Anti-monkey pox virus H3 protein antibody and preparation method thereof

Abstract

The invention relates to an anti-monkey pox virus antibody and a preparation method thereof, and a human monkey pox virus antibody H3-14A7 is obtained by screening memory B cells specifically combined with a fully human antibody humanized mouse anti-H3. The H3-14A7 has the characteristics of strong combining capacity with the monkey pox virus, good in-vitro neutrality, large affinity and in-vivo protection effect.

Description

Technical Field

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

[0002] The monkeypox virus (Mpox virus, MPXV) belongs to the family Poxviridae, subfamily Chordopoxvirinae, and genus Orthopoxvirus (OPXV). The genus Orthopoxvirus has more than 10 members, including variola virus (VARV), vaccinia (smallpox vaccine) virus (VACV), cowpox virus (CPXV), camelpox virus (CMLV), and several new species isolated from infected humans or primates since 2010. Based on the comparison between VACV and MPXV, the surface proteins among poxviruses show approximately 93 - 98% sequence similarity, suggesting that they play similar roles during the process of entering the body.

[0003] In 1958, the monkeypox virus was first discovered in a group of monkeys used for research. In 1970, a 9 - month - old baby boy was diagnosed as the first human case, indicating that the monkeypox virus can infect humans. At this time, the monkeypox epidemic was mainly endemic in countries in the tropical rainforest regions of Africa, and many cases were reported in Central and West African countries, with mortality rates of 3.6% and 10.6% caused by the West African and Central African branches respectively. Although monkeypox has been mainly distributed in Central and West Africa in the past few decades, in recent years, the transmission range of monkeypox has changed, and cases have even emerged in regions outside Africa.

[0004] Monkeypox is a zoonosis, and its natural host has not been clearly identified. It is mainly transmitted among rodents and non - human primates. It can be transmitted through various forms such as contact with damaged skin tissues. In addition, recently, the transmission of the monkeypox virus in non - endemic countries has shown a phenomenon related to sexual contact, and the presence of the monkeypox virus has been detected in body fluids such as semen, but whether it is transmitted through sexual contact is not clear. After infection, symptoms such as swollen lymph nodes, muscle soreness, and permanent scars left after pustules rupture will appear.

[0005] Multiple monkeypox virus strains are spreading in non - endemic countries, with diverse and concealed transmission routes and atypical infection symptoms. We need more research on this virus to better prevent and treat monkeypox.

[0006] To date, neutralizing antibodies have proven to be an effective method for treating 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] It is very important and necessary to develop new monkeypox virus antibodies with strong antigen affinity and good in vitro neutralization. Summary of the Invention

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

[0009] The technical solution includes:

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

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

[0012] In one embodiment, it further includes a heavy chain constant region and a light chain constant region.

[0013] An embodiment of the present application also provides a nucleic acid molecule, including a nucleotide sequence encoding the antibody.

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

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

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

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

[0018] Culturing the host cell and collecting the antibody from the obtained cell culture.

[0019] One embodiment of the present application also provides a detection reagent or a detection kit comprising the antibody.

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

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

[0022] One embodiment of the present application also provides the application of the antibody in preparing a detection product for monkeypox virus.

[0023] One embodiment of the present application also provides the application of the antibody in preparing a drug for preventing or treating monkeypox virus and / or vaccinia virus.

[0024] Compared with the traditional technology, the present application has the following beneficial effects:

[0025] The humanized monoclonal antibody of the present application has strong binding ability to monkeypox virus, good in vitro neutralization, large affinity, and has in vivo protective effect. It is completely different from the reported monkeypox virus antibody sequences, providing products for the detection and neutralization of monkeypox virus, and providing the possibility for providing monkeypox virus products with in vivo protective effect. Description of the Drawings

[0026] In order to more clearly illustrate the technical solutions in the embodiments of the present application and more completely understand the present application and its beneficial effects, the following will briefly introduce the drawings required for the description of the embodiments. Obviously, the following drawings are only some embodiments of the present application, and those skilled in the art can obtain other drawings based on these drawings without creative efforts.

[0027] Figure 1 It is the SDS-PAGE result of the purification molecular sieve for the H3 protein of monkeypox virus;

[0028] Figure 2 It is the kinetic curve result of the H3 antibody and H3;

[0029] Figure 3 It is the neutralization curve result of the H3 antibody against VACV-WR;

[0030] Figure 4 It is a schematic diagram of the experiment and the curves of the survival and weight changes of mice after virus infection. Among them, the upper figure is the schematic diagram of the experiment implementation, and the lower figure is the curves of the survival and weight changes of mice after virus infection. Specific implementation manners

[0031] To make the above objects, features, and advantages of the present application more obvious and understandable, the following will give a detailed description of the specific implementation manners of the present application. Many specific details are set forth in the following description in order to fully understand the present application. However, the present application can be implemented in many other ways different from those described herein. Those skilled in the art can make similar improvements without departing from the connotation of the present application. Therefore, the present application is not limited by the specific embodiments disclosed below.

[0032] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the technical field to which this application belongs. The terms used in the specification of this application herein are only for the purpose of describing specific embodiments and are not intended to limit this application.

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

[0034] Aiming at the existing problems, the present application screens memory B cells that specifically bind to humanized mouse anti-H3 of a fully human antibody, obtains a human-derived high neutralizing activity MPXV antibody H3-14A7, and finally screens out a new humanized monoclonal antibody against monkeypox virus that is specific, has good in vitro neutralization against monkeypox virus, and has strong affinity.

[0035] The specific technical solutions include:

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

[0037] Through the expression and purification of monkeypox virus H3 protein, the sorting of specific memory B cells that bind to H3 protein, and the expression and purification of monkeypox virus H3 antibody, 1 kind of monkeypox virus H3 monoclonal antibody is obtained, namely H3-14A7. The variable region of the light chain and the variable region of the heavy chain of this monkeypox virus monoclonal antibody are different from the existing sequences and are specific.

[0038] II. Performance verification of monoclonal antibody

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

[0040] 2. Surface plasmon resonance technology detects the affinity of the antibody and antigen: H3-14A7 has good affinity with the antigen.

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

[0042] 4. Animal protection assay: In the case of virus infection, the H3-14A7 antibody was administered, and H3-14A7 provided partial protection to mice infected with monkeypox virus.

[0043] The implementation scheme of the present application will be described in detail below in conjunction with the embodiments. It should be understood that these embodiments are only used to illustrate the present application and not to limit the scope of the present application. For the experimental methods without specific conditions in the following embodiments, the guidance given in the present application shall be preferentially referred to, and it can also be carried out according to the experimental manuals or conventional conditions in the art, or according to the conditions recommended by the manufacturer, or by referring to the experimental methods known in the art.

[0044] In the following specific embodiments, for the measurement parameters of the raw material components, if there is no special description, there may be slight deviations within the weighing accuracy range. For the temperature and time parameters, acceptable deviations caused by instrument test accuracy or operation accuracy are allowed.

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

[0046] Key drugs and reagents: Humanized mouse of whole human antibody, model RenMab, Beijing Biocytogen Co., Ltd.; 14K chip, model 750-00021, Berkeley lights.

[0047] Main instrument: Beacon cell 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 gene of H3 was constructed into the eukaryotic expression vector pCAGGS and transfected into 293F cells. The H3 protein was secreted and expressed by the cells. The cell supernatant was collected and purified by affinity chromatography and molecular sieve, and identified by SDS-PAGE.

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

[0052] AAVKTPVIVVPVIDRPPSETFPNVHEHINDQKFDDVKDNEVMQEKRDVVIVNDDPDHYKDYVFIQWTGGNIRDDDKYTHFFSGFCNTMCTEETKRNIARHLALWDSKFFTELENKNVEYVVIIENDNVIEDITFLRPVLKAIHDKKIDILQMREIITGNKVKTELVIDKDHAIFTYTGGYDVSLSAYIIRVTTALNIVDEIIKSGGLSSGFYFEIARIENEMKINRQIMDNSAKYVEHDPRLVAEHRFETMKPNFWSRIGTVAAKRYPGVMYTFTTPLISF

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

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

[0055] The gene of H3 was constructed into the mRNA vector respectively. After in vitro transcription and capping, the concentration was measured and the quality was identified. Humanized mice with full human antibodies (RenMab) were immunized 3 times at a dose of 2.5 μg per mouse. Serum was collected before immunization and 12 days after each immunization to detect the antibody titer. When the antibody titer reached an appropriate level, after a booster immunization, the lymph nodes of the humanized mice were taken, ground into single cells, incubated with antigen molecules, and then specific single B cells were sorted using a Beacon cell sorter. The variable region gene sequences of the antibodies were obtained after reverse transcription and PCR amplification.

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

[0057] Monoclonal antibody H3-14A7:

[0058] Amino acid sequence of the heavy chain variable region, 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 GTGGGACAGAATTCACTCTCACCATCAGCAGCCTGCAGTCTGAAGATTTTGCAGTTTATTACTGTCAGCAGTATAATAACTGGCCGCTCAGTTTCGGCGGAGGGACCAAGGTGGAAATCAAA

[0066] Among them, 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. Expression and purification of the antibody

[0072] 3.1 Antibody cloning and construction: The heavy chain variable region gene (VH) of the antibody was fused with the IgG1 constant region gene (CH) and constructed into the pCAGGS vector (named pCAGGS-Heavy chain-full length), and the light chain variable region gene (VL) of the antibody was fused with the constant region gene (CL) and constructed into the pCAGGS vector (named pCAGGS-Light chain-fulllength).

[0073] 3.2 Antibody expression and purification: When co-transfecting 100 mL of 293F cells with light chain and heavy chain plasmids, take 36 μg of heavy chain plasmid and 75 μg of light chain plasmid, dissolve them in 1 mL of HBS, and let it stand for 5 min. Take 300 μg of PEI and dissolve it in 1 mL of HBS, and also let it stand for 5 min. Mix the plasmid and PEI, let it stand for 20 min, and then add it to the cells. Culture in a constant temperature shaker at 37°C. After 5 days, centrifuge at 8000 r for 90 min and harvest the supernatant. The supernatant is passed through an affinity column Protein A, and the antibody is obtained after elution with 0.1 M Glycine, pH 3.0 using an AKTA Purifier machine, and concentrated and exchanged to PBS. The purified antibody is detected by SD-PAGE, with high purity. The size of the heavy chain after antibody denaturation is approximately 50 kDa, and the size of the light chain is approximately 25 kDa.

[0074] Example 2 Detection of the affinity between antibody and antigen by surface plasmon resonance technology

[0075] Surface plasmon resonance (SPR) experiments can be used to detect the interaction between proteins in vitro, and the kinetic parameters of the binding of two proteins can be known by detecting the change in the response value of the mobile phase flowing through the surface of the chip. It is carried out on the instrument BIACORE 8K. In surface plasmon resonance experiments, usually one of the two proteins to be analyzed is immobilized on the surface of the metal chip, and the other is used as the mobile phase to flow through the surface of the chip to detect the change in the response value it causes. Before the experiment, PBST should be filtered through a 0.22 μM filter membrane and then autoclaved. The antibody concentration is 10 μg / mL (can be adjusted appropriately), and the antigen concentration titers are 400 nM, 200 nM, 100 nM, 50 nM, 25 nM (can be adjusted appropriately). The characteristics of the H3 antigen-antibody affinity detection are shown in Figure 2 .

[0076] Example 3 Antibody neutralization test

[0077] The neutralization activity of the antibody was detected by the plaque method, which is briefly described as follows: The initial concentration of the antibody was 200 μg / mL, and it was serially diluted 4-fold for 10 gradients. 400 μL of each gradient was mixed with an equal volume of the dilution of vaccinia virus VACV-WR (containing 10% complement), and incubated at 37 °C for 1 hour. Take out a 12-well plate pre-inoculated with Vero cells, at this time the cell confluence was ~100%, and the cells were rinsed twice. 400 μL of the mixture of the antibody and the virus was added to each well, and two replicate wells were set for each gradient. A control well with only the virus without the antibody was set, and incubated at 37 °C for 1 hour. Discard the mixture of the antibody and the virus, rinse the cells once, and add 1 mL / well of the fixative, which was prepared by mixing methyl cellulose and high-glucose culture medium (2×) at a volume ratio of 1:1. Place the cell plate in the cell culture incubator. After 48 hours, fix with 4% tissue cell fixative, then add crystal violet for staining and counting, and calculate the neutralization activity PRNT50 value of the antibody.

[0078] As Figure 3 shown, for the neutralization curve result of the H3 antibody against VACV-WR, the PRNT50 value of H3-14A7 was 0.135 μg / mL.

[0079] Example 4 Animal protection test

[0080] To further evaluate the applicability of the antibody, H3-14A7 was injected into mice, and the protective effect was evaluated at the mouse level. Since animal experiments on monkeypox virus infection need to be carried out in a high-level biosafety level 3 laboratory, initially, the vaccinia virus (belonging to the same family of poxviruses as monkeypox virus) Western Reserve (VACV-WR) strain was used for evaluation. Mice aged 7-8 weeks were infected with 5 LD50 of virus VACV-WR by nasal drip, and 200 μg / mouse of the antibody was intraperitoneally injected 4 hours before and 4 hours after infection, respectively.

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

[0082] The technical features of the above-described embodiments can be combined arbitrarily. For the sake of brevity of description, 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, it should be considered as the scope recorded in this specification.

[0083] The above-described embodiments merely represent several implementation manners of the present application. The description thereof is relatively specific and detailed, but it should not be construed as a limitation on the scope of the invention patent. It should be noted that for those of ordinary skill in the art, without departing from the concept of the present application, several modifications and improvements can still be made, and these all fall within the protection scope of the present application. Therefore, the protection scope of the patent of the present application shall be subject to 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: The heavy chain variable region thereof 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 the light chain variable region thereof 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 shown in SEQ ID NO:1, and the light chain variable region shown in SEQ ID NO:

2.

3. The antibody according to claim 1 or 2, characterized in that Also included are the heavy chain constant region and the light chain constant region.

4. A nucleic acid molecule, characterized in that The method comprises a 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 an antibody according to any one of claims 1 to 3, characterized in that: The steps include: Cultivating the host cell according to claim 6, and collecting antibodies in the obtained cell culture.

8. A detection reagent or a detection kit, characterized in that: The invention comprises the antibody according to any one of claims 1 to 3.

9. A drug, characterized in that The invention comprises the antibody according to any one of claims 1 to 3.

10. 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 monkeypox virus detection products; (3) Use in the preparation of drugs for preventing or treating monkeypox virus and / or vaccinia virus.

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