Anti-orthopoxvirus neutralizing monoclonal antibody B36B9 and application thereof

Abstract

The invention provides an anti-orthopoxvirus neutralizing monoclonal antibody B36B9 and application thereof, the monoclonal antibody is obtained by screening through a flow cytometry sorting technology and a single cell PCR technology, has a unique CDR partition, and is mainly used for recognizing L1R (M1R) antigen protein of orthopoxvirus. The affinity of the antibody to VARV (Vector Activated Receptor Virus), VACV (Vector Activated Receptor Virus), CPXV (Cytopoxvirus) and MPXV (Metapoxvirus) of orthopoxvirus is 0.21 nM, 0.39 nM, 0.03 nM and 0.12 nM respectively. In a virus neutralization experiment, the IC50 of the antibody and mature virions in vaccinia virus cells is 41.13 nM. In a monkey pox virus infected mouse model, the antibody can significantly reduce mouse lung virus titer. The monoclonal antibody provided by the invention has the characteristics of high expression, high affinity and broad spectrum, and has application prospects in preparation of medicines for treating and preventing the monkey pox virus or kits for detecting the monkey pox virus.

Description

Technical Field

[0001] The invention discloses an antibody, belonging to the technical field of proteins or polypeptides. Background Art

[0002] Orthopoxviruses (OPXV) belong to the Poxviridae family, of which four are primarily associated with human pathogenic infections: Variola virus (VARV), Monkeypox virus (MPXV), Vaccina virus (VACV), and Cowpox virus (CPXV). OPXV produces two infectious viral particles during replication: the intracellular mature virus (IMV) and the extracellular enveloped virus (EEV). OPXV is a double-stranded DNA virus with a genome of approximately 200 kb, encoding up to 200 viral proteins. Protein sequence identity between different OPXV species exceeds 90%. OPXV has six surface antigenic proteins that are important targets for neutralizing antibodies, including the intracellular mature viral surface antigens L1R (the homologous protein in monkeypox virus is M1R), A27L, H3L, and D8L, and the extracellular envelope viral surface antigens B5R and A33R (Cell. 2016, 167, 3, 684-694). The L1R protein is associated with the virally encoded multiprotein fusion complex and plays a key role in viral entry, mediating membrane fusion and binding to target cells (J Virol. 2008, 82, 17, 8687-8694).

[0003] Neutralizing monoclonal antibodies (mAbs) are highly promising therapeutic agents compared to small molecule drugs. They offer advantages such as a well-defined mechanism of action, high specificity, rapid onset of action, and minimal cross-reactivity, making them an important research strategy for the prevention and treatment of orthopoxvirus infections. Compared to vaccines, neutralizing antibodies can provide protection in immunocompromised individuals and during the vaccination window (the period between vaccination and antibody stimulation), making them an effective vaccine alternative. Compared to the FDA-approved vaccinia immune globulin (VIG), neutralizing antibodies offer broader efficacy, lower development costs, and less batch-to-batch variability in potency, making them more effective against viral infections. Several studies have proposed the use of mAbs as an alternative to VIG (Int J Infect Dis. 2006, 10, 3, 193-201; Nat Commun. 2024, 15, 1, 3265). However, most current research on mAbs against orthopoxviruses remains in the preclinical stage, and no neutralizing mAbs have entered clinical trials or received approval.

[0004] Current monoclonal antibody production technologies have evolved from traditional hybridoma technology to a variety of efficient and precise engineering approaches, including phage display, humanized transgenic mouse technology, single B cell clone screening, and novel yeast and ribosome display technologies. Single B cell clone screening involves isolating B cells from the peripheral blood of immunized individuals (vaccine recipients, recovered patients, or experimental animals), sorting antigen-specific B cells using flow cytometry, and then obtaining antibodies through single-cell PCR and in vitro gene recombination and protein expression. Single B cell clone screening offers advantages such as low immunogenicity, retention of natural antibody light and heavy chain pairing, and rapid response, making it a core tool in antibody development.

[0005] The present invention proposes to use a method that combines flow cytometry sorting technology and single-cell PCR technology to isolate monoclonal antibodies with excellent broad-spectrum neutralizing activity from the peripheral blood of crab-eating macaques vaccinated with recombinant orthopoxvirus antigen proteins. The purpose is to provide therapeutic monoclonal antibodies with good protective effects against orthopoxviruses, provide research support for the prevention, treatment and diagnosis of orthopoxvirus infections, and respond to possible epidemics of orthopoxvirus infections. Summary of the Invention

[0006] Based on the above-mentioned purpose of the invention, first, the present invention combines flow cytometry sorting technology and single-cell PCR technology to screen a monoclonal antibody against orthopoxvirus, and the amino acid sequences of the CDR1, CDR2 and CDR3 regions of the heavy chain variable region of the monoclonal antibody are shown in the amino acid sequences of 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 the amino acid sequences of positions 23-36, 52-58, and 91-102 of SEQ ID NO: 5, respectively.

[0007] The heavy and light chains of the monoclonal antibodies described herein both include variable and constant regions. The variable regions have three complementarity determining regions (CDRs): CDR1, CDR2, and CDR3, which are highly variable and diverse. The sequence diversity of the heavy and light chain CDR regions determines the specificity and affinity of the antibody, 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-orthopoxvirus 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-orthopoxvirus monoclonal antibody is named "B36B9" in the present invention.

[0009] In a more preferred embodiment, the amino acid sequence of the heavy chain constant region of the anti-orthopoxvirus 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-orthopoxvirus monoclonal antibody. The sequence of the polynucleotide encoding the heavy chain variable region of the anti-orthopoxvirus monoclonal antibody is shown in SEQ ID NO: 2, and the sequence of the polynucleotide encoding the light chain variable region of the anti-orthopoxvirus monoclonal antibody is shown in SEQ ID NO: 6.

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

[0012] Third, the present invention also provides a functional element for expressing the above-mentioned polynucleotide encoding the heavy chain and light chain of the monoclonal antibody against orthopoxvirus. The expression functional element described in the present invention refers to a component combination that has the function of expressing the encoding polynucleotide as a protein, for example, a conventional plasmid expression vector in this field.

[0013] In a preferred embodiment, the functional element is a linear expression cassette. The Linear Expression Cassette (LEC) described herein 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 of the necessary gene expression elements, without a plasmid backbone (such as a bacterial replication origin or resistance genes). Core components include: a promoter (such as CMV or T7) that drives transcription of downstream genes; a target gene that encodes the target protein; and a terminator that signals transcription termination (such as BGH polyA or SV40 polyA). Optional elements may also be included, such as 5' / 3' UTRs, a signal peptide sequence (for secretory expression), and a tag sequence (such as a His-tag or FLAG-tag).

[0014] Fourthly, the present invention also provides a host cell containing the above linear expression cassette, which is used to express and obtain the above anti-OTPV monoclonal antibody.

[0015] In a preferred embodiment, the host cell is a HEK 293F cell. In another preferred embodiment, the cell is a CHO cell. The present invention can construct a CHO stable engineered cell line to achieve industrialized cultivation of the antibody.

[0016] Finally, the present invention also provides the use of the above-mentioned anti-OPXV monoclonal antibody in the preparation of a therapeutic drug or preventive drug for orthopoxvirus disease, or a kit for detecting orthopoxvirus.

[0017] The monoclonal antibodies provided by the present invention have excellent binding activity and neutralizing activity with the L1R protein of the orthopoxvirus and can specifically target the L1R antigen protein. Therefore, these properties of the monoclonal antibodies can be used to specifically inhibit the binding and fusion of orthopoxviruses with host cell membranes, or to specifically target therapeutic drugs to infected lesions or pathogens to exert clinical treatment or prevent infection. Therefore, the present invention provides the use of the above-mentioned monoclonal antibodies in the preparation of drugs for treating and / or preventing monkeypox virus.

[0018] The monoclonal antibodies provided by the present invention have excellent affinity for the L1R protein of orthopoxviruses and can be used to detect orthopoxvirus particles containing the L1R protein that may be present in a sample. This detection can be a single-antibody assay, where the monoclonal antibody acts as a primary antibody and specifically binds to the pathogen, followed by detection of this binding using a secondary antibody. Alternatively, a dual-antibody assay can be used.

[0019] The monoclonal antibody provided by the present invention was obtained by flow cytometry sorting and single-cell PCR screening. It has a unique CDR partitioning and mainly recognizes the L1R (M1R) antigen protein of orthopoxvirus. The antibody has an affinity of 0.21 nM, 0.39 nM, 0.03 nM, and 0.12 nM for VARV, VACV, CPXV, and MPXV of the orthopoxvirus genus, respectively. In the virus neutralization experiment, the IC of the antibody against mature virions of vaccinia virus cells was 0. 50 The antibody significantly reduced viral titers in the lungs of monkeypox virus-infected mice. The monoclonal antibody disclosed in this invention exhibits high expression, high affinity, and broad-spectrum activity, making it suitable for industrial production and potentially useful in addressing future outbreaks of orthopoxvirus infections. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 Flow cytometry single cell sorting diagram; Figure 2 Capillary electrophoresis analysis of H, κ, and λ chain genes after nested PCR amplification. Figure 3 . Graph showing the binding activity of antibody expression supernatant to M1R protein; Figure 4 Output of search results for the variable region sequence of monoclonal antibody B36B9; Figure 5 ELISA to detect the cross-binding activity of monoclonal antibody B36B9 with orthopoxvirus L1R (M1R) protein; Figure 6 BLI detection of the affinity of monoclonal antibody B36B9 for the orthopoxvirus L1R (M1R) protein; Figure 7 Neutralizing activity of the B36B9 antibody against vaccinia virus; Figure 8 . Protective effect of B36B9 antibody on monkeypox virus-infected mice. DETAILED DESCRIPTION

[0021] 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 the description proceeds. 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.

[0022] Example 1: Screening and preparation of orthopoxvirus monoclonal antibodies 1. Isolation of peripheral blood mononuclear cells from cynomolgus monkeys: 10 ml blood samples were collected from cynomolgus monkeys 2 weeks after the third immunization, and peripheral blood mononuclear cells were isolated by Ficoll density gradient centrifugation.

[0023] 2. Flow cytometry sorting of memory B cells: (1) Antigen labeling: 100 μg of antigen protein was transferred to PBS using a desalting column, and the amount of NHS-biotin recommended in the instructions 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 the mixture was stored in the dark at 4°C until use. (2) Single staining tubes: Prepare 7 flow cytometry tubes, with 500,000 cells in each tube. Add the antibodies listed in Table 1 to 6 flow cytometry tubes according to the concentrations recommended in the instructions. The remaining tube serves as a bare cell control. (3) Incubation of primary antibodies in sample tubes: 1 million cells per tube, use 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) to stain the cells according to the recommended dosage in the instructions, and incubate in the dark at 4°C for 1 hour; (4) Incubation of secondary antibodies in sample tubes: After repeated washing with 2% FPBS twice, the cells were stained with fluorescently labeled streptavidin (Streptavidin-AF488, Streptavidin-BV421) according to the dosage recommended in the instructions, and incubated in the dark at 4°C for 30 min; (5) Cell sorting: After washing the cells with 2% FPBS, resuspend the cells with FPBS, pass them through a 40 μm cell sieve, and use a cell sorter (SONY, MA900) to sort out antigen-specific single memory B cells. Figure 1 As 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. Single cells were sorted into 96-well plates, each well containing 20 M RNase inhibitor and 20 μL RNase-free water, and stored at -80°C until use.

[0024] Table 1. Fluorescent antibodies for flow cytometry .

[0025] 3. Single-cell PCR amplification of antibody variable region genes: (1) Reverse transcription PCR: Follow the instructions for the one-step kit and add the mixed primers and reagents directly to the 96-well plate containing the single cells for PCR reaction. See Table 2 for primer sequences and Table 3 for reagents. Reaction conditions: 42°C / 10 min, 25°C / 10 min, 50°C / 60 min, 94°C / 5 min.

[0026] Table 2. Reverse transcription PCR primer sequences (SEQ ID NO. 9-45) .

[0027] Table 3. Reverse transcription PCR reaction system .

[0028] (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°C / 5 min, 40 cycles (95°C / 15 s, 57°C / 15 s, 72°C / 45 s), 72°C / 5 min.

[0029] Table 4. Nested PCR primer sequences (SEQ ID NO. 46-84) .

[0030] Table 5. Nested PCR reaction system .

[0031] (3) Capillary electrophoresis: The products after nested PCR amplification reaction were subjected to capillary electrophoresis using QIAxcel DNA Fast Analysis Cartridge. The results are as follows: Figure 2 PCR products with positive amplification results were subjected to DNA sequencing, and the sequencing results were analyzed using the IMGT / V-QUEST website. Clones with positive results for both light and heavy chains were recorded as paired clones.

[0032] 4. Construction of antibody linear expression cassette: The antibody variable region genes obtained by single-cell PCR amplification are connected with the promoter-leader sequence and constant region-poly A tail sequence through overlap extension PCR to construct a linear expression cassette for efficient and rapid antibody screening.

[0033] (1) Amplification of the promoter-leader sequence fragment: Using the pcDNA-H plasmid as a template, primers CMV-UP and 3'Leader-H were used to amplify the promoter-leader sequence of the heavy chain; using the pcDNA-λ plasmid as a template, primers CMV-UP and 3'Leader-L were used to amplify the promoter-leader sequence of the light chain. After agarose gel electrophoresis, the PCR products were excised and recovered. The primer sequence information is shown in Table 6. The promoter-leader sequence fragment amplification reaction system is shown in Table 7. Reaction conditions: 95°C / 10 min, 30 cycles (95°C / 30 s, 60°C / 30 s, 72°C / 1 min), 72°C / 10 min.

[0034] Table 6. Primer sequences for amplifying the linear expression cassette of antibodies (SEQ ID NOs. 85-109) .

[0035] Table 7. Promoter-leader sequence amplification reaction system .

[0036] 2) Amplification of the constant region-poly A sequence fragment: Using the pcDNA-H plasmid as a template, primers 5'CH and TK-POLYA were used to amplify the heavy chain constant region-poly A tail fragment. Using the pcDNA-λ plasmid as a template, primers 5'Cλ and TK-POLYA were used to amplify the λ chain constant region-poly A tail fragment. PCR products were subjected to agarose gel electrophoresis, and the target fragments were excised and recovered. Primer sequences are detailed in Table 6. The constant region-poly A tail fragment amplification reaction system is detailed in Table 8. Reaction conditions: 95°C / 10 min, 30 cycles of (95°C / 30 s, 60°C / 30 s, 72°C / 2 min), and 72°C / 10 min.

[0037] Table 8. Constant region-poly A tail fragment amplification reaction system

[0038] (3) Amplification of variable region fragments: Using the single-cell nested PCR product with successful light and heavy chain pairing as a template, add the gene linkers required for overlap extension PCR at both ends of the antibody variable region gene. The primers are dissolved in deionized water to 100 μM, and the primers corresponding to the H chain and the λ chain are mixed in equal volumes. The corresponding primer mixtures are used to amplify the H chain and the λ chain respectively. The variable region primer sequence information is detailed in Table 6. The variable region fragment amplification reaction system is detailed in Table 9. Reaction conditions: 30 cycles (98 ℃ / 10 s, 55 ℃ / 5 s, 72 ℃ / 10 s).

[0039] Table 9. Antibody variable region amplification reaction system .

[0040] (4) Amplification of linear expression cassettes: Using the amplified promoter-leader sequence fragment, constant region-poly A 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 chain and λ chain, 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°C / 10 s, 55°C / 5 s, 72°C / 30 s).

[0041] Table 10. Full-length linear expression cassette amplification reaction system

[0042] 5. Cell co-transfection: (1) HEK293T cells were seeded in a 24-well plate, with 20,000 cells per well, and cultured overnight in a cell culture incubator; (2) Take 1 μg of each heavy chain linear expression cassette and light chain linear expression cassette and add them to Opti-MEM medium, mix well, add TurboFect (Thermo Scientific, R0531) transfection reagent, mix well, and incubate at room temperature for 20 minutes; (3) Add the mixture from the previous step dropwise to the cells, mix gently, and incubate in the incubator for 48 h; (4) Collect the cell supernatant, centrifuge at 4°C, 8000 rpm, 15 min, and freeze the supernatant for later use.

[0043] 6. ELISA primary screening of binding antibodies: (1) Coating: coat the ELISA plate with monkeypox virus M1R antigen protein at a concentration of 1 μg / ml and incubate at 4°C overnight; (2) Blocking: Wash the plate three times with PBST, add 2% BSA, 100 μl / well, and incubate at 37°C for 1 h; (3) Sample incubation: Wash the plate three times with PBST, add a 1:1 mixture of cell transfection supernatant and diluent, 100 μl / well, and incubate at 37 °C for 1 h; (4) Secondary antibody incubation: Wash the plate three times with PBST, add goat anti-human IgG (HRP) antibody (Abcam, ab97225, 1:10000 dilution), and incubate at 37 °C for 1 h; (5) Color development: Wash the plate three times with PBST, add color development solution (Solabo, PR1200), 100 μl / well, incubate at 37°C in the dark for 3 minutes, and then add 50 μl stop solution (Solabo, C1058) to stop color development; (6) Microplate reader reading: Use a SpetraMax ABS Plus instrument to detect the absorbance at 450 nm with a reference wavelength of 630 nm. The wells without the test sample are used as negative controls. The detection limit of the positive value is defined as 2.1 times the reading of the negative control well.

[0044] 7. Expression and purification of monoclonal antibodies: Construct light and heavy chain expression plasmids for expression and preparation of monoclonal antibodies.

[0045] (1) Construction of light and heavy chains in the pCDNA3.4 vector: Using the linear expression frame as a template, the light and heavy chains were amplified, and the light and heavy chain fragments of 0.7 kb and 1.4 kb were recovered from the gel. The light and heavy chain fragments were then connected to the vector fragment by homologous recombination (NEBuilder HiFi DNA Assembly Master Mix, E2621L). After competent cells were transformed, single clones were picked for sequencing and identification. Finally, the light chain expression vector pCDNA3.4-B36B9-L and the heavy chain expression vector pCDNA3.4-B36B9-H were constructed; (2) Expression and purification of monoclonal antibodies: Antibodies were expressed using the HEK 293F expression system. Following the instructions for the transfection reagent (ThermoFisher Scientific, A14635), 15 μg of the constructed light and heavy chain plasmids were mixed and transfected into 293F cells. After 5-6 days of culture, the cell supernatant containing the antibody was collected and centrifuged at 12,000 x g 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-HCl buffer (pH = 3.0).

[0046] Results: 106 monoclonal antibodies were expressed and their binding activity to orthopoxvirus L1R antigen protein was determined. The results showed that 5 antibodies could specifically bind to L1R protein. Figure 3 The light and heavy chain genes of the five antibodies were constructed into the pCDNA3.4 plasmid vector, and the monoclonal antibodies were expressed, purified, and stored at low temperatures.

[0047] 8. Sequence analysis The DNA sequence of the PCR amplified product of the screened clone B36B9 was determined and analyzed, and the variable region search was performed on the IMGT website (http: / / www.imgt.org / IMGT_vquest / analysis). The sequence was a typical antibody sequence, which was consistent with expectations. The search results are as follows: Figure 4 As shown, Figure 4 Figure A shows the search results of the heavy chain variable region of antibody B36B9. The highest homology in the V region is 96.14%, the highest homology in the J region is 98.08%, and the D region uses reading frame 3. Figure 4 Figure B shows the search results for the light chain of antibody B36B9, with the highest homology in the V region being 99.31% and the highest homology in the J region being 97.30%. The sequence of monoclonal antibody B36B9 was analyzed. The amino acid sequence encoding the heavy chain variable region is shown in SEQ ID NO: 1, the polynucleotide sequence of the heavy chain variable region is shown in SEQ ID NO: 2, and the amino acid sequences of the CDR1, CDR2, and CDR3 regions of the heavy chain variable region are shown in amino acid sequences at positions 31-35, 50-65, and 98-111 of SEQ ID NO: 1, respectively. The amino acid sequence encoding the light chain variable region is shown in SEQ ID NO: 5, the polynucleotide sequence of the light chain variable region is shown in SEQ ID NO: 6, and 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.

[0048] Example 2: Identification of cross-binding activity of antibody B36B9 Cross-binding activity of B36B9 against the L1R (M1R) proteins of orthopoxviruses associated with human infection (VARV, MPXV, VACV, and CPXV).

[0049] (1) Coating: Coat the ELISA plate with the orthopoxvirus L1R (M1R) antigen protein at a concentration of 2 μg / ml, 100 μl / well, and incubate at 4°C overnight. (2) Blocking: Wash the plate three times with PBST, add 2% BSA blocking solution, 100 μl / well, and incubate at 37°C for 1 h; (3) Primary antibody incubation: Wash the plate three times with PBST, add 1 μg / ml antibody to the first well, dilute it 4-fold, set up three replicate wells, 100 μl / well, and incubate at 37°C for 1 h; (4) Secondary antibody incubation: Wash the plate three times with PBST, add goat anti-human IgG (HRP) antibody (Abcam, ab97225, 1:10000 dilution), 100 μl / well, and incubate at 37 °C for 1 h; (5) Color development: Wash the plate three times with PBST, add TMB color development solution (Solyb, PR1200), 100 μl / well, incubate in the dark for 6 minutes, and then add 50 μl stop solution (Solyb, C1058) to stop color development; (6) Microplate reader reading: Use a SpetraMax ABS Plus instrument to detect the absorbance at 450 nm with a reference wavelength of 630 nm. The wells without the test sample are used as negative controls. The detection limit of the positive value is defined as 2.1 times the reading of the negative control well.

[0050] Results: The binding activity of B36B9 with orthopoxvirus antigen proteins of different species was detected. Figure 5 , EC of B36B9 to VACV 50 The EC value for binding to VARV is 4.45 ng / ml. 50 The EC value for binding to CPXV was 4.71 ng / ml. 50 The EC value for binding to MPXV was 3.06 ng / ml. 50 The results showed that B36B9 specifically binds to the L1R (M1R) protein of different orthopoxvirus species in a dose-response relationship.

[0051] Example 3: Determination of the affinity of B36B9 for L1R (M1R) antigen using biomembrane interferometry (BLI) (1) Reagent preparation: Prepare PBST as the experimental buffer and 0.01 M glycine-HCl buffer (pH = 1.7) as the regeneration solution; (2) Sample preparation: Dilute B36B9 to 10 μg / ml and dispense into the sample test plate, 200 μl / well, dilute L1R (M1R) antigen protein, the first well is 100 nM, and then dilute it 2-fold to 100 nM, 50 nM, 25 nM, 12.5 nM, 6.25 nM, 3.125 nM and 1.5625 nM, respectively, 200 μl / well; (3) Turn on the Octet R8 instrument, place the sample plate and the AHC2 (Sartorius, 18-5142) probe plate in the instrument, and perform sample detection using the kinetic and affinity detection methods. 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; (4) Result processing: Open Octet Analysis Studio software and run the result file: in the Preprocessed Data interface, deduct the reference well result value, Baseline and inter-step calibration; in the Kinetics Analysis interface, select 1:1 Binding for Binding Model and Global (Group) for Fitting type, and click Apply to perform data fitting; record the binding kinetics data ka, kdis, KD, etc.

[0052] Results: Table 11 shows the binding kinetic data ka, kdis, and KD of monoclonal antibody B36B9 to L1R (M1R) antigens from different species. Figure 6 Affinity constants for B36B9 binding to L1R (M1R) antigens from VARV, MPXV, VACV, and CPXV strains are shown. Results show that B36B9 has excellent affinity for L1R (M1R) antigens from various orthopoxvirus species, with affinities of 0.21 nM, 0.39 nM, 0.03 nM, and 0.12 nM for VACV, VARV, CPXV, and MPXV, respectively.

[0053] Table 11. Binding kinetics of B36B9 to L1R (M1R) antigenic proteins of different orthopoxvirus species .

[0054] Example 4: Identification of Neutralizing Activity of Antibody B36B9 against Vaccinia Virus (VACV) (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 h; (2) Antibody dilution: Use MEM + 2% FBS + PBS as the diluent, the antibody concentration in the first well is 100 μg / ml, 4-fold gradient dilution, set up 3 replicate wells, 60 μl / well; (3) Virus dilution: Dilute the VACV_LUC virus suspension to an appropriate titer with diluent and mix evenly with ultrasound; (4) Add 60 μl of diluted VACV_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; (5) Remove the culture medium in the 96-well plate and add the antibody-virus mixture at 100 μl / well. Incubate in a cell culture incubator for 2 h. Then, replace and discard the antibody-virus mixture and add 100 μl / well of MEM + 2% FBS + PBS. Incubate in a cell culture incubator overnight. (6) After 24 h, the cell culture supernatant was discarded, 100 μl PBS was added to wash the cells, and then 50 μl / well lysis buffer (Promega, E1531) was added. The cells were shaken and lysed for 15 min. 30 μl of cell lysate was transferred to a white microplate, 30 μl luciferase reaction substrate (Promega, E1501) was added and mixed evenly, and the luciferase signal value was read using a Glomax Navigator detector. The antibody neutralization rate was: (1 – sample reading / positive control reading) × 100%. Graphpad Prism 8.0 was used to make a fitting curve and calculate the IC 50 value.

[0055] Results: Neutralization test results are detailed in Figure 7 , IC of monoclonal antibody B36B9 against vaccinia virus 50 The results showed that B36B9 has good neutralizing activity against vaccinia virus.

[0056] Example 5: Protective effect of antibody B36B9 on monkeypox virus (MPXV)-infected mice 1. Monkeypox virus challenge and protection experiment in mice: (1) BALB / C mice aged 6-8 weeks were randomly divided into three groups, with 6 animals in each group: PBS control group, low-dose B36B9 treatment group (5 mg / kg / mouse), and high-dose B36B9 treatment group (10 mg / kg / mouse); (2) Mice were challenged with 70,000 PFU of monkeypox virus by intranasal injection; (3) One day after challenge, mice in the treatment group were intraperitoneally injected with 5 mg / kg or 10 mg / kg of B36B9 monoclonal antibody, while mice in the control group were intraperitoneally injected with an equal volume of PBS. On the sixth day after challenge, lung tissues were collected and ground to determine the lung virus titer.

[0057] 2. Determination of virus titer in lung tissue: (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 store it in a cryogenic freezer. (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 h; (3) Prepare diluent (MEM + 2% FBS + P / S), dilute the lung tissue grinding solution 10 times in a gradient, and the dilution range is 10 -2to 10 -5 , 500 μl / well, 3 replicates / sample; (4) Remove the cell culture medium, add the gradient diluted lung tissue grinding solution, and incubate in a cell culture incubator for 2 hours; (5) Discard the tissue grinding solution and add 1 ml / well of 1% agar-MEM mixture. Place the plate in a cell culture incubator. After the agar solidifies, invert the cell culture plate and culture for 4-5 days. (6) After plaques have grown, remove the 12-well plate from the incubator and add 4% paraformaldehyde for fixation (1 ml / well) and place at room temperature overnight. (7) Discard the 4% paraformaldehyde, remove the agar gel in the well plate, add 500 μl / well crystal violet, and incubate at room temperature for 10 minutes; (8) Discard the crystal violet solution, rinse the plate with plenty of tap water, dry it, and count the number of plaques; (9) Data processing: Divide the number of plaques by 0.05 ml and multiply by the corresponding dilution factor to obtain the corresponding virus plaque titer (PFU / mL). The detection limit is 100 PFU / ml.

[0058] Results: The results of virus titer determination in mouse lung tissue were as follows: Figure 8 As shown, the virus titer in the lungs of mice treated with the low-dose B36B9 monoclonal antibody (mean, 2811 PFU / g) was significantly lower than that in the control group (mean, 81,444 PFU / g). In the high-dose B36B9 monoclonal antibody group, the virus titer in the lungs of mice was undetectable. These results demonstrate that the 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 anti-orthopoxvirus monoclonal antibody are shown in the 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 the amino acid sequences at positions 23-36, 52-58, and 91-102 of SEQ ID NO: 5, respectively.

2. The anti-orthopoxvirus monoclonal antibody according to claim 1, characterized in that The amino acid sequence of the heavy chain variable region of the anti-orthopoxvirus 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.

3. The anti-orthopoxvirus monoclonal antibody according to claim 2, characterized in that The amino acid sequence of the heavy chain constant region of the anti-orthopoxvirus 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.

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

6.

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

8.

6. An expression vector comprising a functional element encoding the polynucleotide of claim 4.

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

8. A host cell comprising the linear expression cassette of claim 7.

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

10. Use of the anti-orthopoxvirus monoclonal antibody according to any one of claims 1 to 3 in the preparation of a therapeutic drug or preventive drug for monkeypox virus disease, or a kit for detecting monkeypox virus.

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