Antibodies against monkeypox virus h3 protein and methods of making the same

Abstract

The application relates to an antibody against monkeypox virus and a preparation method thereof, and the humanized monkeypox virus antibody H3-14A7 is obtained by screening memory B cells of a human antibody against H3 specific binding mouse. The H3-14A7 has the characteristics of strong monkeypox virus binding capacity, good in-vitro neutralization, large affinity and in-vivo protection.

Description

Technical Field

[0001] This application belongs to the field of antibody engineering, specifically relating to antibodies against monkeypox virus H3 protein and their preparation methods. Background Technology

[0002] Monkeypox virus (MPXV) belongs to the family Poxviridae, subfamily Chordopoxvirinae, and genus Orthopoxvirus (OPXV). The genus Orthopoxvirus contains more than 10 members, including smallpox virus (VARV), cowpox vaccine virus (VACV), rinderpest virus (CPXV), camelpox virus (CMLV), and several new species isolated from infected humans or primates since 2010. Based on comparisons between VACSV and MPXV, the surface proteins of poxviruses show approximately 93–98% sequence similarity, suggesting they play similar roles in the process of entering the body.

[0003] Monkeypox virus was first discovered in a population of monkeys used in research in 1958. In 1970, a nine-month-old boy was diagnosed, marking the first human case and demonstrating that monkeypox could infect humans. At that time, monkeypox outbreaks were mainly localized in countries in the tropical rainforests of Africa, with numerous cases reported in Central and West Africa. The mortality rates for the West African and Central African branches were 3.6% and 10.6%, respectively. Although monkeypox was primarily distributed in Central and West Africa for the past few decades, its spread has changed in recent years, with cases even appearing outside of Africa.

[0004] Monkeypox is a zoonotic disease whose natural host is not yet clearly identified. It primarily spreads among rodents and non-human primates. Transmission can occur through various means, including contact with damaged skin tissue. Furthermore, recent transmission of monkeypox virus in non-endemic countries has shown a link to sexual contact; the virus has been detected in bodily fluids such as semen, although whether sexual transmission is possible remains unclear. Symptoms after infection include swollen lymph nodes, muscle aches, and permanent scarring after the pustules rupture.

[0005] Multiple strains of monkeypox virus circulate in non-endemic countries, with diverse and insidious transmission routes and atypical symptoms. Further research on this virus is needed to better prevent and treat monkeypox.

[0006] To date, neutralizing antibodies have proven to be an effective treatment for viral diseases, including human immunodeficiency virus (HIV), influenza virus, and other flaviviruses. MPXV exists in two forms: extracellular enveloped virus (EEV) and intracellular mature virus (IMV) particles.

[0007] Developing new monkeypox virus antibodies with strong affinity for antigens and good in vitro neutralization properties is very important and necessary. Summary of the Invention

[0008] Based on this, one embodiment of this application provides an antibody with strong affinity and good in vitro neutralization against monkeypox virus H3 protein, as well as its preparation method and application.

[0009] The technical solution includes:

[0010] One embodiment of this application provides an antibody against monkeypox virus H3 protein, comprising a heavy chain variable region and a light chain variable region; wherein the heavy chain variable region includes HCDR1 as shown in SEQ ID NO:5, HCDR2 as shown in SEQ ID NO:6, and HCDR3 as shown in SEQ ID NO:7; and wherein the light chain variable region includes LCDR1 as shown in SEQ ID NO:8, LCDR2 as shown in SEQ ID NO:9, and LCDR3 as shown in SEQ ID NO:10.

[0011] In one embodiment, the amino acid sequence of the heavy chain variable region is shown in SEQ ID No: 1, and the amino acid sequence of the light chain variable region is shown in SEQ ID No: 2.

[0012] In one embodiment, a heavy chain constant region and a light chain constant region are also included.

[0013] One embodiment of this application also provides a nucleic acid molecule including a nucleotide sequence encoding the antibody.

[0014] In one embodiment, the nucleotide sequence comprises a nucleotide sequence encoding the heavy chain variable region and a nucleotide sequence encoding the light chain variable region, the nucleotide sequence being selected from: the heavy chain variable region as shown in SEQ ID NO: 3, and the light chain variable region as shown in SEQ ID NO: 4.

[0015] An embodiment of this application also provides an expression vector comprising the nucleic acid molecule.

[0016] One embodiment of this application also provides a host cell comprising the nucleic acid molecule or the expression vector.

[0017] An embodiment of this application also provides a method for preparing the antibody, comprising the following steps:

[0018] The host cells are cultured, and the antibodies are collected from the resulting cell culture.

[0019] One embodiment of this application also provides a detection reagent or detection kit, including the antibody.

[0020] One embodiment of this application also provides a drug comprising the antibody.

[0021] One embodiment of this application also provides the use of the antibody in the detection of monkeypox virus H3 protein for non-diagnostic purposes.

[0022] One embodiment of this application also provides the use of the antibody in the preparation of a detection product for monkeypox virus.

[0023] One embodiment of this application also provides the use of the antibody in the preparation of medicaments for the prevention or treatment of monkeypox virus and / or vaccinia virus.

[0024] Compared with traditional technologies, this application has the following advantages:

[0025] The humanized monoclonal antibody of this application has strong binding ability to monkeypox virus, good in vitro neutralization, high affinity, and in vivo protective effect. Its sequence is completely different from that of previously reported monkeypox virus antibodies, providing a product for the detection and neutralization of monkeypox virus and making it possible to provide monkeypox virus products with in vivo protective effects. Attached Figure Description

[0026] To more clearly illustrate the technical solutions in the embodiments of this application and to more completely understand this application and its beneficial effects, the drawings used in the description of the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of this application. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0027] Figure 1 SDS-PAGE results of molecular sieve purification of monkeypox virus H3 protein;

[0028] Figure 2 The results show the kinetic curves of the H3 antibody and H3.

[0029] Figure 3 The results show the neutralization curve of H3 antibody against VACV-WR;

[0030] Figure 4 The figures show a schematic diagram of the experiment and curves showing the survival and weight changes of mice after viral infection. The top figure is a schematic diagram of the experiment, and the bottom figure is a curve showing the survival and weight changes of mice after viral infection. Detailed Implementation

[0031] To make the above-mentioned objectives, features, and advantages of this application more apparent and understandable, a detailed description of specific embodiments of this application is provided below. Many specific details are set forth in the following description to provide a thorough understanding of this application. However, this application can be implemented in many other ways different from those described herein, and those skilled in the art can make similar modifications without departing from the spirit of this application. Therefore, this application is not limited to the specific embodiments disclosed below.

[0032] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the application.

[0033] In this document, the term "and / or" includes any and all combinations of one or more of the related listed items.

[0034] To address the existing problems, this application obtained a humanized MPXV antibody H3-14A7 with high neutralizing activity by screening humanized mouse memory B cells that specifically bind to H3 using fully human antibodies. Finally, a novel humanized monoclonal antibody against monkeypox virus with specificity, good in vitro neutralization, and strong affinity was screened out.

[0035] The specific technical solutions include:

[0036] I. Preparation and purification of monkeypox virus H3 monoclonal antibody

[0037] By expressing and purifying the monkeypox virus H3 protein, sorting specific memory B cells that bind to the H3 protein, and expressing and purifying the monkeypox virus H3 antibody, a monkeypox virus H3 monoclonal antibody, namely H3-14A7, was obtained. The light chain variable region and heavy chain variable region of this monkeypox virus monoclonal antibody are different from existing sequences, and have specificity.

[0038] II. Performance Validation of Monoclonal Antibodies

[0039] 1. ELISA detection of H3 antigen and antibody binding activity: H3-14A7 antibody can bind to antigen and has a strong binding ability to antigen.

[0040] 2. Surface plasmon resonance technology for detecting antibody-antigen affinity: H3-14A7 showed good affinity for the antigen.

[0041] 3. Antibody neutralization assay: The neutralizing activity of the antibody was detected by the plaque assay. The neutralization curve of H3 antibody against VCV-WR showed that the PRNT50 value of H3-14A7 was 0.135 μg / mL.

[0042] 4. Animal protection experiment: Under viral infection, H3-14A7 antibody was administered, which can provide partial protection against monkeypox virus infection in mice.

[0043] The embodiments of this application will be described in detail below with reference to examples. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of this application. For experimental methods in the following embodiments where specific conditions are not specified, please refer to the guidelines given in this application, or follow experimental manuals or conventional conditions in the art, or follow the conditions recommended by the manufacturer, or refer to experimental methods known in the art.

[0044] In the specific embodiments described below, the measurement parameters involving raw material components may have slight deviations within the weighing accuracy range unless otherwise specified. For temperature and time parameters, acceptable deviations due to instrument testing accuracy or operational precision are permissible.

[0045] The main experimental materials involved in the following embodiments include, but are not limited to:

[0046] Key pharmaceuticals and reagents: fully humanized antibody mice, model RenMab, Biocytogen Pharmaceuticals, Inc.; 14K chip, model 750-00021, Berkeley Lights.

[0047] Main instrument: Beacon sorter, model Beacon, Berkeley Lights.

[0048] Example 1: Preparation and purification of monkeypox virus monoclonal antibody

[0049] 1. Expression and purification of monkeypox virus H3 protein

[0050] The H3 gene was constructed into the eukaryotic expression vector pCAGGS and transfected into 293F cells. The H3 protein was expressed by cell secretion. The cell supernatant was collected and purified by affinity chromatography and molecular sieve, and then identified by SDS-PAGE.

[0051] Specifically, the A2-F282 gene (SEQ ID NO:17) of H3L was extracted, and after gene sequence optimization, it was constructed into the vector pCAGGS.

[0052] AAVKTPVIVVPVIDRPPSETFPNVHEHINDQKFDDVKDNEVMQEKRDVVIVNDDPDHYKDYVFIQWTGGNIRDDDKYTHFFSGFCNTMCTEETKRNIARHLALWDSKFFTELENKNVEYVVIIENDNVIEDITFLRPVLK AIHDKKIDILQMREIITGNKVKTELVIDKDHAIFTYTGGYDVSLSAYIIRVTTALNIVDEIIKSGGLSSGFYFEIARIENEMKINRQIMDNSAKYVEHDPRLVAEHRFETMKPNFWSRIGTVAAKRYPGVMYTFTTPLISF

[0053] like Figure 1 As shown, the SDS-PAGE results of monkeypox virus H3 protein purification were obtained. When the protein was passed through a Superdex 200GL, a UV 280 nM absorption peak could be detected at a position of ~16 mL. The molecular weight of the protein in the SDS-PAGE was approximately ~32 kDa, and the obtained protein had high purity.

[0054] 2. Sorting of specific memory B cells that bind to H3 protein

[0055] The H3 gene was constructed into an mRNA vector, transcribed in vitro, and capped. Concentration and quality were then measured. Humanized mice (RenMab) received three immunizations at a dose of 2.5 μg / mouse. Serum was collected before immunization and 12 days after each immunization to detect antibody titers. Once the antibody titer reached an appropriate level, a booster immunization was administered. Lymph nodes from the humanized mice were then collected, ground into single cells, incubated with antigen molecules, and specifically sorted using a Beacon sorter. The variable region gene sequence of the antibody was obtained after reverse transcription and PCR amplification.

[0056] A monoclonal antibody against H3-14A7 monkeypox virus was obtained. The amino acid sequences of the antibody light and heavy chains, the variable regions CDR1-3 of the light chain, and the variable regions CDR1-3 of the heavy chain are as follows. The variable regions of the light chain and the variable regions of the heavy chain are different from existing sequences and have specificity.

[0057] H3-14A7 monoclonal antibody:

[0058] Heavy chain variable region amino acid sequence, SEQ ID NO:1:

[0059] QVQLVQSGGGLVQPGGSLRLSCAASGFIFSRYWMHWVRQAPGKGLVWVSRTNSDGSGTSYADSVKGRFTISRDNAKNTLYLQMNSLRAEDTAVYYCTRGGVLRSGMDVWGQGTTVTVSS

[0060] Amino acid sequence of the light chain variable region, SEQ ID NO: 2:

[0061] EIVMPQSPSPLSVFPGERATLSCRASQSVSSNFAWYQQKPGQAPRLLIYGASTRATGIPTRFSGSGCGTEFTLTISSLQSEDFAVYYCQQYNNWPLSFGGGTKVEIK

[0062] Nucleic acid sequence of the heavy chain variable region, SEQ ID NO: 3:

[0063] CAGGTGCAGCTGGTGCAGTCTGGGGGAGGCTTAGTTCAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCATCTTCAGTAGGTACTGGATGCACTGGGTCCGCCAAGCTCCAGGGAAGGGGCTGGTGTGGGTCTCACGTACTAATAGTGATGGGAGTGGCACAAGCTACGCGGACTCCGTGAAGGGCCGATTCACCATATCCAGAGACAACGCCAAGAACACGCTATATCTTCAAATGAACAGTCTGAGAGCCGAGGACACGGCTGTGTATTATTGTACAAGAGGGGGGGTATTACGGAGCGGTATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCA

[0064] Nucleic acid sequence of the light chain variable region, SEQ ID NO: 4:

[0065] GAAATTGTGATGCCGCAGTCTCCATCCCCCCTGTCTGTGTTTCCAGGGGAAAGAGCCACCCTCTCCTGCAGGGCCAGTCAGAGTGTTAGCAGCAACTTTGCCTGGTACCAGCAGAAACCTGGCCAGGCTCCCAGGCTCCTCATTTATGGTGCATCCACCAGGGCCACTGGTATCCCAACCAGGTTCAGTGGCAGTGGGT GTGGGACAGAATTCACTCTCACCATCAGCAGCCTGCAGTCTGAAGATTTTGCAGTTTTACTGTCAGCAGTATAATAACTGGCCGCTCAGTTTCGGCGGAGGGACCAAGGTGGAAATCAAA

[0066] The amino acid sequence of the CDR region of the H3-14A7 antibody is shown in Table 1; the nucleic acid sequence of the CDR region of the H3-14A7 antibody is shown in Table 2.

[0067] Table 1

[0068]

[0069] Table 2

[0070]

[0071] 3. Antibody expression and purification

[0072] 3.1 Antibody Cloning and Construction: The antibody heavy chain variable region gene (VH) and the IgG1 constant region gene (CH) were fused into the pCAGGS vector (named pCAGGS-Heavy chain-full length), and the antibody light chain variable region gene (VL) and the constant region gene (CL) were fused into the pCAGGS vector (named pCAGGS-Light chain-full length).

[0073] 3.2 Antibody Expression and Purification: When co-transfecting 100 mL of 293F cells with both light and heavy chain plasmids, 36 μg of heavy chain plasmid and 75 μg of light chain plasmid were dissolved in 1 mL of HBS and allowed to stand for 5 min. 300 μg of PEI was dissolved in 1 mL of HBS and allowed to stand for 5 min. The plasmid and PEI were mixed, allowed to stand for 20 min, and then added to the cells. The cells were cultured in a shaker at 37°C. After 5 days, the supernatant was harvested after centrifugation at 8000 rpm for 90 min. The supernatant was passed through a Protein A affinity column and eluted with 0.1 M Glycine at pH 3.0 using an AKTA Purifier to obtain the antibody. The antibody was then concentrated and the medium was changed to PBS. SD-PAGE was used to detect the purified antibody. The purity was high, with the heavy chain size after dissociation being approximately 50 kDa and the light chain size approximately 25 kDa.

[0074] Example 2: Detection of antibody-antigen affinity using surface plasmon resonance technology

[0075] Surface plasmon response (SPR) assays can be used to detect interactions between proteins in vitro. The kinetic parameters of protein binding are determined by detecting changes in the response value as the mobile phase flows over the chip surface. This is performed on a BIACORE 8K instrument. In SPR assays, one of the two proteins to be analyzed is typically immobilized on a metal chip surface, while the other is used as the mobile phase flowing over the chip surface, and the resulting change in response value is detected. Before the experiment, PBST should be filtered through a 0.22 μM filter and then autoclaved. The antibody concentration is 10 μg / mL (adjustable as needed), and the antigen titers are 400 nM, 200 nM, 100 nM, 50 nM, and 25 nM (adjustable as needed). The characteristics of H3 antigen-antibody affinity assays are described in [link to relevant documentation]. Figure 2 .

[0076] Example 3 Antibody Neutralization Test

[0077] The neutralizing activity of the antibody was detected using the plaque assay, as briefly described below: The initial antibody concentration was 200 μg / mL, serially diluted 4-fold to create 10 gradients. 400 μL of each gradient was mixed with an equal volume of vaccinia virus VAV-WR dilution buffer (containing 10% complement) and incubated at 37°C for 1 hour. A 12-well plate pre-inoculated with Vero cells (at approximately 100% confluence) was removed, and the cells were washed twice. 400 μL of the antibody-virus mixture was added to each well, with two replicates per gradient and a control well containing only virus and no antibody. The plates were incubated at 37°C for 1 hour. The antibody-virus mixture was discarded, and the cells were washed once. 1 mL of fixative was added per well, prepared by mixing methylcellulose and high-glucose medium (2×) at a 1:1 volume ratio. The cell culture plate was placed in a cell culture incubator. After 48 hours, the cells were fixed with 4% tissue cell fixative, stained with crystal violet, counted, and the PRNT50 value of the antibody's neutralizing activity was calculated.

[0078] like Figure 3 As shown, the neutralization curve of H3 antibody against VACV-WR shows that the PRNT50 value of H3-14A7 is 0.135 μg / mL.

[0079] Example 4: Animal Protection Experiment

[0080] To further evaluate the suitability of the antibody, H3-14A7 was injected into mice to assess its protective efficacy at the mouse level. Because animal experiments involving monkeypox virus infection require a high-level biosafety level 3 laboratory, the Western Reserve (VACV-WR) strain of vaccinia virus (belonging to the same family as monkeypoxvirus) was initially used for evaluation. Seven- to eight-week-old mice were infected intranasally with 5LD50 VAV-WR, and 200 μg of the antibody was injected intraperitoneally per mouse 4 hours before and 4 hours after infection.

[0081] like Figure 4 As shown, after infection with the 5LD50 virus, the body weight of mice in the control antibody group decreased by more than 20% on days 6 and 7, respectively, while injection of H3-14A7 antibody could provide 20% protection for mice.

[0082] The technical features of the above embodiments can be combined in any way. For the sake of brevity, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0083] The embodiments described above are merely illustrative of several implementation methods of this application, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of this application, and these all fall within the protection scope of this application. Therefore, the protection scope of this patent application should be determined by the appended claims, and the specification can be used to interpret the content of the claims.

Claims

1. An antibody against monkeypox virus H3 protein, characterized in that, Its heavy chain variable region includes HCDR1 as shown in SEQ ID NO:5, HCDR2 as shown in SEQ ID NO:6, and HCDR3 as shown in SEQ ID NO:7; and its light chain variable region includes LCDR1 as shown in SEQ ID NO:8, LCDR2 as shown in SEQ ID NO:9, and LCDR3 as shown in SEQ ID NO:

10.

2. The antibody according to claim 1, characterized in that, It includes the heavy chain variable region as shown in SEQ ID NO:1, and the light chain variable region as shown in SEQ ID NO:

2.

3. The antibody according to claim 1 or 2, characterized in that, It also includes the heavy chain constant region and the light chain constant region.

4. A nucleic acid molecule, characterized in that, Includes the nucleotide sequence encoding the antibody according to any one of claims 1 to 3.

5. An expression vector comprising the nucleic acid molecule of claim 4.

6. A host cell comprising the nucleic acid molecule of claim 4 or the expression vector of claim 5.

7. The method for preparing the antibody according to any one of claims 1 to 3, characterized in that, Includes the following steps: The host cells of claim 6 are cultured, and antibodies are collected from the resulting cell culture.

8. A detection reagent or detection kit, characterized in that, The antibody includes any one of claims 1 to 3.

9. A drug, characterized in that, The antibody includes any one of claims 1 to 3.

10. The use of the antibody according to any one of claims 1 to 3 in any of the following: (1) Application in the detection of monkeypox virus H3 protein for non-diagnostic purposes. (2) Application in the preparation of detection products for monkeypox virus; (3) Use in the preparation of medicines for the prevention or treatment of monkeypox virus and / or vaccinia virus.

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