Monoclonal antibody of monkey pox virus and application thereof

Abstract

The invention belongs to the field of biological medicine, and particularly relates to a monoclonal antibody aiming at a monkey pox virus A29 antigen and application of the monoclonal antibody. The invention provides two monoclonal antibodies targeting a monkey pox virus A29 antigen. The monoclonal antibodies have high affinity and high detection sensitivity; furthermore, the monoclonal antibody can be used for simultaneously detecting type I and type II monkey pox viruses, so that broad-spectrum detection of the monkey pox viruses is realized, missing detection is avoided, and the application feasibility is improved; furthermore, in consideration of continuous evolution of the monkey pox virus and higher epitope exposure degree, smaller aggregation degree and higher protein stability of the A29 protein, a series of mutated A29 proteins are designed, verification finds that the monoclonal antibody can recognize various mutated A29 proteins, future evolved strains of the monkey pox virus can be effectively detected, and the monoclonal antibody can be applied to the field of detection of the monkey pox virus. The clinical value is further improved, and the application prospect is larger.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to monkeypox virus monoclonal antibodies targeting monkeypox virus A29 antigen and applications thereof. Background Art

[0002] Monkeypox virus (MPXV), also known as monkeypox, is a zoonotic enveloped double-stranded DNA virus belonging to the Poxviridae family. Poxviruses are the largest class of double-stranded linear DNA viruses, with brick-shaped or oval particles measuring 200 to 400 nm. The virus replicates in the cytoplasm of infected cells. MPXV is a rare, sporadic, smallpox-like zoonotic virus that can cause zoonotic infections. MPXV has the typical morphology of an orthopoxvirus, with a rounded brick or oval shape and a size of 200 to 300 nm. The MPXV genome consists of approximately 197 kb of closed double-stranded DNA, sharing high homology with other orthopoxviruses such as smallpox. The genome terminates in an approximately 6 kb inverted repeat sequence, which forms a hairpin loop. Computer-assisted analysis revealed that the viral genome contains 190 open reading frames, four of which are located within the terminal inverted repeat sequence. The functions and expression status of most of these genes remain unclear.

[0003] Monkeypox virus can be transmitted from animals to humans through direct contact, primarily through blood, body fluids, wounds on the skin or mucous membranes, or through contaminated items such as clothing or bedsheets, needlestick injuries during healthcare, or in community settings such as tattoo parlors. Monkeypox virus can also be present in respiratory droplets, potentially leading to secondary human transmission through airborne droplets. These droplets can infect the mucous membranes of the eyes, nose, and throat, causing monkeypox infection. Early clinical symptoms of monkeypox resemble those of smallpox. The incubation period (the time from exposure to onset) is 5-11 days. Initial symptoms include headache, fever, rash, malaise, superficial lymphadenopathy, fever, stiffness, headache, and muscle aches. A few days later, a rash appears, typically starting on the face and gradually spreading to other parts of the body, including the extremities. The blisters are typically large, measuring 0.5 to 1 cm in diameter, a hallmark of monkeypox. While these symptoms are usually mild, they can be more severe in immunocompromised individuals. According to statistics, the mortality rate of monkeypox virus is about 1%-10%, and the mortality rate is higher among children, young people and immunocompromised people after infection.

[0004] Monkeypox virus has two distinct evolutionary clades: Congo Basin Clade I and West African Clade II. The unexpected occurrence and progression of monkeypox outbreaks necessitate effective detection methods to aid epidemic prevention and control. Nucleic acid diagnostics remain the primary method used both domestically and internationally, but they are highly dependent on personnel, equipment, and facilities. The lack of high-quality bioactive raw materials is one of the main limitations on the use of more convenient immunological detection methods.

[0005] Currently, the main research target for monkeypox antigen detection is the A29 (L) protein. Among the known public information, CN115975012A discloses an antibody against monkeypox A29L, but only discloses one antibody and only evaluates the indirect reactivity with monkeypox antigen. The detection effect of monkeypox antigen in the sandwich method is not reported; CN 116284349 B also discloses a monkeypox A29L monoclonal antibody. Although it has increased the specificity evaluation of cross-reactivity with other antigens, it is still limited to the indirect method. The application effect in the sandwich method is still unknown; CN 117209597 A discloses a sandwich antibody pairing for detecting monkeypox A29L antigen, but only reports the detection sensitivity of the pairing to the self-developed A29L recombinant protein. The source of the A29L protein sequence is not disclosed. Whether the pairing can effectively detect the two evolutionary branches of monkeypox viruses in actual application is obviously uncertain; CN 117700533 Patent B discloses an antigen sandwich paired antibody for detecting A29. The patent emphasizes the high sensitivity of the paired antibodies in detecting the A29 antigen, but also does not disclose the source of the A29 antigen sequence. There is no relevant evidence to support whether this pairing can effectively detect the two evolutionary branches of monkeypox in actual applications.

[0006] The antibody raw materials reported in the current public technology have obvious application limitations, especially the uncertainty of simultaneously detecting type I and type II monkeypox viruses. There is a risk of missed detection affecting the accuracy of the results, which will have an adverse impact on disease diagnosis and epidemic prevention and control. Summary of the Invention

[0007] In response to the shortcomings of the above-mentioned prior art, the present invention uses recombinant monkeypox A29 protein as an immunogen and screens A29 proteins from monkeypox clade I and clade II to obtain monoclonal antibodies with high affinity and high detection sensitivity. Furthermore, the monoclonal antibody can simultaneously detect type I and type II monkeypox viruses, achieving broad-spectrum detection of monkeypox viruses, avoiding missed detections, and improving application feasibility. Furthermore, considering that monkeypox viruses are constantly evolving, the A29 protein may have a higher degree of epitope exposure, a lower degree of aggregation, and higher protein stability, so a series of mutated A29 proteins were designed. Verification found that the monoclonal antibody of the present invention can recognize multiple mutated A29 proteins, which may achieve effective detection of future evolutionary strains of monkeypox viruses, further enhancing clinical value and having greater application prospects.

[0008] The present invention provides a monoclonal antibody or an antigen-binding fragment thereof against monkeypox virus, which is an antibody against the monkeypox virus A29 antigen, comprising:

[0009] (1) a heavy chain variable region having HCDR1, HCDR2, and HCDR3 with amino acid sequences as shown in SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3, respectively, or sequences with at least 70% sequence identity to the sequences shown in SEQ ID NO: 1, 2, or 3, or sequences with one or more amino acid mutations compared to the amino acid sequences shown in SEQ ID NO: 1, 2, or 3;

[0010] and a light chain variable region comprising LCDR1, LCDR2, and LCDR3 having amino acid sequences as shown in SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6, respectively, or sequences having at least 70% sequence identity to the sequences shown in SEQ ID NO: 4, 5, and 6, or sequences having one or more amino acid mutations compared to the amino acid sequences shown in SEQ ID NO: 4, 5, and 6;

[0011] or (2) a heavy chain variable region having HCDR1, HCDR2, and HCDR3 with amino acid sequences as set forth in SEQ ID NO: 7, SEQ ID NO: 8, and SEQ ID NO: 9, respectively, or sequences having at least 70% sequence identity to the sequences set forth in SEQ ID NO: 7, 8, and 9, or sequences having one or more amino acid mutations compared to the amino acid sequences set forth in SEQ ID NO: 7, 8, and 9;

[0012] and a light chain variable region having LCDR1, LCDR2 and LCDR3 with amino acid sequences as shown in SEQ ID NO: 10, SEQ ID NO: 11 and SEQ ID NO: 12, respectively, or sequences having at least 70% sequence identity with the sequences shown in SEQ ID NO: 10, 11, 12, or sequences having one or more amino acid mutations compared to the amino acid sequences shown in SEQ ID NO: 10, 11, 12.

[0013] The above-mentioned sequence with at least 70% sequence identity refers to a sequence that has at least 70% homology with the sequence shown and can cooperate with other sequences to recognize the monkeypox virus A29 antigen epitope and produce a reaction, including but not limited to sequences with at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity.

[0014] The above-mentioned sequence having one or more amino acid mutations refers to a sequence having one or several amino acid substitutions, deletions or additions, and being able to cooperate with other sequences to recognize the monkeypox virus A29 antigen epitope and produce a response, including but not limited to sequences having 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions.

[0015] The term "antigen-binding fragment" refers to an antigen-binding fragment of an antibody and an antibody analog, which generally includes at least a portion of the antigen-binding region or variable region of the parent antibody, such as one or more CDRs. Antibody fragments retain at least some of the binding specificity of the parent antibody.

[0016] Preferably, it comprises:

[0017] (1) A heavy chain variable region having an amino acid sequence as shown in SEQ ID NO: 13, or a sequence having at least 70% sequence identity to the sequence shown in SEQ ID NO: 13, or a sequence having one or more amino acid mutations compared to the amino acid sequence shown in SEQ ID NO: 13

[0018] and a light chain variable region having an amino acid sequence as set forth in SEQ ID NO: 14, or a sequence having at least 70% sequence identity to the sequence set forth in SEQ ID NO: 14, or a sequence having one or more amino acid mutations compared to the amino acid sequence set forth in SEQ ID NO: 14;

[0019] or (2) a heavy chain variable region having an amino acid sequence as set forth in SEQ ID NO: 15, or a sequence having at least 70% sequence identity to the sequence set forth in SEQ ID NO: 15, or a sequence having one or more amino acid mutations compared to the amino acid sequence set forth in SEQ ID NO: 15

[0020] and a light chain variable region with an amino acid sequence as shown in SEQ ID NO: 16, or a sequence having at least 70% sequence identity with the sequence shown in SEQ ID NO: 16, or a sequence having one or more amino acid mutations compared to the amino acid sequence shown in SEQ ID NO: 16.

[0021] The above-mentioned sequence with at least 70% sequence identity refers to a sequence that has at least 70% homology with the sequence shown and can cooperate with other sequences to recognize the monkeypox virus A29 antigen epitope and produce a reaction, including but not limited to sequences with at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity.

[0022] The above-mentioned sequence having one or more amino acid mutations refers to a sequence having one or several amino acid substitutions, deletions or additions, and being able to cooperate with other sequences to recognize the monkeypox virus A29 antigen epitope and produce a response, including but not limited to sequences having 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions.

[0023] Preferably, it comprises:

[0024] (1) A heavy chain having an amino acid sequence as set forth in SEQ ID NO: 17, or a sequence having at least 70% sequence identity to the sequence set forth in SEQ ID NO: 17, or a sequence having one or more amino acid mutations compared to the amino acid sequence set forth in SEQ ID NO: 17

[0025] and a light chain having an amino acid sequence as set forth in SEQ ID NO: 18, or a sequence having at least 70% sequence identity to the sequence set forth in SEQ ID NO: 18, or a sequence having one or more amino acid mutations compared to the amino acid sequence set forth in SEQ ID NO: 18;

[0026] or (2) a heavy chain having an amino acid sequence as set forth in SEQ ID NO: 19, or a sequence having at least 70% sequence identity to the sequence set forth in SEQ ID NO: 19, or a sequence having one or more amino acid mutations compared to the amino acid sequence set forth in SEQ ID NO: 19

[0027] and a light chain with an amino acid sequence as shown in SEQ ID NO: 20, or a sequence having at least 70% sequence identity with the sequence shown in SEQ ID NO: 20, or a sequence having one or more amino acid mutations compared to the amino acid sequence shown in SEQ ID NO: 20.

[0028] The above-mentioned sequence with at least 70% sequence identity refers to a sequence that has at least 70% homology with the sequence shown and can cooperate with other sequences to recognize the monkeypox virus A29 antigen epitope and produce a reaction, including but not limited to sequences with at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity.

[0029] The above-mentioned sequence having one or more amino acid mutations refers to a sequence having one or several amino acid substitutions, deletions or additions, and being able to cooperate with other sequences to recognize the monkeypox virus A29 antigen epitope and produce a response, including but not limited to sequences having 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions.

[0030] Preferably, the antigen-binding fragment is selected from Fab, Fab', Fv, scFv, F(ab')2, diabody, and antibody combination.

[0031] Among them, "Fab" is composed of a light chain, a heavy chain variable region and CH1.

[0032] "Fab'" contains a light chain and a portion of a heavy chain including the variable region and the region between the CH1 or CH1 and CH2 domains, with an interchain disulfide bond forming between the two heavy chains of two Fab' fragments to form an F(ab')2 molecule.

[0033] A "F(ab')2 fragment" contains two light chains and two heavy chains comprising a portion of the constant region between the CH1 and CH2 domains, such that an interchain disulfide bond is formed between the two heavy chains. Thus, a F(ab')2 fragment consists of two Fab' fragments held together by a disulfide bond between the two heavy chains.

[0034] The "Fv region" comprises the variable regions from both the heavy and light chains, but lacks the constant regions.

[0035] "Single-chain Fv antibody (scFv antibody)" refers to an antigen-binding fragment comprising the variable regions of an antibody, wherein these domains are contained in a single polypeptide chain. Generally speaking, scFv comprises a polypeptide linker between the heavy chain variable region and the light chain variable region, which enables the scFv to form the desired structure for antigen binding.

[0036] "Diabodies" are antigen-binding fragments with two antigen-binding sites. The fragments comprise a VH linked to a VL in the same polypeptide chain (VH-VL or VL-VH). By using a linker that is too short to allow pairing between the two domains on the same chain, the domains pair with the complementary domains of another chain and form two antigen-binding sites.

[0037] An "antibody composition" comprises a mixture of multiple antibodies.

[0038] Preferably, the above-mentioned monkeypox virus antibody or antigen-binding fragment thereof is used in the preparation of a medicament for treating and preventing monkeypox virus infection.

[0039] The present invention provides a polynucleotide encoding the above-mentioned monkeypox virus antibody or an antigen-binding fragment thereof.

[0040] The present invention provides an expression vector comprising the polynucleotide.

[0041] The present invention provides a host cell comprising the above expression vector.

[0042] The present invention provides a pharmaceutical composition comprising the monkeypox virus antibody or an antigen-binding fragment thereof and a pharmaceutically acceptable carrier.

[0043] The present invention provides a kit containing the monkeypox virus antibody or antigen-binding fragment thereof, or a conjugate thereof.

[0044] The conjugate includes, but is not limited to, a linked detectable label;

[0045] Preferably, the detectable label is selected from an enzyme (e.g., horseradish peroxidase, alkaline phosphatase), a chemiluminescent reagent (e.g., acridinium ester compounds, luminol and its derivatives, ruthenium derivatives), a fluorescent dye (e.g., fluorescein, fluorescent protein), a radionuclide or biotin.

[0046] The advantages and beneficial effects of the present invention are:

[0047] The envelope protein A29, a key protein of monkeypox virus, has amino acid differences and different protein conformations between type I and type II evolutionary branches. Therefore, it is challenging to screen for highly sensitive monoclonal antibodies that recognize different epitopes in the two strains and can simultaneously detect A29 proteins from both branches.

[0048] Compared to commercially available antibodies, the monkeypox virus antibodies or antigen-binding fragments thereof of the present invention possess higher affinity and higher paired detection sensitivity. Furthermore, they can effectively detect both type I and type II monkeypox viruses, achieving broad-spectrum detection of monkeypox viruses and avoiding missed detections, thereby improving detection effectiveness. Furthermore, the antibodies of the present invention can recognize multiple mutated A29 proteins, potentially enabling effective detection of future evolved strains of monkeypox viruses. These antibodies have broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0049] The present invention will be further described below with reference to the accompanying drawings and embodiments.

[0050] Figure 1 The figure is an SDS-PAGE identification diagram of A29-wild type (Congo type I) and West African type II A29 of the present invention.

[0051] Lane 1: Marker; Lane 2: A29-wild type (Congo type I); Lane 2: West African branch type II A29.

[0052] Figure 2 This is the SDS-PAGE identification diagram of the A29 protein monoclonal antibody of the present invention.

[0053] Lane 1: Marker; Lane 2: 6A7; Lane 3: 8A1; Lane 4: 7D6; Lane 5: 6A4-3; Lane 6: 2A9; Lane 7: 2B1; Lane 8: 7D8; Lane 9: 4B5; Lane 10: 7F3; Lane 11: 7C6; Lane 12: 7A1-2; Lane 13: 6C4-2.

[0054] Figure 3 This is a diagram showing the effect of different A29 antigen 2A9 / 7F3 pairing tests.

[0055] The specific information of each sequence of the present invention is as follows:

[0056] SEQ ID NO: 1: GFNIKDTY

[0057] SEQ ID NO: 2: IDPASANT

[0058] SEQ ID NO: 3: AMSDFDGFLAWFAY

[0059] SEQ ID NO:4: QSIGTT

[0060] SEQ ID NO: 5: YAS

[0061] SEQ ID NO:6:QQTYFWPYT

[0062] SEQ ID NO:7:GYTFADYE

[0063] SEQ ID NO:8:IHPGSGGT

[0064] SEQ ID NO:9:TRRGNY

[0065] SEQ ID NO:10:QSLLHSDGKTF

[0066] SEQ ID NO:11:LVS

[0067] SEQ ID NO:12:WQGTHLPYT

[0068] SEQ ID NO:13:GFNIKDTYIDPASANTAMSDFDGFLAWFAY

[0069] SEQ ID NO:14:QSIGTTYASQQTYFWPYT

[0070] SEQ ID NO:15:GYTFADYEIHPGSGGTTRRGNY

[0071] SEQ ID NO:16:QSLLHSDGKTFLVSWQGTHLPYT

[0072] SEQ ID NO:17:

[0073] RFSCSSLGQTLSQGPQSSCPAQLLASTLKTPIFTGSRGLNRAWSGLEELILRVL

[0074] ILNMTRSSRARPLQQTRPPTQPTYNSAAHLRTLPSITVLCQTLMVSSPGLLTG

[0075] AKGLCT

[0076] SEQ ID NO:18:

[0077] DILLTQSPAILSVSPGERVSFSCRASQSIGTTIHWYQQRTNGSPRLLIKYASESIS

[0078] GIPSRFSGSGSGTDFTLSINSVESEDIANYYCQQTYFWPYTFGGGTRLK

[0079] SEQ ID NO:19:

[0080] QVQLQQSGAELVRPGASVKLSCKALGYTFADYEMHWVKQTPVHGLEWIGA

[0081] IHPGSGGTAYNQKFKGKATLTADKSSSTAYMELSSLTSEDSAVYYCTRRGNY

[0082] WGQGTTLTV

[0083] SEQ ID NO: 20:

[0084] DVVMTQTPLSLSVTIGQPASISCKSSQSLLHSDGKTFLNWLLQRPGQSPERLI

[0085] YLVSKMDSGVPDRFTGSGSGTDFTLKISRVEAEDLGVYYCWQGTHLPYTFG

[0086] GGTKLEIK DETAILED DESCRIPTION

[0087] In order to better understand the present invention, the present invention is further described in detail below with reference to the embodiments and drawings. However, those skilled in the art will understand that the following embodiments are not limitations on the scope of protection of the present invention, and any changes and modifications made on the basis of the present invention are within the scope of protection of the present invention.

[0088] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0089] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0090] Example 1 Preparation of recombinant A29-related proteins

[0091] The amino acid sequence of the monkeypox virus gene ysA29 (Gen Bank: KJ642618.1) is shown in SEQ ID NO:21 (as shown in Table 1), and the full-length nucleotide sequence is shown in SEQ ID NO:22. The ysA29 gene was synthesized by General Biotech and integrated into the pET9a vector. It was expressed and purified using an E. coli expression system to produce the pET-9a-A29 antigen (hereinafter referred to as A29-wild type (Congo type I)), representing the A29 protein from the Congo Basin clade type I strain. A29 mutants were cloned and prepared in-house using A29, with PCR primers provided by General Biotech. The amino acid sequence of the A29 mutant is shown in SEQ ID NO:27, and the full-length nucleotide sequence is shown in SEQ ID NO:28. The target fragment DNA of A29-wild type (Congo type I) was recovered by overlapping PCR amplification, and the target fragment was ligated to the pET-9a expression vector, and further expressed and prepared using the Escherichia coli expression system as the pET-9a-ysA29-H74R-R107H mutant antigen (hereinafter referred to as West African type II A29), that is, the A29 protein of the West African branch type II strain. West African II type A29 was used as a vector to construct mutants A29-N27K (hereinafter referred to as mutant 1), A29-T30A (hereinafter referred to as mutant 2), A29-V36I (hereinafter referred to as mutant 3), A29-Y39D (hereinafter referred to as mutant 4), A29-G40E (hereinafter referred to as mutant 5), A29-D41Y (hereinafter referred to as mutant 6), A29-I61V (hereinafter referred to as mutant 7), A29-T63I (hereinafter referred to as mutant 8), A29-C71A-C72A (hereinafter referred to as mutant 9),

[0092] A29-N27K-T30A-V36I-Y39D-G40E-I61V-C71A-C72A (hereinafter referred to as mutant 10) and all point mutation collections A29-N27K-T30A-V36I-Y39D-G40E-D41Y-I61V (hereinafter referred to as mutant 11) were cloned, and the amino acid sequences were shown as SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, and SEQ ID NO:39, respectively. The full-length nucleotide sequences were shown as SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, and SEQ ID NO:48. NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50. The mutant antigen was expressed and purified using an E. coli expression system. The specific construction process is as follows:

[0093] Using molecular cloning technology, the target gene fragment of the synthesized wild-type ysA29 was used as a template, and primers were designed to amplify the H74R point mutation fragment, i.e., aa1-74 (the 74th amino acid mutant sequence of ysA29 is shown in SEQ ID NO: 23, and the nucleotide mutant sequence is shown in SEQ ID NO: 24). Primers were designed to amplify the R107H point mutation fragment, i.e., aa75-110 (the 107th amino acid mutant sequence of ysA29 is shown in SEQ ID NO: 25, and the nucleotide mutant sequence is shown in SEQ ID NO: 26). The target fragments were recovered and the sequences of SEQ ID NO: 24 and SEQ ID NO: 26 were amplified and spliced ​​by overlapping PCR, i.e., aa1-110 (the 74th and 107th amino acid mutant sequences of ysA29 are shown in SEQ ID NO: 27, and the nucleotide mutant sequence is shown in SEQ ID NO: 28). NO:28), the target fragment was recovered and connected to the pET-9a expression vector by seamless cloning to prepare the pET-9a-ysA29-H74R-R107H mutant protein. The results are shown in FIG. Figure 1 shown.

[0094] The target fragment DNA of mutant strain 1, mutant strain 2, mutant strain 3, mutant strain 4, mutant strain 5, mutant strain 6, mutant strain 7, mutant strain 8, mutant strain 9, mutant strain 10, and mutant strain 11 was recovered by overlapping PCR amplification, and the target fragment was connected to the pET30a expression vector for further expression and preparation of mutant antigens. Using the correctly sequenced pET-9a-ysA29-1-110 mutant clone plasmid as a template, primers were designed to amplify mutant 1, mutant 2, mutant 3, mutant 4, mutant 5, mutant 6, mutant 7, mutant 8, and mutant 9 point mutation fragments, and the target fragments were recovered. The complete sequences were amplified and spliced ​​by overlapping PCR, as shown in SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, and SEQ ID NO:48; each mutation site was sequentially superimposed and mutated to recover fragments, and finally overlapped PCR was used to amplify and splice to obtain a comprehensive mutant clone mutant 11, the complete sequence of which is shown in SEQ ID NO:50; using the target gene fragment of A29-wild type (Congo type I) as a template, primers were designed to amplify mutant 10 point mutation fragments, and the target fragments were recovered. The complete sequence was amplified and spliced ​​by overlapping PCR, as shown in SEQ ID NO:49. The corresponding sequences are shown in Table 1.

[0095] Table 1: A29-related nucleic acid and protein sequences

[0096]

[0097]

[0098]

[0099]

[0100]

[0101]

[0102]

[0103] Example 2 Preparation of anti-A29 protein mouse monoclonal antibody

[0104] 1. Preparation of Immunogen: For the immunogen, A29-wild type (Congo type I) prepared in Example 1 was diluted with 10 mmol / L PBS. The corresponding antigen was mixed with equal volumes of Freund's adjuvant to a final concentration of 0.33 mg / mL to form a water-in-oil emulsion. Freund's complete adjuvant was used for the primary immunization, and Freund's incomplete adjuvant was used for the booster immunization.

[0105] 2. Basic immunization: Select 6-8 week old BALB / c female mice for subcutaneous multiple injection immunization. The immunogen injection dose is 300 μL / mouse / time. The immunization interval is 2 weeks. The complete immunization program consists of 4 injections. Two weeks after the fourth immunization, orbital blood collection and separation of serum are used for indirect ELISA to determine the immune titer. After determining that the mouse serum titer has reached the plateau phase, subcutaneous immunization is performed with 100 μg / mouse A29-wild type (Congo type I) 72 hours before cell fusion.

[0106] 3. Immune monitoring: A29-wild type (Congo type I), West African type II A29 and carrier antigens were used for detection, and the detection method was indirect ELISA. The specific method was to coat West African type II A29, 200ng / mL, 100ul per well, add 100uL of the corresponding mouse serum, incubate at 37°C for 30min, wash the plate 5 times with a plate washer, add 100ul 1:5K prepared GAM-HRP, incubate at 37°C for 30min, wash the plate 5 times with a plate washer, add 100ul / well colorimetric solution, incubate at 37°C for 15min, terminate the reaction with a stop solution, place in an enzyme reader and use dual wavelengths to read the signal value. Based on the experimental results, determine whether fusion can be carried out. The immune titer test is shown in Table 2: The results show that the serum of A29-wild type (Congo type I) immunized mice diluted 1*10 6 The mice reacted with both wild-type A29 (Congolese type I) and West African type II A29, and showed no cross-reactivity with the carrier antigen. The mice had high immune titers and were suitable for fusion development.

[0107] Table 2: Immunization effect in mice

[0108]

[0109]

[0110] 4. Hybridoma cell screening:

[0111] 4.1 Preparation of feeder macrophage cells:

[0112] (i) BALB / c mice, approximately 6 weeks old, were sacrificed by cervical dislocation and soaked in 75% alcohol solution for 5 min. The mice were removed and placed in a sterile dish pre-placed on a clean bench. Hemostats were used to adjust the mouse's posture so that its abdomen was facing upward, allowing for a more relaxed posture. The mice were then placed sideways. Hemostats were used to clamp the skin near the lower abdomen of the mouse. The skin was then suddenly pulled apart in the opposite direction to fully expose the abdomen.

[0113] (ii) Use sterile ophthalmic curved forceps to lift the peritoneum. Then, use a 5 mL syringe to inject an appropriate amount of culture medium into the abdominal cavity, ensuring that the abdomen is fully inflated. Place the syringe down, hold the hemostatic forceps with both hands, and lift the contralateral limbs of the mouse, gently shaking them to allow sufficient infiltration of macrophages in the peritoneal cavity. Afterwards, use the same syringe to aspirate the macrophages and inject them into the pre-prepared 1640HT medium containing 20% ​​fetal bovine serum for later use.

[0114] 4.2 Preparation of thymocyte feeder cells:

[0115] (i) Three-week-old BALB / c mice were sacrificed by cervical dislocation and soaked in 75% alcohol solution for 5 min. The mice were removed and placed in a sterile dish pre-placed on a clean bench. Hemostats were used to adjust the mouse's posture so that its abdomen was facing upward, allowing for a more relaxed posture. The mice were then placed sideways. Hemostats were used to clamp the skin just below the chest. The skin was then suddenly pulled apart in the opposite direction to fully expose the chest and abdomen.

[0116] (ii) Use sterile curved forceps in your left hand to grasp the sternal tip through the chest and abdominal skin. Cut along the outer edge of the sternum to fully expose the thoracic diaphragm. Keep grasping the sternal tip to expose the chest cavity as much as possible.

[0117] (iii) Using clean ophthalmic scissors in the right hand, cut open the thoracic diaphragm to fully expose the thoracic cavity. Using curved forceps, reach into the thoracic cavity, grasp the thymus at the base, and remove the thymus intact. Grind the thymus through a 200-mesh sieve that has been pre-placed on a plate and soaked in culture medium to obtain thymic feeder cell fluid. Transfer the entire thymus to the aforementioned 1640HT medium supplemented with 20% fetal bovine serum for later use.

[0118] 4.3 Preparation of mouse myeloma cells: Resuscitate mouse myeloma cells 5 days before fusion. Each fusion requires approximately one bottle of 225 cm 2 90%-100% density of mouse myeloma cells.

[0119] 4.4 Preparation of splenocytes:

[0120] (i) Orbital blood was collected from BALB / C mice to be fused. After bleeding stopped, the mice were killed by cervical dislocation. The mice were immersed in 75% alcohol solution for 5 minutes and then placed on a sterile plate in a clean bench in the right lateral decubitus position.

[0121] (ii) Expose the abdominal cavity as described in 4.1, open the abdominal cavity aseptically, remove the spleen, and grind it through a 200-mesh sieve pre-placed on a plate and soaked in culture medium. Prepare a spleen cell suspension and transfer it to a 50 mL sterile centrifuge tube.

[0122] (iii) Add an appropriate amount of RPMI-1640 culture medium (30-35 mL). After removing obvious fat aggregates and other impurities using a curved pipette, collect spleen cells by centrifugation at 1500 rpm for 5 min each time. Remove the supernatant by centrifugation and add 30-35 mL of new culture medium. Repeat this process twice to wash the spleen cells.

[0123] (iv) Resuspend the cells in RPMI-1640 medium and count them.

[0124] 4.5 Cell fusion:

[0125] (i) Before fusion, pre-warm 1 mL of PEG-1450, 35 mL of RPMI-1640 serum-free medium, and 200 mL of HAT complete medium containing 20% ​​fetal bovine serum to 37°C.

[0126] (ii) Prepared myeloma cells and spleen cells in a ratio of 1*10 8 Splenocytes and 1*10 7 Myeloma cells were mixed in a 50 mL centrifuge tube at a ratio of approximately 10:1 and centrifuged at 1500 rpm for 5 min. After centrifugation, the supernatant was discarded as much as possible and the bottom of the tube was gently tapped to loosen the cells into a paste.

[0127] (iii) Pipette 1 mL of PEG into the centrifuge tube using a 1 mL pipette. Gently pipette and mix thoroughly for approximately 60 s. Immediately, add 35 mL of pre-warmed RPMI-1640 complete culture medium to terminate the fusion reaction.

[0128] (iv) After standing for 1-5 minutes, centrifuge at 1000 rpm for 5 minutes. Carefully discard the supernatant. Gently flick the cells with your fingers. Add the above complete medium to fully resuspend the cells. Transfer all cells to complete medium supplemented with feeder cells. After mixing, plate 200 μl / well into a 96-well cell culture plate and culture in a CO2 incubator.

[0129] (v) After 7 days, 100% of the cell supernatant in the wells was replaced with 15% HT complete medium; after 7 days, the supernatant was aspirated for detection.

[0130] 4.6 Screening of hybridomas: A29-wild type (Congo type I) was used for initial screening. The screening method was indirect ELISA. The specific method was to coat A29-wild type (Congo type I) at 200 ng / mL, coat 100 ul per well, add 30 ul of cell supernatant, incubate at 37°C for 30 min, wash the plate 5 times with a plate washer, add 100 ul of 1:5K prepared GAM-HRP, incubate at 37°C for 30 min, wash the plate 5 times with a plate washer, add 100 ul / well colorimetric solution, incubate at 37°C for 15 min, terminate the reaction with stop solution, place the plate in a microplate reader and read the signal value using dual wavelength. According to the experimental results, select the corresponding positive well cells for the next step.

[0131] 4.7 Hybridoma Cell Cloning: Using the limiting dilution method, cells were first diluted at a specific concentration and then seeded into each well of a 96-well cell culture plate, ensuring that only one cell grew within each well. Hybridoma monoclonal positive cell lines were cloned at least three times, with 100% positive results in the final round confirming them as stable clones. The results of the final screening of stable clones are shown in Table 3.

[0132] Table 3: Reactivity of cell supernatants

[0133] serial number name A29-wild type (Congo type I) 1 2A9 3.4240 2 2B1 3.2210 3 4B5 3.4390 4 6A4-3 3.4780 5 6A7 3.3690 6 6C4-2 3.5710 7 7A1-2 3.0590 8 7C6 3.6670 9 7D6 3.1190 10 7D8 3.2880 11 7F3 3.5080 12 8A1 3.4270

[0134] 5. Monoclonal Antibody Preparation:

[0135] 5.1 Monoclonal Antibody Ascites Preparation: BALB / c mice were sensitized. One week later, the stable hybridoma cells in the logarithmic growth phase were resuspended and centrifuged at 1500 rpm for 5 min to collect the cells. The precipitated cells were suspended in serum-free culture medium and the cell count was adjusted to 1×10 6 / mL, and 1 mL was injected intraperitoneally into each mouse to induce ascites. After 7-10 days, when the mouse abdomen was noticeably distended, the ascites was collected. The collected ascites was placed in a centrifuge tube and centrifuged at 12,000 rpm for 10 minutes. The supernatant was collected and set aside.

[0136] 5.2 Monoclonal Antibody Preparation: The collected ascites was precipitated with ammonium sulfate and purified by Protein A affinity chromatography (purchased from GE, USA). The purified monoclonal antibodies were identified by 12% SDS-PAGE and the purity was above 90%, indicating that the prepared monoclonal antibodies were of good purity. Figure 2 shown.

[0137] Example 3: Enzyme-linked immunosorbent assay (EIA) screening of mouse monoclonal antibodies against A29 protein

[0138] 1. A29 paired antibody detection screening: 20mmol / L PB7.4 was used as the coating buffer solution, and the first antibody was coated on the irradiated plate at 5ug / mL, 100ul / well, and incubated at 37°C for 2h; the second antibody was labeled with horseradish peroxidase HRP at a ratio of 1:1, with a final labeled concentration of 1mg / mL and a labeled working concentration of 1:500. The 12 antibodies obtained in Example 2 were paired in sequence, and a 12*12 antibody orthogonal pairing evaluation was performed. The evaluation samples were 250pg / mL A29-wild type (Congo type I), 250pg / mL West African type II A29, and 10mmol / L PBS diluent. The evaluation results are shown in Table 4. The paired screening screening criteria were: A29-wild type (Congo type I) or West African type II A29 must be detected, and PBS <0.1. The higher the detection sensitivity of A29-wild type (Congo type I) and West African type II A29, the better.

[0139] Table 4: A29 protein mouse monoclonal antibody paired detection

[0140]

[0141]

[0142]

[0143]

[0144]

[0145] Among all the pairs, the pair with the best reaction sensitivity with A29-wild type (Congo type I) and West African type II A29 was selected: 2A9 as coating and 7F3 as marker.

[0146] 2. 2A9 / 7F3 Paired Sensitivity Testing: 2A9 antibody was coated onto irradiated plates using 20 mmol / L PB7.4 as coating buffer at 5 μg / mL, 100 μL / well, and incubated at 37°C for 2 h. 7F3 antibody was labeled with horseradish peroxidase (HRP) at a 1:1 ratio to a final concentration of 1 mg / mL, using a 1:500 concentration. Evaluation samples included wild-type A29 (Congolese type I) and West African type II A29 diluted to 250 pg / mL and then folded into eight dilutions. Using PBS as a reference, an OD value greater than twice that of PBS was considered valid. The 2A9 / 7F3 pairing achieved a sensitivity of 3.91 pg / mL on the ELISA platform. The specific test results are shown in Table 5. At the same time, the detection sensitivity of the paired pair on the colloidal gold platform was evaluated. The evaluation sample was A29-wild type (Congo type I) diluted to 50 pg / mL and then diluted three times. Its detection sensitivity on the colloidal gold platform can reach 12.5 pg / ml. The specific test results are shown in Table 6.

[0147] Table 5: 2A9 / 7F3 Paired Sensitivity Detection (ELISA)

[0148]

[0149] Table 6: 2A9 / 7F3 Paired Sensitivity Detection (ICA)

[0150] Coating 2A9 mark 7F3 50 0.43 25 0.26 12.5 0.20 blank 0.00

[0151] 3. Detection of Multiple Mutated A29 Antigens Using the 2A9 / 7F3 Pairing: 2A9 was used as the coating and 7F3 as the label. A double-antibody sandwich assay was used to detect multiple mutant A29 antigens, namely, mutants 1 through 11. The specific detection method was the same as the sensitivity test for the 2A9 / 7F3 pairing. The evaluation samples were A29-wild type (Congo type I), West African type II A29, and mutants 1 through 11, diluted to 100 pg / mL, 50 pg / mL, and 25 pg / mL, respectively. The specific test results are shown in Table 7. The 2A9 / 7F3 pairing showed good reactivity with multiple mutant A29 antigens, indicating that the 2A9 / 7F3 pairing is more likely to effectively detect future evolutionary strains of monkeypox virus.

[0152] Table 7: Detection of various mutant A29 antigens by 2A9 / 7F3 pairing

[0153]

[0154] In summary, the present invention uses recombinant monkeypox A29 protein as an immunogen, and prepares and obtains two monoclonal antibodies targeting the monkeypox virus A29 antigen through mouse hybridoma technology. The monoclonal antibodies have high affinity and high detection sensitivity; further, the monoclonal antibodies can simultaneously detect type I and type II monkeypox viruses, achieving broad-spectrum detection of monkeypox viruses, avoiding missed detections, and improving application feasibility; further, considering that the monkeypox virus is constantly evolving, the A29 protein may have a higher degree of epitope exposure, a lower degree of aggregation, and higher protein stability. Therefore, a series of mutated A29 proteins were designed, and verification found that the monoclonal antibodies of the present invention can recognize multiple mutated A29 proteins, which may achieve effective detection of future evolved strains of monkeypox virus, further enhance clinical value, and have greater application prospects.

[0155] Although the specific embodiments of the present invention are described above, those skilled in the art should understand that the specific embodiments described are merely illustrative and are not intended to limit the scope of the present invention. Equivalent modifications and changes made by those skilled in the art in accordance with the spirit of the present invention should be included within the scope of protection of the claims of the present invention.

Claims

1. A monoclonal antibody or antigen-binding fragment thereof against monkeypox virus, characterized in that: It is an antibody against the monkeypox virus A29 antigen, containing: (1) a heavy chain variable region having HCDR1, HCDR2, and HCDR3 with amino acid sequences as shown in SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3, respectively, or a sequence having at least 70% sequence identity with the sequences shown in SEQ ID NO: 1, 2, or 3, or a sequence having one or more amino acid mutations compared to the amino acid sequences shown in SEQ ID NO: 1, 2, or 3; and a light chain variable region comprising LCDR1, LCDR2, and LCDR3 having amino acid sequences as shown in SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6, respectively, or sequences having at least 70% sequence identity to the sequences shown in SEQ ID NO: 4, 5, and 6, or sequences having one or more amino acid mutations compared to the amino acid sequences shown in SEQ ID NO: 4, 5, and 6; or (2) a heavy chain variable region having HCDR1, HCDR2, and HCDR3 with amino acid sequences as set forth in SEQ ID NO: 7, SEQ ID NO: 8, and SEQ ID NO: 9, respectively, or sequences having at least 70% sequence identity to the sequences set forth in SEQ ID NO: 7, 8, and 9, or sequences having one or more amino acid mutations compared to the amino acid sequences set forth in SEQ ID NO: 7, 8, and 9; and a light chain variable region having LCDR1, LCDR2 and LCDR3 with amino acid sequences as shown in SEQ ID NO: 10, SEQ ID NO: 11 and SEQ ID NO: 12, respectively, or sequences having at least 70% sequence identity with the sequences shown in SEQ ID NO: 10, 11, 12, or sequences having one or more amino acid mutations compared to the amino acid sequences shown in SEQ ID NO: 10, 11, 12.

2. The monoclonal antibody or antigen-binding fragment thereof against monkeypox virus according to claim 1, wherein It includes: (1) A heavy chain variable region having an amino acid sequence as shown in SEQ ID NO: 13, or a sequence having at least 70% sequence identity to the sequence shown in SEQ ID NO: 13, or a sequence having one or more amino acid mutations compared to the amino acid sequence shown in SEQ ID NO: 13 and a light chain variable region having an amino acid sequence as set forth in SEQ ID NO: 14, or a sequence having at least 70% sequence identity to the sequence set forth in SEQ ID NO: 14, or a sequence having one or more amino acid mutations compared to the amino acid sequence set forth in SEQ ID NO: 14; or (2) a heavy chain variable region having an amino acid sequence as set forth in SEQ ID NO: 15, or a sequence having at least 70% sequence identity to the sequence set forth in SEQ ID NO: 15, or a sequence having one or more amino acid mutations compared to the amino acid sequence set forth in SEQ ID NO: 15 and a light chain variable region with an amino acid sequence as shown in SEQ ID NO: 16, or a sequence having at least 70% sequence identity with the sequence shown in SEQ ID NO: 16, or a sequence having one or more amino acid mutations compared to the amino acid sequence shown in SEQ ID NO:

16.

3. The monoclonal antibody or antigen-binding fragment thereof against monkeypox virus according to claim 1, wherein It includes: (1) A heavy chain having an amino acid sequence as set forth in SEQ ID NO: 17, or a sequence having at least 70% sequence identity to the sequence set forth in SEQ ID NO: 17, or a sequence having one or more amino acid mutations compared to the amino acid sequence set forth in SEQ ID NO: 17 and a light chain having an amino acid sequence as set forth in SEQ ID NO: 18, or a sequence having at least 70% sequence identity to the sequence set forth in SEQ ID NO: 18, or a sequence having one or more amino acid mutations compared to the amino acid sequence set forth in SEQ ID NO: 18; or (2) a heavy chain having an amino acid sequence as set forth in SEQ ID NO: 19, or a sequence having at least 70% sequence identity to the sequence set forth in SEQ ID NO: 19, or a sequence having one or more amino acid mutations compared to the amino acid sequence set forth in SEQ ID NO: 19 and a light chain with an amino acid sequence as shown in SEQ ID NO: 20, or a sequence having at least 70% sequence identity with the sequence shown in SEQ ID NO: 20, or a sequence having one or more amino acid mutations compared to the amino acid sequence shown in SEQ ID NO:

20.

4. The monoclonal antibody or antigen-binding fragment thereof against monkeypox virus according to any one of claims 1 to 3, wherein: The antigen-binding fragment is selected from Fab, Fab', Fv, scFv, F(ab')2, diabody, and antibody combination.

5. Use of the monkeypox virus antibody or antigen-binding fragment thereof according to any one of claims 1 to 4 in the preparation of a medicament for treating and / or preventing monkeypox virus infection.

6. A polynucleotide encoding the monkeypox virus antibody or antigen-binding fragment thereof according to any one of claims 1 to 4. An expression vector comprising the polynucleotide according to claim 5 . A host cell comprising the expression vector according to claim 6 .

9. A pharmaceutical composition comprising the monkeypox virus antibody or antigen-binding fragment thereof according to any one of claims 1 to 4 and a pharmaceutically acceptable carrier.

10. A kit comprising the monkeypox virus antibody or antigen-binding fragment thereof, or a conjugate thereof according to any one of claims 1 to 4.

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