal tract, and can lead to death in severe cases. Currently, clinical treatment for Mpox mainly relies on anti-smallpox drugs, and there is no effective treatment to control the infection. With the cessation of smallpox vaccination programs globally, an increasing number of people lack the adaptive immunity to rapidly respond to Mpox infection. Therefore, there is an urgent need to develop novel therapeutics against Mpox. Neutralizing antibodies show promising clinical application prospects in inhibiting viral infection and clearing the virus from the body, as seen in cases such as Ebola hemorrhagic fever, respiratory syncytial infection, and COVID-19. Currently, screening for high-affinity anti-Mpox virus neutralizing antibodies is of significant clinical value in improving treatment outcomes for patients with Mpox infection and preventing its spread.

[0003] B6R is one of the potential protective antigenic proteins of monkeypox virus and a homologous protein of vaccinia virus (VACV) B5R. B6R is expressed on the surface of EEV-type viruses and is crucial for multiple stages of viral infection, including the integrity of the EEV envelope, the formation of the actin tail, and maintaining normal plaque size and virulence. For example, Chan WM et al., “Increased interaction between vaccinia virus proteins A33 and B5 is detrimental to infectious extracellular enveloped virion production.” Journal of Virology vol. 86, 15(2012):8232-44. In addition, studies have shown that the six protective antigens of B5R, including A27L, A33R, D8L, H3R and L1R, can induce a strong immune response in mice and generate high levels of neutralizing antibodies, as seen in Heraud, JM et al., “Subunit recombinant vaccine protects against monkeypox.” Journal of Immunology, 2006, 177(4):2552-64.

[0004] In the development of neutralizing antibodies, due to the similar biological activities and immunogenicity among homologous proteins, most antibodies with anti-monkeypoxvirus activity are currently induced and screened by VCV-related antigens. For example, Runchu Zhao et al. screened two neutralizing antibodies targeting the MPXV surface protein B6R from VCV vaccine recipients. Both showed broad-spectrum anti-orthopoxvirus infection effects in mouse models and have the potential to become antibody drugs for treating monkeypoxvirus 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). Cross-immunity among orthopoxvirus family members suggests the broad-spectrum neutralizing effect of anti-B6R antibodies.

[0005] Currently, there are few reports on directly using MPXV antigens, especially those targeting B6R, for immunization or screening of monoclonal antibodies. Yuanyuan Qu et al. prepared neutralizing antibodies binding to MPXV surface proteins B6R and M1R (homogeneous with VAV B5R and L1R) from a human antibody library using phage display technology. Prophylactic and therapeutic administration of either antibody alone provided partial protection against VAV in mice, while the combination of the two antibodies further enhanced the antiviral efficacy (Qu Y et al., “Generation and characterization of neutralizing antibodies against M1R and B6R proteins of monkeypox virus.” *Nature Communications*, 2025, 16(1):3100). Developing MPXV-specific neutralizing antibodies against B6R is of great significance for the control of monkeypox outbreaks. Summary of the Invention

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

[0007] To achieve the above-mentioned objectives, the present invention adopts the following technical solution:

[0008] In a first aspect, the present invention provides a monoclonal antibody that specifically binds to monkeypox B6R 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 in SEQ ID No. 7, and the amino acid sequence of the heavy chain variable region is shown in SEQ ID No. 8.

[0011] Among them, SEQ ID No. 1: KSLLHSNGNTY;

[0012] SEQ ID No. 2: RMS;

[0013] SEQ ID No. 3: MQHLEYPYT;

[0014] SEQ ID No. 4: GYTFTDNK;

[0015] SEQ ID No. 5: ISTYSGNT;

[0016] SEQ ID No. 6: ARGETWFAY;

[0017] SEQ ID No. 7:

[0018] DIVMTQAAPSVPVTPGESVSISCRSSKSLLHSNGNTYLYWFLQRPGQSP QLLIYRMSNLASGVPDRFSGSGSGTAFTLRISRVEAEDVGVYYCMQHLEYP YTFGGGTKLEIK;

[0019] SEQ ID No. 8:

[0020] QVQLQQSGPELVRPGVSVKISCKGSGYTFTDNKMHWVKQSHAKSLE WIGIISTYSGNTNYNQKFKGKATMTVDKSSSTAYMELARLTSEDSAIYYCAR GETWFAYWGQGTLVTVSA.

[0021] As a preferred embodiment, the monoclonal antibody that specifically binds to monkeypox B6R protein further comprises a constant region, which includes a heavy chain constant region and / or a light chain constant region.

[0022] As a preferred embodiment, the heavy chain constant region is selected from the heavy chain constant regions of any one of IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE and IgD.

[0023] The light chain constant region is selected from the κ-type or λ-type light chain constant region.

[0024] In a second aspect, the present invention also provides a polynucleotide encoding a monoclonal antibody that specifically binds to the monkeypox B6R protein as described in any of the preceding claims.

[0025] Thirdly, the present invention also provides an expression vector comprising the polynucleotides described above.

[0026] Fourthly, the present invention also provides a host cell comprising the expression vector as described above. Fifthly, the present invention also provides the use of the monoclonal antibody as described in any of the preceding embodiments 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] In a sixth aspect, the present invention also provides the use of the monoclonal antibody as described in any of the preceding embodiments in the preparation of an immunoassay tool for detecting monkeypox virus.

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

[0030] In a seventh aspect, the present invention also provides a kit for detecting monkeypox virus, the kit comprising a monoclonal antibody that specifically binds to the monkeypox B6R protein as described in any of the preceding claims.

[0031] Compared with the prior art, the beneficial effects of this invention are:

[0032] This invention uses the surface protein B6R of recombinant monkeypox virus (MPox virus) as an antigen and employs an immune library for screening to obtain a specific monoclonal antibody, B6-30 mAb, targeting the antigen. This antibody exhibits good affinity for the recombinant monkeypox virus surface protein B6R and simultaneously inhibits the infection efficiency of VCV replication-deficient strains and MPXV in VERO cells. In animal protection experiments, B6-30 mAb demonstrates good in vivo anti-MPXV efficacy and can inhibit monkeypox virus infection, showing potential pharmaceutical and diagnostic value. Attached Figure Description

[0033] Figure 1 This is a flowchart of the mouse immunization process in Example 1 of the present invention;

[0034] Figure 2 This is an ELISA curve of the immune effect of recombinant B6R on three mice, as measured in Example 1 of this invention.

[0035] Figure 3 This is a flow cytometry diagram of the B6R-positive B cell population separated by flow cytometry in Example 1 of the present invention;

[0036] Figure 4 This is an agarose gel electrophoresis image of the target antibody fragment (30VH, 30KV) obtained by double enzyme digestion in Example 1 of the present invention;

[0037] Figure 5 This is a bar chart of the ELISA detection of the reactivity of candidate antibody 30 to five recombinant antigens in Example 1 of the present invention;

[0038] Figure 6 This is an SDS-PAGE electrophoresis image of B6-30 mAb in cell culture supernatant as detected by Example 2 of the present invention;

[0039] Figure 7 This is an ELISA affinity curve of B6-30 mAb and recombinant B6R in Example 3 of the present invention;

[0040] Figure 8 This is a ForteBio affinity curve of B6-30 mAb and recombinant B6R in Example 3 of the present invention;

[0041] Figure 9 This is a bar chart illustrating the inhibition of VCV-GFP infection in VERO cells by B6-30 mAb in Example 4 of this invention;

[0042] Figure 10 This is a bar chart showing the inhibition of MPXV infection in VERO cells by B6-30 mAb in Example 5 of the present invention;

[0043] Figure 11 This is a time-weight line graph showing how B6-30 mAb reduced weight loss in mice after MPXV challenge in Example 5 of this invention.

[0044] Figure 12 This is a bar chart showing the reduction of viral load in mouse lung tissue after MPXV challenge using B6-30 mAb in Example 5 of this invention. Detailed Implementation

[0045] The technical solution of the present invention will be further explained and illustrated below through specific embodiments.

[0046] The 6-week-old male BALB / c mice used in this invention were purchased from Shanghai Vital River Laboratory Animal Technology Co., Ltd.; recombinant B6R antigen, FITC-labeled B6R antigen, and Freund's incomplete adjuvant were obtained from laboratory storage using conventional methods; Freund's complete adjuvant was purchased from Sigma Aldrich; Alexa Fluor 594-labeled goat anti-mouse IgG was purchased from Jackson Immuno; pcDNA3.4 carrying antibody heavy / light chain variable region sequences was synthesized by General Biotechnology (Anhui) Co., Ltd.; pcDNA3.4 carrying antibody heavy / light chain constant region sequences were obtained from laboratory storage using conventional methods; E. coli DH5α cells were purchased from Xinsaimei Biotechnology Co., Ltd.; HEK-293T cells, Expi-293F cells, and VERO-E6 cells were obtained from laboratory storage using conventional methods; HRP-labeled goat anti-human IgG was purchased from Cell Signaling; MPXV was obtained from the P3 laboratory of Changchun Veterinary Research Institute and donated by them.

[0047] The screening of monoclonal antibodies that specifically bind to the monkeypox B6R protein in this embodiment of the invention includes the following process:

[0048] I. Mouse Immunization

[0049] Six-week-old male BALB / c mice were used as immunization subjects, with recombinant B6R as the antigen. The first immunization was performed using Freund's complete adjuvant emulsified with the antigen. Seven days later, the second, third, and fourth immunizations were performed using Freund's incomplete adjuvant emulsified with the antigen, with each immunization spaced seven days apart. Three days after the fourth immunization, blood samples were collected to verify the immunization effect. Serum anti-B6R antibody titers greater than 8000-10000 times were sufficient for further experiments.

[0050] II. B cell enrichment and flow cytometry sorting

[0051] Mice were euthanized after four immunizations, and their spleens were harvested and a spleen B-cell suspension was prepared. Non-B cells were separated and B cells were enriched from the spleen cell suspension using a mouse B-cell isolation kit (Stemcell). Flow cytometry was then used to further separate B cells that specifically bind to antigens. The enriched B-cell suspension was stained with FITC-labeled B6R antigen and AlexaFluor 594-labeled goat anti-mouse IgG. During the sorting process, FITC- and AlexaFluor 594-positive cell populations were collected.

[0052] III. Sequencing Results Analysis and Sequence Design for Single B Cells

[0053] The sorted B cells were sequenced using the 10×Genomics platform. The sequencing results were analyzed using NCB1gblast to identify the antibody variable region sequence, and full-length antibodies were designed and plasmids constructed according to the sequence frequency. The N-terminus of the antibody variable region sequence was fused with a human IgG kappa chain signal peptide, and these peptides were synthesized sequentially. Subsequently, pCDNA3.4 carrying the antibody heavy / light chain constant region sequences was used as a vector, and the variable and constant regions were fused through double enzyme digestion and ligation to prepare full-length IgG1-type chimeric antibody heavy / light chain plasmids.

[0054] IV. Expression Trial and Antigen Validation

[0055] The candidate antibody was first expressed in HEK-293T cells to determine its normal expression in the cells and its target antigen. The full-length light and heavy chain plasmids of the antibody were co-transfected into HEK-293T cells via transient transfection, and the cell culture supernatant was collected 2 days later. The supernatant was added to polystyrene 96-well plates coated with recombinant B6R, with HRP-labeled goat anti-human IgG as the secondary antibody, and untransfected plain HEK-293T cell culture supernatant as a negative control.

[0056] Example 1:

[0057] The screening process for mouse monoclonal antibodies in this embodiment includes the following steps:

[0058] 1. Mouse immunization

[0059] Mouse immunization procedure as follows Figure 1 As shown, three 6-week-old male BALB / c mice were used as immunization subjects, and recombinant B6R was used as the antigen. For the first immunization (day 0), the antigen was emulsified with an equal volume of Freund's complete adjuvant using ultrasound, and immunization was performed via subcutaneous injection at multiple sites, with each mouse receiving 100 μg of antigen. Seven days later, the antigen was emulsified with Freund's incomplete adjuvant, and the second and third immunizations were performed using the same dosage and method, with each immunization spaced 7 days apart. Three days after the third immunization, mouse serum was collected, and the immunization effect was verified using enzyme-linked immunosorbent assay (ELISA). A fourth immunization was performed after the serum anti-B6R antibody titer was greater than 8000-10000 times. The fourth immunization was performed via intraperitoneal injection, with each mouse receiving 300 μg of antigen. Three days after the fourth immunization, spleens were collected for B cell enrichment and flow cytometry sorting. Figure 2 As shown, after three immunizations, the highest dilution at which mouse serum produced a positive reaction with the recombinant B6R antibody was 76,800 (a positive reaction was defined as OD). 450The reading was greater than 2.1 times that of the negative control, indicating that the titer of anti-B6R antibody in the mouse was 76,800. After immunization, the mouse produced a strong immune response, which met the requirements for preparing monoclonal antibodies.

[0060] 2. B cell enrichment and flow cytometry sorting

[0061] On the day of sorting, mice were sacrificed after blood collection on day 3 following four immunizations. The spleen was removed, and a spleen B cell suspension was prepared. The density of the spleen cell suspension was adjusted to 1×10⁻⁶. 8 Non-B cells were isolated and removed from the suspension using a mouse B cell isolation kit (Stemcell). The number of enriched B cells was generally 2-2.5 × 10⁻⁶ cells / ml. 7 The B cells were collected, with a purity between 94% and 97%. The enriched B cells were washed three times with FACS at 1000 rpm for 7 minutes each time, and the cell density was adjusted to 1 × 10⁶ cells / year. 7 Cells / mL. Cells were divided into four groups according to Table 1: blank group, antigen-staining group, IgG-staining group, and experimental group. Fluorescently labeled antigens / antibodies (as shown in Table 1) were added, and the cells were incubated at room temperature in the dark for 30 min. Then, each group of cells was washed three times with FACS for later use. The target cell population was collected using an Aria III sorting flow cytometer. FITC and Alexa Fluor 594 were excited at wavelengths of 488 nm and 405 nm, respectively. 530 / 30 and 610 / 20 filters were used for the receiving channels, and an 85 μm nozzle was used. The voltages of FCS, SSC, and the fluorescence channel were adjusted using the blank group, and the other fluorescence channel was calibrated using the single-staining group. Subsequently, the experimental group was used to delineate the FITC and Alexa Fluor 594 double-positive cell population, and the purity mode was selected for sorting, controlling the sorting speed to within 1000 events / s. The flow cytometry results of the B6R-positive B cell population separated by flow cytometry are shown below. Figure 3 As shown.

[0062] Table 1. Antibody incubation protocol for flow cytometry sorting

[0063]

[0064] 3. Sequencing, sequence design, and plasmid construction of single B cells

[0065] The sorted target cell population was sequenced using a 10×Genomics platform, and the sequencing results were analyzed using NCBI Igblast to identify the antibody variable region sequence. The target fragment (pcDNA3.4 carrying the antibody heavy / light chain variable region sequence) plasmid and the vector plasmid (pcDNA3.4 carrying the antibody heavy / light chain constant region sequence) were double-digested with Xba I and Nhe I restriction endonucleases, respectively. After digestion, the target fragment was first separated and purified by agarose gel electrophoresis, and then the gel was recovered using a gel extraction kit. The vector was purified using a clean-up kit. The purified target fragment and vector were ligated using T4 DNA ligase, maintaining a target fragment to vector molar ratio of approximately 5:1, and incubated overnight at 16°C. The ligation product was transformed into *E. coli* DH5α cells and seeded onto LB agarose plates containing 50 μg / mL ampicillin, incubated overnight at 37°C. Transformed single colonies were sequenced for verification. Strains with correct sequencing were preserved, and endotoxin-free plasmids were extracted in small quantities to obtain heavy chain and light chain expression plasmids for each candidate antibody. The double-enzyme digestion agarose gel electrophoresis results for candidate antibody #30 are shown below. Figure 4 As shown, the results indicate that the digestion products of the heavy chain variable region and the light chain variable region (respectively...) Figure 4 Both 30VH and 30KV bands showed a single band at 400bp, with sizes consistent with theoretical values ​​(397-411bp). After gel extraction and recovery of the target bands, they were inserted into vector plasmids carrying the constant regions of the antibody heavy and light chains. Sequencing verification confirmed the successful construction of the light chain expression plasmid and heavy chain expression plasmid for candidate antibody 30.

[0066] 4. Expression trial and antigen verification

[0067] Each candidate antibody was first expressed in HEK-293T cells on a small scale. A transfection mixture (containing 0.5 μg heavy chain expression plasmid, 0.5 μg light chain expression plasmid, and 1.6 μL Lipo8000) was prepared using serum-free DMEM and gently mixed before incubation at room temperature for 15 min. The transfection mixture was then added to a monolayer of HEK-293T cells in 12-well plates and cultured at 37°C. Two days after transfection, the cell culture supernatant was collected. Recombinant B6R was coated at 0.2 μg / well onto 96-well polystyrene plates. The cell supernatant was used as the primary antibody, and HRP-labeled goat anti-human IgG (1:15000) was used as the secondary antibody. Untransfected ordinary HEK-293T cell culture supernatant served as a negative control.

[0068] The reactivity of candidate antibody cell culture supernatants to five recombinant antigens was detected by ELISA. Figure 5The results showed that candidate antibody No. 30 exhibited binding activity against recombinant B6R, indicating that it is a B6R-targeting antibody, and it was named B6-30 mAb. The amino acid sequence of the light chain variable region (V region) of the above-mentioned monoclonal antibody B6-30 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. Among them, after bioinformatics analysis, the amino acid sequence of the light chain CDR1 of the monoclonal antibody B6-30 mAb 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 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.

[0069] Among them, SEQ ID No. 1: KSLLHSNGNTY;

[0070] SEQ ID No. 2: RMS;

[0071] SEQ ID No. 3: MQHLEYPYT;

[0072] SEQ ID No. 4: GYTFTDNK;

[0073] SEQ ID No. 5: ISTYSGNT;

[0074] SEQ ID No. 6: ARGETWFAY;

[0075] SEQ ID No. 7:

[0076] DIVMTQAAPSVPVTPGESVSISCRSSKSLLHSNGNTYLYWFLQRPGQSPQLLIYRMSNLASGVPDRFSGSGSGTAFTLRISRVEAEDVGVYYCMQHLEYPYTFGGGTKLEIK;

[0077] SEQ ID No. 8:

[0078] QVQLQQSGPELVRPGVSVKISCKGSGYTFTDNKMHWVKQSHAKSLEWIGIISTYSGNTNYNQKFKGKATMTVDKSSSTAYMELARLTSEDSAIYYCARGETWFAYWGQGTLVTVSA.

[0079] Example 2:

[0080] This embodiment provides the expression and purification of B6-30 mAb, specifically including the following steps:

[0081] After a small-scale expression trial of the B6-30 mAb antibody, Expi-293F cells were rapidly transfected using a PEI-mediated transient transfection method to express the target antibody. A large amount of the target plasmid was extracted, and Expi-293F cells were transfected at a concentration of 1.5 × 10⁻⁶ cells per cell line the day before transfection. 6 Cells were passaged at a density of 3 × 10⁶ cells / mL, and the culture volume was maintained at 200 mL. The next day, when the cell density reached 3 × 10⁶ cells / mL... 6 When the cell count is [number] cells / mL, prepare the transfection mixture according to Table 2, mix gently, and incubate at room temperature for 15 min. Add the transfection mixture to the cell culture and culture the cells at 37°C and 220 rpm in a shaker. Add 10 mL of feed on the first day after transfection. When the cell viability drops to 60% around the fifth day after transfection, collect the cell supernatant by centrifugation at 4°C and 4000 rpm for 10 min. Centrifuge the supernatant again at 4°C and 7000 rpm for 30 min, and collect the supernatant for purification.

[0082] Table 2 Preparation of 293F transfection mixture

[0083]

[0084] Antibodies in cell culture supernatants were purified using a Protein A (MabSelect SuRe LX) column on an AKTA Avant chromatography system. The column was equilibrated with 10 column volumes of phosphate-buffered saline (pH 7.4), and the cell culture supernatant was loaded at a rate of 1 mL / min. After loading, the column was equilibrated again with 10 column volumes of phosphate-buffered saline. Following equilibration, the target protein was eluted with 0.1 M citrate buffer (pH 3.0), and the pH of the eluted product was immediately adjusted to 7.4 with 1 M Tris-HCl buffer (pH 9.0). Each eluted fraction was mixed with SDS loading buffer containing / without β-mercaptoethanol and heated at 95°C for 5 min to obtain reduced and non-reduced samples. The eluted target protein was placed in a dialysis bag with a pore size of 30 kDa and dialyzed in PBS buffer (4°C). Fresh PBS buffer was replaced every 4-6 hours, and this process was repeated three times to obtain the target protein B6-30 mAb. An SDS-PAGE electrophoresis image of the target protein B6-30 mAb is shown below. Figure 6 As shown, lane 1 is for the loaded sample, lane 2 is for the flow-through sample, and lane 3 is for the eluted sample. The results show that the reduced sample of the eluted fraction after Protein A affinity chromatography purification (…) Figure 6In the reduced 3 lanes, there are two single bands at 55kDa and 25kDa, representing the antibody heavy chain and light chain protein bands, respectively; the non-reducing sample ( Figure 6 In the non-reduced antibody fraction, there is a single band at 180 kDa, which is the protein band of the full-length antibody. The elution fraction contains few contaminating proteins, indicating high antibody purity, which meets the requirements for subsequent experiments.

[0085] Example 3:

[0086] The target protein B6-30 mAb obtained in Example 2 was subjected to an affinity test with the antigen, including the following steps:

[0087] 1. Enzyme-linked immunosorbent assay (ELISA) to test the affinity of B6-30 mAb for the antigen.

[0088] The specific binding ability of purified B6-30 mAb to recombinant B6R was detected using an ELISA assay. Recombinant B6R was coated onto an ELISA plate at a concentration of 2 μg / mL and incubated overnight at 4°C. After discarding the coating solution, the plate was washed three times with PBST and blocked with 3% BSA at 37°C for 30 min. The liquid in the wells was discarded, and a three-fold serially diluted B6-30 mAb was added to the treatment group, incubating at 37°C for 1 h. The liquid in the wells was discarded, and the plate was washed three times with BST. The binding reaction was initiated using HRP-labeled goat anti-human IgG (1:15000) as a secondary antibody, incubated at 37°C for 1 h, and the liquid in the wells was discarded again, washing three times with PBST. TMB substrate was added, and the plate was placed in the dark at room temperature. After 5-10 min, ELISA stop solution was added to terminate the colorimetric reaction, and the OD was measured. 450 The concentration-dependent binding relationship between B6-30mAb and recombinant B6R was analyzed using GraphPad Prism 9 software, and a fitting curve was plotted, as shown below. Figure 7 The affinity curve shown indicates an EC50 value of 27.54 ng / mL, demonstrating that B6-30 mAb exhibits good binding activity with the antigen. 2. FoeteBio method for testing the affinity between B6-30 mAb and the antigen.

[0089] The equilibrium dissociation constant (KD) of B6-30 mAb and recombinant B6R was determined using the ForteBio method. Recombinant B6R was biotinylated using a biotinylated reagent kit (Tongren Chemical) and then immobilized at a concentration of 2 μg / mL on a pre-wetted streptavidin sensor (SA sensor). After equilibration with PBST, it was then bound to serially diluted B6-30 mAb (20 nmol / L, 10 nmol / L, 5 nmol / L, 2.5 nmol / L, 1.25 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 using 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. The concentration-dependent binding relationship of B6-30 mAb to recombinant B6R was analyzed using ForteBio software, and fitted curves were plotted (blue represents the original curve, red represents the fitted curve for 1:1 binding). The results are shown below. Figure 8 The affinity curve shown indicates that its equilibrium dissociation constant KD is 3.81 × 10⁻⁶. -11 M is a high-affinity antibody.

[0090] Example 4:

[0091] The inhibitory test of the target protein B6-30 mAb obtained in Example 2 against VCV replication-deficient oncolytic virus is as follows:

[0092] The in vitro neutralizing activity of B6-30 mAb was investigated using VACCV (Vaccinia virus) replication-deficient oncolytic virus (VACV-GFP, Wuhan Shumi). The virus was diluted to 2000 PFU / ml in DMEM medium containing 2% FBS (inactivated in a 55°C water bath for 30 min), and B6-30 mAb was diluted to 200 μg / ml. The resulting virus dilution was mixed with an equal volume of antibody dilution and incubated at 37°C for 2 h. A negative control group (NC) without antibody and a positive control group (PC) without virus were also included. The culture medium of the monolayer of VERO-E6 cells seeded in 96-well plates was removed, and 100 μl / well of antibody-virus mixture was added to each well, with three replicates, and the cells were infected at 37°C for 1 h. The mixture in the wells was removed, the cells were washed once with PBS, and fresh DMEM medium containing 10% FBS was added. The cells were then incubated at 37°C with 5% CO2. Fluorescence data under 488nm excitation light were acquired using an Amersham Typhoon laser scanning imager 24 hours later, and the data were analyzed according to the following formula.

[0093]

[0094] Cells express green fluorescent protein after being infected by a virus. The ability of antibodies at different concentrations to inhibit viral infection of cells can be detected by measuring the relative fluorescence intensity. Figure 9 The results showed that at a working concentration of 100 ug / ml, B6-30 mAb neutralized VACV in vitro by 16%, indicating that B6-30 mAb exhibited partial inhibitory effects at the tested concentration.

[0095] Example 5:

[0096] The inhibition test of MPXV by the target protein B6-30 mAb obtained in Example 2 is as follows:

[0097] The in vitro neutralizing activity of B6-30 mAb was investigated using MPXV. In a biosafety level 3 laboratory, B6-30 mAb was serially diluted twofold with neutralizing medium (DMEM + 3% FBS + 5% complement), with 50 μL added to each well of a 96-well plate. Three replicates were set up for each concentration, including a virus group (antibody concentration of 0 mg / mL) and a cell group (without antibody or virus). MPXV was diluted to 100 PFU / mL with neutralizing medium, and 50 μL was added to each well, mixing thoroughly with the antibody dilution. The antibody-virus mixture was incubated at 37°C for 1 h. VERO-E6 cells were collected by digestion and adjusted to a density of 2 × 10⁶ cells with neutralizing medium. 5 Cells / mL, 100 μL of cell suspension was added to each well. The 96-well plates were incubated at 37°C and 5% CO2. After 24 h, the number of plaques in each well was counted under a microscope, and PRNT was calculated. 50 .like Figure 10 As shown, the maximum dilution factor at which B6-30 mAb achieves 50% inhibition of MPXV is 40, and its PRNT... 50 The value was 2.39 μg / mL.

[0098] The neutralizing activity of B6-30 mAb in mice was investigated using MPXV. Fifteen female BALb / c mice were housed in a biosafety level 3 laboratory and randomly divided into three groups (experimental group, virus group, and control group), with five mice in each group. Initial body weight was recorded. Mice were anesthetized and intraperitoneally injected with 200 μg B6-30 mAb (experimental group) or an equal volume of PBS (virus group and control group). One day later, they were given 2 × 10⁻⁶ PBS. 6Mice were challenged with pfu / ml MPXV (450 μL intraperitoneal injection, 50 μL nasal drip), while the control group received no challenge. One day after challenge, mice were intraperitoneally injected with 200 μg B6-30 mAb (experimental group) or an equal volume of PBS (virus group and control group). Mouse weight and survival were observed daily. Mice were sacrificed when their body weight decreased by 25%, and lung tissue was collected for MPXV viral load detection by qPCR. The time-weight loss line graph for each group is shown below. Figure 11 As shown in the figure, the results indicated that after MPXV challenge in mice, administration of B6-30 mAb reduced weight loss in the experimental group, suggesting that B6-30 mAb has an in vivo protective effect against MPXV in mice. Seven days after MPXV challenge, the viral load of MPXV in the lungs of mice in each group was detected by qPCR. Figure 12 As shown, the results indicated that administration of B6-30 mAb significantly reduced viral load in mouse lung tissue. B6R is crucial for the integrity of the EEV envelope, the formation of the actin tail, and maintaining normal plaque size and virulence. Based on its important role in the intercellular transmission of monkeypox virus, the monkeypox neutralizing antibody B6-30 mAb targeting B6R of this invention was demonstrated by ELISA and BLI experiments to have good antigen-binding ability to B6R at the molecular level. This antibody exhibited a certain neutralizing ability against VCV in an in vitro neutralization model and shows promise as a novel antibody drug for the treatment of monkeypox virus infection. In MPXV challenge experiments, B6-30 mAb reduced the weight loss in mice and significantly reduced the viral titer in their lung tissue, indicating that B6-30 mAb has excellent in vivo anti-MPXV ability.

[0099] 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. The drug is a liquid injection or a dry powder.

[0100] This invention also provides the application of the above-mentioned monoclonal antibody in the preparation of an immunoassay tool for detecting monkeypox virus. The immunoassay tool may be a reagent, kit, chip, or test strip.

[0101] The above description is merely a detailed explanation of preferred embodiments and principles of the present invention. For those skilled in the art, there may be changes in specific implementation methods based on the ideas provided by the present invention, and these changes should also be considered within the scope of protection of the present invention.

Claims

1. A monoclonal antibody that specifically binds to monkeypox B6R 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 that specifically binds to monkeypox B6R protein according to claim 1, characterized in that, The amino acid sequence of the light chain variable region of the monoclonal antibody is the sequence shown in SEQ ID No. 7 or its homologous sequence, and the amino acid sequence of the heavy chain variable region is the sequence shown in SEQ ID No. 8 or its homologous sequence.

3. A polynucleotide, characterized in that, The polynucleotide encodes the monoclonal antibody that specifically binds to monkeypox B6R protein as described in any one of claims 1-2.

4. An expression carrier, characterized in that, The expression vector comprises the polynucleotide as described in claim 3.

5. A host cell, characterized in that, The host cell contains the expression vector as described in claim 4.

6. The use of a monoclonal antibody that specifically binds to the monkeypox B6R protein as described in any one of claims 1-2 in the preparation of a medicament for neutralizing monkeypox virus.

7. The application according to claim 6, characterized in that, The drug is either a liquid injection or a dry powder.

8. The use of a monoclonal antibody that specifically binds to the monkeypox B6R protein as described in any one of claims 1-2 in the preparation of an immunoassay tool for detecting monkeypox virus.

9. The application according to claim 8, characterized in that, The immunoassay tool is a reagent, reagent kit, chip, or test strip.

10. A kit for detecting monkeypox virus, characterized in that, The kit includes a monoclonal antibody that specifically binds to the monkeypox B6R protein as described in any one of claims 1-2.

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