Anti-orthopoxvirus neutralizing monoclonal antibody B36B9 and its application

Abstract

This invention provides a neutralizing monoclonal antibody B36B9 against orthopoxvirus and its applications. The monoclonal antibody, obtained through flow cytometry sorting and single-cell PCR, possesses a unique CDR region and primarily recognizes the L1R (M1R) antigen protein of orthopoxvirus. The antibody exhibits affinities of 0.21 nM, 0.39 nM, 0.03 nM, and 0.12 nM for VARV, VACV, CPXV, and MPXV, respectively. In virus neutralization experiments, the antibody showed an IC50 value of [missing information - likely related to viral neutralization]. 50 The titer was 41.13 nM. In a mouse model of monkeypox virus infection, the antibody significantly reduced the viral titer in the lungs of mice. The monoclonal antibody provided by this invention has the characteristics of high expression, high affinity, and broad spectrum, and has the potential for application in the preparation of therapeutic and preventive drugs for monkeypox virus or kits for detecting monkeypox virus.

Description

Technical Field

[0001] This invention discloses an antibody, belonging to the field of protein or polypeptide technology. Background Technology

[0002] Orthopoxvirus (OPXV) belongs to the Poxviridae family, which includes four main types of viruses associated with human pathogenic infection: Variola virus (VARV), monkeypox virus (MPXV), vaccinia virus (VACV), and cowpox virus (CPXV). During replication, OPXV produces two types of infectious viral particles: intracellular mature virus (IMV) and extracellular enveloped virus (EEV). OPXV is a double-stranded DNA virus with a genome length of approximately 200 kb, encoding up to 200 viral proteins. Different species of OPXV share over 90% homology in their protein sequences. Six antigenic proteins on the surface of OPXV are important targets for neutralizing antibodies, including the intracellular mature viral surface antigen protein L1R (homophore M1R in monkeypox virus), A27L, H3L, and D8L, and the extracellular envelope viral surface antigen proteins B5R and A33R (Cell. 2016, 167, 3, 684-694). Among them, the L1R protein is associated with the virus-encoded multi-protein fusion complex and plays an important role in the process of viral entry into cells, mediating membrane fusion and binding to target cells (J Virol. 2008, 82, 17, 8687-8694).

[0003] Neutralizing monoclonal antibodies are promising therapeutic agents compared to small molecule drugs, possessing advantages such as a clear mechanism of action, high specificity, rapid onset of action, and low cross-reactivity, making them an important research strategy for the prevention and treatment of orthopoxvirus infection. Compared to vaccines, neutralizing antibodies can provide protection for immunocompromised patients and during the vaccination window (post-vaccination and antibody challenge period), serving as an effective vaccine alternative. Compared to FDA-approved vaccinia immunoglobulin (VIG), neutralizing antibodies offer broader efficacy, lower development costs, and smaller batch-to-batch efficacy variations, enabling more effective responses to viral infections. Several studies have proposed using monoclonal antibodies as an alternative to VIG (Int J Infect Dis. 2006,10, 3, 193-201; Nat Commun. 2024, 15, 1, 3265). However, current research on orthopoxvirus monoclonal antibodies is mostly still in the preclinical stage, with no neutralizing monoclonal antibodies yet entering clinical trials or receiving approval.

[0004] Current monoclonal antibody preparation technologies have evolved from traditional hybridoma techniques to a variety of efficient and precise engineered methods, including phage display, humanized transgenic mice, single-B-cell clone screening, and novel yeast and ribosome display technologies. The principle of single-B-cell clone screening is to isolate B cells from the peripheral blood of immunized individuals (vaccinated individuals, recovered patients, or laboratory animals), sort antigen-specific B cells using flow cytometry, and then obtain antibodies through single-cell PCR and in vitro gene recombination and protein expression techniques. Single-B-cell clone screening offers advantages such as low immunogenicity, preservation of natural antibody light and heavy chain pairing characteristics, and rapid response, and has become a core tool in antibody development.

[0005] This invention aims to isolate monoclonal antibodies with excellent broad-spectrum neutralizing activity from the peripheral blood of cynomolgus monkeys inoculated with recombinant orthopoxvirus antigen protein by combining flow cytometry and single-cell PCR. The goal is to provide therapeutic monoclonal antibodies with good protective effects against orthopoxvirus, providing research support for the prevention, control and diagnosis of orthopoxvirus infection, and to cope with possible orthopoxvirus infection epidemics. Summary of the Invention

[0006] Based on the above-mentioned objectives, this invention firstly screens a monoclonal antibody against orthopoxvirus by combining flow cytometry and single-cell PCR. The amino acid sequences of the CDR1, CDR2, and CDR3 regions of the heavy chain variable region of the monoclonal antibody are shown as amino acid sequences at positions 31-35, 50-65, and 98-111 of SEQ ID NO:1, respectively; the amino acid sequences of the CDR1, CDR2, and CDR3 regions of the light chain variable region are shown as amino acid sequences at positions 23-36, 52-58, and 91-102 of SEQ ID NO:5, respectively.

[0007] The monoclonal antibody described in this invention comprises variable and constant regions in both its heavy and light chains. The variable regions have three complementarity determining regions (CDRs): CDR1, CDR2, and CDR3, exhibiting high variability and diversity. The sequence diversity of the CDR regions in both the heavy and light chains determines the antibody's specificity and affinity, as they recognize and bind to specific antigenic determinants through interaction with the antigen.

[0008] In a preferred embodiment, the amino acid sequence of the heavy chain variable region of the anti-orchiopeptidosis virus monoclonal antibody is shown in SEQ ID NO: 1, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 5. The anti-orchiopeptidosis virus monoclonal antibody is named "B36B9" in this invention.

[0009] In a more preferred embodiment, the amino acid sequence of the heavy chain constant region of the anti-orchiopycosis virus monoclonal antibody is shown in SEQ ID NO:3, and the amino acid sequence of the light chain constant region is shown in SEQ ID NO:7.

[0010] Second, the present invention also provides a polynucleotide encoding the heavy chain and light chain of the above-mentioned anti-orchicine virus monoclonal antibody, wherein the sequence of the polynucleotide encoding the variable region of the heavy chain of the anti-orchicine virus monoclonal antibody is shown in SEQ ID NO:2, and the sequence of the polynucleotide encoding the variable region of the light chain of the anti-orchicine virus monoclonal antibody is shown in SEQ ID NO:6.

[0011] In a preferred embodiment, the polynucleotide sequence encoding the heavy chain constant region of the anti-orchicine virus monoclonal antibody is shown in SEQ ID NO: 4, and the polynucleotide sequence encoding the light chain constant region of the anti-orchicine virus monoclonal antibody is shown in SEQ ID NO: 8.

[0012] Third, the present invention also provides a functional element expressing the heavy and light chains of the polynucleotides encoding the above-mentioned monoclonal antibody against orthopoxvirus. The functional element expressed in the present invention refers to a combination of components that have the function of expressing the polynucleotide as a protein, such as plasmid expression vectors conventional in the art.

[0013] In a preferred embodiment, the functional element is a linear expression cassette (LEC). The LEC described in this invention is a tool for simplifying gene delivery in genetic engineering, commonly used in in vitro transcription, cell transfection, or cell-free protein synthesis systems. It contains a linear DNA fragment with essential gene expression elements, without a plasmid backbone (such as bacterial replication origin, resistance genes, etc.). Its core components include: a promoter (such as CMV, T7, etc.): driving the transcription of downstream genes; a target gene: a sequence encoding the target protein; and a terminator: a signal to terminate transcription (such as BGH polyA, SV40 polyA). It may also include optional elements, such as a 5'UTR / 3'UTR, a signal peptide sequence (for secretory expression), and a tag sequence (such as a His-tag, FLAG-tag).

[0014] Fourth, the present invention also provides a host cell containing the above-described linear expression frame. The host cell is used to express the above-described anti-orchiopycosis virus monoclonal antibody.

[0015] In a preferred embodiment, the host cell is HEK 293F cell. In another preferred embodiment, the cell is CHO cell, and the present invention can construct a stable CHO engineered cell line to realize the industrial culture of the antibody.

[0016] Finally, the present invention also provides the application of the above-mentioned anti-orchiopyvirus monoclonal antibody in the preparation of orchiopyvirus disease treatment drugs, preventive drugs, or orchiopyvirus detection kits.

[0017] The monoclonal antibody provided by this invention has excellent binding and neutralizing activity with the L1R protein of orthopoxvirus and can specifically target the L1R antigen protein. Therefore, by utilizing these characteristics of the monoclonal antibody, it can be used to specifically inhibit the binding and fusion of orthopoxvirus with the host cell membrane, or to specifically target the lesion or pathogen of infection with a therapeutic drug to exert a clinical therapeutic or preventive effect. Therefore, this invention provides the application of the above-mentioned monoclonal antibody in the preparation of drugs for treating and / or preventing monkeypox virus.

[0018] Based on the excellent affinity of the monoclonal antibody provided by this invention for the L1R protein of orthopoxvirus, it can be used to detect orthopoxvirus particles with the L1R protein that may be present in a sample. The detection can be a single-antibody detection, i.e., the monoclonal antibody specifically binds to the pathogen as a primary antibody, and then a secondary antibody is used to detect the binding; or it can be a combination detection of two antibodies.

[0019] The monoclonal antibody provided by this invention was obtained through flow cytometry sorting and single-cell PCR screening. It possesses a unique CDR region and primarily recognizes the L1R (M1R) antigen protein of orthopoxvirus. The antibody exhibits affinities of 0.21 nM, 0.39 nM, 0.03 nM, and 0.12 nM for VARV, VACV, CPXV, and MPXV, respectively. In virus neutralization experiments, the antibody showed an IC50 value of [missing information - likely related to viral neutralization]. 50 The titer is 41.13 nM. In a mouse model of monkeypox virus infection, the antibody significantly reduced the viral titer in the lungs of mice. The monoclonal antibody disclosed in this invention has the characteristics of high expression, high affinity, and broad spectrum, and can be used for industrial production and can be applied to respond to possible future outbreaks of orthopox virus infection. Attached Figure Description

[0020] Figure 1 Single-cell sorting diagram from flow cytometry;

[0021] Figure 2 Capillary electrophoresis identification of the H, κ, and λ strand genes after nested PCR amplification;

[0022] Figure 3 A diagram showing the binding activity of antibody expression supernatant with M1R protein;

[0023] Figure 4 Output image of search results for the variable region sequence of monoclonal antibody B36B9;

[0024] Figure 5 ELISA was used to detect the cross-binding activity of monoclonal antibody B36B9 with orthopoxvirus L1R (M1R) protein;

[0025] Figure 6 BLI assay was used to detect the affinity of monoclonal antibody B36B9 for orthopoxvirus L1R (M1R) protein.

[0026] Figure 7 The neutralizing activity of the B36B9 antibody against vaccinia virus;

[0027] Figure 8 The protective effect of B36B9 antibody against monkeypox virus infection in mice. Detailed Implementation

[0028] The present invention will be further described below with reference to specific embodiments, and the advantages and features of the present invention will become clearer as a result of the description. However, these embodiments are merely exemplary and do not constitute any limitation on the scope of protection defined by the claims of the present invention.

[0029] Example 1: Screening and preparation of monoclonal antibodies against orthopoxvirus

[0030] 1. Isolation of peripheral blood mononuclear cells from cynomolgus monkeys:

[0031] Two weeks after the third immunization of cynomolgus monkeys, 10 ml of blood samples were collected, and peripheral blood mononuclear cells were separated by Ficoll density gradient centrifugation.

[0032] 2. Flow cytometry sorting of memory B cells:

[0033] (1) Antigen labeling: 100 μg of antigen protein was transferred to PBS using a desalting column, and the recommended amount of NHS biotin was added. The mixture was incubated at room temperature in the dark for 30 min. The remaining NHS biotin was removed using a desalting column and stored at 4 °C in the dark for later use.

[0034] (2) Single staining tubes: Prepare 7 flow cytometry tubes, each containing 500,000 cells. Add the antibodies in Table 1 to 6 flow cytometry tubes according to the concentration recommended in the instructions. Use the remaining tube as a naked cell control.

[0035] (3) Primary antibody incubation in each sample tube: 1 million cells per tube, stain the cells with fluorescent dyes (PE-Anti Human IgG, Alexa Fluor 700-Anti Human CD19, PerCP-Anti Human CD3, PE Cy7-Anti Human CD27) and biotin-labeled antigens (E174-biotin, MT3-L1-A33-biotin) according to the recommended dosage in the instructions, and incubate at 4°C in the dark for 1 h;

[0036] (4) Secondary antibody incubation in sample tubes: After washing twice with 2% FPBS, stain the cells with fluorescently labeled streptavidin (Streptavidin-AF488, Streptavidin-BV421) according to the recommended dosage in the instructions, and incubate at 4 ℃ in the dark for 30 min.

[0037] (5) Cell sorting: After washing the cells with 2% FPBS, resuspend the cells in FPBS, pass them through a 40 μm cell sieve, and then use a cell sorter (SONY, MA900) to sort out antigen-specific single memory B cells. Figure 1As shown: Lymphocytes were circled by FSC and SSC. CD3- / CD19+ were B cells, IgG+ / CD27+ were memory B cells, and AF488+ were M1R protein-specific memory B cells. Individual cells were sorted into 96-well plates, each containing 20 M RNase inhibitor and 20 μL of RNase-free water, and stored at -80℃ for later use.

[0038] Table 1. Flow cytometry sorting of fluorescent antibodies

[0039] .

[0040] 3. Single-cell PCR amplification of antibody variable region genes:

[0041] (1) Reverse transcription PCR: Follow the instructions of the one-step kit and directly add the mixed primer and reagent reaction system to a 96-well plate containing a single cell for PCR reaction. Primer sequences are detailed in Table 2, and reagent reaction system is detailed in Table 3. Reaction conditions: 42 ℃ / 10 min, 25 ℃ / 10 min, 50 ℃ / 60 min, 94 ℃ / 5 min.

[0042] Table 2. Reverse transcription PCR primer sequences (SEQ ID NO. 9-45)

[0043] .

[0044] Table 3. Reverse Transcription PCR Reaction System

[0045] .

[0046] (2) Nested PCR: Using the reverse transcription PCR product as a template, the variable region gene sequences of the antibody H, κ, and λ chains were amplified using the primer sequences in Table 4. The nested PCR reaction system is detailed in Table 5. Reaction conditions: 95 ℃ / 5 min, 40 cycles (95 ℃ / 15 s, 57 ℃ / 15 s, 72 ℃ / 45 s), 72 ℃ / 5 min.

[0047] Table 4. Nested PCR primer sequences (SEQ ID NO.46-84)

[0048] .

[0049] Table 5. Nested PCR reaction system

[0050] .

[0051] (3) Capillary electrophoresis: The products after nested PCR amplification were subjected to capillary electrophoresis using the QIAxcel DNA Fast Analysis Cartridge. The results are as follows: Figure 2 As shown. PCR products with positive amplification results were subjected to DNA sequencing. The sequencing results were analyzed using the IMGT / V-QUEST website. Clones with positive results for both light and heavy chains were designated as paired clones.

[0052] 4. Construction of antibody linear expression cassettes:

[0053] The antibody variable region gene obtained by single-cell PCR amplification is linked to the promoter-leader sequence and the constant region-polyA tail sequence by overlapping extension PCR to construct a linear expression cassette for efficient and rapid antibody screening.

[0054] (1) Amplification of promoter-leader sequence fragments: Using pcDNA-H plasmid as a template, the promoter-leader sequence of the heavy chain was amplified using primers CMV-UP and 3'Leader-H; using pcDNA-λ plasmid as a template, the promoter-leader sequence of the light chain was amplified using primers CMV-UP and 3'Leader-L. After agarose gel electrophoresis, the PCR products were excised and recovered. Primer sequence information is detailed in Table 6. The promoter-leader sequence fragment amplification reaction system is detailed in Table 7. Reaction conditions: 95 ℃ / 10 min, 30 cycles (95 ℃ / 30 s, 60 ℃ / 30 s, 72 ℃ / 1 min), 72 ℃ / 10 min.

[0055] Table 6. Primer sequences for antibody linear expression cassette amplification (SEQ ID NO. 85-109)

[0056] .

[0057] Table 7. Promoter-leader sequence amplification reaction system

[0058] .

[0059] 2) Amplification of the constant region-polyA tail sequence fragment: Using pcDNA-H plasmid as a template, the constant region-polyA tail fragment of the heavy chain was amplified using primers 5'CH and TK-POLYA; using pcDNA-λ plasmid as a template, the constant region-polyA tail fragment of the λ chain was amplified using primers 5'Cλ and TK-POLYA. After agarose gel electrophoresis, the target fragment was excised and recovered. Primer sequence information is detailed in Table 6. The amplification reaction system for the constant region-polyA tail fragment is detailed in Table 8. Reaction conditions: 95 ℃ / 10 min, 30 cycles (95 ℃ / 30 s, 60 ℃ / 30 s, 72 ℃ / 2 min), 72 ℃ / 10 min.

[0060] Table 8. Constant Region-Polymer A-Tail Fragment Amplification Reaction System

[0061]

[0062] (3) Amplification of the variable region fragment: Using the successfully paired single-cell nested PCR product of the light and heavy chains as a template, gene adapters required for overlapping extension PCR were added to both ends of the antibody variable region gene. The primers were dissolved in deionized water to a concentration of 100 μM, and the primers corresponding to the H chain and λ chain were mixed in equal volumes. The corresponding primer mixtures were used to amplify the H chain and λ chain, respectively. The primer sequence information for the variable region is detailed in Table 6. The reaction system for amplifying the variable region fragment is detailed in Table 9. Reaction conditions: 30 cycles (98 ℃ / 10 s, 55 ℃ / 5 s, 72 ℃ / 10 s).

[0063] Table 9. Antibody variable region amplification reaction system

[0064] .

[0065] (4) Amplification of linear expression cassettes: Using the amplified promoter-leader sequence fragment, constant region-polyA tail fragment, and variable region fragment as templates, and CMV-UP and TK-POLYA as primers, overlap extension PCR was performed to amplify the linear expression cassettes of the H and λ chains, respectively. Primer sequence information is detailed in Table 6. The linear expression cassette amplification reaction system is detailed in Table 10. Reaction conditions: 30 cycles (98 ℃ / 10 s, 55 ℃ / 5 s, 72 ℃ / 30 s).

[0066] Table 10. Full-length linear expression cassette amplification reaction system

[0067]

[0068] 5. Cell co-transfection:

[0069] (1) Seed HEK293T cells into 24-well plates, 20,000 cells per well, and culture overnight in a cell culture incubator;

[0070] (2) Take 1 μg of heavy chain linear expression cassette and light chain linear expression cassette respectively and add them to Opti-MEM medium. After mixing, add TurboFect (Thermo Scientific, R0531) transfection reagent, mix well and incubate at room temperature for 20 min.

[0071] (3) Add the mixture after incubation in the previous step dropwise to the cells, mix gently, and incubate in an incubator for 48 h;

[0072] (4) Collect the cell supernatant, centrifuge at 4 ℃, 8000 rpm, for 15 min, and freeze the supernatant for later use.

[0073] 6. ELISA initial screening of binding antibodies:

[0074] (1) Plate coating: The monkeypox virus M1R antigen protein was coated onto the ELISA plate at a concentration of 1 μg / ml and incubated overnight at 4 ℃;

[0075] (2) Blocking: Wash the plate with PBST 3 times, add 2% BSA, 100 μl / well, and incubate at 37℃ for 1 h;

[0076] (3) Sample incubation: Wash the well plate 3 times with PBST, add 100 μl of a 1:1 mixture of cell transfection supernatant and diluent to each well, and incubate at 37 °C for 1 h;

[0077] (4) Secondary antibody incubation: Wash the well plate 3 times with PBST, add goat anti-human IgG (HRP) antibody (Abcam, ab97225, 1:10000 dilution), and incubate at 37 ℃ for 1 h;

[0078] (5) Color development: Wash the plate with PBST 3 times, add color development solution (Solepro, PR1200), 100 μl / well, incubate at 37 ℃ in the dark for 3 minutes, and then add 50 μl of stop solution (Solepro, C1058) to stop the color development;

[0079] (6) Microplate reader readings: The absorbance at 450 nm was detected using a SpetraMax ABS Plus instrument with 630 nm as the reference wavelength. The wells without the test sample were used as negative controls. The detection limit for positive values ​​was defined as 2.1 times the reading of the negative control wells.

[0080] 7. Expression and purification of monoclonal antibodies:

[0081] Light and heavy chain expression plasmids were constructed for monoclonal antibody expression and preparation.

[0082] (1) Construction of light and heavy chains of pCDNA3.4 vector: Using the linear expression frame as a template, light and heavy chains were amplified, and light and heavy chain fragments of 0.7 kb and 1.4 kb in size were recovered by gel cutting. Then, the light and heavy chain fragments were ligated with the vector fragments by homologous recombination (NEBuilder HiFi DNA Assembly Master Mix, E2621L). After the competent cells were transformed, single clones were picked for sequencing identification. Finally, the light chain expression vector pCDNA3.4-B36B9-L and the heavy chain expression vector pCDNA3.4-B36B9-H were constructed.

[0083] (2) Expression and purification of monoclonal antibodies: Antibody expression was performed using the HEK 293F expression system. Following the instructions for the transfection reagent (ThermoFisher Scientific, A14635), 15 μg each of the constructed light and heavy chain plasmids were mixed and transfected into 293F cells. After culturing for 5-6 days, the cell supernatant containing the antibody was collected and centrifuged at 12000 xg for 10 minutes. The antibody in the cell supernatant was then purified by affinity chromatography using a Protein A column (Cytiva, 17040201). The loading buffer was 20 mM PBS, and the protein elution buffer was 0.1 M glycine-hydrochloric acid buffer (pH = 3.0).

[0084] Results: 106 monoclonal antibodies were expressed, and their binding activity to the orthopoxvirus L1R antigen protein was measured. The results showed that 5 antibodies could specifically bind to the L1R protein. Figure 3 As shown. The light and heavy chain genes of the five antibodies were constructed into the pCDNA3.4 plasmid vector, and after monoclonal antibody expression and purification, they were stored at low temperature.

[0085] 8. Sequence Analysis

[0086] The DNA sequence of the PCR amplification product of the screened clone B36B9 was determined and analyzed. A variable region search was performed on the IMGT website (http: / / www.imgt.org / IMGT_vquest / analysis), revealing a typical antibody sequence, consistent with expectations. The search results are as follows: Figure 4 As shown, Figure 4 Image A shows the search results for the variable region of the heavy chain of antibody B36B9. Region V showed the highest homology at 96.14%, region J showed the highest homology at 98.08%, and region D used reading frame 3. Figure 4 The search results for the light chain of antibody B36B9 are shown in section B. Region V showed the highest homology (99.31%), and region J showed the highest homology (97.30%). Sequence analysis of monoclonal antibody B36B9 revealed the following amino acid sequences: encoding the heavy chain variable region (SEQ ID NO: 1), polynucleotide sequence (SEQ ID NO: 2), and amino acid sequences of CDR1, CDR2, and CDR3 regions (SEQ ID NO: 1, positions 31-35, 50-65, and 98-111), respectively. Similarly, encoding the light chain variable region (SEQ ID NO: 5), polynucleotide sequence (SEQ ID NO: 6), and amino acid sequences of CDR1, CDR2, and CDR3 regions (SEQ ID NO: 5, positions 23-36, 52-58, and 91-102), respectively, are also shown.

[0087] Example 2: Identification of cross-binding activity of antibody B36B9

[0088] Cross-binding activity of L1R (M1R) proteins against B36B9-associated human infection-related orpoxviruses (VARV, MPXV, VACV, and CPXV).

[0089] (1) Plate coating: The orthopoxvirus L1R (M1R) antigen protein was coated onto the ELISA plate at a concentration of 2 μg / ml, 100 μl / well, and incubated overnight at 4 ℃;

[0090] (2) Blocking: Wash the plate with PBST 3 times, add 2% BSA blocking solution, 100 μl / well, and incubate at 37℃ for 1 h;

[0091] (3) Primary antibody incubation: Wash the plate with PBST 3 times, add 1 μg / ml of antibody to the first well, dilute 4-fold serially, set up 3 replicates, 100 μl / well, and incubate at 37 ℃ for 1 h;

[0092] (4) Secondary antibody incubation: Wash the well plate 3 times with PBST, add 100 μl of goat anti-human IgG (HRP) antibody (Abcam, ab97225, 1:10000 dilution) to each well, and incubate at 37 °C for 1 h;

[0093] (5) Color development: Wash the plate with PBST 3 times, add 100 μl of TMB color development solution (Solepro, PR1200) per well, incubate in the dark for 6 minutes, and then add 50 μl of stop solution (Solepro, C1058) to stop the color development.

[0094] (6) Microplate reader readings: The absorbance at 450 nm was detected using a SpetraMax ABS Plus instrument with 630 nm as the reference wavelength. The wells without the test sample were used as negative controls. The detection limit for positive values ​​was defined as 2.1 times the reading of the negative control wells.

[0095] Results: The binding activity of B36B9 to antigen proteins of different species of orthopoxvirus was detected, as detailed in the following figures. Figure 5 B36B9 for VAV EC 50 The concentration was 4.45 ng / ml, combined with VARV's EC. 50 The concentration was 4.71 ng / ml, combined with the EC of CPXV. 50 The concentration was 3.06 ng / ml, and the EC50 of MPXV was [missing information]. 50 The concentration was 5.65 ng / ml. The results showed that B36B9 specifically binds to the L1R (M1R) protein of different species of orthopoxviruses, exhibiting a dose-response relationship.

[0096] Example 3: Determination of the affinity between B36B9 and L1R (M1R) antigen using biomembrane interferometry (BLI).

[0097] (1) Reagent preparation: Prepare PBST as experimental buffer and 0.01 M glycine-hydrochloric acid buffer (pH = 1.7) as regeneration solution;

[0098] (2) Sample preparation: Dilute B36B9 to 10 μg / ml and dispense into the sample detection plate, 200 μl / well. Dilute L1R (M1R) antigen protein, the first well is 100 nM, and then serially dilute 2-fold to 100 nM, 50 nM, 25 nM, 12.5 nM, 6.25 nM, 3.125 nM and 1.5625 nM, 200 μl / well.

[0099] (3) Turn on the Octet R8 instrument, place the sample plate and the AHC2 (Sartorius, 18-5142) probe plate inside the instrument, and use kinetic and affinity detection methods to detect the sample. The specific parameters and times for each experimental step are as follows: Baseline time is 60 s; B36B9 antibody loading height is 1 nm; Association time is 200 s; Dissociation time is 600 s; Regeneration and Neutralization cycles are set to 3 times.

[0100] (4) Results processing: Open Octet Analysis Studio software and run the results file: In the Preprocessed Data interface, subtract the reference well result value, Baseline and inter-step calibration; In the Kinetics Analysis interface, select 1:1 Binding for Binding Model, select Global (Group) for Fitting type, and click apply to fit the data; Record the binding kinetic data ka, kdis, KD, etc.

[0101] Results: Table 11 shows the binding kinetic data ka, kdis, and KD of monoclonal antibody B36B9 with L1R (M1R) antigens from different species. Figure 6 The figures show the affinity constants of B36B9 with L1R (M1R) antigens of VARV, MPXV, VACV, and CPXV strains. The results indicate that B36B9 exhibits good affinity for the L1R (M1R) antigens of different orthopoxvirus species, with affinities of 0.21 nM, 0.39 nM, 0.03 nM, and 0.12 nM with VACV, VARV, CPXV, and MPXV, respectively.

[0102] Table 11. Binding kinetics data of B36B9 with L1R (M1R) antigen proteins of different species of orthopoxvirus

[0103] .

[0104] Example 4: Identification of the neutralizing activity of antibody B36B9 against vaccinia virus (VACV)

[0105] (1) Cell plating: BS-C-1 cells were plated in 96-well plates, with 15,000 cells per well, and incubated in an incubator for 24 hours;

[0106] (2) Antibody dilution: MEM + 2% FBS + PBS was used as the diluent. The initial concentration of antibody in each well was 100 μg / ml. The antibody was serially diluted 4 times and set up 3 replicates, with 60 μl / well.

[0107] (3) Virus dilution: Dilute the VCV_LUC virus suspension to a suitable titer with diluent and mix thoroughly by ultrasound;

[0108] (4) Add 60 μl of diluted VCV_LUC virus solution to the serially diluted antibody (positive control is the well without antibody treatment), mix thoroughly, and incubate in a cell culture incubator for 1 h;

[0109] (5) Remove the culture medium from the 96-well plate, add 100 μl of antibody-virus mixture per well, and incubate in a cell culture incubator for 2 h. Then, replace and discard the antibody-virus mixture, add 100 μl of MEM + 2% FBS + PBS per well, and incubate in a cell culture incubator overnight.

[0110] (6) After 24 h, discard the cell culture supernatant, add 100 μl PBS to wash the cells, then add 50 μl / well of lysis buffer (Promega, E1531), shake and lyse for 15 min, aspirate 30 μl of cell lysis buffer and transfer to a white microplate, add 30 μl of luciferase reaction substrate (Promega, E1501) and mix well, then use a Glomax Navigator to read the luciferase signal value. The antibody neutralization rate is: (1 – sample reading / positive control reading) × 100%. Use Graphpad Prism 8.0 to fit the curve and calculate the IC50. 50 value.

[0111] Results: See details of the neutralization experiment. Figure 7 The IC50 of monoclonal antibody B36B9 against vaccinia virus 50The result was 41.13 nM, indicating that B36B9 has good neutralizing activity against vaccinia virus.

[0112] Example 5: Protective effect of antibody B36B9 against monkeypox virus (MPXV) infection in mice.

[0113] 1. Mouse monkeypox virus challenge protection experiment:

[0114] (1) 6-8 week old BALB / C mice were randomly divided into 3 groups, with 6 animals in each group: PBS control group, B36B9 low-dose treatment group (5 mg / kg / mouse) and B36B9 high-dose treatment group (10 mg / kg / mouse).

[0115] (2) Mouse were challenged by intranasal administration of monkeypox virus at a dose of 70,000 PFU;

[0116] (3) One day after challenge, mice in the treatment group were treated with intraperitoneal injection of 5 mg / kg / mouse or 10 mg / kg / mouse of B36B9 monoclonal antibody, while mice in the control group were treated with an equal volume of PBS. On the 6th day after challenge, lung tissue of the mice was collected and ground to determine the viral titer in the lungs.

[0117] 2. Determination of viral titer in lung tissue:

[0118] (1) Lung tissue grinding: Place the EP tube containing lung tissue into a tissue grinder, set the parameters to 30 Hz, 60 s, repeat 3 times, then centrifuge at 8000 rpm for 10 min, transfer the supernatant to a 1.5 ml EP tube, and freeze at low temperature;

[0119] (2) Cell plating: BS-C-1 cells were plated in 12-well plates, with 450,000 cells per well, and incubated in an incubator for 24 hours;

[0120] (3) Prepare diluent (MEM + 2% FBS + P / S), and serially dilute the lung tissue homogenate 10-fold, with a dilution range of 10. -2 Up to 10 -5 500 μl / well, 3 replicates / sample;

[0121] (4) Remove the cell culture medium, add the serially diluted lung tissue homogenate, and incubate in a cell culture incubator for 2 hours;

[0122] (5) Remove the tissue homogenate, add 1 ml / well of 1% agar-MEM mixture, place in a cell culture incubator, and after the agar solidifies, invert the cell culture plate and culture for 4-5 days;

[0123] (6) After the empty spots grow, remove the 12-well plate from the incubator and add 4% paraformaldehyde for fixation, 1 ml / well, and place at room temperature overnight;

[0124] (7) Remove 4% paraformaldehyde, remove agar gel from the well plate, add 500 μl / well of crystal violet, and incubate at room temperature for 10 minutes;

[0125] (8) Discard the crystal violet solution, rinse the well plate with plenty of tap water and let it dry, then count the number of empty spots;

[0126] (9) Data processing: Divide the number of empty spots by 0.05 ml and multiply by the corresponding dilution factor to obtain the corresponding viral plaque titer (PFU / mL). The detection limit is 100 PFU / mL.

[0127] Results: The viral titer assay results in mouse lung tissue are as follows: Figure 8 As shown, the viral titer in the lungs of mice treated with low-dose B36B9 monoclonal antibody (mean 2811 PFU / g) was significantly lower than that in the control group (mean 81444 PFU / g), and the viral titer in the lungs of mice treated with high-dose B36B9 monoclonal antibody was undetectable. The results indicate that monoclonal antibody B36B9 can significantly protect mice from monkeypox virus infection.

Claims

1. A monoclonal antibody against orthopoxvirus, characterized in that, The amino acid sequences of the CDR1, CDR2, and CDR3 regions of the heavy chain variable region of the monoclonal antibody against orthopoxvirus are shown in amino acid sequences at positions 31-35, 50-65, and 98-111 of SEQ ID NO:1, respectively; the amino acid sequences of the CDR1, CDR2, and CDR3 regions of the light chain variable region are shown in amino acid sequences at positions 23-36, 52-58, and 91-102 of SEQ ID NO:5, respectively.

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

5.

3. The monoclonal antibody against orthopoxvirus according to claim 2, characterized in that, The amino acid sequence of the heavy chain constant region of the monoclonal antibody against orthopoxvirus is shown in SEQ ID NO:3, and the amino acid sequence of the light chain constant region is shown in SEQ ID NO:

7.

4. A polynucleotide encoding a monoclonal antibody against orthopoxvirus as described in any one of claims 1-3, characterized in that, The polynucleotide sequence encoding the heavy chain variable region of the monoclonal antibody against orthopoxvirus is shown in SEQ ID NO: 2, and the polynucleotide sequence encoding the light chain variable region of the monoclonal antibody against orthopoxvirus is shown in SEQ ID NO:

6.

5. The polynucleotide according to claim 4, characterized in that, The sequence of the polynucleotide encoding the heavy chain constant region of the monoclonal antibody against orthopoxvirus is shown in SEQ ID NO: 4, and the sequence of the polynucleotide encoding the light chain constant region of the monoclonal antibody against orthopoxvirus is shown in SEQ ID NO:

8.

6. A functional element comprising the polynucleotide of claim 4.

7. The functional element according to claim 6, characterized in that, The functional element is a linear expression box.

8. A host cell containing the linear expression frame of claim 7.

9. The host cell according to claim 8, characterized in that, The host cells are HEK 293F cells or CHO cells.

10. The use of the monoclonal antibody against orthopoxvirus according to any one of claims 1-3 in the preparation of therapeutic drugs, preventive drugs, or kits for detecting monkeypoxvirus.

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